4-arylamidobenzyl substituted 5-bromomethylene-2(5H)-furanones for chronic bacterial infection

Article abstract of DOI:10.1016/j.ejmech.2017.11.085

Bacterial quorum-sensing (QS) can cause bacterial biofilm formation, thus induce antibiotic resistance and inflammation in chronic bacterial infections. A series of novel 4-arylamidobenzyl substituted 5-bromomethylene-2(5H)-furanones were designed by intr

Full text of DOI:10.1016/j.ejmech.2017.11.085

European Journal of Medicinal Chemistry 144 (2018) 164e178
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
4-arylamidobenzyl substituted 5-bromomethylene-2(5H)-furanones
for chronic bacterial infection
Xing-Jun Xu a, , Fang Wang a b , Ting Zeng , Jing Lin , Jun Liu , Yi-Qun Chang ,
1
, ,
1
a
a
,
*
a
a
a
a, **
Ping-Hua Sun , Wei-Min Chen
a
College of Pharmacy, Jinan University, Guangzhou, 510632, PR China
Department of Scientific Research, Qingdao Stomatological Hospital, Qingdao, 266001, PR China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 September 2017
Received in revised form
Bacterial quorum-sensing (QS) can cause bacterial biofilm formation, thus induce antibiotic resistance
and inflammation in chronic bacterial infections. A series of novel 4-arylamidobenzyl substituted 5-
bromomethylene-2(5H)-furanones were designed by introducing of brominated furanones into rosigli-
tazone skeleton, and their potential application in the treatment of chronic bacterial infection was
evaluated with regard to their disruption of quorum sensing and anti-inflammatory activities in vitro as
well as in animal infection model. Compound 2e displayed both potent QS inhibitory activity and anti-
inflammatory activity. Further mechanism studies revealed that the biological effects of 2e and 2k could
11 November 2017
Accepted 27 November 2017
Available online 2 December 2017
Keywords:
be attributed, at least in part, to their interaction with PPAR
g, and consequent suppression of the acti-
Chronic bacterial infections
Quorum sensing inhibitor
Anti-inflammation
vation of NF- B and MAPK cascades. Importantly, pretreatment with 2e significantly protects mice from
k
lethal-dose LPS challenge. Thus, these data suggest that the dual effective derivative 2e may serve as a
PPAR
g
valuable candidate for the treatment of chronic bacterial infection.
©
2017 Elsevier Masson SAS. All rights reserved.
1. Introduction
most gram-negative opportunistic pathogen, such as Pseudomonas
aeruginosa (P. aeruginosa) [4].
The increasing prevalence of persistent chronic bacterial in-
fections, such as venous leg ulcers, diabetic foot ulcers or medical
device-related infection, which frequently lead to high morbidity
and mortality [1,2], calls for the development of new therapeutic
strategies. Chronic wounds often harbor bacterial infections, and
bacterial resistance that caused by biofilm formation is rather
common in chronic wound infections. In general, biofilm is an
extracellular polymeric matrix consisting of proteins, lipids, uncleic
acids and polysaccharides, which could protect bacteria from the
host immune response and cause high intrinsic resistance
On the other hand, it is well-known that bacterial endotoxin
such as lipopolysaccharides (LPS), a component of the cell wall of
gram-negative bacteria, as well as 3-oxododecanoyl homoserine
lactone (3OC12-HSL), a signaling compound in QS circuits, are the
potent microbial initiators of inflammation [5]. Thus, long-term
bacterial infection accompanied by biofilm often induces aggra-
vated inflammation in host tissues that with chronic wound in-
fections, and subsequently lead to chronic inflammatory responses
and irreversible organ damage [6]. Consequently, anti-
inflammation is also an important aspect for the treatment of
chronic bacterial infections.
(
10e1000 fold higher tolerance) against various antibiotic agents
and thus result in the extremely difficult to eradicate the chronic
wound infections [3]. Quorum-sensing (QS) circuits, including Las,
Rhl, and Pqs pathways are the core systems that responsible for the
regulation of biofilm formation and virulence factors secretion in
Accumulating studies have shown that substance with both QS
inhibitory and anti-inflammatory effects could be effective in the
treatment of chronic bacterial infections. LL-37, a human host de-
fense peptide, which could modulate inflammatory responses by
inhibiting the release of the proinflammatory cytokine tumor ne-
crosis factor
a (TNF-a) in LPS-stimulated human monocytic cells,
*
Corresponding author.
and inhibit the formation of bacterial biofilms in vitro, was proved
to be effective in enhancing healing of hard-to-heal venous leg
ulcers. The clinical applicability of this rather large compound has
already been demonstrated in a phase I and II clinical trial showing
** Corresponding author.
E-mail addresses: linjing_jnu@163.com (J. Lin), twmchen@jnu.edu.cn
(
W.-M. Chen).
1
These authors contributed equally.
https://doi.org/10.1016/j.ejmech.2017.11.085
223-5234/© 2017 Elsevier Masson SAS. All rights reserved.
0

X.-J. Xu et al. / European Journal of Medicinal Chemistry 144 (2018) 164e178
165
-
promising results for topical treatment of patients with chronic leg
paper, in order to develop preferable agents for the treatment of
chronic bacterial infections, molecular backbone replacements
have been carried out by scaffold hopping with rosiglitazone. As
shown in Fig. 1, the drug-design strategy involved: (a) the thiazo-
lidinedione moiety of rosiglitazone being replaced by brominated
furanyl group, which is an important pharmacophore with QS
inhibitory activity that was identified in our earlier work [21e23];
(b) the section C being replayed with a substituted benzene ring or
aromatic heterocyclic ring to increase the structural diversity of the
compounds; (c) the section C and B being linked with an amide
bond with the aim of improving the solubility of the compounds.
Synthesis of compounds. The synthesis of the designed com-
pounds in Fig. 1 was accomplished as outlined in Scheme 1 (1a-1m
and 2a-2m). In general, the target compounds were obtained via a
seven-step strategy. Firstly, the starting materials, benzylacetone
and glyoxylic acid were converted into (E)-3-benzyl-4-oxopent-2-
enoic acid (1) via the cross Aldol condensation in orthophos-
phoric acid [26]. Then, 3-benzyl-4-oxopentanoic acid (2) was pre-
pared from the reaction of compound 1 with zinc dust as reducing
agent in acetic acid. Compound 2 reacted with fuming nitric acid to
yield 3-(4-nitrobenzyl)-4-oxo-pentan-oic acid (3) intermediates via
a nitration reaction [27]. Bromination of compound 3 was easily
accomplished, and the product was then reacted with phosphorus
pentoxide to give the intermediate 5-(dibromomethylene)-4-(4-
nitrobenzyl)furan-2(5H)-one (4) and (Z)-5-(bromomethylene)-4-
(4-nitrobenzyl)furan-2(5H)-one (5) in dry dichloromethane
[26,28]. The subsequent synthetic procedure typically involved the
reduction of the nitro group to form the 4-(4-aminobenzyl)-5-
(dibromomethylene)- furan-2(5H)-one (6) and (Z)-4-(4- amino-
benzyl)-5-(bromomethylene)furan-2(5H)-one (7) using reduced
iron powder and ammonium chloride in acetone under a nitrogen
atmosphere [29,30]. Finally, compound 6 or 7 was connected with
different acyl chloride derivatives via acylation with dry pyridine as
base in dichloromethane to form the amide end-products [31,32] as
shown in Scheme 1.
ulcers [7e9]. Furthermore, treatment with honey, which was vali-
dated with both anti-microbial and anti-inflammatory activity
[10e12], has presented significantly healing and contraction effects
on burns [13,14]. Thus, development of compounds that with both
QS inhibitory and anti-inflammatory activities may be an effective
therapeutic strategy for the treatment of chronic bacterial
infections.
It has been reported [15e18] that rosiglitazone, a well-known
peroxisome proliferator-activated receptor
shown significant anti-inflammatory properties because of its
ability to activate PPAR , and consequently to suppress nuclear
factor B (NF- B), leading to an alteration in the expression of pro-
inflammatory genes such as TNF- and interleukin-6 (IL-6). More-
over, the proinflammatory effect of 3OC12-HSL, a singnalling com-
pound in QS sircuits, in lung epithelial cells can be blocked by the
PPARg agonist rosiglitazone [19], Which suggests that rosiglitazone
is likely to be a potential leading compound for the development of
agents used in the treatment of inflammation caused by bacterial
infections. Meanwhile, most QS systems of gram-negative bacteria
employ N-acyl homoserine lactones (AHL) as the signaling molec-
ular to control biofilm formation [16,17], and it has been reported
that compounds bearing the AHL analogue moiety-halogenated
furanone have excellent QS inhibitory activities [20]. Our previous
work showed that compounds bearing a brominated furanone
moiety have excellent QS inhibitory activity [21e23]. Based on the
above facts, we hypothesized that introduction of brominated fur-
anones into rosiglitazone by scaffold hopping strategy may lead to
compounds potentially with both anti-inflammatory and QS
inhibitory activity.
g (PPARg) agonist, has
g
k
k
a
Therefore, in the present study, using a scaffold hopping strat-
egy,
a
series of novel 4-arylamidobenzyl substituted 5-
bromomethylene-2(5H)-furanones were designed by introducing
of brominated furanones (QS inbibitory moiety) into rosiglitazone
skeleton (anti-inflammatory agent) (Fig. 1), and their potential
application in the treatment of chronic wound infection were
evaluated with regard to their QS inhibitory and anti-inflammatory
activities in vitro as well as in animal infection model. Western blot
experiments and docking studies were also performed in an
attempt to clarify the anti-inflammation mechanism.
A notable problem from a chemical point of view was that Z and
E geometrical isomers around the exocyclic double bond (CH]C)
1
were possible in compounds 2a-2m. The H NMR spectrum of 2a-
2m had only one signal for the 5-(bromomethylene)furan-2-one
proton showing that the chemical shifts were basically in the
same range from 6.24 to 6.27 ppm for 2a, 2h, 2i (in deuterated
dimethylsulfoxide solution) and from 6.06 to 6.14 ppm for 2b-2g,
2
. Chemistry
2j-2m (in deuterated acetone solution). It has been reported that
the chemical shift value for the exocyclic olefinic proton in 5-
Design of compounds. Scaffold hopping (also termed chemo-
(
bromomethylene)furan-2-ones are characteristic for either the
Z)-isomer (typically 6.24 ppm) or the (E)-isomer (typically
6.57 ppm) [33]. Additionally, the Z-configuration had been re-
ported to be thermodynamically more stable than the E-
type switching), a medicinal chemistry method for molecular
backbone replacements, is an integral component of the drug dis-
covery process and is an effective drug-design strategy for devel-
opment of novel molecules with potent activity [24,25]. In this
(
d
d
Fig. 1. Drug design by scaffold hopping strategy from rosiglitazone.

166
X.-J. Xu et al. / European Journal of Medicinal Chemistry 144 (2018) 164e178
△
△
ꢀ
Scheme 1. General synthesis of compounds 1a-1m and 2a-2m
.
Reagents and conditions: (a) phosphoric acid, 85 C, 4 h; (b) zinc dust, AcOH/H
2
O, reflux; (c) fuming nitric
acid, ꢁ20 ꢀC; (d) Br
, CHCl
, reflux; (e) P
O, reflux; (g) Et
N, DCM, 0 C to r.t.
ꢀ
2
3
2
O
5
, DCM, reflux; (f) Fe, NH
4
Cl, acetone/H
2
3
configuration [34,35]. It is also important to note that the alkyl
chain at C4 of 5-(bromomethylene)furan-2-one discourages for-
mation of the transition state leading to the (E)-isomer, due to steric
clashes with the exocyclic bromine atom. These results therefore
suggest that the preferred configuration of synthesized compounds
was the Z configuration.
3. Results and discussion
Inhibition of Quorum Sensing Systems on P. Aeruginosa. It is
becoming increasingly clear that control of bacterial quorum
sensing development is a potential approach to reduce of biofilm
associated antibiotic resistance and chronic inflammation occur-
ring in infectious diseases [36]. In this paper, all of the designed
compounds were consequently firstly evaluated for their QS
inhibitory activity using QS monitors lasB-gfp, rhlA-gfp, and pqsA-
gfp which can indicate the activity of the promoter of lasB, rhlA, and
All the synthesized compounds were fully analyzed and char-
acterized by 1H, C nuclear magnetic resonance (NMR), mass
spectrometry (MS) and high resolution mass spectrometry (HRMS)
and HPLC purity analysis before beginning biological evaluation.
13

X.-J. Xu et al. / European Journal of Medicinal Chemistry 144 (2018) 164e178
Table 1
167
pqsA gene based on expression of green fluorescence protein (GFP)
Inhibition rates of the synthesized compounds to the expression of gfp(ASV) in
PAO1-lasB-gfp, PAO1-rhlA-gfp, and PAO1-pqsA-gfp reporter strains.
in P. aeruginosa PAO1. At here, P. aeruginosa, which can cause a wide
range of infections and inflammations through the formation of
biofilm in a variety of hosts thus were chosen as the model strain
37]. The experiments were performed using a fixed concentration
of 10 М, and rosiglitazone and the brominated-2(5H)-furanone
Fig. 2) were taken as standard drugs.
As depicted in Table 1, the results of QS inhibitory activity
reflexed by the gfp (ASV) expression levels in QS reporter strains
showed that all the 4-arylamidobenzyl substituted 5-
bromomethylene-2(5H)-furanones (1a-1m and 2a-2m) almost
exhibited an improved inhibitory effect on all the three QS systems
of P. aeruginosa compared to that of the reference compound rosi-
glitazone. Notably, some newly synthesized compounds even
showed a better QS inhibitory activity than the positive control
agent brominated-2(5H)-furanone, especially for pqs system, in
which, compounds 2e and 2h displayed promising QS inhibitory
activities in pqs system, with the inhibition rates up to
Compound
Fura
PAO1-lasB-gfp
PAO1-rhlA-gfp
PAO1-pqsA-gfp
[
a
21.43 ± 2.55
7.43 ± 1.40
*
24.03 ± 1.08
15.51 ± 0.07
*
20.19 ± 3.03
9.19 ± 0.24
*
Rosib
m
1a
2a
1b
2b
21.19 ± 0.41*
20.25 ± 4.34*
28.23 ± 0.87
34.54 ± 3.20
34.64 ± 4.04
20.37 ± 3.21
26.44 ± 1.91
20.33 ± 1.02
24.83 ± 2.00
25.82 ± 1.72
19.99 ± 3.01
50.29 ± 1.07
20.55 ± 1.15
26.44 ± 1.91
24.50 ± 2.86
29.83 ± 1.02
27.92 ± 2.93
40.75 ± 6.42
19.29 ± 2.29
17.62 ± 2.53
22.89 ± 0.25
20.60 ± 1.20
16.22 ± 0.19
23.09 ± 1.44
21.92 ± 3.99
24.30 ± 1.17
19.53 ± 3.11
20.28 ± 2.37
**
(
*
*
16.13 ± 0.40
17.20 ± 2.47
22.89 ± 0.31*
23.57 ± 4.57*
**
*
17.33 ± 0.81
19.97 ± 5.04*
10.22 ± 0.92
19.21 ± 1.01*
18.01 ± 2.92*
9.49 ± 3.29
15.20 ± 2.05
11.76 ± 2.29
14.75 ± 0.37
14.22 ± 0.47
19.02 ± 4.92*
10.41 ± 2.38
24.87 ± 4.61*
19.13 ± 2.49*
16.91 ± 4.51
11.01 ± 3.21
14.94 ± 0.01
9.48 ± 5.47
13.64 ± 2.48
19.57 ± 3.65
23.93 ± 2.20*
19.22 ± 2.93*
17.28 ± 1.82
15.93 ± 0.92
14.71 ± 4.97
13.77 ± 4.27
11.81 ± 3.49
16.37 ± 3.30
**
*
1c
2c
*
1d
2d
1e
2e
**
**
*
1f
*
*
*
2
1
2
2
f
g
g
h
*
**
**
**
*
18.22 ± 3.92
20.01 ± 2.49*
5
0.29 ± 1.07% and 40.75 ± 6.42% respectively. Our results suggest
2h
1i
17.17 ± 3.87
19.20 ± 2.30*
15.17 ± 2.88
19.50 ± 2.63*
that introduction of a halogenated furanone, e.g. a brominated
furanone into the molecule would increase its QS inhibitory
activity.
2
1
2
1
i
j
j
k
*
*
15.25 ± 3.61
Inhibition of NO Production in (LPS)-Stimulated RAW264.7
Cells [38,39]. On the purpose of designing dual-effect molecular
that with both QS inhibitory activity and anti-inflammatory effect
to overcome chronic bacterial infection problems, next, the anti-
inflammatory activity of all the synthesized compounds were
therefore evaluated for their inhibitory activity against lipopoly-
saccharide (LPS)-induced nitric oxide (NO) release in
RAW264.7 cells for NO has been widely recognized as a ubiquitous
pro-inflammatory mediator that plays key roles in various
inflammation-associated diseases. The macrophages were pre-
treated with compounds for 2 h, then LPS was added and the sys-
tem was incubated for 48 h. The amount of NO was determined by
the Griess method. Rosiglitazone and indomethacin (Fig. 2) were
taken as standard drugs for anti-inflammation studies.
As depicted in Table 2, most of the 4-arylamidobenzyl
substituted 5-bromomethylene-2(5H)-furanones (1a-1m and 2a-
m) displayed improved NO inhibitory activity compared to rosi-
glitazone and indomethacin. In particular, compounds 2e and 2k
demonstrated a reasonably good anti-inflammatory activity at a
concentration of 10 mM. Their NO inhibition rates were 73.7 ± 3.3%
18.07 ± 7.62*
*
*
*
*
*
2k
11.84 ± 4.32
5.03 ± 0.92
12.23 ± 6.66
9.79 ± 1.17
12.02 ± 1.09
12.93 ± 1.48
10.56 ± 2.01
7.66 ± 5.99
5.57 ± 3.44
1
2
1
2
l
l
m
m
8.53 ± 0.79
*
*P < 0.01, *P < 0.05 versus the blank (cultured with bacteria only) group.
Results are shown as means ± SD (n ¼ 3) of three independent experiments.
a
Fura: brominated-2(5H)-furanone.
Rosi: rosiglitazone.
b
benzyl-5-monobromomethylene-furan-2(5H)-one structure is
conducive to improvement of anti-inflammatory activity (2a-2m
compared to 1a-1m, for example, 2a compared to 1a); (b) the anti-
inflammatory activity increases gradually with an increase of the
length of carbon chain between the ring B and ring C (Fig. 1), and a
two-atom linker could be the most favorable (2j compared to 2d
and 2a). Moreover, the introduction of a double bond increased the
activity further (2k compared to 2j); (c) if C ring is substituted with
2
a
heterocyclic ring rather than a benzene ring, the anti-
inflammatory effect is better (2b and 2c compared to 2a); (d)
electron-withdrawing substituents (fluorine, chlorine or bromine)
in the phenyl ring C would increase the NO inhibitory activity, and
introduction of fluorine led to the best activity (2e, 2h and 2i
and 70.0 ± 3.5%, respectively.
Based on these results, some preliminary structure-activity re-
lationships (SAR) of the 4-arylamidobenzyl substituted 5-
bromomethylene-2(5H)-furanones can be summarized: (a) the 4-
Fig. 2. Structures of rosiglitazone, indomethacin, brominated-2(5H)-furanone, 2e and 2k.

168
X.-J. Xu et al. / European Journal of Medicinal Chemistry 144 (2018) 164e178
Table 2
inhibitory activities were further confirmed by evaluating their
in vitro TNF- and IL-6 inhibitory effects using TNF- and IL-6
specific enzyme linked immunosorbent assay (ELISA) experiment
in LPS-induced RAW264.7 cells. Rosiglitazone and indomethacin
(Fig. 2) were used as positive reference drugs. In the current study,
both these two experiments were performed using a fixed con-
centration of 10 mM and in a time-dependent manner.
The results are shown in Table 4 and Fig. 3. It was shown that 2e
and 2k induce a significant decrease in both the LPS-induced pro-
The inhibitory effects of the synthesized compounds on NO production in LPS-
stimulated RAW264.7 cells.
a
a
Compounds
Rosib
NO inhibition (%)a
Compounds
NO inhibition (%)a
**
Indoc
2a
2b
2c
2d
2e
2f
2g
2h
2i
**
16.6 ± 3.8
29.4 ± 4.7
46.2 ± 2.9
59.9 ± 5.8
54.2 ± 4.3
59.5 ± 3.7
73.7 ± 3.3
39.3 ± 4.2
29.4 ± 5.0
64.9 ± 3.0
66.6 ± 3.8
64.8 ± 5.2
70.0 ± 3.5
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
**
1
1
1
1
1
1
1
1
1
1
1
1
1
a
b
c
d
e
f
g
h
i
30.3 ± 3.7
44.5 ± 9.1
39.9 ± 6.6
44.1 ± 2.9
59.2 ± 3.7
25.2 ± 3.6
19.0 ± 3.7
50.7 ± 2.9
53.5 ± 3.3
48.1 ± 4.6
54.4 ± 4.3
44.3 ± 6.4
21.2 ± 1.8
**
**
**
**
**
duction of TNF-
exhibited evidently weaker inhibitory effects on either TNF-
production compared with those of 2e and 2k at all three
detection times. For example, without treatment, after induction by
LPS for 24 h, the concentration of TNF- in RAW264.7 cells was
24216.0 ± 498.7 pg/mL, while the concentration of TNF- was
decreased to 18322.0 241.6 pg/mL (p 0.01) and
a
and IL-6, while the positive control drugs
**
a
or IL-
**
6
**
**
j
k
l
2j
2k
2l
**
a
**
61.0 ± 5.3
a
**
m
2m
34.3 ± 2.7
±
<
*
*
*
P < 0.01.
P < 0.05 versus the LPS (treated with LPS only) group.
Results were showed as means ± SD (n ¼ 4) of at least three independent
19550.0 ± 292.1 pg/mL (p < 0.01) respectively in 2e and 2k pre-
treated RAW264.7 cells. These concentrations were evidently
lower than those in cells pre-treated with the standard drugs
a
experiments.
b
rosiglitazone and indomethacin, whose TNF-
22226.0 ± 588.0 pg/mL (p < 0.05) and 20772.0 ± 558.9 pg/mL
0.01), respectively (all values vs. LPS alone,
4216.0 ± 498.7 pg/mL). Moreover, the experimental data show
that 2e and 2k also possess remarkable inhibitory activity on LPS-
induced IL-6 production and on TNF- production. As shown in
a levels were
Rosi: rosiglitazone.
Indo: indomethacin.
c
(p
<
p
2
compared to 2d); (e) an electron-donating substituent (methyl or
methoxyl) in the phenyl ring C decreases it's NO inhibitory activity
a
(2f, 2g and 2m compared to 2d). Consequently, these results will be
Table 4, rosiglitazone and indomethacin inhibited IL-6 production
from 407.6 ± 16.7 pg/mL (LPS alone) to 345.6 ± 25.6 pg/mL
(p < 0.05) and 286.6 ± 19.5 pg/mL (p < 0.01) respectively at 24 h,
while 2e and 2k induced a decrease in IL-6 production to the
concentration of 145.2 ± 16.6 pg/mL (p < 0.01) and 158.2 ± 19.1 pg/
useful in the future to guide the design and modification of new
anti-inflammatory agents.
Cytotoxicity in RAW264.7 Cells. To investigate whether the
effects of 2e and 2k on suppression of the production of NO were
related to cell viability, these compounds were further exposed to
RAW264.7 macrophages to explore their cytotoxicity using a
methyl thiazolyl tetrazolium (MTT) assay. The results of the cyto-
toxicity assay are shown in Table 3 and clearly indicate that all the
agents (rosiglitazone, indomethacin, 2e, 2k, GW9662 and LPS) at
the concentrations measured here had no obvious cytotoxicity, and
the relative cell viabilities of the treated cells were all more than
mL (p
< 0.01), respectively (all p values vs. LPS alone,
407.6 ± 16.7 pg/mL). From the above results, it becomes increas-
ingly clear that 2k, and especially 2e, are excellent anti-
inflammatory agents, which prompted us to investigate the
possible anti-inflammatory mechanism of 2e and 2k by analyzing
inflammatory-associated signaling pathways.
Western Blot Analysis for Interpretation of Possible Mecha-
nisms. NF-kB pathway is an important signaling pathway associ-
9
5%. The NO inhibitory effects of 2e and 2k could probably be
attributed to the interaction of these two compounds with their
specific target. Accordingly, on the basis of cellular viability in vitro,
these non-toxic concentrations were used and evaluated in the
following experimental processes.
ated with the process of inflammation [43]. Generally, LPS-induced
Table 4
The inhibitory effects of rosiglitazone, indomethacin, 2e and 2k on LPS-induced
TNF-a and IL-6 production in RAW264.7 cells.
Inhibition of TNF-
a and IL-6 Production in RAW264.7 Cells.
TNF- and IL-6 are two crucial pro-inflammatory mediators, their
a
TNFꢁa (pg/mL)a
blockers are recognized as effective agents for the treatment of
Compounds
inflammatory disease [40,41]. The ability of an agent to inhibit the
6
h
12 h
24 h
LPS-induced increases in TNF-a and IL-6 production is an important
Blank
LPS
135.3 ± 20.1
174.7 ± 37.8
238.0 ± 45.4
criterion with which to assess the potential anti-inflammatory ef-
fects of drug candidates [42]. Consequently, the anti-inflammatory
effects of compounds 2e and 2k that showed significant NO
##
##
##
*
11922.0 ± 628.4
11311.3 ± 823.0
10850.0 ± 691.5
7524.7 ± 50.6
7750.0 ± 89.4
21561.3 ± 857.0
20616.0 ± 655.7
19664.0 ± 576.1
13362.7 ± 129.4
24216.0 ± 498.7
22226.0 ± 588.0
20772.0 ± 558.9
18322.0 ± 241.6
19550.0 ± 292.1
b
LPS þ Rosi
c
*
**
**
**
LPS þ Indo
*
*
**
LPSþ2e
LPSþ2k
**
**
15037.3 ± 1033.9
IL-6 (pg/mL)a
Table 3
6
h
12 h
24 h
Effects of compounds on the viability of RAW264.7 cells.
Cell viability (%)a
Blank
LPS
1.2 ± 0.3
1.7 ± 0.2
175.2 ± 10.2
140.0 ± 6.2
120.7 ± 2.7
66.3 ± 17.8
75.3 ± 20.3
3.1 ± 1.1
407.6 ± 16.7
345.6 ± 25.6
286.6 ± 19.5
145.2 ± 16.6
158.2 ± 19.1
Compounds
Rosib
Concentrations
#
#
##
##
94.6 ± 5.9
64.8 ± 4.2
59.6 ± 6.1
17.0 ± 1.2
19.6 ± 1.7
b
**
**
**
*
10
10
10
10
mM
mM
mM
mM
99.0 ± 6.9
100.8 ± 7.5
97.0 ± 4.6
103.7 ± 5.0
99.1 ± 6.3
99.4 ± 4.7
103.6 ± 3.0
LPS þ Rosi
c
c
**
**
Indo
LPS þ Indo
*
*
*
*
**
**
2e
LPSþ2e
LPSþ2k
**
**
2k
GW9662
LPS
LPS
5 mM
500 ng/mL
100 ng/mL
#
#
#P < 0.01.
P < 0.05 versus the blank (cultured with fresh medium only) group.
P < 0.01.
**
a
*
Results were expressed as means
±
SD (n
¼
4) of three independent
P < 0.05 versus the LPS (treated with LPS only) group.
a
experiments.
Results were showed as means ± SD (n ¼ 3) of three independent experiments.
b
c
b
c
Rosi: rosiglitazone.
Indo: indomethacin.
Rosi: rosiglitazone.
Indo: indomethacin.

X.-J. Xu et al. / European Journal of Medicinal Chemistry 144 (2018) 164e178
169
Fig. 3. The effects of rosiglitazone, indomethacin, 2e and 2k on the LPS-induced production of TNF-a and IL-6 in RAW264.7 cells. RAW264.7 cells were treated with rosiglitazone
M) and LPS (100 ng/mL) for 6 h, 12 h and 24 h. Data are presented as means ± SD (n ¼ 3). P < 0.01, #P < 0.05 versus the
##
(
10 mM), indomethacin (10 mM), 2e (10 mM) and 2k (10 m
**
*
blank (cultured with fresh medium only) group; P < 0.01, P < 0.05 versus the LPS (treated with LPS only) group.
activation of NF-
kappa B alpha (I
degradation of I
plays a key regulatory role in the occurrence of inflammation
44e46]. Meanwhile, the activation of MAPK pathways which
mainly involve extracellular regulated protein kinases (ERK1/2, or
p42/44) and p38 MAPK is also often observed in LPS-stimulated
macrophages. It is well accepted that the up-regulation of the
synthesis of inflammatory mediators induced by LPS, involves NF-
k
k
k
B pathway involving phosphorylation of inhibitor
B- ) kinase, and subsequently ubiquitination and
B- , as well as the phosphorylation of NF- B p65,
As shown in Fig. 4, phosphorylation of NF-kB p65, IkBa, ERK1/2
a
a
and p38 MAPK was significantly increased by treatment with LPS,
which is commonly used as a pro-inflammatory stimulus, and the
levels of the phosphorylation forms of these proteins decreased in
varying degrees by treatment with 2e and 2k. Specifically, we found
that 2k, and especially 2e significantly inhibits the phosphorylation
k
[
of NF-
compared with LPS-induced macrophages, while 2e and 2k had a
slightly antagonistic effect on phosphorylation of I . Additionally,
to determine whether PPAR participates in the influence of the
phosphorylation of these proteins, a highly selective and irrevers-
ible PPAR antagonist GW9662 [52], was used prior to treatment
with 2e or 2k. Application of GW9662 (5 M) can reverse the
suppressive effects of 2e and 2k on the protein phosphorylations,
which suggests that PPAR plays a role in the regulation of phos-
kB p65, IkBa, ERK1/2 and p38 MAPK protein expression when
kBa
k
B and MAPK activation, and is modulated by PPAR
g, and the MAPK
g
and NF- B activation can be inhibited by pre-treatment with PPAR
k
g
activator, such as rosiglitazone [47e51]. Therefore, in the present
study, to determine whether the anti-inflammatory effects of 2e
and 2k are associated with the PPARg mediated activation of NF-kB
and MAPK as leading compound rosiglitazone, the expression
levels of these two pathways related proteins in RAW264.7 cells,
with and without treatments by 2e or 2k were analyzed.
g
m
g
phorylation of related proteins.
In view of these findings, it may be speculated that anti-

170
X.-J. Xu et al. / European Journal of Medicinal Chemistry 144 (2018) 164e178
Fig. 4. The effects of compounds 2e and 2k on a LPS-induced phosphorylation of NF-
k
B p65, I
B p65, IkBa, ERK1/2, and p38 MAPK proteins, and their phosphorylated forms were analyzed
** *
kBa, ERK1/2 and p38 MAPK in RAW264.7 cells. RAW264.7 cells were treated with 2e
(
10
m
M), 2k (10
mM), GW9662 (5
m
M), and LPS (500 ng/mL) for 4 h. The levels of NF-
k
using western blotting. Data are presented as means ± SD (n ¼ 3). P < 0.01, #P < 0.05 versus the blank (cultured with fresh medium only) group; P < 0.01, P < 0.05 versus the
#
#
LPS (treated with LPS only) group.
inflammatory effects of 2e and 2k were due at least in part, to their
interaction with PPAR , thereby suppressing activation of the NF-
B and MAPK cascades, and leading to NO, TNF- and IL-6 levels
being decreased in LPS-stimulated RAW264.7 macrophages.
Nonetheless, further studies are needed to confirm this conjecture.
Docking Analysis. To confirm the above speculation that 2e and
known PPAR
structure information of PPAR
was obtained from the Protein Data Bank (PDB ID: 2PRG). Table 5
showed the docking scores of the four tested molecules with
PPARg.
g
activators, were used as reference molecules. The
protein used in the docking studies
g
g
k
a
Interestingly, the docking scores correlated well with the
2
k were likely to act as PPAR
g
agonists, docking studies were
pharmacological testing results, the compounds 2e and 2k showing
performed using the SYBYL 8.1 (Tripos, Inc., St. Louis, MO, USA)
program package to investigate the interaction of 2e and 2k with
high docking scores with PPAR
glitazone and indomethacin. These results imply the possibility of
the direct interaction of 2e and 2k with PPAR . Furthermore, the
g, even higher than those for rosi-
PPARg. Here, rosiglitazone and indomethacin, which are two
g

X.-J. Xu et al. / European Journal of Medicinal Chemistry 144 (2018) 164e178
171
Table 5
Docking scores for the combination of rosiglitazone, indomethacin, 2e and 2k to
PPAR
modes between 2e, 2k and PPAR
g
, which reveals the strong binding
ability of 2e and 2k to PPAR
g, and provides strong evidence for the
g.
assumption that PPAR
g was at least one of the specific targets of 2e
Compounds
Scores
Rosia
6.94
Indob
4.92
2e
2 k
and 2k.
7.96
7.71
Pretreatment with 2e Protects Mice from Lethal-Dose LPS
Challenge. Before the animal experiment, compounds 2e and 2k,
which exhibit the best anti-inflammatory activity were evaluated
the growth effect of 2e and 2k on P. aeruginosa strain PAO1. Just as
we expected, 2e and 2k would not affect the growth of bacteria,
a
Rosi: rosiglitazone.
Indo: indomethacin.
b
MOLCAD (Molecular Computer Aided Design) program was
employed to visualize the binding mode between rosiglitazone,
even at 100 mM Fig. 7 (A B). It means that our compounds may
hardly emerge tolerance since it will not threaten germs' living. We
further explored by a dose-dependent assay for their QS inhibitory
activities used the most effected QS system-pqs. As shown in
Fig. 7(C D), in line with the screening results, 2e displayed excellent
inhibitory activity on the expression of pqs-gfp in a concentration
dependent manner, while 2k almost has no effect on QS system.
Therefore, 2e with both good QS inhibitory activity and anti-
inflammatory activity was forward to next animal model evalua-
tion. In this study, the mouse model of LPS-injection-induced sepsis
shock [53,54] was employed to determine whether 2e can be able
to attenuate endotoxin shock through inhibition of LPS-induced
inflammatory response. Mice were daily treated with rosiglita-
zone or 2e or LPS. On day 5, LPS (40 mg/kg) was intravenously
intraperitoneally injected 1 h after the last administration. Survival
rates are shown in Fig. 8. LPS (40 mg/kg) administration resulted in
the death rate of 82% within one day, and death of all the mice in
the model group within 2 days. In the positive group, administra-
tion of rosiglitazone (20 mg/kg, 1 mg/kg) shows the 45.5% and
36.5% survival rate on day 7. Similarly, administration of 2e (high
dose: 20 mg/kg and low dose: 5 mg/kg) also significantly improved
indomethacin, 2e and 2k and the PPAR
MOLCAD cavity depth (CD) potential surfaces structure between
the PPAR -binding pocket and four compounds. The cavity depth
g pocket. Fig. 5 displayed the
g
color ramp ranged from blue (low depth values ¼ outside of the
pocket) to orange (high depth values ¼ cavities deep inside the
pocket). Fig. 6 shows the hydrogen bonding of these four com-
pounds with PPAR
dashed lines.
g, and the hydrogen bonds are showing as dark
As shown in Fig. 5, four ligands are able to bind to PPAR
g
at
different depths. Rosiglitazone and indomethacin were mostly
located in a blue region, which indicates a relatively low depth,
while compounds 2e and 2k were in a light yellow region with
good flexibility, which revealed that the majority parts of these two
molecules were anchored deep inside the pocket. In addition, as
shown in Fig. 6, several strong hydrogen bonds were formed be-
tween 2e, 2k and PPARg. Compound 2e formed H-bonds with the
imino groups of residues Ser289 and Arg288, and 2k formed H-
bonds with the Cys285 and Ser342 residues.
Taken together, the docking studies highlighted the binding
Fig. 5. The MOLCAD Multi-Channel surface structures displayed with cavity depth of the PPARg within the (A) rosiglitazone, (B) indomethacin, (C) 2e and (D) 2k. The color ramp for
cavity depth ranged from blue (outside of the pocket; most negative) to orange (deep inside the pocket; most positive).

172
X.-J. Xu et al. / European Journal of Medicinal Chemistry 144 (2018) 164e178
Fig. 6. Hydrogen bonds formed between PPAR
g
and (A) rosiglitazone, (B) indomethacin, (C) 2e and (D) 2k. Hydrogen bonds (HB) are shown as dark dashed lines. Hydrogen atoms
are omitted for clarity.
Fig. 7. (A) (B) Growth curves of compound 2e and 2k incubated with the PAO1, (C) (D) Dose-dependent inhibition curves of compound 2e and 2k incubated with the QS monitors
PAO1-pqsA-gfp.
the survival rate of the LPS-injected mice (45.5% and 36.5% survival
on day 7 in high dose group and in low dose group, respectively,
P < 0.05 in both groups vs. LPS group). Thus, pretreated with 2e can
prolong survival in LPS-induced acute inflammatory.
4. Conclusion
In this study, a series of novel 4-arylamidobenzyl substituted 5-
bromomethylene- 2(5H)-furanones have been designed and

X.-J. Xu et al. / European Journal of Medicinal Chemistry 144 (2018) 164e178
173
Procedure for Synthesis of (E)-3-benzyl-4-oxopent-2-enoic
acid (1). Glyoxylic acid (15.5 g, 0.21 mol) and benzylacetone
(
94.4 mL, 0.63 mol) were added to orthophosphoric acid (30 mL).
ꢀ
The mixture was heated at 85 C for 4 h and then stirred at room
temperature overnight. Water (30 mL) was added to the mixture
which was extracted with EtOAc (3 ꢂ 100 mL). The combined
organic layer was washed with H
2
O and brine, dried over anhy-
drous Na SO and evaporated to give dark brown oil. The crude
2
4
product was chromatographed on a silica gel column using petro-
leum ether/EtOAc/AcOH (5:1:0.03). Final product 1 was obtained as
1
a pale yellow oil (22.3 g, 52.1%). H NMR (300 MHz, acetone-d
6
)
d
7.35 (d, J ¼ 4.4 Hz, 4H), 7.30e7.16 (m, 1H), 6.94 (s, 1H), 4.28 (s, 2H),
13
2
1
C
.47 (s, 3H). C NMR (75 MHz, acetone-d
6
)
d
200.8, 168.3, 153.6,
þ
40.5, 130.4, 129.9, 128.7, 127.7, 32.9, 27.7. HRMS (ESI ) calcd for
þ
H
12 13
O
3
[Mþ1] 205.0859, found 205.0854.
Procedure for Synthesis of 3-benzyl-4-oxopentanoic acid (2).
(
E)-3-Benzyl-4-oxopent-2-enoic acid (1) (22.3 g, 0.16 mol) was
dissolved in AcOH (200 mL) and H O (7.5 mL) mixture. Zinc dust
21.0 g, 0.32 mol) was added to the mixture slowly, then the
2
(
Fig. 8. Effects of 2e on the mortality of mouse model of LPS-induced sepsis shock.
Survival was recorded for 7 days after LPS injection at an interval of 12 h. n ¼ 11
animals in each group. * means p < 0.05 vs LPS group, and ***means p < 0.001 vs LPS
group.
mixture was heated to reflux and kept at this temperature for 2 h.
The mixture was filtered with a sand core funnel. Water (200 mL)
was added to the filtrate and the mixture was extracted with DCM
(
3 ꢂ 150 mL). The combined DCM phase was washed to neutral,
dried over anhydrous Na SO and evaporated under reduced
pressure to give a brown oil (32.5 g, 97.9%). H NMR (300 MHz,
acetone-d 10.52 (s, 1H), 7.39e7.13 (m, 5H), 3.25 (m, 1H), 2.95
dd, J ¼ 13.4, 6.8 Hz, 1H), 2.65 (dd, J ¼ 13.9, 7.7 Hz, 2H), 2.34 (dd,
2
4
1
synthesized based on rosiglitazone by scaffold hopping strategy,
and their QS inhibitory and anti-inflammatory activities were
evaluated. Among these compounds, a promising leading com-
pound (2e) that not only shown potent QS inhibitory activity, but
also excellent anti-inflammatory activity was identified. Further-
more, western blot and docking results revealed that the anti-
inflammatory activity of this type of compound could also be
6
)
d
d
(
13
J ¼ 17.2, 4.3 Hz, 1H), 2.07 (s, 3H). C NMR (75 MHz, acetone-d
6
)
d
209.7, 173.2, 139.5, 129.4, 128.8, 126.8, 49.8, 37.4, 34.9, 29.6. HRMS
þ
þ
(
ESI ) calcd for C12
H
15
O
3
[MþH] 207.1016, found 207.1017.
Procedure for Synthesis of 3-(4-nitrobenzyl)-4-oxopentanoic
acid (3). Fuming nitric acid (50 mL) was added to a 100 mL round
bottom flask, then placed in a low temperature reaction apparatus.
attributed at least in part, to their interaction with PPARg, thereby
suppressing activation of NF-kB and MAPK cascades and leading to
3
-Benzyl-4-oxopentanoic acid (2) (32.5 g, 0.16 mol) was added in
the decrease of NO, TNF-
a
and IL-6 levels in LPS-stimulated
ꢀ
batches and the mixture was stirred at ꢁ15 C for 4 h, then poured
RAW264.7 macrophages. This study provides a valuable candidate
e, which could serve as a potential agent for the treatment of
into ice water (100 mL), extracted with DCM (3 ꢂ 100 mL). The
2
2
combined organic layer was washed with H O and brine, dried over
chronic bacterial infection. Meanwhile, these results revealed that a
combination of the structural features with quorum sensing
inhibitory activity into the potential anti-inflammatory agents are
likely to be a valuable strategy to obtain drugs with both quorum
sensing inhibitory and anti-inflammatory effects which induced by
the chronic bacterial infections.
2 4
anhydrous Na SO and evaporated to give a yellow powder. The
crude product was chromatographed on a silica gel column using
petroleum ether/EtOAc/AcOH (2:1:0.03). The final product 3 was
ꢀ
ꢀ
obtained as a yellow powder (28.6 g, 72.1%). mp 142.7 C-144.1 C.
1
H NMR (300 MHz, CDCl
3
)
d
8.18 (d, J ¼ 8.7 Hz, 2H), 7.34 (d,
J ¼ 8.7 Hz, 2H), 3.40e3.18 (m,1H), 3.06 (dd, J ¼ 13.5, 7.2 Hz,1H), 2.78
(
ddd, J ¼ 7.41, 13.5, 8.5 Hz, 2H), 2.37 (dd, J ¼ 17.4, 4.7 Hz, 1H), 2.14 (s,
13
5
. Experimental section
3H). C NMR (75 MHz, CDCl
1
3
)
d
200.0, 171.6, 158.8, 124.8, 26.8, 20.5,
þ
[MþH]þ 258.0866, found
3.7. HRMS (ESI ) calcd for C12
H14NO
5
General Chemistry. All reagents and solvents were purchased in
258.0866.
analytical grade from commercial suppliers. Flash column chro-
matography was carried out on silica gel obtained from Qindao
Procedure for the Synthesis of 5-(dibromomethylene)-4- (4-
nitrobenzyl)furan-2(5H)-one (4) and (Z)-5-(bromomethylene)-
Haiyang Chem. Solvents used including petroleum ether, EtOAc,
4-(4-nitrobenzyl) furan-2(5H)-one (5)
(17.5 mL, 0.35 mol) in dry CHCl (40 mL) was added slowly to an ice-
cooled solution of 3-(4-nitrobenzyl)-4-oxopentanoic acid (28.6 g,
0.114 mol) in dry CHCl (100 mL). Then the mixture was heated to
reflux for 3 h then the resulting solution was washed with aqueous
sodium sulfate and evaporated. The crude product was used for
next step without further purification. Phosphorus pentoxide (80 g,
0.57 mol) was added with stirring to a solution of crude product in
dry DCM (200 mL). The mixture was heated to reflux for 2 h, and
cooled to room termperature. The resulting mixture was filtered,
.
A solution of bromine
1
DCM, MeOH, and CHCl
3
were purified and redistilled. H NMR and
3
13
C NMR spectra were recorded on a Bruker Avance DPX300
300 M) spectrometer with TMS as the baseline and were deter-
mined in DMSO-d and acetone-d solvent peaks as the internal
(
3
6
6
reference. Chemical shifts are reported in parts per million (ppm)
relative to the reference signal, and coupling constant (J) values are
reported in hertz (Hz). Melting points were measured on an Opti-
melt automated melting point system and are uncorrected. Thin
layer chromatography (TLC) was performed on precoated silica gel
plates, and spots were visualized with UV light or potassium per-
manganate coloration. High-resolution mass spectra (HRMS) were
recorded on an Agilent TOF/MS instrument equipped with an ESI
interface. Detailed characterization data for the synthesized com-
pounds are available in the supporting information of this
manuscript.
2
washed with H O and brine successively, dried over sodium sulfate
and evaporated to produce a pale yellow oil. The crude product was
chromatographed on a silica gel column using petroleum ether/
EtOAc (10:1). Compound 4 was isolated as a pale solid (16.1 g,
ꢀ
ꢀ
1
36.3%). mp 125.3 C-126.9 C. H NMR (300 MHz, CDCl
3
) d 8.26 (d,
J ¼ 8.6 Hz, 2H), 7.38 (d, J ¼ 8.6 Hz, 2H), 5.88 (t, J ¼ 1.6 Hz, 2H), 4.27

174
X.-J. Xu et al. / European Journal of Medicinal Chemistry 144 (2018) 164e178
(
1
C
d, J ¼ 1.6 Hz, 1H). 13C NMR (75 MHz, CDCl
3
)
d
165.1, 155.3, 143.0,
chloride, the product was yellow solid (250 mg, 89%). mp 199.1
þ
ꢀ ꢀ
1
30.5, 130.0, 124.5, 124.5, 122.0, 90.92, 36.1. HRMS (ESI ) calcd for
C-199.8 C. H NMR (300 MHz, DMSO-d ) d 10.25 (s, 1H), 8.02 (d,
6
7
9
þ
12
H
8
Br
2
NO
4
[Mþ1] 387.8820, found 387.8817. Compound 5 was
J ¼ 3.2 Hz, 1H), 7.85 (d, J ¼ 4.8 Hz, 1H), 7.72 (d, J ¼ 8.4 Hz, 2H), 7.23
ꢀ
ꢀ
1
13
the yellow solid (18.3 g, 51.8%). mp 116.7 C-118.1 C. H NMR
300 MHz, CDCl
8.21 (d, J ¼ 8.5, 2H), 7.40 (d, J ¼ 8.5, 2H), 6.13 (d,
J ¼ 0.5 Hz, 1H), 5.92 (d, J ¼ 0.5 Hz, 1H), 3.94 (s, 2H). C NMR
(dd, J ¼ 10.0, 6.5 Hz, 3H), 6.20 (s, 1H), 4.14 (s, 2H). C NMR (75 MHz,
(
3
)
d
DMSO-d
6
)
d
165.6, 160.0, 157.6, 149.3, 140.1, 137.7, 132.0, 131.4, 129.3,
13
þ
129.2, 128.2, 121.3, 120.6, 81.5, 34.5. HEMS (ESI ) calcd for
7
9þ81
þ
(
75 MHz, CDCl
3
)
d
167.0, 152.3, 147.5, 142.8, 129.8, 124.4, 118.5, 90.9,
C
17
H
12
Br
2
NO
3
S [MþH] 469.8879, found 469.8893.
þ
79
[MþH]þ 309.9709, found
3
3
2.2. HEMS (ESI ) calcd for C12
09.9709.
H
9
BrNO
4
N-(4-((2-(Dibromomethylene)-5-oxo-2,5-dihydrofuran-3-yl)
methyl)phenyl)-2-phenylacetamide (1d). 1d was prepared
through acylation from compound 6 and phenylacetyl chloride, the
Procedure for Synthesis of 4-(4-aminobenzyl)-5- (dibromo-
methylene)furan-2(5H)-one (6). Reduced iron powder (7 g,
0
0
solution of compound 5 (16.1 g, 0.041 mol) in Me
added dropwise and the mixture was refluxed for 4 h. Then the
ꢀ
ꢀ
1
product was white solid (200 mg, 98%). mp 188.1 C-189.9 C. H
NMR (300 MHz, CDCl
7.44 (d, J ¼ 8.4 Hz, 2H), 7.35 (dd, J ¼ 13.4,
6.1 Hz, 3H), 7.06 (d, J ¼ 8.4 Hz, 2H), 5.75 (t, J ¼ 1.6 Hz, 1H), 4.03 (d,
.125 mol) was added to a solution of ammonium chloride (2.2 g,
.041 mol) in H O (30 mL) under a nitrogen atmosphere. Then a
CO (50 mL) was
3
) d
2
1
3
2
J ¼ 1.1 Hz, 2H), 3.72 (s, 2H). C NMR (75 MHz, CDCl
3
) d 169.4, 165.7,
157.9, 149.6, 137.2, 134.4, 131.4, 129.6, 129.3, 127.8, 121.4, 120.5, 81.0,
44.9, 36.1. HEMS (ESI ) calcd for C20
þ
79
þ
mixture was extracted with EtOAc. After that, the combined organic
layer was basified by addition of a saturated solution of NaHCO
H
16Br
2
NO
3
[MþH] 475.9492,
3
and
found 475.9491.
the solvent was removed under reduced pressure. The crude
product was chromatographed on a silica gel column using petro-
leum ether/EtOAc (5:1). The final product 6 was a yellow powder
N-(4-((2-(Dibromomethylene)-5-oxo-2,5-dihydrofuran-3-yl)
methyl)phenyl)-2-(4-fluorophenyl)acetamide (1e). 1e was pre-
pared through acylation from compound
6
and 4-
ꢀ ꢀ
1
(
11.9 g, 81.1%). mp 95.3 C-97.1 C. H NMR (300 MHz, CDCl ) d 6.95
3
fluorophenylacetyl chloride, the product was white powder
ꢀ
ꢀ
1
(
3
1
d, J ¼ 8.4 Hz, 2H), 6.68 (d, J ¼ 8.4 Hz, 2H), 5.82 (t, J ¼ 1.8 Hz, 1H),
(180 mg, 90%). mp 190.6 C-191.6 C. H NMR (300 MHz, DMSO-d
6
)
13
.98 (d, J ¼ 1.8 Hz, 2H). C NMR (75 MHz, CDCl
3
)
d
165.9, 159.1,
d
10.22 (s, 1H), 7.56 (d, J ¼ 8.5 Hz, 2H), 7.32 (dd, J ¼ 8.5, 5.7 Hz, 2H),
þ
13
49.8, 145.8, 130.0, 125.2, 121.3, 115.7, 80.7, 36.0. HRMS (ESI ) calcd
7.21e7.10 (m, 4H), 6.17 (s, 1H), 4.10 (s, 2H), 3.62 (s, 2H). C NMR
8
1
þ
for C12
H
10Br
NO
2 2
[MþH] 357.9072, found 357.9073.
(75 MHz, DMSO-d
6
)
d
169.4, 163.2, 160.0, 158.1, 149.6, 138.5, 131.2,
þ
Procedures for Synthesis of (Z)-4-(4-aminobenzyl)-5- (bro-
momethylene)furan-2(5H)-one (7). The synthesis procedure for 7
followed that of 6. The final product was a yellow powder (14.9 g,
129.7, 121.5, 119.7, 115.6, 115.3, 115.2, 81.9, 42.7, 34.8. HEMS (ESI )
7
9
þ
calcd for C20
H
15Br
2
FNO
3
[MþH] 493.9397, found 493.9397.
N-(4-((2-(Dibromomethylene)-5-oxo-2,5-dihydrofuran-3-yl)
methyl)phenyl)-2-(4-methoxyphenyl)acetamide (1f). 1f was
ꢀ
ꢀ
1
9
0.1%). mp 86.3 C-88.1 C. H NMR (300 MHz, CDCl
3
) d 6.94 (d,
J ¼ 8.4 Hz, 2H), 6.65 (d, J ¼ 8.4 Hz, 2H), 6.08 (s, 1H), 5.89 (s, 1H), 3.67
prepared through acylation from compound
6
and 4-
13
(
3
s, 2H). C NMR (75 MHz, CDCl ) d 167.7, 157.8, 152.7, 145.9, 129.7,
methoxyphenylacetyl chloride, the product was yellow powder
þ
79
ꢀ
ꢀ
1
1
25.0, 117.7, 115.7, 90.3, 32.0. HRMS (ESI ) calcd for C12
H
11BrNO
2
(186 mg, 85%). mp 201.0 C-202.9 C. H NMR (300 MHz, DMSO-d
10.15 (s, 1H), 7.58 (d, J ¼ 8.5 Hz, 2H), 7.25 (d, J ¼ 8.6 Hz, 2H), 7.18 (d,
J ¼ 8.5 Hz, 2H), 6.88 (d, J ¼ 8.7 Hz, 2H), 6.14 (s, 1H), 4.09 (s, 2H), 3.72
6
)
þ
[MþH] 279.9967, found 279.9968.
d
General Procedures for Synthesis of 1a-1m and 2a-2m. The
13
acyl chloride (0.22 mmol) was added dropwise to a mixture of 6 (or
6
(s, 3H), 3.55 (s, 2H). C NMR (75 MHz, DMSO-d ) d 169.6, 165.6,
ꢀ
7
) (0.2 mmol) and dry pyridine (0.24 mmol) in DCM at 0 C. The
158.1,157.7, 149.3,138.3,130.8,130.2,129.3,128.0,121.2,119.4, 113.8,
þ
79
[MþH]þ
solution was stirred at room temperature overnight, then saturated
NH Cl solution (10 mL) was added to the final mixture, then
81.5, 55.1, 42.6, 34.5. HEMS (ESI ) calcd for C21
H
18Br
2
NO
4
4
505.9597, found 505.9597.
extracted with EtOAc (3 ꢂ 15 mL). Afer the combined organic phase
was washed and dried, the solvent was removed. The crude product
was chromatographed on a silica gel column using DCM/EtOAc
N-(4-((2-(Dibromomethylene)-5-oxo-2,5-dihydrofuran-3-yl)
methyl)phenyl)-2-(p-tolyl)acetamide (1g). 1g was prepared
through acylation from compound 6 and 4-methylphenylacetyl
chloride, the product was white solid (193 mg, 90%). mp 186.5
(
10:1).
N-(4-((2-(Dibromomethylene)-5-oxo-2,5-dihydrofuran-3-yl)
ꢀ
ꢀ
1
6
C-189.1 C. H NMR (300 MHz, acetone-d ) d 9.33 (s, 2H), 7.66 (d,
methyl)phenyl)benamide (1a). 1a was prepared by acylation from
compound 6 and benzoyl chloride, the product was white solid
J ¼ 8.5 Hz, 2H), 7.24 (t, J ¼ 8.6 Hz, 4H), 7.12 (d, J ¼ 7.9 Hz, 2H), 6.00 (s,
13
1H), 4.17 (d, J ¼ 1.2 Hz, 2H), 3.63 (s, 2H), 2.29 (s,3H). C NMR
(75 MHz, acetone-d 170.1, 166.1, 159.1, 150.8, 139.5, 136.9, 133.8,
ꢀ
ꢀ
1
(
d
7
4
1
1
4
195 mg, 95%). mp 196.2 C-198.0 C. H NMR (300 MHz, acetone-
9.58 (s, 2H), 8.03e7.96 (m, 2H), 7.88 (d, J ¼ 8.5 Hz, 2H),
.60e7.47 (m, 3H), 7.32 (d, J ¼ 8.5 Hz, 2H), 6.06 (t, J ¼ 1.6 Hz, 1H),
6
) d
6
)
d
131.9, 130.3, 130.0, 129.9, 122.1, 120.4, 80.2, 44.4, 36.0, 21.1. HEMS
þ
79
þ
(ESI ) calcd for C21
H
18Br
2
NO
3
[MþH] 489.9648, found 489.9644.
13
.23 (d, J ¼ 1.4 Hz, 2H). C NMR (75 MHz, acetone-d
6
)
d
166.3, 166.1,
N-(4-((2-(Bromomethylene)-5-oxo-2,5-dihydrofuran-3-yl)
59.0, 150.8, 139.4, 136.3, 132.4, 132.3, 130.3, 129.3, 128.3, 122.1,
methyl)phenyl)-2-(4-bromophenyl)acetamide (1h). 1h was pre-
þ
79
[MþH]þ
21.3, 80.2, 36.0. HRMS (ESI ) calcd for C19
61.9340; found 461.9335.
H
14Br
2
NO
3
pared through acylation from compound
6
and 4-
bromophenylacetyl chloride, the product was yellow powder
ꢀ
ꢀ
1
N-(4-((2-(Dibromomethylene)-5-oxo-2,5-dihydrofuran-3-yl)
(173 mg, 91%). mp 192.5 C-194.6 C. H NMR (300 MHz, DMSO-d
10.22 (s,1H), 7.57 (d, J ¼ 8.5 Hz, 2H), 7.52 (d, J ¼ 8.3 Hz, 2H), 7.29 (d,
J ¼ 8.4 Hz, 2H), 7.19 (d, J ¼ 8.5 Hz, 2H), 6.16 (s, 1H), 4.10 (s, 2H), 3.62
6
)
methyl)phenyl)furan-2-carboxamide (1b). 1b was prepared
through acylation from compound 6, the product was light yellow
d
ꢀ
ꢀ
1
13
solid (156 mg, 90%). mp 185.3 C-187.1 C. H NMR (300 MHz,
DMSO-d
10.21 (s, 1H), 7.94 (dd, J ¼ 1.6, 0.7 Hz, 1H), 7.74 (d,
J ¼ 8.6 Hz, 2H), 7.33 (dd, J ¼ 3.5, 0.7 Hz, 1H), 7.24 (d, J ¼ 8.5 Hz, 2H),
6
(s, 2H). C NMR (75 MHz, DMSO-d ) d 168.8, 165.6, 157.7, 149.0,
6
)
d
138.0, 135.5, 131.5, 131.3, 130.9, 129.4, 121.2, 119.9, 119.4, 42.6, 34.4.
þ
79
[MþH]þ 553.8597, found
HEMS (ESI ) calcd for C20
553.8591.
H
15Br
3
NO
3
1
3
6
.70 (dd, J ¼ 3.5, 1.7 Hz, 1H), 6.20 (s, 1H), 4.11 (d, J ¼ 16.6 Hz, 2H).
C
NMR (75 MHz, DMSO-d 165.6, 157.6, 156.3, 149.3, 147.6, 145.8,
6
)
d
N-(4-((2-(Dibromomethylene)-5-oxo-2,5-dihydrofuran-3-yl)
methyl)phenyl)-2-(2,6-dichlorophenyl)acetamide (1i). 1i was
1
37.5, 131.4, 129.3, 121.3, 120.6, 114.8, 112.3, 81.6, 55.0, 34.4. HRMS
þ
81
þ
(
ESI ) calcd for C17
H12Br NO
2 4
[MþH] 453.9108; found 453.9117.
prepared through acylation from compound
6
and 2,6-
N-(4-((2-(Dibromomethylene)-5-oxo-2,5-dihydrofuran-3-yl)
dichlorophenylacetyl acid, yellow powder was got (231 mg, 78%).
ꢀ
ꢀ
1
methyl)phenyl)thiophene-2-carboxamide (1c). 1c was prepared
through acylation from compound 6 and 2-thiophenecarbonyl
mp 201.4 C-202.9 C. H NMR (300 MHz, acetone-d
6
) d 9.51 (s, 1H),
7.67 (d, J ¼ 6.7 Hz, 2H), 7.45 (d, J ¼ 8.6 Hz, 2H), 7.32 (t, J ¼ 5.9 Hz,1H),

X.-J. Xu et al. / European Journal of Medicinal Chemistry 144 (2018) 164e178
175
7
2
.25 (d, J ¼ 10.8 Hz, 2H), 6.04 (t, J ¼ 1.6 Hz, 1H), 4.20 (d, J ¼ 1.2 Hz,
6
acetone-d ) d 168.0, 158.8, 157.0, 153.6, 149.1, 145.9, 138.7, 132.8,
H), 4.15 (s, 2H). 13C NMR (75 MHz, DMSO-d
)
d
166.5, 165.6, 157.7,
130.1, 121.3, 118.2, 115.4, 113.1, 90.9, 32.1. HEMS (ESI ) calcd for
þ
6
7
9
þ
149.2, 138.0, 135.7, 132.2, 130.9, 129.6, 129.4, 128.2, 81.6, 34.1, 13.8.
C
17
H
13BrNO
4
[MþH] 374.0023, found 374.0023.
þ
79 35
[MþH]þ 543.8712, found
HEMS (ESI ) calcd for C20
43.8708.
H
14Br
2
Cl
2
NO
3
(Z)-N-(4-((2-(Bromomethylene)-5-oxo-2,5-dihydrofuran-3-
5
yl)methyl)phenyl)thiophene-2-carboxamide (2c). 2c was pre-
pared through acylation from compound and 2-
thiophenecarbonyl chloride, he product was yellow solid
N-(4-((2-(Dibromomethylene)-5-oxo-2,5-dihydrofuran-3-yl)
7
methyl)phenyl)-3-phenylpropanamide (1j). 1j was prepared
through acylation from compound 6 and hydrocinnamoyl chloride,
ꢀ
ꢀ
1
(159 mg, 84%). mp 188.1 C-189.1 C. H NMR (300 MHz, acetone-
9.57 (s,1H), 7.89 (d, J ¼ 3.1 Hz,1H), 7.77 (dd, J ¼ 6.6, 4.6 Hz, 3H),
7.32 (d, J ¼ 8.4 Hz, 2H), 7.22e7.11 (m, 1H), 6.64 (s, 1H), 6.12 (s, 1H),
ꢀ
ꢀ
the product was white solid (183 mg, 82%). mp 193.4 C-194.9 C.
6
d ) d
1
H NMR (300 MHz, DMSO-d
6
)
d
9.95 (s, 1H), 7.57 (d, J ¼ 8.5 Hz, 2H),
13
7.32e7.21 (m, 4H), 7.22e7.13 (m, 3H), 6.16 (s, 1H), 4.09 (s, 2H), 2.91
6
3.97 (s, 2H). C NMR (75 MHz, acetone-d ) d 168.0, 160.8, 158.8,
13
(
d
t, J ¼ 7.6 Hz, 2H), 2.62 (t, J ¼ 7.7 Hz, 2H). C NMR (75 MHz, DMSO-
153.6, 141.4, 139.0, 132.7, 132.2, 130.1, 129.2, 128.7, 121.3, 118.2, 90.9,
þ
79
S [MþH]þ 389.9834, found
6
)
d
170.5, 165.6, 157.8, 149.3, 141.3, 138.2, 130.6, 129.3, 128.4, 128.4,
32.1. HEMS (ESI ) calcd for C17
389.9830.
H
13BrNO
3
þ
126.1, 121.2, 119.3, 81.5, 38.1, 34.5, 30.9. HEMS (ESI ) calcd for
7
9
þ
C
21
H
18Br
2
NO
3
[MþH] 489.9648, found 489.9451.
(Z)-N-(4-((2-(Bromomethylene)-5-oxo-2,5-dihydrofuran-3-
yl)methyl)phenyl)-2-phenylacetamide (2d). 2d was prepared
through acylation from compound 7 and phenylacetyl chloride, and
N-(4-((2-(Dibromomethylene)-5-oxo-2,5-dihydrofuran-3-yl)
methyl)phenyl)cinnamamide (1k). 1k was prepared through
acylation from compound 6 and cinnamoyl chloride, the product
ꢀ
ꢀ
1
the yield was 98%, white solid 187 mg mp 188.6 C-190.4 C. H
NMR (300 MHz, acetone-d
9.39 (s, 1H), 7.64 (d, J ¼ 8.5 Hz, 2H),
7.36 (t, J ¼ 7.9 Hz, 2H), 7.34e7.27 (m, 2H), 7.24 (dd, J ¼ 9.1, 2.4 Hz,
ꢀ
ꢀ
1
was brown solid (250 mg, 90%). mp 193.0 C-195.1 C. H NMR
300 MHz, acetone-d
9.49 (s, 1H), 7.80 (d, J ¼ 8.5 Hz, 2H), 7.69 (d,
6
) d
(
6
) d
13
J ¼ 15.6 Hz, 1H), 7.62 (dd, J ¼ 7.6, 1.9 Hz, 2H), 7.46e7.36 (m, 2H), 7.29
3H), 6.60 (s, 1H), 6.07 (s, 1H), 3.90 (s, 2H), 3.69 (s, 2H). C NMR
(
4
1
d, J ¼ 8.5 Hz, 2H), 6.87 (d, J ¼ 15.6 Hz, 1H), 6.04 (t, J ¼ 1.6 Hz, 1H),
(75 MHz, acetone-d
6
)
d
169.8, 167.9, 158.7, 153.4, 139.3, 136.7, 132.2,
13
þ
.21 (d, J ¼ 1.3 Hz, 2H). C NMR (75 MHz, acetone-d
6
)
d
166.2, 164.6,
130.0, 129.9, 129.1, 127.4, 120.4, 118.0, 90.8, 44.6, 32.0. HEMS (ESI )
7
9
þ
59.1, 150.8, 141.8, 139.6, 136.1, 132.2, 130.6, 130.4, 129.9, 128.7,
calcd for C20
H
17 BrNO
3
[MþH] 398.0386, found 398.0400.
þ
123.0, 122.2, 120.6, 80.3, 36.1. HEMS (ESI ) calcd for
(Z)-N-(4-((2-(Bromomethylene)-5-oxo-2,5-dihydrofuran-3-
yl)methyl)phenyl)-2-(4-fluorophenyl)acetamide (2e). 2e was
7
9þ81
[MþH]þ 487.9491, found 487.9494.
C
21
H
16 2 3
Br NO
N-(4-((2-(Dibromomethylene)-5-oxo-2,5-dihydrofuran-3-yl)
prepared through acylation from compound
7
and 4-
methyl)phenyl)-3-(4-methoxyphenyl)acrylamide (1l). 1l was
prepared through acylation from compound and 4-
methoxycinnamic acid, the product was white solid (125 mg,
fluorophenylacetyl chloride, the product was white solid (241 mg,
ꢀ ꢀ
1
6
88%). mp 188.1 C-189.6 C. H NMR (300 MHz, acetone-d ) d 9.41
6
(s, 1H), 7.64 (d, J ¼ 8.6 Hz, 2H), 7.40 (dd, J ¼ 8.7, 5.5 Hz, 2H), 7.25 (d,
J ¼ 8.6 Hz, 2H), 7.06 (dd, J ¼ 12.3, 5.5 Hz, 2H), 6.60 (s, 1H), 6.08 (s,
ꢀ ꢀ
1
7
1
2
4
0%). mp 188.6 C-190.4 C. H NMR (300 MHz, DMSO-d ) d 10.16 (s,
6
13
H), 7.69 (d, J ¼ 8.6 Hz, 2H), 7.60e7.51 (m, 4H), 7.21 (d, J ¼ 8.6 Hz,
H), 7.00 (d, J ¼ 8.8 Hz, 2H), 6.69 (d, J ¼ 15.5 Hz, 1H), 6.20 (s, 1H),
6
1H), 3.92 (s, 2H), 3.63 (s, 2H). C NMR (75 MHz, acetone-d ) d 169.7,
167.9,164.2,160.9,158.7,153.4,139.2,132.8,132.7,132.2,132.1,131.9,
131.8, 131.7, 130.0, 120.4, 118.0, 115.8, 115.7, 115.6, 115.5, 90.8, 43.5,
13
6
.13 (s, 2H), 3.80 (s, 3H). C NMR (75 MHz, DMSO-d ) d 166.0, 164.3,
þ
79
[MþH]þ 416.0292, found
1
61.1, 158.1, 149.6, 140.4, 138.7, 131.2, 129.8, 129.8, 127.7, 120.1, 119.7,
32.0. HEMS (ESI ) calcd for C20
416.0295.
H
16BrFNO
3
þ
81
119.8, 114.9, 81.9, 55.8, 34.8. HRMS (ESI ) calcd for C22
H18Br
2
NO
4
þ
[MþH] 519.9578, found 519.9588.
(Z)-N-(4-((2-(Bromomethylene)-5-oxo-2,5-dihydrofuran-3-
yl)methyl)phenyl)-2-(4-methoxyphenyl)acetamide (2f). 2f was
N-(4-((2-(Dibromomethylene)-5-oxo-2,5-dihydrofuran-3-yl)
methyl)phenyl)-2-(3,4,5-trimethoxyphenyl)acetamide (1m). 1m
was prepared through acylation from compound 6 and 3,4,5-
trimethoxyphenylacetic chloride, the product was white powder
prepared through acylation from compound
7
and 4-
methoxyphenylacetyl chloride, the product was light yellow solid
ꢀ
ꢀ
1
(253 mg, 89%). mp 175.8 C-178.1 C. H NMR (300 MHz, acetone-
9.34 (s, 1H), 7.63 (d, J ¼ 8.5 Hz, 2H), 7.28 (d, J ¼ 8.6 Hz, 2H), 7.24
(d, J ¼ 8.5 Hz, 2H), 6.86 (d, J ¼ 8.7 Hz, 2H), 6.59 (s, 1H), 6.06 (s, 1H),
ꢀ
ꢀ
1
(
212 mg, 92%). mp 195.6 C-199.3 C. H NMR (300 MHz, acetone-
9.96 (s, 1H), 7.89 (d, J ¼ 8.6 Hz, 2H), 7.62 (s, 2H), 7.38 (d,
J ¼ 8.6 Hz, 2H), 6.08 (t, J ¼ 1.6 Hz,1H), 4.27 (d, J ¼ 1.3 Hz, 2H), 3.90 (s,
H), 3.86 (s, 3H). 13C NMR (75 MHz, DMSO-d
175.6, 171.3, 169.1,
6
d ) d
6
d ) d
13
3.90 (s, 2H), 3.75 (s, 3H), 3.61 (s, 2H). C NMR (75 MHz, acetone-d
6
)
6
6
)
d
d
170.2, 167.9, 159.4, 158.7, 153.3, 139.2, 132.0, 130.9, 129.9, 128.5,
þ
1
66.0,158.0,149.6,138.4,135.8,131.9,131.6,131.3,129.7,121.6,120.2,
120.3, 117.9, 114.5, 90.8, 55.3, 43.7, 31.9. HEMS (ESI ) calcd for
þ
79
[MþH]þ
79
þ
119.8, 81.9, 42.9. HEMS (ESI ) calcd for C23
H22Br
2
NO
6
C
21
H
19BrNO
4
[MþH] 428.0492, found 428.0291.
5
65.9808, found 565.9806.
(Z)-N-(4-((2-(Bromomethylene)-5-oxo-2,5-dihydrofuran-3-
(
Z)-N-(4-((2-(Bromomethylene)-5-oxo-2,5-dihydrofuran-3-
yl)methyl)phenyl)-2-(p-tolyl)acetamide (2g). 2g was prepared
through acylation from compound 7 and 4-methylphenylacetyl
chloride, the product was light brown solid (168 mg, 79%). mp
yl)methyl)phenyl)benzamide (2a). 2a was prepared through
acylation from compound 7 and benzoyl chloride, the product was
white solid (230 mg, 90%). mp 185.1 C-186.6 C. H NMR (300 MHz,
DMSO-d
ꢀ
ꢀ
1
ꢀ
ꢀ
1
171.4 C-173.1 C. H NMR (300 MHz, acetone-d
6
) d 9.33 (s, 1H), 7.63
6
)
d
10.28 (s, 1H), 7.95 (d, J ¼ 7.1 Hz, 2H), 7.76 (d, J ¼ 8.1 Hz,
(d, J ¼ 8.5 Hz, 2H), 7.25 (d, J ¼ 8.2 Hz, 4H), 7.12 (d, J ¼ 7.9 Hz, 2H),
13
2
6
H), 7.55 (dt, J ¼ 14.2, 7.0 Hz, 3H), 7.31 (d, J ¼ 8.1 Hz, 2H), 6.91 (s,1H),
6.61 (s, 1H), 6.08 (s, 1H), 3.91 (s, 2H), 3.63 (s, 2H), 2.28 (s, 3H).
C
13
.27 (s, 1H), 3.89 (s, 2H). C NMR (75 MHz, DMSO-d
6
)
d
167.6, 165.6,
NMR (75 MHz, acetone-d 170.1, 168.1, 158.9, 153.6, 139.5, 136.9,
6
) d
1
9
58.2,152.0, 138.2, 135.0, 131.9, 131.7, 129.2, 128.5, 127.8, 120.7, 117.0,
133.8, 132.2, 130.1, 130.0, 129.9, 129.8, 120.5, 118.1, 90.9, 44.4, 32.1,
þ
79
[MþH]þ 384.0230,
þ
79
[MþH]þ 412.0498, found
2.1, 30.9. HEMS (ESI ) calcd for C19
H
15BrNO
3
21.1. HEMS (ESI ) calcd for C21
H
19BrNO
3
found 384.0229.
412.0498.
(
Z)-N-(4-((2-(Bromomethylene)-5-oxo-2,5-dihydrofuran-3-
(Z)-N-(4-((2-(Bromomethylene)-5-oxo-2,5-dihydrofuran-3-
yl)methyl)phenyl)-2-(4-bromophenyl)acetamide (2h). 2h was
yl)methyl)phenyl)furan-2-carboxamide (2b). 2b was prepared
through acylation from compound 7 and 2-furoyl chloride, he
product was yellow solid (159 mg, 84%). mp 183.9 C-186.1 C. H
NMR (300 MHz, acetone-d
prepared through acylation from compound
7
and 4-
ꢀ
ꢀ
1
bromophenylacetic acid, the product was yellow solid (241 mg,
ꢀ ꢀ
1
6
)
d
9.46 (s, 1H), 7.84 (d, J ¼ 8.5 Hz, 2H),
78%). mp 183.5 C-185.1 C. H NMR (300 MHz, DMSO-d ) d 10.20 (s,
6
7
6
.75 (s, 1H), 7.33 (d, J ¼ 8.5 Hz, 2H), 7.21 (d, J ¼ 3.5 Hz, 1H),
1H), 7.55 (d, J ¼ 8.5 Hz, 2H), 7.50 (d, J ¼ 8.4 Hz, 2H), 7.33 (d,
J ¼ 8.4 Hz, 2H), 7.26 (d, J ¼ 8.5 Hz, 2H), 6.96 (s, 1H), 6.24 (s, 1H), 3.85
13
.68e6.59 (m, 2H), 6.13 (s, 1H), 3.97 (s, 2H). C NMR (75 MHz,

176
X.-J. Xu et al. / European Journal of Medicinal Chemistry 144 (2018) 164e178
(
1
4
s, 2H), 3.62 (s, 2H). 13C NMR (75 MHz, DMSO-d
6
)
d
169.1, 167.9,
single colony was cultured in Luria-Bertani (LB) broth with 300
m
g/
ꢀ
58.5, 152.4, 138.4, 135.8, 131.9, 131.6, 129.6, 120.2, 119.8, 117.3, 92.4,
mL carbenicillin and 60
mg/mL gentamicin for 18 h at 37 C.
þ
79
[MþH]þ 477.9472,
3.9, 31.2. HEMS (ESI ) calcd for C20
H
16Br
2
NO
3
P. aeruginosa QS Inhibition Assays. To assess the impact of the
compounds on QS signaling, the P. aeruginosa reporter strain PAO1-
lasB-gfp strain PAO1-rhlA-gfp strain and PAO1-pqsA-gfp strain,
which harbors a fusion of the lasB promoter or rhlA promoter or
pqsA promoter to an unstable gfp(ASV) gene and responds to the
signalling molecules respectly, was used. The test compounds were
dissolved in DMSO to 10 mM for storage and diluted in LB medium
found 477.9471.
(
Z)-N-(4-((2-(Bromomethylene)-5-oxo-2,5-dihydrofuran-3-
yl)methyl)phenyl)-2-(2,6-dichlorophenyl)acetamide (2i). 2i was
prepared through acylation from compound and 2,6-
dichlorophenylacetic acid, the product was white solid (120 mg,
7
ꢀ
ꢀ
1
7
5%). mp 182.6 C-184.9 C. H NMR (300 MHz, DMSO-d
6
) d 10.33 (s,
1
H), 7.54 (d, J ¼ 8.4 Hz, 2H), 7.48 (d, J ¼ 8.0 Hz, 2H), 7.36e7.30 (t,1H),
to a final concertration of 10
mM for assay. Furanone C-30 was
7.25 (d, J ¼ 8.4 Hz, 2H), 6.95 (s, 1H), 6.24 (s, 1H), 4.04 (s, 2H), 3.85 (s,
selected as positive control. Bacteria were cultured in Luria-Bertani
1
3
ꢀ
2
H). C NMR (75 MHz, DMSO-d
6
)
d
167.5, 166.5, 158.1, 151.9, 138.0,
broth (LB) at 37 C with shaking at 200 rpm for 18 h and then an
1
35.6, 132.2, 131.4, 129.5, 129.2, 128.2, 119.3, 116.9, 92.0, 38.5, 30.7.
inoculating culture was prepared by diluting the overnight culture
þ
81 37
[MþH]þ 467.9584, found
HEMS (ESI ) calcd for C20
67.9561.
H
15Br Cl
2
NO
3
1:100 into fresh LB medium. Finally 100
mL of bacterial suspension
4
and 100 L diluted compounds were added to the wells. DMSO
m
(
Z)-N-(4-((2-(Bromomethylene)-5-oxo-2,5-dihydrofuran-3-
control (0.1% final concentration) and blank control were used. The
gfp(ASV) expression (fluorescence, excitation 485 nm, emission
528 nm) was measured every 15 min at 37 C by the use of a
microplate reader (Bio-Rad Laboratories, CA, USA) for at least 12 h.
The inhibition assay for all test compounds and controls were done
in triplicate manner [55].
yl)methyl)phenyl)-3-phenylpropanamide (2j). 2j was prepared
through the acylation from compound 7 and hydrocinnamoyl
chloride, the product was yellow solid (198 mg, 95%). mp 178.4 C-
ꢀ
ꢀ
ꢀ
1
1
79.9 C. H NMR (300 MHz, acetone-d
6
) d 9.20 (s, 1H), 7.64 (d,
J ¼ 8.5 Hz, 2H), 7.22e7.33 (m, 6H), 7.16 (d, J ¼ 8.5 Hz, 2H), 6.62 (s,
1
H), 6.09 (s, 1H), 3.92 (s, 2H), 2.99 (t, J ¼ 7.7 Hz, 2H), 2.68 (t,
Growth measurement. The steps to measure the growth of
P. aeruginosa was similar to QS inhibition assays. The overnight
cultured P. aeruginosa strain PAO1 was diluted with fresh LB me-
dium and the compounds were also diluted to appropriate con-
centration. Bacterial suspension and diluted compounds were
equally added to the 96-well plates. Last the growth curve was
13
J ¼ 7.7 Hz, 2H). C NMR (75 MHz, acetone-d
6
)
d
170.7, 167.6, 158.4,
1
3
53.1, 141.9, 139.0, 131.6, 129.6, 128.8, 126.4, 119.9, 117.6, 90.4, 38.9,
þ
79
[MþH]þ 412.0575,
1.7, 31.6. HEMS (ESI ) calcd for C21
found 412.0571.
H19BrNO
3
(Z)-N-(4-((-2-(Bromomethylene)-5-oxo-2,5-dihydrofuran-3-
ꢀ
yl)methyl)phenyl)cinnamamide (2k). 2k was prepared through
the acylation from compound 7 and cinnamoyl chloride, the
product was white solid (253 mg, 94%). mp 184.6 C-186.3 C. H
NMR (300 MHz, acetone-d
evaluated every 15 min at 37 C by measuring OD600 using a
microplate reader (Bio-Rad Laboratories, CA, USA) for at least 12 h.
Cell Culture. The murine RAW264.7 macrophage cells were
obtained from the American Type Culture Collection (ATCC) and
cultured in Dulbecco's modified Eagle's medium (DMEM) supple-
ꢀ
ꢀ
1
6
)
d
9.44 (s, 1H), 7.77 (d, J ¼ 8.5 Hz, 2H),
7
4
6
1
1
.69 (d, J ¼ 15.6 Hz, 1H), 7.62 (dd, J ¼ 7.5, 1.9 Hz, 2H), 7.49e7.36 (m,
H), 7.31 (d, J ¼ 8.5 Hz, 3H), 6.85 (d, J ¼ 15.6 Hz, 1H), 6.65 (s, 1H),
mented with 10% fetal bovine serum, penicillin (100 units/mL) and
13
ꢀ
.12 (s, 1H), 3.96 (s, 2H). C NMR (75 MHz, acetone-d
6
)
d
167.7,
streptomycin (100
atmosphere.
m
g/mL) at 37 C in a 5% CO
2
humidified
64.1, 158.5, 153.3, 141.4, 139.2, 135.7, 132.1, 130.3, 129.9, 129.5,
28.3, 122.5, 120.2, 117.8, 90.5, 31.8. HEMS (ESI ) calcd for
H
þ
Assay for NO Production. Nitrite level, an index for NO pro-
duction, was measured in the supernatant of RAW264.7 cells by the
Griess methol [56]. RAW 264.7 cells were inoculated to 96-well
7
9
þ
C
21
17 BrNO
3
[MþH] 410.0386, found 410.0385.
(Z)-N-(4-((-2-(Bromomethylene)-5-oxo-2,5-dihydrofuran-3-
4
yl)methyl)phenyl)-3- (4-methoxyphenyl)acrylamide (2l). 2l was
prepared through the acylation reaction of compound 7 and 4-
methoxycinnamic acid, the product was white solid (132 mg,
plates at the density of 5 ꢂ 10 cells per well and cultured for
18 h. Different compounds at the concentration of 10
mM were
added to each well and then was additionally treated the LPS
ꢀ
ꢀ
1
ꢀ
7
8%). mp 185.4 C-187.3 C. H NMR (300 MHz, acetone-d
6
)
d
9.37
(100 ng/mL) and incubated at 37 C for 48 h. After stimulated for
(
s, 1H), 7.77 (d, J ¼ 8.5 Hz, 2H), 7.64 (d, J ¼ 15.6 Hz, 1H), 7.55 (d,
48 h by LPS, the supernatant of the cell culture medium and the
same volume of Griess reagent (1% sulfanilamide and 0.1% N-(1-
naphthyl)ethylenediamine dihydrochloride in 5% phosphoric acid)
were added to a 96-well plate for 10 min, and nitrite was measured
at 540 nm (OD540) using a microplate reader (Bio-Rad Laboratories,
CA, USA). Meanwhile, the results were evaluated from three inde-
pendent experiments.
J ¼ 8.7 Hz, 2H), 7.55 (d, J ¼ 8.7 Hz, 2H), 7.29 (d, J ¼ 8.5 Hz, 2H), 6.96
(
3
1
1
d, J ¼ 8.8 Hz, 2H), 6.71 (d, J ¼ 15.6 Hz, 1H), 6.64 (s, 1H), 6.11 (s, 1H),
13
6
.95 (s, 2H), 3.83 (s, 3H). C NMR (75 MHz, acetone-d ) d 168.1,
64.9, 162.1, 158.9, 153.6, 141.5, 139.7, 132.2, 130.3, 130.2, 128.6,
þ
20.6, 120.3, 118.2, 115.2, 90.9, 55.8, 32.2. HEMS (ESI ) calcd for
7
9
þ
C
22
H
(
19BrNO
4
[MþH] 440.0492, found 440.0486.
Z)-N-(4-((2-(Bromomethylene)-5-oxo-2,5-dihydrofuran-3-
NO inhibition rate (%) ¼ [control (OD540) - compound (OD540)]/
yl)methyl)phenyl)-2-(3,4,5-trimethoxyphenyl)acetamide (2m).
m was prepared through acylation from compound 7 and 3,4,5-
trimethoxyphenylacetic chloride, the product was white solid
[control (OD540) - blank (OD540)] ꢂ 100.
2
Control: the cells treated with LPS only.
Compound: the cells treated with LPS and compounds.
Blank: the cells cultured with fresh medium only.
ꢀ ꢀ
(
230 mg, 94%). mp 190.1 C-191.3 C. 1H NMR (300 MHz, DMSO-d )
6
d
10.11 (s, 1H), 7.56 (d, J ¼ 8.5 Hz, 2H), 7.16 (d, J ¼ 8.4 Hz, 2H), 6.63 (s,
H), 6.13 (s, 1H), 4.07 (s, 2H), 3.74 (s, 6H), 3.61 (s, 3H), 3.53 (s, 2H).
Cell Cytotoxicity. Cell cytotoxicity was assessed by the 3-[4,5-
dimethylthiazol-2-yl]-2,5- diphenyltetrasolium bromide (MTT)
2
1
3
3
C NMR (75 MHz, DMSO-d
6
)
d
169.5, 166.0, 158.1, 153.1, 149.6,
assay. In brief, RAW264.7 cells (4 ꢂ 10 cells/well) were seeded in
1
4
38.5, 136.7, 131.9, 131.2, 129.7, 121.5, 119.8, 106.9, 81.8, 60.4, 56.3,
96-well plate containing DMEM supplemented with 10% FBS and
treated with different compounds which were diluted in DMEM for
þ
79
[MþH]þ 488.0703,
4.0, 34.8. HEMS (ESI ) calcd for C23H23BrNO
6
found 488.0714.
48 h. After treatment, 20
and incubated with in the dark for 4 h. Then, the medium was
removed and 150 L DMSO was added to dissolve formazone
mL of 0.5 mg/mL MTT reagent was added
Bacterial Culture. In this work, P. aeruginosa reporter strain
PAO1-lasB-gfp strain PAO1-rhlA-gfp strain and PAO1-pqsA-gfp
strain, which was kindly provided by Singapore Centre for Envi-
m
crystals for measurement at 570 nm (OD570) with a microplate
reader. Cell viability was given as relative value to the control in
percent from three independent experiments.
ꢀ
ronmental Life Sciences Engineering, stored at ꢁ80 C was used to
study the effects of new compounds by QS Inhibition Assays. A

X.-J. Xu et al. / European Journal of Medicinal Chemistry 144 (2018) 164e178
177
Cell viability (%) ¼ compound (OD570)/control (OD570) ꢂ 100.
Pharmaceutical University.
Control: the cells cultured with fresh medium only.
Compound: the cells treated with compounds or LPS.
LPS-Induced Inflammatory Mortality in SPF Mice. Compounds
were dissolved in DMSO, then stroke-physiological saline solution
was added to the concentration of 20 mg/kg. Male SPF mice were
randomly divided into five groups (n ¼ 10 mice/group) and i.v.
injection with high dose (20 mg/kg), low dose (5 mg/kg) of 2e, or
20 mg/kg of rosiglitazone. 6 days later, LPS was i.v. injected (20 mg/
kg). Control group received a similar volume of vehicle. Then, the
mortality was recorded for 7 days.
Statistical Analysis. All data in the text were presented as the
arithmetic mean ± standard deviation (SD) of at least three inde-
pendent experiments, and values of p < 0.05 and p < 0.01 were
considered to be statistically significant.
Measurement of TNF-a and IL-6. Production of the pro-
inflammatory cytokines was evaluated by an enzyme-linked
5
immunosorbent assay (ELISA). RAW264.7 cells (5 ꢂ 10 cells/
well) were pretreated with or without 10 mM of compounds in 24-
well plates for 2 h, and then the cells were stimulated with 100 ng/
mL LPS and cultured for 6 h, 12 h and 24 h to assay TNF- and IL-6
production. The levels of TNF- and IL-6 were measured using a
specific ELISA kit (TNF- : MultiSciences, EK2822; IL-6: Multi-
a
a
a
Sciences, EK2062) according to the manufacturer's instructions,
and were read at 450 nm (OD450) in a microplate reader. LPS was
used as the positive control in parallel experiments. The results
were calculated from three independent experiments.
Acknowledgment
6
Western Blot Analysis. RAW264.7 cells (2 ꢂ 10 cells/well)
were pretreated with or without compounds for 1 h in 6-well
plates. After treatment, cells were stimulated with LPS (500 ng/
mL) for 4 h. The cells were collected and washed with ice-cold
phosphate buffered saline (PBS), and the extract proteins were
lysed with IP buffer (Beyotime, P0013) containing with 1 mM
phenylmethanesulfonyl fluoride (PMSF: Beyotime, ST506) for
This work was supported by the National Natural Science
Foundation of China (81072554, 81673336) and the Fundamental
Research Funds for the Central University (No. 21617478).
Appendix A. Supplementary data
ꢀ
ꢀ
3
0 min at 4 C. Then cell lysates were centrifuged at 4 C to collect
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.ejmech.2017.11.085.
the supernatant. Total protein concentration was quantitated with
the BCA protein assay kit (Thermo Scientific, 23227). The samples
(
1
20
mg of protein) were mixed with loading buffer, separated on
Abbreviations used
2.5% SDS polyacrylamide gels, and transferred onto polyvinylidene
fluoride (PVDF) membranes (Millipore, USA). The PVDF membranes
were blocked at room temperature with 5% non-fat dry milk in Tris-
buffered saline Tween-20 (TBST) buffer and then were incubated
while shaking with targeted primary antibodies, including anti-
phosphorylation of ERK1/2 (Thr202/Tyr204), anti-ERK1/2, anti-
phosphorylation of p38 (Thr180/Tyr182), anti-p38, anti-phos-
QS, quorum-sensing; NF-
mitogen-activated protein kinase; PPAR
activated receptor ; LPS, lipopolysaccharides; 3OC12-HSL, 3-
oxododecanoyl homoserine lactone; TNF- , tumor necrosis factor
; PPAR , Peroxisome proliferator-activated receptor gamma; IL-6,
interleukin-6; AHL, N-acyl homoserine lactones; NMR, nuclear
magnetic resonance; MS, mass spectrometry; HRMS, high resolu-
tion mass spectrometry; NO, nitric oxide; MTT, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ELISA,
k
B, nuclear factor-kappa B; MAPK,
g, peroxisome proliferator-
g
a
a
g
phorylation of I
k
B
a
(Ser32/36), anti-I
k
B
a
, anti-phosphorylation of
ꢀ
NF- B p65, anti-NF-
k
k
B p65, and
b-actin, overnight at 4 C overnight.
All the primary antibodies were purchased from Cell Signaling
Technology. Then, the PVDF membrane was washed three times
with TBST buffer and incubated with anti-rabbit or anti-mouse
HRP-conjugated secondary antibody for 90 min at room tempera-
ture. Finally, the membranes were washed with TBST and exposed
to ECL reagents. Protein level was normalized to the matching
densitometric value of the internal control.
enzyme linked immunosorbent assay; IkB-a, inhibitor kappa B
alpha; ERK, extracellular regulated protein kinases; DCM,
dichloromethane; DMSO, dimethyl sulfoxide; LB, Luria-Bertani;
DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate
buffered saline; PMSF, phenylmethanesulfonyl fluoride; PVDF,
polyvinylidene fluoride; TBST, Tris-buffered saline Tween-20;
MOLCAD, Molecular Computer Aided Design.
Molecular Modeling. In present work, we employed the
structure of PPAR
g (PDB ID: 2PRG) to complete this docking
experiment. First, the structures of 2e, 2k, rosiglitazone and indo-
methacin were built by the sketching program in SYBYL 8.1 mo-
lecular modeling package of Tripos, and the minimized energy
structures of these four ligands were performed by using the Tripos
molecular mechanics force field. Second, all ligands were removed
and the polar hydrogen atoms were added. Other parameters were
established by defaults in the software. Third, the docking calcu-
lation was performed using the empirical scoring function in
Surflex-Dock. The Molecular Computer Aided Design (MOLCAD)
program was also developed and displayed with cavity depth po-
tential and hydrogen bond site to further explore the interaction
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