Design, synthesis and anti-inflammatory evaluation of novel 5-benzylidene-3,4-dihalo-furan-2-one derivatives

Article abstract of DOI:10.1016/j.ejmech.2013.10.074

Rosiglitazone has shown promising anti-inflammation effect. To develop preferable anti-inflammatory agents, twenty-two rosiglitazone analogs were synthesized and their anti-inflammatory activity was evaluated. Among these compounds, 6i and 6k displayed excellent inhibitory activities on the production of inflammatory mediators, including nitric oxide (NO), tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). Furthermore, 6i and 6k showed suppression effects on the nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways, and this suppression effects could be partially reversed by GW9662, which is a peroxisome proliferator-activated receptor γ (PPARγ) antagonist. Additionally, our docking results exhibited the well combination of 6i and 6k to PPARγ. So the anti-inflammation activity of 6i and 6k was due at least in part, to their interaction with PPARγ.

Full text of DOI:10.1016/j.ejmech.2013.10.074

European Journal of Medicinal Chemistry 72 (2014) 35e45
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and anti-inflammatory evaluation of novel
-benzylidene-3,4-dihalo-furan-2-one derivatives
5
Fang Wang, Jun-Rong Sun, Mei-Yan Huang, Hui-Ying Wang, Ping-Hua Sun, Jing Lin,
*
Wei-Min Chen
Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, College of Pharmacy, Jinan University, Huangpu
Dadao West, Guangzhou 510632, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 September 2013
Received in revised form
Rosiglitazone has shown promising anti-inflammation effect. To develop preferable anti-inflammatory
agents, twenty-two rosiglitazone analogs were synthesized and their anti-inflammatory activity was
evaluated. Among these compounds, 6i and 6k displayed excellent inhibitory activities on the production
28 October 2013
of inflammatory mediators, including nitric oxide (NO), tumor necrosis factor-alpha (TNF-
interleukin-6 (IL-6). Furthermore, 6i and 6k showed suppression effects on the nuclear factor-kappa B
NF- B) and mitogen-activated protein kinase (MAPK) pathways, and this suppression effects could be
partially reversed by GW9662, which is a peroxisome proliferator-activated receptor (PPAR ) antag-
onist. Additionally, our docking results exhibited the well combination of 6i and 6k to PPAR . So the anti-
inflammation activity of 6i and 6k was due at least in part, to their interaction with PPAR
a) and
Accepted 29 October 2013
Available online 20 November 2013
(
k
g
g
Keywords:
g
3,4-Dihalo-furan-2-one
g
.
Synthesis
Anti-inflammation
Action mechanism
Ó 2013 Elsevier Masson SAS. All rights reserved.
PPAR
g
1
. Introduction
and pioglitazone, are capable of reducing the expression of in-
flammatory genes in macrophages [12,13]. In particular, rosigli-
tazone has reported to have promising anti-inflammatory effect
Inflammation is a fundamental protective response of the im-
mune system to pathogens or harmful irritants and the classical
signs of inflammation are redness, swelling, heat, and pain [1,2].
Paradoxically, the inflammatory process itself may cause tissue
damage and lead to a host of diseases while it is a fundamental
protective response. For example, excessive production of inflam-
matory mediators, such as nitric oxide (NO), interleukin-6 (IL-6)
through the mechanisms that inhibition of TNF-
duction, as well as activation of PPAR receptor and inhibition the
NF- B activation [14e17]. Thus to develop small molecules to
activate PPAR , thereby inhibiting the production of inflammatory
mediators, as well as suppressing activation of NF- B and MAPK
a and IL-6 pro-
g
k
g
k
cascades has been shown to be a feasible anti-inflammation
strategy.
and tumor necrosis factor-alpha (TNF-
a) [3e5], as well as aberrant
activation of nuclear factor-kappa B (NF-
protein kinase (MAPK) pathways may be observed after infection or
injury, which have been implicated in many inflammatory diseases
k
B) and mitogen-activated
In order to develop novel agents that are preferable in anti-
inflammation, at here, a variety of structural modifications of the
rosiglitazone have been carried out. Based on the result of our
previous work that compounds bearing a halogenated furanone
moiety had excellent anti-bacterial activity, as well as the fact that
bacterial infection is an important factor for inflammation, we hy-
pothesized that using halogenated furanones to transform rosigli-
tazone’s skeleton may lead to more active compounds [18e20]. In
this regard, according to the bioisosterism, a series of rosiglitazone
analogs with the thiazolidinedione moiety replaced by halogenated
furanone were designed, synthesized, and their anti-inflammation
activities were examined with regard to their effects on the pro-
[
6,7].
Recently, accumulating evidences have illustrated that perox-
isome proliferator-activated receptor (PPAR ), which is origi-
g
g
nally known as a nuclear receptor and functions as transcription
factor, has emerged as a potential target for the treatment of in-
flammatory diseases such as ulcerative colitis, atherosclerosis,
asthma and rheumatoid arthritis [8e12]. It is well known that the
PPAR
g agonists-thiazolidinediones (TZDs), such as rosiglitazone
duction of inflammatory mediators, including NO, TNF-a and IL-6 in
lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Moreover,
western blot experiments and docking studies were performed in
*
Corresponding author. Tel.: þ86 20 8522 4497; fax: þ86 20 8522 4766.
E-mail address: twmchen@jnu.edu.cn (W.-M. Chen).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.10.074

36
F. Wang et al. / European Journal of Medicinal Chemistry 72 (2014) 35e45
an attempt to illustrate the anti-inflammation mechanism of 6i and
k, which were two compounds with dramatically improved anti-
inflammatory activity.
under alkaline condition [29]. The latter compounds and thiazo-
lidine-2,4-dione were dissolved in anhydrous toluene and refluxed
for 2 h under nitrogen to give four compounds (as shown in
Scheme 3).
6
There was a problem that E and Z geometrical isomers around
2
. Chemistry
the exocyclic double bond (CH]C) were possible for 6ae6w and
1
7
ae7d. H NMR spectrum of the twenty-two compounds showed
In order to develop preferable anti-inflammatory agents, a va-
only one signal for the benzyl fork proton that the chemical shifts
were basically in the same range from 6.33 to 6.40 ppm and from
.70 to 7.78 ppm for 6ae6w (in deuterated chloroform solution)
riety of structural modifications of the rosiglitazone have been
carried out according to the bioisosterism. As shown in Scheme 1,
the modifications include: (a) replaced the thiazolidinedione moi-
ety (Scheme 1: section A) of rosiglitazone with halogenated fur-
anone, which was an important structure that could improve the
drug activity because of the role inhibition of bacteria [18e20]; (b)
introduction of electron-donating substituent (methoxyl or
ethyoxyl) into the benzene ring (Scheme 1: section B); (c) replayed
the section C (Scheme 1) with pyridyl group or tetramethylpyr-
7
and 7ae7d (in deuterated dimethylsulfoxide solution), respec-
tively. Additionally, the Z-configuration had been reported ther-
modynamically more stable than the E-configuration [30,31]. Our
results as well as the judgment of similar compounds in the liter-
atures [32,33] suggested that the configuration of synthesized
compounds was single Z configuration.
1
All the compounds were fully analyzed and characterized by H,
azinyl group which showed PPAR
activity [21,22].
g agonist and anti-inflammatory
13
C nuclear magnetic resonance (NMR), mass spectrometry (MS)
and high resolution mass spectrometry (HRMS) before entering the
biological evaluation.
The synthesis of the designed compounds in Scheme 1 was
accomplished as outlined in Scheme 2 (6ae6w) and Scheme 3 (7a-
7
d).
In brief, mucochloric acid or mucobromic acid as starting ma-
3. Results and discussion
terials, were converted into halogenated furanones via a reaction
employed sodium borohydride as reducing agent and concen-
trated sulfuric acid as dehydrating agent in absolute methanol
3
.1. Inhibition of NO production in (LPS)-stimulated RAW264.7 cells
Nitric oxide (NO) is a significant pro-inflammatory mediator.
[23,24]. The halogenated furanones then reacted with hydroxyl
Excessive production of NO was found to be associated with the
pathogenesis of inflammation diseases, and it is generally
accepted that NO inhibitors may offer potential opportunity to
identify new therapeutic method for the inflammatory diseases
benzaldehydes to yield 3,4-dihalo-5-(4-hydroxybenzylidene)
furan-2(5H)-one intermediates via Knoevenagel reaction [24,25].
This condensation was carried out in refluxing toluene, containing
a catalytic amount of piperidinium acetate. Finally the 3,4-dihalo-
[34]. So first all the synthesized compounds were investigated
5
-(4-hydroxybenzylidene)furan-2(5H)-ones were connected to
for their inhibitory activity against lipopolysaccharide (LPS)-
induced NO release in RAW264.7 cells (As shown in Fig. S1, LPS
treatment caused significant changes in cell morphologies, which
indicated that inflammation could be induced by LPS). Here
rosiglitazone and indomethacin (Fig. 1) were chosen as positive
controls. As depicted in Table 1, most of the 5-benzylidene-3,4-
dihalo-furan-2-one derivatives (6ae6w) displayed improved NO
inhibitory activity compared to rosiglitazone and indomethacin.
In particular, two compounds (6i and 6k) exhibited excellent
activity, and their NO inhibition rates exceeded 75% at the con-
form ether with (3,5,6-trimethylpyrazin-2-yl)methanol (TMP-OH)
or pyridine derivatives via Mitsunobu coupling [26,27] to produce
end-products with a yield of 70e80% (as shown in Scheme 2).
Besides, 5-benzylidenethiazolidine-2,4-dione derivatives were
synthesized from 2,3,5,6-tetramethylpyrazine (TMP) and N-bro-
mosuccinimide (NBS) via free radical reaction [28]. The typical
subsequent synthetic procedure involved the combination of 2-
(
bromomethyl)-3,5,6-trimethylpyrazine (TMP-Br) and hydroxyl
benzaldehyde derivatives through the formation of ether bonds
centration of 10 mM. Another four 5-benzylidenethiazolidine-2,4-
dione derivatives (7ae7d) with TMP substituted were slightly
less active.
Based on these results, some preliminary structureeactivity
relationship (SAR) of the rosiglitazone analogs could be summa-
rized: (a) 3,4-dihalo-furan-2-one structure was conducive to anti-
inflammatory activity (6ae6w compared to 7ae7d, such as 6w
compared to 7b); (b) a two-atom linker between the ring B and ring
C (Scheme 2) would be favorable (6h compared to 6n); (c) electron-
donating substituent (methoxyl or ethyoxyl) at phenyl ring would
increase its NO inhibitory activity (6h and 6j compared to 6a); (d)
TMP substitution almost had no effect on the anti-inflammatory
activity (6v compared to 6b). These results will be really useful in
the future to guide the design and modification of new candidate
anti-inflammatory agents.
3.2. Cytotoxicity in RAW264.7 cells
To investigate whether the NO inhibitory activities of 6i and 6k
were related to cell viability, their cytotoxicities in RAW264.7 cells
were examined by methyl thiazolyl tetrazolium (MTT) assay. As
shown in Table 2, all the agents (rosiglitazone, indomethacin, 6i, 6k,
GW9662 and LPS) at the concentrations we detected here had no
obviously cytotoxicity in RAW264.7 cells, and the relative cell
Scheme 1. Design strategy for rosiglitazone analogs.

F. Wang et al. / European Journal of Medicinal Chemistry 72 (2014) 35e45
37
3 3
Scheme 2. General synthesis of compounds 6ae6w. Reagents and conditions: (a) 2-methyl piperidine, CH COOH, toluene, 2 h; (b) PPh , DEAD, THF, 24 h.
2 2 4 2 3 3
Scheme 3. General synthesis of compounds 7ae7d. Reagents and conditions: (a) NBS, (PhCO) O , CCl , 12 h; (b) K CO , DMF, 10 h; (c) 2-methyl piperidine, CH COOH, toluene, 2 h.
Fig. 1. Chemical structures of indomethacin, rosiglitazone, 6i and 6k.
viabilities of the treated cells were all more than 90%. These results
indicated that the NO inhibitory effects of 6i and 6k were likely to
be attributed to the interaction of these two compounds and their
specific target. These non-toxic concentrations were further used in
subsequent experiment processes.
treatment, and their inhibitory effects were evidently better than
that of the positive control drugs treatment on both TNF- and IL-6
production at all three detection time. Noticeably, 6k exhibited
significant improvement in inhibition of IL-6 production compared
to control drugs. All the above results suggested that 6i, especially
a
6k, were excellent agents in anti-inflammation effect. Next, a
plenty of efforts were performed to explore the possible anti-
inflammatory mechanism of 6i and 6k.
3.3. Inhibition of TNF-
a and IL-6 production in RAW264.7 cells
TNF- and IL-6 are two crucial pro-inflammatory cytokines
a
which are of paramount importance in inflammatory diseases, and
widely recognized that inhibition of their generations would be an
effective method for treatment of inflammation [35]. So next, the
3.4. Western blot for interpreting possible mechanism
It has been reported that NF-kB and MAPK activation (phos-
inhibitory effects of 6i and 6k on TNF-
a
and IL-6 production were
phorylation) was quite significant in the regulation of inflammation
investigated. Rosiglitazone and indomethacin (Fig. 1) were also
used as positive reference drugs. The effects of these four com-
because of their crucial roles in the mediation of the production of
NO, TNF-a, IL-6, and other inflammatory mediators in activated
pounds were evaluated using a fixed concentration of 10
a time-dependent manner.
mM and in
macrophages [36e40]. And it is well accepted that the up-
regulation of the synthesis of inflammatory mediators induced by
The results (Table 3) showed that the LPS-induced production of
TNF- and IL-6 were significantly decreased by 6i and 6k
LPS, derived involved NF-
k
B and MAPK activation, was modulated
B activation could be inhibited by
a
by PPAR , and the MAPK and NF-k
g

38
F. Wang et al. / European Journal of Medicinal Chemistry 72 (2014) 35e45
Table 1
3.5. Docking analysis
The inhibitory effects of the synthesized compounds on NO production in LPS-
stimulated RAW264.7 cells.
To confirm the above hypothesis that 6i and 6k were likely to act
as PPAR agonist, we performed some docking analysis to investi-
gate the interaction of 6i and 6k with PPAR . For the docking
studies, rosiglitazone and indomethacin, which are two identified
Compounds
Rosib
NO inhibition (%)a
Compounds
NO inhibition (%)a
g
**
g
16.7 ꢀ 3.9
6p
6q
6r
6s
6t
6u
6v
6w
7a
7b
7c
7d
40.8 ꢀ 5.1*
16.3 ꢀ 3.4*
50.4 ꢀ 2.6*
36.8 ꢀ 3.2*
38.9 ꢀ 5.9*
36.4 ꢀ 1.7**
30.1 ꢀ 2.4**
37.2 ꢀ 7.1*
19.3 ꢀ 8.0*
24.9 ꢀ 6.1*
30.7 ꢀ 4.9*
23.5 ꢀ 2.2**
c
**
Indo
31.3 ꢀ 2.9
*
6
6
6
6
6
6
6
6
6
6
a
b
c
g
h
i
j
k
l
38.0 ꢀ 6.7
PPAR
PPAR
g
g
agonists, were used as reference molecules. The structure of
used in the docking studies was obtained from the Protein
*
29.2 ꢀ 9.5
*
*
*
*
*
*
*
*
*
*
43.6 ꢀ 2.8
20.2 ꢀ 6.1
54.7 ꢀ 2.3
75.8 ꢀ 4.3
62.9 ꢀ 6.8
80.1 ꢀ 4.1
42.2 ꢀ 3.7
33.9 ꢀ 4.7
Data Bank (PDB ID: 2PRG) and all docking experiments were per-
formed using SYBYL 8.1 program package of Tripos [49]. The
docking scores of all four molecules with PPAR
Table 4.
g were shown in
In line with the western blot results, 6i and 6k could well interact
with PPAR , even with higher docking scores than that for rosigli-
tazone and indomethacin, which implied the possibility of the
directly interaction of 6i and 6k with PPAR . Furthermore, the
g
n
*
*P < 0.01, *P < 0.05 versus the LPS (treated with LPS only) group.
a
g
Results were showed as means ꢀ SD (n ¼ 4) of at least three independent
experiments.
MOLCAD (Molecular Computer Aided Design) Multi-Channel sur-
faces structures displayed with cavity depth potential or hydrogen
bonds of the PPARg-binding site within the four compounds were
b
Rosi: rosiglitazone.
Indo: indomethacin.
c
also developed to explore the ligandereceptor interaction details.
Fig. 3 depicted the MOLCAD cavity depth potential surface of the
PPARg pocket within four compounds, the cavity depth color ramp
Table 2
Effects of compounds on the viability of RAW264.7 cells.
Compounds
Concentrations
Cell viability (%)a
ranged from blue (low depth values ¼ outside of the pocket) to or-
ange (high depth values ¼ deep inside the pocket). Fig. 4 displayed
Blank
Rosib
100.0 ꢀ 5.8**
99.7 ꢀ 6.9*
100.8 ꢀ 7.4*
98.1 ꢀ 7.2*
93.1 ꢀ 5.6**
101.0 ꢀ 6.3*
99.4 ꢀ 4.7**
103.6 ꢀ 3.0**
10
10
10
10
mM
mM
mM
mM
the hydrogen bonds formed between PPARg and four compounds,
c
Indo
the hydrogen bonds were showed as yellow dashed lines.
As shown in Fig. 3, four ligands were able to bind to PPAR
6
6
i
k
g
while with different depths. Rosiglitazone and indomethacin were
stayed in a blue region which indicated a relative low depth,
while compounds 6i and 6k were oriented in a light yellow region
which demonstrated that the majority parts of these two mole-
cules were anchored deep inside the pocket. And as shown in
GW9662
LPS
LPS
5 mM
500 ng/mL
100 ng/mL
**P < 0.01, *P < 0.05 compared to the blank (cultured with fresh medium only)
group.
a
Results were expressed as means
experiments.
ꢀ
SD (n
¼
5) of three independent
Fig. 4, several hydrogen bonds formed between 6i, 6k and PPAR
In summary, our docking results exhibited the strong binding
ability of 6i and 6k to PPAR , which provided strong evidence for
was, at least one of the specific targets
g.
b
Rosi: rosiglitazone.
Indo: indomethacin.
c
g
the hypothesis that PPAR
g
of 6i and 6k.
4. Conclusions
pre-treatment with PPARg agonists, such as rosiglitazone [41].
Therefore, in this study, western blot analysis was employed to
investigate whether the anti-inflammatory effects of 6i and 6k
In this study, a series of novel 5-benzylidene-3,4-dihalo-furan-
-one derivatives, obtained from the structural modifications of the
2
were associated with the PPAR
MAPK. The proteins we detected here included two NF-
proteins: NF- B p65 and inhibitor kappa B alpha (I ) kinase [42e
4], as well as three MAPK cascades: extracellular regulated protein
g
mediated activation of NF-kB and
k
B pathway
Table 3
k
kB
a
The inhibitory effects of rosiglitazone, indomethacin, 6i and 6k on LPS-induced TNF-
a and IL-6 production in RAW264.7 cells.
4
kinases (ERK1/2, or p42/44), c-Jun N-terminal kinases (JNK) and
p38 MAPK [45e47].
As shown in Fig. 2, LPS significantly increased the levels of
phosphorylated NF-kB p65, IkBa, JNK, ERK1/2 and p38 MAPK, and
both 6i and 6k treatment could lead to a decrease in activating of
these proteins in varying degrees. Furthermore, the phosphoryla-
(pg/mL)a
TNF-a
Compounds
Blank
6
h
12 h
11.8 ꢀ 1.2
24 h
38.3 ꢀ 3.3
5.9 ꢀ 0.5
LPS
455.4 ꢀ 41.1**
416.9 ꢀ 19.7**
366.5 ꢀ 55.1*
347.1 ꢀ 35.8**
661.7 ꢀ 59.4**
631.3 ꢀ 68.0*
612.6 ꢀ 54.6*
575.6 ꢀ 39.6*
519.8 ꢀ 42.1**
1474.0 ꢀ 65.7*
1293.8 ꢀ 94.9*
1242.0 ꢀ 80.3**
1110.5 ꢀ 84.5*
1044.2 ꢀ 132.8*
LPS + Rosib
c
LPS + Indo
tions of NF-
inhibited after treatment with 6i and 6k, while they had slightly
antagonized effect on phosphorylation of I . Moreover, 6k
kB p65, I
k
Ba
, ERK1/2 and p38 MAPK were obviously
LPS+6i
LPS+6k
323.8 ꢀ 46.3*
a
IL-6 (pg/mL)
kBa
significantly inhibited the phosphorylation of JNK. Additionally, it
was worth to notice that the suppressive effects of 6i and 6k on the
6 h
12 h
24 h
Blank
7.2 ꢀ 0.9
13.9 ꢀ 2.6
45.1 ꢀ 5.6
protein phosphorylations could be reversed by GW9662 (5
mM),
LPS
3065.4 ꢀ 166.4**
2783.9 ꢀ 136.1**
2313.5 ꢀ 218.0*
1705.9 ꢀ 124.1**
598.1 ꢀ 117.1**
4014.2 ꢀ 244.8*
3477.9 ꢀ 145.9**
2989.6 ꢀ 228.9*
2528.4 ꢀ 170.2*
902.2 ꢀ 137.8**
9546.6 ꢀ 595.0*
8388.6 ꢀ 446.9*
7645.9 ꢀ 369.1**
6360.0 ꢀ 635.5*
2360.2 ꢀ 482.1*
LPS + Rosib
which is a highly selective and irreversible PPAR antagonist [48].
g
c
LPS + Indo
LPS + 6i
LPS + 6k
Combination of all the western blot results, we hypothesized that
anti-inflammation effects of 6i and 6k were due at least in part, to
their interaction with PPAR
g, thereby suppressing activation of NF-
*
*P < 0.01, *P < 0.05 versus the LPS (treated with LPS only) group.
kB and MAPK cascades, resulting in decreased NO, TNF-
a
and IL-6
a
Results were showed as means ꢀ SD (n ¼ 3) of three independent experiments.
levels. Nonetheless, further studies were needed to confirm this
hypothesis.
b
Rosi: rosiglitazone.
c
Indo: indomethacin.

F. Wang et al. / European Journal of Medicinal Chemistry 72 (2014) 35e45
39
Fig. 2. The effects of compounds 6i and 6k on the LPS-induced phosphorylation of NF-
k
B p65, I
kBa, JNK, ERK1/2 and p38 MAPK in RAW264.7 cells. RAW264.7 cells were treated
with 6i (10
m
M), 6k (10
m
M), GW9662 (5
m
M), and LPS (500 ng/mL) for 4 h. The levels of NF- B p65, IkBa, JNK, ERK1/2, and p38 MAPK proteins, and their phosphorylated forms were
k
analyzed using western blotting. Data were 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.
PPAR
g
agonist-rosiglitazone, were synthesized and their bio-
halogenated furanone structure, which was an important structure
of some bacterial inhibitors, generally exhibited improved anti-
activities were evaluated. Among these compounds, two promising
compounds (6i and 6k) for the treatment of inflammatory diseases
were identified. Mechanism studies revealed that, like rosiglita-
zone, anti-inflammation activity of 6i and 6k was due at least in
part, to their interaction with PPAR
tion of NF- B and MAPK cascades, resulting in decreased NO, TNF-
and IL-6 levels. These results further confirmed the previous pro-
posal that PPAR might be a feasible anti-inflammatory target.
inflammation activity compared to PPAR
which suggested that combination of the structural features
with anti-bacterial activity into the potential PPAR agonists to
obtain molecules with both anti-bacterial and PPAR activation
g agonist-rosiglitazone,
g
g, thereby suppressing activa-
g
k
a
properties would be an effective strategy to develop preferable
anti-inflammatory agents.
g
Moreover, SAR analysis illustrated that compounds with the
5. Experimental section
5.1. Chemistry
Table 4
Docking scores for the combination of rosiglitazone, indomethacin, 6i and 6k to
PPAR
g
.
5.1.1. General procedure for synthesis of halogenated furanone
derivatives (A
The 3,4-dihalo-furan-2(5H)-ones (A
to our previously reported methods [29].
1
)
Compounds
Scores
Rosi
6.94
Indo
4.92
6i
6k
1
) were prepared according
8.07
8.52

40
F. Wang et al. / European Journal of Medicinal Chemistry 72 (2014) 35e45
Fig. 3. The MOLCAD Multi-Channel surface structures displayed with cavity depth of the PPARg within the (A) rosiglitazone, (B) indomethacin, (C) 6i and (D) 6k. The color ramp for
cavity depth ranged from blue (outside of the pocket; most negative) to orange (deep inside the pocket; most positive). (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)
5
.1.2. General procedure for synthesis of 3,4-dihalo-5-(4-
hydroxybenzylidene)furan- 2(5H)-one intermediates (A
Benzaldehyde derivatives (B , 1.0 mmol) and halogenated fur-
anones (A ,1.0 mmol) were dissolved in anhydrous toluene (30 mL),
NMR (75 MHz, DMSO-d
117.2, 116.7, 113.4. ESI-MS m/z: 255.2 [M ꢂ H] .
6
) d 162.6, 160.1, 142.5, 141.3, 133.6, 123.3,
-
1 1
B )
1
1
5
.1.2.3. (Z)-3,4-Dibromo-5-(4-hydroxy-3-methoxybenzylidene)
and then a catalytic amount of 2-methyl piperidine and glacial
acetic acid were slowly dropped to the solution under the atmo-
sphere of nitrogen. The reaction mixture was refluxed at 130 C for
1
furan-2(5H)-one. Yellow solid; yield 33.0%. H NMR (300 MHz,
DMSO-d
9.88 (s, 1H), 7.44 (s, 1H), 7.39 (d, J ¼ 1.8 Hz, 1H), 6.87 (d,
J ¼ 1.8 Hz, 1H), 6.58 (s, 1H), 3.81 (s, 3H). C NMR (75 MHz, DMSO-
163.9, 149.8, 148.2, 144.0, 138.0, 125.8, 123.9, 116.6, 115.2, 114.9,
6
) d
ꢁ
13
2
h until the disappearance of starting materials (TLC analysis), and
6
d ) d
then cooled to room temperature. The toluene was removed under
reduced pressure and the residue was dissolved in methanol. This
solution was concentrated and purified by silica gel column chro-
matography with ethyl acetate-petroleum (1:12) as eluent to pro-
duce resultant intermediates.
-
111.2, 56.1. ESI-MS m/z: 375.4 [M ꢂ H] .
5
2
d
.1.2.4. (Z)-3,4-Dichloro-5-(4-hydroxy-3-methoxybenzylidene)furan-
(5H)-one. Yellow solid; yield 36.1%. H NMR (300 MHz, DMSO-d
1
6
)
9.91 (s, 1H), 7.41 (d, J ¼ 1.8 Hz, 1H), 7.37 (d, J ¼ 1.8 Hz, 1H), 6.90 (d,
13
1
6
H), 6.62 (s, 1H), 3.81 (s, 3H). C NMR (75 MHz, DMSO-d ) d 162.5,
1
49.8, 148.3, 142.5, 141.4, 126.0, 123.7, 117.2, 116.6, 115.2, 113.7, 56.1.
5
.1.2.1. 3,4-Dibromo-5-(4-hydroxybenzylidene)furan-2(5H)-one
ꢂ
ESI-MS m/z: 285.3 [M ꢂ H] .
1
(
d
Z:E ¼ 92:8). Yellow solid; yield 35.7%. H NMR (300 MHz, DMSO-
) Z type
10.28 (s, 1H), 7.72 (d, J ¼ 8.7 Hz, 2.07H), 6.86 (d,
J ¼ 8.7 Hz, 2.06H), 6.57 (s, 0.99H); E type 10.28 (s, 1H), 7.35 (d,
6
d
5
.1.2.5. (Z)-3,4-Dibromo-5-(3-ethoxy-4-hydroxybenzylidene)furan-
d
1
13
2(5H)-one. Red solid; yield 30.1%. H NMR (300 MHz, DMSO-d6)
d
J ¼ 8.8 Hz, 0.10H), 7.16 (s, 0.09H), 6.81 (d, J ¼ 8.8 Hz, 0.12H).
C
9.83 (s, 1H), 7.44 (s, 1H), 7.40 (d, J ¼ 1.8 Hz, 1H), 6.90 (d, J ¼ 1.8 Hz,
NMR (75 MHz, DMSO-d
6
)
d
163.9, 160.1, 143.9, 138.0, 133.5, 123.6,
13
ꢂ
1H), 6.58 (s, 1H), 4.08 (q, J ¼ 7.2 Hz, 2H), 1.36 (t, J ¼ 7.2 Hz, 3H).
C
116.7, 114.6, 111.3. ESI-MS m/z: 345.2 [M ꢂ H] .
6
NMR (75 MHz, DMSO-d ) d 163.9, 150.0, 147.4, 144.0, 138.0, 125.9,
1
24.0, 116.7, 116.4, 114.9, 111.2, 64.4, 15.1. ESI-MS m/z: 389.4
-
[M ꢂ H] .
5
.1.2.2. 3,4-Dichloro-5-(4-hydroxybenzylidene)furan-2(5H)-one
1
(
Z:E ¼ 92:8). Yellow solid; yield 37.3%. H NMR (300 MHz, DMSO-
) Z type
10.27 (s, 1H), 7.71 (d, J ¼ 8.7 Hz, 1.98H), 6.88 (d,
J ¼ 8.7 Hz, 1.96H), 6.62 (s, 0.97H); E type 10.27 (s, 1H), 7.40 (d,
J ¼ 9.0 Hz, 0.10H), 7.31 (s, 0.08H), 6.82 (d, J ¼ 9.0 Hz, 0.12H).
d
6
d
5.1.2.6. (Z)-3,4-Dichloro-5-(3-ethoxy-4-hydroxybenzylidene)furan-
1
d
2(5H)-one. Red solid; yield 37.1%. H NMR (300 MHz, DMSO-d
6
)
13
C
d
9.83 (s, 1H), 7.40 (d, J ¼ 1.8 Hz, 1H), 7.35 (d, J ¼ 1.8 Hz, 1H), 6.90

F. Wang et al. / European Journal of Medicinal Chemistry 72 (2014) 35e45
41
Fig. 4. The hydrogen bonds formed between PPAR
g
and (A) rosiglitazone, (B) indomethacin, (C) 6i and (D) 6k. Hydrogen bonds (HB) were showed as yellow dashed lines. Hydrogen
atoms omitted for clarity.
(
d, 1H), 6.62 (s, 1H), 4.07 (q, J ¼ 7.2 Hz, 2H), 1.37 (t, J ¼ 7.2 Hz, 3H).
5.1.4.2. (Z)-3,4-Dibromo-5-(4-(pyridin-2-ylmethoxy)benzylidene)
furan-2(5H)-one (6b). Yellow solid; yield 87.1%. H NMR (300 MHz,
1
3
1
C NMR (75 MHz, DMSO-d
6
)
d
162.5, 150.1, 147.4, 142.5, 141.3,
1
26.0, 123.7, 117.2, 116.7, 116.5, 113.7, 64.4, 15.1. ESI-MS m/z: 299.4
CDCl
3
)
d
8.61 (d, J ¼ 4.8 Hz, 1H), 7.77 (d, J ¼ 9.0 Hz, 2H), 7.73 (d,
-
[M ꢂ H] .
J ¼ 7.8, 4.8 Hz,1H), 7.50 (d, J ¼ 7.8 Hz,1H), 7.25e7.21 (m, 1H), 7.02 (d,
13
J ¼ 9.0 Hz, 2H), 6.39 (s, 1H), 5.25 (s, 2H). C NMR (75 MHz, CDCl
3
)
d
163.5, 159.9, 156.6, 149.4, 144.4, 137.2, 136.9, 133.0, 125.2, 122.8,
5
.1.3. General procedure for synthesis of (3,5,6-trimethylpyrazin-2-
yl)methanol (C
Compound C
50].
þ
1
21.4, 115.5, 114.0, 111.8, 70.7. ESI-MS m/z: 436.2 [M þ H] . HRMS
2
)
þ
(ESI) m/z: 435.9165 [M þ H] ; Calcd for C17
H11Br
2
NO
3
(M þ H)
2
was synthesized according to reported methods
435.9185.
[
5
.1.4. General procedures for the synthesis of 6ae6w
To a solution of A (1.0 mmol) and triphenylphosphine
1.5 mmol) in anhydrous tetrahydrofuran (3 mL), added C or C
2.0 mmol) and dropwise added diethyl azodicarboxylate (DEAD,
5
.1.4.3. (Z)-3,4-Dibromo-5-(4-(2-(5-ethylpyridin-2-yl)ethoxy)benzy-
1
B
1
1
lidene)furan-2(5H)-one (6c). Yellow solid; yield 78.1%. H NMR
300 MHz, CDCl
8.40 (s, 1H), 7.73 (d, J ¼ 9.0 Hz, 2H), 7.47 (d,
(
(
1
2
(
3
) d
J ¼ 7.8 Hz,1H), 7.19 (d, J ¼ 7.8 Hz,1H), 6.93 (d, J ¼ 9.0 Hz, 2H), 6.38 (s,
1.5 mmol) in anhydrous and anoxybiotic conditions. The reaction
1
2
H), 4.40 (t, J ¼ 6.7 Hz, 2H), 3.25 (t, J ¼ 6.7 Hz, 2H), 2.64 (q, J ¼ 7.6 Hz,
13
ꢁ
mixture was stirred at ꢂ2 C for 30 min, and then stirred at room
H), 1.25 (t, J ¼ 7.6 Hz, 3H). C NMR (75 MHz, CDCl
3
) d 163.5, 160.7,
temperature for 24 h. After completion of reaction was monitored
through TLC, the reaction mixture was filtered and washed with
ether and saturated salt water to get products because there would
be a solid precipitate.
155.3, 148.9, 144.1, 137.3, 136.0, 133.0, 132.1, 124.6, 123.4, 115.1, 114.4,
þ
1
11.5, 67.4, 37.3, 25.8, 15.5. ESI-MS m/z: 478.3 [M þ H] . HRMS (ESI)
þ
m/z: 477.9640 [M þ H] ; Calcd for C20
H17Br
2
NO
3
(M þ H) 477.9655.
5
.1.4.1. (Z)-3,4-Dibromo-5-(4-(2-(pyridin-2-yl)ethoxy)benzylidene)
5.1.4.4. (Z)-3,4-Dichloro-5-(4-(2-(pyridin-2-yl)ethoxy)benzylidene)
furan-2(5H)-one (6g). Yellow solid; yield 80.6%. H NMR (300 MHz,
1
1
furan-2(5H)-one (6a). Yellow solid; yield 70.0%. H NMR (300 MHz,
CDCl
8.56 (d, J ¼ 5.6 Hz, 1H), 7.74 (d, J ¼ 11.6 Hz, 2H), 7.62 (dd,
J ¼ 10.4, 2.4 Hz, 1H), 7.28 (d, J ¼ 10.4 Hz, 1H), 7.16 (dd, J ¼ 5.6, 2.4 Hz,
H), 6.92 (d, J ¼ 11.6 Hz, 2H), 6.37 (s,1H), 4.43 (t, J ¼ 8.8 Hz, 2H), 3.28
3
)
d
CDCl
3
)
d
8.58 (dd, J ¼ 4.9, 0.8 Hz, 1H), 7.74 (d, J ¼ 8.8 Hz, 2H), 7.66
(dd, J ¼ 7.5, 2.1 Hz, 1H), 7.36 (m, 1H), 7.19 (td, J ¼ 7.5, 4.9, 0.8 Hz, 1H),
1
6.95 (d, J ¼ 8.8 Hz, 2H), 6.36 (s, 1H), 4.46 (d, J ¼ 6.6 Hz, 2H), 3.30 (t,
13
13
(
1
t, J ¼ 8.8 Hz, 2H). C NMR (75 MHz, CDCl
3
)
d
163.5, 160.5, 158.0,
J ¼ 6.6 Hz, 2H). C NMR (75 MHz, CDCl
3
) d 162.5, 160.5, 158.0,149.4,
49.4, 144.0, 137.2, 136.3, 132.8, 124.6, 123.7, 121.6, 115.1, 114.2, 111.3,
142.7, 141.7, 136.5, 132.9, 124.4, 123.8, 121.7, 117.9, 115.2, 112.8, 67.2,
þ
þ
6
7.2, 37.8. ESI-MS m/z: 450.2 [M þ H] . HRMS (ESI) m/z: 449.9315
37.8. ESI-MS m/z: 362.2 [M þ H] . HRMS (ESI) m/z: 362.0349
þ
þ
[M þ H] ; Calcd for C18
H13Br
2
NO
3
(M þ H) 449.9342.
[M þ H] ; Calcd for C18
H13Cl
2
NO
3
(M þ H) 362.0352.

42
F. Wang et al. / European Journal of Medicinal Chemistry 72 (2014) 35e45
þ
5
.1.4.5. (Z)-3,4-Dibromo-5-(3-methoxy-4-(2-(pyridin-2-yl)ethoxy)
56.1. ESI-MS m/z: 468.3 [M þ H] . HRMS (ESI) m/z: 465.9281
1
þ
benzylidene)furan- 2(5H)-one (6h). Yellow solid; yield 71.1%.
NMR (300 MHz, CDCl
8.56 (d, J ¼ 4.5 Hz, 1H), 7.68 (td, J ¼ 7.5,
.5 Hz, 1H), 7.41 (d, J ¼ 1.8 Hz, 1H), 7.35 (d, J ¼ 7.5 Hz, 1H), 7.29 (dd,
J ¼ 8.4, 1.8 Hz, 1H), 7.21 (dd, J ¼ 4.5, 1.5 Hz, 1H), 6.93 (d, J ¼ 8.4 Hz,
H), 6.36 (s, 1H), 4.48 (t, J ¼ 6.9 Hz, 2H), 3.9 (s, 3H), 3.38 (t,
H
[M þ H] ; Calcd for C17
H11Br NO
2 3
(M þ H) 465.9291.
3
) d
1
5
.1.4.11. (Z)-3,4-Dichloro-5-(4-(2-(5-ethylpyridin-2-yl)ethoxy)ben-
1
zylidene)furan- 2(5H)-one (6p). Yellow solid; yield 54.3%. H NMR
300 MHz, CDCl
8.40 (s, 1H), 7.73 (d, J ¼ 9.0 Hz, 2H), 7.47 (d,
1
(
3
) d
13
J ¼ 6.9 Hz, 2H). C NMR (75 MHz, CDCl
3
) d 163.5, 160.3, 157.5, 150.3,
J ¼ 7.8 Hz,1H), 7.19 (d, J ¼ 7.8 Hz,1H), 6.93 (d, J ¼ 9.0 Hz, 2H), 6.34 (s,
149.5, 148.5, 144.3, 137.3, 125.8, 125.2, 124.3, 122.0, 114.5, 113.5,
1
H), 4.40 (t, J ¼ 6.7 Hz, 2H), 3.25 (t, J ¼ 6.7 Hz, 2H), 2.64 (q, J ¼ 7.6 Hz,
13
þ
1
12.9, 111.6, 68.1, 56.1, 37.4. ESI-MS m/z: 480.2 [M þ H] . HRMS (ESI)
2
H), 1.25 (t, J ¼ 7.6 Hz, 3H). C NMR (75 MHz, CDCl
3
) d 162.5, 160.5,
þ
m/z: 479.9435 [M þ H] ; Calcd for C19
H15Br
2
NO
4
(M þ H) 479.9447.
1
1
55.1, 148.9, 142.6, 141.7, 137.3, 136.0, 132.9, 131.9, 124.4, 123.4, 115.2,
þ
12.8, 67.4, 37.3, 25.7, 15.3. ESI-MS m/z: 390.4 [M þ H] . HRMS (ESI)
þ
5
.1.4.6. (Z)-3,4-Dibromo-5-(4-(2-(5-ethylpyridin-2-yl)ethoxy)-3-
m/z: 390.0639 [M þ H] ; Calcd for C20
H17Cl
2
NO
3
(M þ H) 390.0665.
methoxybenzylidene) furan-2(5H)-one (6i). Yellow solid; yield
8
1
1
1.3%. H NMR (300 MHz, CDCl
3
)
d
8.39 (s, 1H), 7.47 (d, J ¼ 7.9 Hz,
5
.1.4.12. (Z)-3,4-Dichloro-5-(4-(pyridin-2-ylmethoxy)benzylidene)
H), 7.42 (d, J ¼ 2.1 Hz, 1H), 7.30 (dd, J ¼ 8.4, 2.1 Hz, 1H), 7.22 (d,
1
furan-2(5H)-one (6q). Yellow solid; yield 71.0%. H NMR (300 MHz,
CDCl
J ¼ 7.9 Hz, 1H), 6.94 (d, J ¼ 8.4 Hz, 1H), 6.38 (s, 1H), 4.46 (t, J ¼ 7.1 Hz,
H), 3.90 (s, 3H), 3.32 (t, J ¼ 7.1 Hz, 2H), 2.64 (q, J ¼ 7.6 Hz, 2H), 1.25
3
)
d
8.60 (d, J ¼ 4.8 Hz, 1H), 7.76 (d, J ¼ 8.9 Hz, 2H), 7.70 (m, 1H),
2
7
6
.50 (d, J ¼ 7.8 Hz, 1H), 7.25e7.21 (m, 1H), 7.02 (d, J ¼ 8.9 Hz, 1H),
13
(
1
1
t, J ¼ 7.6 Hz, 3H). C NMR (75 MHz, CDCl
49.5, 148.8, 144.2, 137.3, 137.2, 136.0, 125.7, 125.0, 123.5, 114.5,
13.5, 112.8, 111.5, 68.3, 56.1, 37.2, 25.7, 15.3. ESI-MS m/z: 508.3
3
)
d
163.5, 155.1, 150.5,
13
3
.34 (s, 1H), 5.24 (s, 1H). C NMR (75 MHz, CDCl ) d 162.4, 159.9,
1
1
3
56.4, 149.3, 142.6, 141.9, 136.9, 133.0, 124.9, 122.8, 121.3, 118.2,
þ
15.5, 112.5, 70.7. ESI-MS m/z: 348.1 [M þ H] . HRMS (ESI) m/z:
þ
þ
[
C
M þ H] . HRMS (ESI) m/z: 507.9752 [M þ H] ; Calcd for
þ
48.0181 [M þ H] ; Calcd for C17
H
11Cl
2
NO
3
(M þ H) 348.0196.
21
H19Br
2
NO
4
(M þ H) 507.9760.
5
.1.4.13. (Z)-3,4-Dichloro-5-(3-ethoxy-4-(2-(pyridin-2-yl)ethoxy)
5
.1.4.7. (Z)-3,4-Dibromo-5-(3-ethoxy-4-(2-(pyridin-2-yl)ethoxy)
1
benzylidene)furan- 2(5H)-one (6r). Yellow solid; yield 76.3%.
NMR (300 MHz, CDCl
8.54 (d, J ¼ 4.8 Hz, 1H), 7.62 (td, J ¼ 7.5,
.5 Hz, 1H), 7.39 (d, J ¼ 1.8 Hz, 1H), 7.33 (d, J ¼ 7.5 Hz, 1H), 7.29 (dd,
J ¼ 8.4, 1.8 Hz, 1H), 7.15 (dd, J ¼ 4.8, 1.5 Hz, 1H), 6.92 (d, J ¼ 8.4 Hz,
H), 6.31 (s, 1H), 4.45 (t, J ¼ 6.8 Hz, 2H), 4.06 (q, J ¼ 7.0 Hz, 2H), 3.33
H
1
benzylidene)furan- 2(5H)-one (6j). Yellow solid; yield 76.3%.
NMR (300 MHz, CDCl
8.55 (d, J ¼ 4.8 Hz, 1H), 7.63 (td, J ¼ 7.7,
.8 Hz, 1H), 7.41 (d, J ¼ 2.1 Hz, 1H), 7.34 (d, J ¼ 7.7 Hz, 1H), 7.32e7.27
H
3
) d
3
) d
1
1
(
1
m, 1H), 7.16 (dd, J ¼ 4.8, 1.8 Hz, 1H), 6.93 (d, J ¼ 8.7 Hz, 1H), 6.36 (s,
1
H), 4.45 (t, J ¼ 6.8 Hz, 2H), 4.07 (q, J ¼ 7.0 Hz, 2H), 3.33 (t, J ¼ 6.8 Hz,
13
13
(
d
t, J ¼ 6.8 Hz, 2H), 1.43 (t, J ¼ 7.0 Hz, 3H). C NMR (75 MHz, CDCl
162.4, 158.2, 150.9, 149.3, 148.9, 142.6, 141.7, 136.3, 125.8, 124.8,
24.0, 121.6, 117.9, 115.4, 113.1, 113.1, 68.1, 64.8, 37.8, 14.7. ESI-MS m/
3
)
2
H), 1.44 (t, J ¼ 7.0 Hz, 3H). C NMR (75 MHz, CDCl
3
) d 163.5, 158.2,
150.9, 149.3, 148.9,144.1, 137.2, 136.4,125.8, 125.1, 124.0, 121.7, 115.4,
1
þ
1
14.6, 113.1, 111.4, 68.1, 64.8, 37.8, 14.7. ESI-MS m/z: 494.2 [M þ H] .
þ
þ
z: 406.4 [M þ H] . HRMS (ESI) m/z: 406.0594 [M þ H] ; Calcd for
þ
HRMS (ESI) m/z: 493.9590 [M þ H] ; Calcd for C20
2 4
H17Br NO
C
20
H17Cl
2
NO
4
(M þ H) 406.0614.
(
M þ H) 493.9596.
5
.1.4.14. (Z)-3,4-Dichloro-5-(3-ethoxy-4-(pyridin-2-ylmethoxy)ben-
5
.1.4.8. (Z)-3,4-Dibromo-5-(3-ethoxy-4-(2-(5-ethylpyridin-2-yl)
1
zylidene)furan- 2(5H)-one (6s). Yellow solid; yield 64.9%. H NMR
(300 MHz, CDCl
1
2
ethoxy)benzylidene) furan-2(5H)-one (6k). Yellow solid; yield
1
3
)
d
8.61 (d, J ¼ 4.5 Hz, 1H), 7.74 (td, J ¼ 7.8, 1.8 Hz,
H), 7.58 (d, J ¼ 7.8 Hz, 1H), 7.47 (d, J ¼ 1.8 Hz, 1H), 7.26e7.23 (m,
H), 6.93 (d, J ¼ 8.4 Hz, 1H), 6.34 (s, 1H), 5.34 (s, 2H), 4.20 (q,
76.3%. H NMR (300 MHz, CDCl
3
)
d
8.39 (s, 1H), 7.47 (d, J ¼ 7.5 Hz,
H), 7.41 (d, J ¼ 1.5 Hz, 1H), 7.30e7.27 (m, 2H), 6.92 (d, J ¼ 8.4 Hz,
H), 6.36 (s, 1H), 4.43 (t, J ¼ 6.8 Hz, 2H), 4.07 (q, J ¼ 7.0 Hz, 2H), 3.31
1
1
13
J ¼ 7.0 Hz, 2H), 1.53 (t, J ¼ 7.0 Hz, 3H). C NMR (75 MHz, CDCl
162.3, 156.8, 150.2, 149.1, 142.6, 141.9, 136.9, 125.5, 125.3, 122.7,
21.1, 118.2, 114.9, 113.7, 112.9, 112.4, 71.3, 64.7, 14.7. ESI-MS m/z:
3
)
(
(
1
1
t, J ¼ 6.8 Hz, 2H), 2.64 (q, J ¼ 7.6 Hz, 2H), 1.44 (t, J ¼ 7.0 Hz, 3H), 1.24
13
d
t, J ¼ 7.6 Hz, 3H). C NMR (75 MHz, CDCl
48.9, 148.8, 144.1, 137.2, 136.9, 135.9, 125.8, 125.0, 123.7, 115.4,
14.6, 113.1, 111.4, 68.2, 64.8, 37.4, 25.7, 15.3, 14.7. ESI-MS m/z: 522.2
3
) d 163.5, 155.3, 151.0,
1
3
C
þ
þ
92.4 [M þ H] . HRMS (ESI) m/z: 392.0442 [M þ H] ; Calcd for
þ
þ
19
H15Cl
2
NO
4
(M þ H) 392.0458.
[
C
M þ H] . HRMS (ESI) m/z: 521.9913 [M þ H] ; Calcd for
22
H
21Br
2
NO
4
(M þ H) 521.9917.
5
.1.4.15. (Z)-3,4-Dichloro-5-(3-methoxy-4-(2-(pyridin-2-yl)ethoxy)
1
benzylidene)furan- 2(5H)-one (6t). Yellow solid; yield 79.9%.
NMR (300 MHz, CDCl
8.57 (d, J ¼ 4.8 Hz, 1H), 7.65 (td, J ¼ 7.7,
.0 Hz, 1H), 7.40 (d, J ¼ 2.1 Hz, 1H), 7.33 (d, J ¼ 7.7 Hz, 1H), 7.30 (d,
J ¼ 8.7, 2.1 Hz, 1H), 7.22e7.15 (m, 1H), 6.95 (d, J ¼ 8.7 Hz,1H), 6.34 (s,
H
5
.1.4.9. (Z)-3,4-Dibromo-5-(3-ethoxy-4-(pyridin-2-ylmethoxy)ben-
1
3
) d
zylidene)furan- 2(5H)-one (6l). Yellow solid; yield 83.4%. H NMR
300 MHz, CDCl
2
(
1
2
3
)
d
8.58 (d, J ¼ 4.5 Hz, 1H), 7.71 (td, J ¼ 7.8, 1.8 Hz,
H), 7.56 (d, J ¼ 7.8 Hz, 1H), 7.44 (d, J ¼ 1.8 Hz, 1H), 7.26e7.17 (m,
H), 6.90 (d, J ¼ 8.4 Hz, 1H), 6.34 (s, 1H), 5.31 (s, 2H), 4.17 (q,
13
1
H), 4.49 (t, J ¼ 7.0 Hz, 2H), 3.90 (s, 3H), 3.37 (t, J ¼ 7.0 Hz, 2H).
NMR (75 MHz, CDCl 162.4, 157.9, 150.4, 149.5, 149.1, 142.6, 141.8,
36.7, 125.7, 124.8, 124.0, 121.8, 113.6, 113.0, 112.8, 112.6, 68.1, 56.1,
C
13
3
) d
J ¼ 7.0 Hz, 2H), 1.50 (t, J ¼ 7.0 Hz, 3H). C NMR (75 MHz, CDCl
163.4, 156.8150.1, 149.0, 144.3, 137.2, 136.9, 125.5, 122.7, 121.1,
14.91, 114.4, 113.7, 111.6, 71.3, 64.7, 14.8. ESI-MS m/z: 480.3
3
)
1
d
þ
3
7.6. ESI-MS m/z: 392.3 [M þ H] . HRMS (ESI) m/z: 392.0456
1
þ
þ
þ
[M þ H] ; Calcd for C19
H15Cl
2
NO
4
(M þ H) 392.0458.
[
C
M þ H] . HRMS (ESI) m/z: 479.9417 [M þ H] ; Calcd for
19
H
15Br
2
NO
4
(M þ H) 479.9447.
5
.1.4.16. (Z)-3,4-Dichloro-5-(3-methoxy-4-(pyridin-2-ylmethoxy)
1
5
.1.4.10. (Z)-3,4-Dibromo-5-(3-methoxy-4-(pyridin-2-ylmethoxy)
benzylidene)furan- 2(5H)-one (6u). Yellow solid; yield 81.3%.
NMR (300 MHz, CDCl
H
1
benzylidene)furan- 2(5H)-one (6n). Yellow solid; yield 80.8%.
NMR (300 MHz, CDCl
8.58 (d, J ¼ 4.5 Hz, 1H), 7.70 (td, J ¼ 7.7,
.8 Hz, 1H), 7.52 (d, J ¼ 7.7 Hz, 1H), 7.43 (d, J ¼ 1.8 Hz, 1H), 7.25e7.16
m, 2H), 6.90 (d, J ¼ 8.4 Hz,1H), 6.35 (s,1H), 5.31 (s, 2H), 3.95 (s, 3H).
H
3
)
d
8.61 (d, J ¼ 4.5 Hz, 1H), 7.73 (td, J ¼ 7.8,
3
)
d
1.8 Hz, 1H), 7.55 (d, J ¼ 7.8 Hz, 1H), 7.45 (d, J ¼ 1.8 Hz, 1H), 7.27 (d,
J ¼ 8.4, 1.8 Hz, 1H), 7.24 (d, J ¼ 1.8 Hz, 1H), 6.93 (d, J ¼ 8.4 Hz, 1H),
1
13
(
3
6.34 (s, 1H), 5.34 (s, 2H), 3.98 (s, 3H). C NMR (75 MHz, CDCl )
1
3
C NMR (75 MHz, CDCl
3
)
d
163.4, 156.6, 149.8, 149.6, 149.2, 144.3,
d
162.3, 156.5, 149.8, 149.6, 149.2, 142.6, 142.0, 136.9, 125.5, 125.2,
137.2, 136.9, 125.5, 125.5, 122.8, 121.3, 114.3, 113.3, 113.3, 111.7, 71.3,
122.8, 121.3, 118.2, 113.4, 113.3, 112.8, 71.3, 56.1. ESI-MS m/z: 378.2

F. Wang et al. / European Journal of Medicinal Chemistry 72 (2014) 35e45
43
þ
þ
[
C
M þ H] . HRMS (ESI) m/z: 378.0283 [M þ H] ; Calcd for
129.9, 123.2, 121.5, 113.4, 69.8, 21.4, 21.1, 20.3. ESI-MS m/z: 257.3
[M þ H] .
þ
H13Cl
18 2
NO
4
(M þ H) 378.0302.
5
.1.4.17. (Z)-3,4-Dibromo-5-(4-((3,5,6-trimethylpyrazin-2-yl)
5.1.7. General procedures for synthesis of 7ae7d
methoxy)benzylidene) furan-2(5H)-one (6v). Yellow solid; yield
To compound thiazolidinedione (A
2
, 1.2 mmol) in toluene
1
8
2.0%. H NMR (300 MHz, CDCl
J ¼ 8.1 Hz, 1H), 6.37 (s, 1H), 5.19 (s, 1H), 2.57 (s, 3H), 2.51 (s, 6H).
NMR (75 MHz, CDCl 163.6, 160.2, 151.6, 149.9, 148.8, 145.1, 144.3,
37.2, 132.9, 125.1, 115.4, 114.1, 111.6, 69.9, 21.7, 21.4, 20.6. ESI-MS m/
3
)
d
7.74 (d, J ¼ 8.1 Hz, 1H), 7.03 (d,
(40 mL) was added B (1.0 mmol), and the reaction mixture was
2 3
C
13
C
stirred under the atmosphere of nitrogen. Then a catalytic amount
of 2-methyl piperidine and glacial acetic acid were added dropwise
to the solution over a period of 10 min, heated at 130 C for 2 h. A lot
of solid would precipitate out when the solution cooled to room
temperature. At last, the reaction mixture was filtered and washed
with toluene and saturated salt water to get products 7ae7d.
3
) d
ꢁ
1
þ
þ
z: 481.5 [M þ H] . HRMS (ESI) m/z: 480.9585 [M þ H] ; Calcd for
19 2 2 3
C H16Br N O
(M þ H) 480.9587.
5
2
.1.4.18. (Z)-3,4-Dibromo-5-(3-methoxy-4-((3,5,6-trimethylpyrazin-
-yl)methoxy) benzylidene)furan-2(5H)-one (6w). Yellow solid;
5.1.7.1. (Z)-5-(4-((3,5,6-Trimethylpyrazin-2-yl)methoxy)benzylidene)
thiazolidine-2,4-dione (7a). White solid; yield 81.5%. H NMR
1
1
yield 80.3%. H NMR (300 MHz, CDCl
3
)
d
7.36 (d, J ¼ 2.1 Hz, 1H), 7.30
(
2
d
dd, J ¼ 8.4, 2.1 Hz, 1H), 7.08 (d, J ¼ 8.4 Hz, 1H), 6.33 (s, 1H), 5.26 (s,
(300 MHz, DMSO-d
J ¼ 8.9 Hz, 2H), 5.23 (s, 2H), 2.48 (s, 3H), 2.45 (d, 6H). C NMR
(75 MHz，DMSO-d 168.6, 168.3, 160.4, 151.6, 149.7, 148.8, 145.3,
132.4, 131.8, 126.4, 121.4, 116.0, 69.9, 21.7, 21.4, 20.5. ESI-MS m/z:
6
)
d
7.73 (s, 1H), 7.56 (d, J ¼ 8.9 Hz, 2H), 7.19 (d,
13
13
H), 3.89 (s, 1H), 2.61 (s, 1H), 2.51 (s, 6H). C NMR (75 MHz, CDCl
162.3, 151.4, 150.1, 150.1, 149.8, 148.6, 145.1, 142.6, 141.9, 125.4,
25.2, 118.2, 113.7, 113.6, 112.9, 70.8, 56.0, 21.7, 21.4, 20.7. ESI-MS m/
3
)
6
) d
1
þ
þ
þ
þ
z: 511.3 [M þ H] . HRMS (ESI) m/z: 510.9734 [M þ H] ; Calcd for
356.3 [M þ H] . HRMS (ESI) m/z: 356.1066 [M þ H] ; Calcd for
C
20
H
18Br
2
N
2
O
4
(M þ H) 510.9693.
18 17 3 3
C H N O
S (M þ H) 356.1071.
5
.1.5. General procedure for synthesis of 2-(bromomethyl)-3,5,6-
trimethylpyrazine (C
Compound C was prepared according to a published method
51].
5.1.7.2. (Z)-5-(3-Methoxy-4-((3,5,6-trimethylpyrazin-2-yl)methoxy)
benzylidene) thiazolidine-2,4-dione (7b). White solid; yield 89.6%.
3
)
1
3
H NMR (300 MHz, DMSO-d
6
)
d
7.71 (s, 1H), 7.27 (d, J ¼ 8.4 Hz, 1H),
[
7.20 (d, J ¼ 1.9 Hz, 1H), 7.16 (dd, J ¼ 8.4, 1.9 Hz, 1H), 5.20 (s, 2H), 3.79
13
(
s, 3H), 2.48 (s, 3H), 2.45 (s, 3H), 2.44 (s, 3H). C NMR (75 MHz，
DMSO-d 168.9, 168.9, 151.6, 150.0, 149.9, 149.7, 148.8, 145.4,
131.8, 126.9, 123.8, 122.1, 114.2, 113.9, 70.4, 56.0, 21.7, 21.4, 20.5. ESI-
5
.1.6. General procedures for synthesis of B
To a solution of benzaldehyde derivatives (B
DMF (30 mL) was added potassium carbonate (K
and C
2
C
3
6
) d
2
, 1.0 mmol) in dry
þ
þ
2
CO
3
, 2.0 mmol)
MS m/z: 386.3 [M þ H] . HRMS (ESI) m/z: 386.1165 [M þ H] ; Calcd
ꢁ
3
(1.2 mmol). And the mixture was stirred for 10 h at 85 C,
for C19
19 3 4
H N O
S (M þ H) 386.1176.
then it was cooled to room temperature. The reaction mixture was
filtered, washed with water and the filtrate was extracted with
5.1.7.3. (Z)-5-(3-Ethoxy-4-((3,5,6-trimethylpyrazin-2-yl)methoxy)
CH
2
Cl
2
. The organic layer was dried over Na
2
SO
4
overnight and
benzylidene) thiazolidine-2,4-dione (7c). White solid; yield 88.7%.
1
evaporated to give a residue which was chromatographed using
ethyl acetate/petroleum ether (1:6, v/v) to get the intermediate
products B
H NMR (300 MHz, DMSO-d
6
)
d
7.78 (s, 1H), 7.24 (d, J ¼ 8.7 Hz, 1H),
7.19 (d, J ¼ 1.8 Hz, 1H), 7.13 (dd, J ¼ 8.7, 1.8 Hz, 1H), 5.21 (s, 2H), 4.06
(q, J ¼ 6.9 Hz, 2H), 2.49 (s, 3H), 2.46 (s, 3H), 2.45 (s, 3H), 1.31 (t,
2 3
C .
13
J ¼ 6.9 Hz, 3H). C NMR (75 MHz，DMSO-d
6
) d 168.4, 168.2, 151.5,
5.1.6.1. 4-((3,5,6-Trimethylpyrazin-2-yl)methoxy)benzaldehyde.
150.1, 149.6, 149.0, 148.6, 145.6, 129.3, 128.7, 127.8, 125.81, 123.4,
1
þ
White solid; yield 81.1%. H NMR (300 MHz, CDCl
.83 (d, J ¼ 8.7 Hz, 2H), 7.12 (d, J ¼ 8.7 Hz, 2H), 5.24 (s, 2H), 2.59 (s,
H), 2.53 (s, 6H). 13C NMR (75 MHz, CDCl
190.8, 163.5, 151.8,
3
)
d
9.89 (s, 1H),
114.8, 71.0, 64.4, 21.7, 21.4, 20.6, 15.0. ESI-MS m/z: 400.4 [M þ H] .
-
7
3
HRMS (ESI) m/z: 398.1183 [M ꢂ H] ; Calcd for C20
H17Br
2
NO
4
(M ꢂ H)
3
)
d
398.1172.
1
50.0, 148.8, 144.8, 132.0, 130.3, 115.2, 70.1, 21.7, 21.4, 20.6. ESI-MS
þ
m/z: 257.3 [M þ H] .
5.1.7.4. (Z)-5-(3-((3,5,6-Trimethylpyrazin-2-yl)methoxy)benzylidene)
1
thiazolidine-2,4e dione (7d). White solid; yield 81.8%. H NMR
5
.1.6.2. 3-Methoxy-4-((3,5,6-trimethylpyrazin-2-yl)methoxy)benzal-
(300 MHz, DMSO-d
(m, 3H), 5.21 (s, 2H), 2.49 (s, 3H), 2.45 (s, 6H). C NMR (75 MHz,
DMSO-d 169.0, 168.9, 159.1, 151.5, 149.7, 148.7, 145.6, 135.1, 131.2,
130.8, 125.5, 123.0, 117.4, 116.1, 69.9, 21.6, 21.4, 20.6. ESI-MS m/z:
6
)
d
7.70 (s, 1H), 7.44 (t, J ¼ 8.1 Hz, 1H), 7.25e7.09
1
13
3
dehyde. White solid; yield 76.3%. H NMR (300 MHz, CDCl ) d 9.85
(
5
s, 1H), 7.45 (d, J ¼ 1.8 Hz, 1H), 7.42 (s, 1H), 7.18 (d, J ¼ 1.8 Hz, 1H),
6
) d
13
.31 (s, 2H), 3.91 (s, 3H), 2.62 (s, 3H), 2.52 (s, 6H). C NMR (75 MHz,
þ
ꢂ
CDCl
3
)
d
190.9, 153.5, 151.5, 150.2, 150.1, 148.7, 144.9, 130.5, 126.5,
356.3 [M þ H] . HRMS (ESI) m/z 354.0914 [M ꢂ H] ; Calcd for
þ
112.7, 109.4, 70.8, 56.0, 21.6, 21.3, 20.6. ESI-MS m/z: 287.3 [M þ H] .
C
20
H17Br
2
NO
4
(M ꢂ H) 354.0910.
5
.1.6.3. 3-Ethoxy-4-((3,5,6-trimethylpyrazin-2-yl)methoxy)benzalde-
5.2. Biological assays and experimental procedures
1
hyde. White solid; yield 81.3%. H NMR (300 MHz, CDCl
H), 7.43 (d, J ¼ 1.8 Hz, 1H), 7.40 (s, 1H), 7.18 (d, J ¼ 1.8 Hz, 1H), 5.31
s, 2H), 4.14 (q, J ¼ 7.0 Hz, 2H), 2.64 (s, 3H), 2.53 (s, 6H), 1.45 (t,
3
) d 9.84 (s,
1
(
5.2.1. Cell culture
RAW264.7 murine macrophages were cultured in DMEM con-
13
J ¼ 7.0 Hz, 3H). C NMR (75 MHz, CDCl
3
)
d
190.9, 153.6, 151.4, 150.2,
taining 10% new-born calf serum, 100 units/mL penicillin, and
ꢁ
149.6, 148.5, 145.1, 130.6, 126.2, 113.2, 110.8, 71.0, 64.5, 21.7, 21.3,
100
m
g/mL streptomycin at 37 C in a 5% CO
2
humidified
þ
2
0.7, 14.6. ESI-MS m/z: 301.2 [M þ H] .
atmosphere.
5
.1.6.4. 3-((3,5,6-Trimethylpyrazin-2-yl)methoxy)benzaldehyde.
5.2.2. Assay for NO production
1
4
White solid; yield 75.4%. H NMR (300 MHz, CDCl
3
)
d
9.65 (s, 1H),
RAW264.7 cells were inoculated at 5 ꢃ 10 cells per well in 96-
7
.27 (s, 1H), 7.15 (d, J ¼ 1.8 Hz, 1H), 7.13 (d, J ¼ 1.8 Hz, 1H), 7.00 (dd,
well plate and cultured for 18 h. The cells were then pre-treated
13
J ¼ 1.8, 1.8 Hz, 1H), 4.94 (s, 2H), 2.32 (s, 3H), 2.22 (s, 6H). C NMR
with 10
mM compounds which were prepared in serum-free me-
(
300 MHz, CDCl 191.4, 158.9, 151.1, 149.6, 148.4, 144.9, 137.6,
3
) d
dium for 2 h before stimulation with LPS (100 ng/mL). After

4
4
F. Wang et al. / European Journal of Medicinal Chemistry 72 (2014) 35e45
stimulated for 48 h by LPS, the NO produced in the culture medium
was quantified by Greiss reagent, in brief, added 100 L of Griess
reagent (1% sulfanilamide and 0.1% N-(1-naphthyl)ethylenedi-
amine dihydrochloride in 5% phosphoric acid) to 100 L of super-
natant medium and incubated for 10 min at room temperature,
then measured absorbance of the samples at 540 nm (OD540) in
a microplate reader (Bio-Rad Laboratories, CA, USA). Rosiglitazone
and indomethacin were used as positive controls. NO inhibition
overnight (all the primary antibodies were purchased from Cell
Signaling Technology and diluted in TBST at the ratio of 1:1000),
washed 3 times in TBST for 5 min each, incubated with anti-rabbit
or anti-mouse secondary antibody (1:1000 in TBST, Cell Signaling
Technology) for 90 min, washed in TBST and exposed to ECL
reagents.
m
m
5.2.6. Molecular modeling
rate
(
¼
[control (OD540
OD540) ꢂ blank (OD540)] ꢃ 100%.
Control: treated with LPS only.
)
ꢂ
compound (OD540)]/[control
First, using the Sketch Molecule module in SYBYL 8.1 program
package of Tripos, the minimized energy structures of four ligands
were built by using the standard Tripos molecular mechanics force
Compound: treated with LPS and compounds.
Blank: cultured with fresh medium only.
field and Gasteiger-Hückel charge [52,53]. The structure of PPARg
was obtained from the Protein Data Bank (PDB ID: 2PRG), all ligands
and water molecules were removed and the polar hydrogen atoms
were added. The docking calculation was performed using the
empirical scoring function and the patented search engine in
Surflex-Dock. The Molecular Computer Aided Design (MOLCAD)
program was employed to display cavity depth potential or
5
.2.3. Cell cytotoxicity
Cell cytotoxicity was evaluated by methyl thiazolyl tetrazolium
3
(
MTT) assay. RAW264.7 cells were inoculated at 4 ꢃ 10 cells per
well in 96-well plate. After cultured for 16 h, the cells were treated
with different compounds which were diluted in DMEM for 48 h.
hydrogen bonds of the PPAR
compounds.
g binding site within the four
Then 20
incubated for 4 h. After 4 h, cell culture was removed and then
50 L DMSO was added to dissolve the formazan. The optical
mL of 0.5 mg/mL MTT reagent was added into the cells and
1
m
Acknowledgment
density was measured at 570 nm (OD570). Cell viability was calcu-
lated from three independent experiments. The density of for-
mazan formed in blank group was set as 100% of viability. Cell
viability (%) ¼ compound (OD570)/blank (OD570) ꢃ 100%
Blank: cultured with fresh medium only.
This work was supported by the National Natural Science
Foundation of China (81072554).
Appendix A. Supplementary data
Compound: treated with compounds or LPS.
These data include MOL files and InChiKeys of the most
5
.2.4. Measurement of TNF-
a
and IL-6
important compounds described in this article.
5
RAW264.7 cells (5 ꢃ 10 cells/well) were cultured in 24-well
plate and pretreated with 10 M of compounds for 2 h, and then
LPS was added. The production of TNF- and IL-6 was stimulated by
the addition of 100 ng/mL LPS and incubated for 6 h, 12 h and 24 h.
The levels of TNF- and IL-6 in the supernatant were determined
using the mouse ELISA kit (TNF- : MultiSciences, EK2822; IL-6:
m
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