Discovery and evaluation of piperid-4-one-containing mono-carbonyl analogs of curcumin as anti-inflammatory agents

Article abstract of DOI:10.1016/j.bmc.2013.03.057

We previously reported the design and discovery of three series of 5-carbon linker-containing mono-carbonyl analogs of curcumin (MCACs) as excellent anti-inflammatory agents. In continuation of our ongoing research, we designed and synthesized the fourth

Full text of DOI:10.1016/j.bmc.2013.03.057

Bioorganic & Medicinal Chemistry 21 (2013) 3058–3065
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery and evaluation of piperid-4-one-containing
mono-carbonyl analogs of curcumin as anti-inflammatory agents
Jianzhang Wu a,b, , Yali Zhang a, , Yuepiao Cai b, Jian Wang c, Bixia Weng a, Qinqin Tang b, Xiangjian Chen c,
Zheer Pan c, Guang Liang b, , Shulin Yang a,
⇑
⇑
a School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing, Jiangsu 210094, China
b Bioorganic & Medicinal Chemistry Research Center, School of Pharmaceutical Sciences, Wenzhou Medical College, University Town, Wenzhou, Zhejiang 325035, China
c Department of Surgery, The 1st Affiliated Hospital, Wenzhou Medical College, Wenzhou, Zhejiang 325035, China
a r t i c l e i n f o
a b s t r a c t
Article history:
We previously reported the design and discovery of three series of 5-carbon linker-containing mono-car-
bonyl analogs of curcumin (MCACs) as excellent anti-inflammatory agents. In continuation of our ongo-
ing research, we designed and synthesized the fourth series of MCACs, whose central linker is a piperid-4-
one. Their inhibitory effects against IL-6 production were evaluated in lipopolysaccharide (LPS)-stimu-
lated macrophages. Among them, compounds F8, F29, F33, F35, and F36 exhibited the IC50 values under
Received 13 February 2013
Revised 18 March 2013
Accepted 20 March 2013
Available online 30 March 2013
5
l
M. The structure–activity relationship was discussed. Mechanistically, F35 and F36 dose-dependently
Keywords:
Curcumin
Mono-carbonyl analogue of curcumin
IL-6
Anti-inflammation
Sepsis
prevented LPS-induced NF- B and ERK activation. Finally, pretreatment with F35 and F36 significantly
j
protected the C57B/L6 mice from LPS-induced septic death. Together, these data present a series of
new analogs of curcumin as promising anti-inflammatory agents.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Although this molecule has already been the subject of several
clinical trials for development as a drug, the rapid plasma clearance
and low bioavailability significantly limit its therapeutic useful-
ness.7 For the purpose of finding novel derivatives with increased
systemic bioavailability and enhanced pharmacological activity,
chemical modifications as well as synthesis of curcumin analogues
have been attempted by many research groups to find a better
treatment for various diseases linked to inflammation.7–13 The
b-diketone moiety in curcumin’s structure has been considered
to be responsible for its instability, fast metabolism and poor
bioavailability.7 In our previous study, 3 series of 5-carbon lin-
ker-containing mono-carbonyl analogues of curcumin (MCACs),
1,5-diaryl-1,4-pentadiene-3-ones, together with cyclopentanone
and cyclohexanone analogues, were designed by displacing
b-diketone moiety with a single carbonyl group.8–13 They were
evaluated the anti-inflammatory activity, and some exhibit prom-
ising pharmacological effects and are undergoing preclinical study.
Our lab works for the discovery of new anti-inflammatory agents
from MCACs. In continuation of our ongoing research, we present
here the fourth series of MCACs in our lab, whose central linker
is a piperid-4-one structure (Fig. 1). We evaluated here the inhibi-
tory effects of 34 piperidone-containing analogs against IL-6 pro-
duction in lipopolysaccharide (LPS)-stimulated macrophages.
Further, active compounds were selected for the study of anti-
inflammatory mechanism and in vivo therapeutic effects against
LPS-induced septic death in mouse models. These results suggest
their potential to serve as new anti-inflammatory agents.
Inflammatory response is a common pathological process of a
variety of diseases. Besides immunological diseases and inflamma-
tion, many important diseases in the preliminary stage such as car-
diovascular disease, atherosclerosis, cancer, and type II diabetes
can be initiated and aggravated by over-expression and activation
of pro-inflammatory cytokines such as interleukin-6 (IL-6) and tu-
mor necrosis factor-
a (TNF-a
).1–4 As a result, pharmacological
intervention of new small molecules, which are capable of reduc-
ing the expression of inflammatory cytokines, has been recently
proposed as a possible method for the prevention and treatment
of these diseases.1,3
Curcumin (Fig. 1) is a yellow natural product isolated from tur-
meric.5 Research shows curcumin is a highly pleiotropic molecule capa-
ble of interacting with numerous molecular targets involved in
inflammation.5 Based on early cell culture and animal research, clinical
trials indicate curcumin may have potential as a therapeutic agent in
diseases such as inflammatory bowel disease, pancreatitis, arthritis,
and chronic anterior uveitis, as well as certain types of cancer.6 Curcu-
minmodulates the inflammatoryresponsebyinhibitingtheproduction
of the inflammatory cytokines such as TNF-
a
and IL-1, -2, -6, and -12.5,6
⇑
Corresponding authors. Tel.: +86 577 86699524; fax: +86 577 86699527 (G.L.).
E-mail addresses: wzmcliangguang@163.com (G. Liang), bioshuliny@yahoo.
com.cn (S. Yang).
These authors contribute equally to this work.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.03.057

J. Wu et al. / Bioorg. Med. Chem. 21 (2013) 3058–3065
3059
O
O
is an important structural component of the outer membrane of
Gram-negative bacteria, and it is a well-studied immunostimulator
that induces a systemic inflammation response, especially the
expression of proinflammatory cytokines such as IL-6.14 Thus, the
anti-inflammatory activity of these synthetic compounds was
characterized by their inhibition on LPS-induced IL-6 secretion in
mouse RAW264.7 macrophages. The macrophages were pre-trea-
O
OCH3
OH
H3CO
HO
R
R
n
Curcumin
MCACs
O
O
Comp.
F1
n
HO
HO
CHO
CHO
HO
HO
OH
OH
2
F2
3
0
+
n
n
F3
ted with 10
lM compounds for 2 h and then incubated with
O
O
0.5 g/mL LPS for 22 h. The amount of IL-6 in media was detected
l
R2
R2
R2
F4 F36
-
+
Comps:
through enzyme-linked immunosorbant assays (ELISA) and nor-
malized by protein concentration of cells harvested in homologous
culture plates.
N
R
N
R
The result of anti-inflammatory evaluation is shown in Fig-
ure 2A. A majority of piperid-4-one-containing MCACs exhibited
high inhibition against LPS-induced IL-6 expression. Among 34
tested compounds, 26 compounds showed stronger activity than
curcumin at the same dosage, and 23 compounds showed inhibi-
tory rates higher than 50%. Compound F8, F18, F29, F35 and F36
showed the strongest inhibitory effect on LPS-induced IL-6 release
and their inhibitory rates reached 90%, 90%, 89%, 96%, and 98%,
respectively, compared to the LPS control.
Compounds F1–F3 only showed moderate inhibition of IL-6
expression. Combined with the anti-inflammatory screening data
on MCACs that we previously published,8–13 it is generally ob-
served that piperid-4-one-containing MCACs are more effective
against LPS-induced IL-6 expression than previous three series of
5-carbon linker-containing mono-carbonyl analogues of curcumin
(MCACs). In addition, electron-withdrawing halogen substituents
in the benzene rings appear to increase the anti-inflammatory
activity. Compounds (F5, F13, F15, F24 and F28) with halogen in
the 2-position of the benzene rings showed good activity. The ef-
fects of electron-donating substituents in the benzene rings on
the anti-inflammatory activity of analogs varied from the type of
groups. The methoxyl group is always favorable to the anti-inflam-
matory effects, while the hydroxyl group and N,N-dimethyl amino
group show uncertain roles in bioactivity. For piperid-4-one deriv-
atives, the N-substitution with different groups should play an
important role in the IL-6-inhibitory activity. We observed that
N-methyl and N-propyl substitution seem to slightly increase the
activity; however, the SAR profile of the substitution with N-
methyl, N-ethyl, N-propyl and hydrogen on the anti-inflammatory
actions of piperid-4-one-containg MCACs is still confused and re-
quire further investigation according to our results.
Figure 1. Structures of synthetic compounds. Reagents and conditions: (I)
CH3CH2OH, HCl, rt or reflux; (II) CH3CH2OH/H2O, NaOH or HCl, rt or reflux.
2. Result and discussion
2.1. Chemistry
In continuation of our ongoing research on potential anti-
inflammatory agents from MCACs, we designed 4 series of 5-car-
bon-linker mono-carbonyl analogues as follows: 1,5-diaryl-1,4-
pentadiene-3-ones, 2,6-(diarylidene) cyclohexanone, 2,5-(diarylid-
ene) cyclopentanone, and 3,5-(diarylidene) piperid-4-one. Herein,
we present the analogues containing the structure of 3,5-(diarylid-
ene) piperid-4-one. In this skeleton, various substitutes on the ben-
zene rings were used for the structure–activity relationship
analysis. The synthesis and structures of compounds F1–F36 are
shown in Figure 1. Compounds were synthesized by direct aldol
condensation of substituted benzaldehyde with the ketones in
acidic or alkaline media. Compounds with hydroxy groups in the
benzene rings (F1–F4, F6, F11, F14, F29, F32 and F33) were synthe-
sized using HCl as a catalyst; among these compounds, F3, F4, F6
and F11 were prepared in a condition of reflux, while F1, F2, F14,
F29, F32 and F33 were obtained at room temperature. Other com-
pounds were synthesized using NaOH as catalyst at 4–8 °C. Com-
pound F8, F17, F18, F22 and F26 were purified by re-
crystallization, and others were purified by silica gel column chro-
matography. The yields of pure products were in the range of 12–
96%. Details of yields, melting points, and spectral analysis in ESI-
MS and 1H NMR of these compounds are described in Section 4.
2.3. Active compounds inhibit LPS-induced IL-6 release in a dose
dependent manner
Among active compounds above, five of the most active com-
pounds F8, F29, F33, F35, and F36 were selected for further
dose-dependent evaluation and they exhibited no obvious cytotox-
icity in macrophages (data not shown). Cells were pretreated with
active compounds at 1, 5, and 10
lM for 2 h, followed by incuba-
tion with LPS (0.5 g/mL) for 22 h. The results are shown in Fig-
l
ure 2B. All of five compounds exhibit good dose-dependent
inhibition of LPS-induced IL-6 release, with the IC50 values under
5
1
l
M. Compound F35 exhibits the best activity and its IC50 is under
lM. This result further suggests the potential of the piperid-4-
one-containg MCACs as anti-inflammatory agents.
2.4. Active compounds show different mechanism in NF-
MAPK pathways
jB and
2.2. Inhibitory effects of these compounds on LPS-induced IL-6
release
LPS is recognized by toll-like receptor 4 (TLR4) and activate
MyD88-dependent pathways, one of which culminates in activa-
IL-6 is a well-characterized cytokine that plays an important
role in many inflammatory diseases caused by endotoxins.14 LPS
tion of the transcription factor nuclear factor (NF)-
other leads to activation of mitogen-activated protein kinase
jB and the

3060
J. Wu et al. / Bioorg. Med. Chem. 21 (2013) 3058–3065
150
100
50
IL-6
A
*
*
*
*
*
*
*
*
*
*
**
*
*
*
**
*
**
*
**
*
**
**
**
**
**
**
**
**
0
n
i
10
14
17
29
35
F1 F2 F3 F4 F5 F6 F8 F9
SO
m
F
F11 F12 F13 F
F15 F16 F
F18 F19 F20 F21 F22 F23 F24 F25 F26 F27 F28 F F30 F31 F32 F33 F
F36
LPS
rcu
DM
LPS 0.5µg/ml
Cu
B
IC50=2.04µM
IC50=2.73µM
IC50=4.19µM
IC50<1.0µM
IC50=2.68µM
100
50
0
*
*
**
**
**
**
**
**
**
**
**
**
**
(µM)
1
5
F8
10
1
5
F29
10
1
5
F33
10
1
5
F35
10
1
5
F36
10
DMSO
LPS 0.5µg/ml
Figure 2. Compounds inhibited LPS-induced IL-6 release in Mouse RAW 264.7 macrophages. Mouse RAW 264.7 macrophages were plated with a density of 1.2 Â 106/plate
for overnight. (A) An inhibitory screening of compounds at 10 lM. (B) A dose-dependent evaluation of active compounds at 1, 5, and 10 lM. Cells were treated with vehicle
(DMSO) or curcumin or compounds for 2 h, then incubated with LPS (0.5 lg/mL) for 22 h. The culture media and the cells were collected, respectively. The IL-6 levels in
culture media were determined with an ELISA kit and were normalized by the total protein concentration of cells. The results were showed in the percent of LPS control. Each
bar represents mean SE of 3–6 independent experiments. Statistical significance relative to LPS was expressed, ⁄p <0.05, ⁄⁄p < 0.01.
(MAPKs).14,15 Both of these two could regulate the expression of
inflammatory factors including IL-6 at the transcriptional level in
response to various inflammatory stimuli containing LPS. In NF-
observed in F36’s inhibition on JNK phosphorylation in a dose-
dependent manner (Fig. 3E). In addition, pretreatment with curcu-
min at 10 lM exhibited obvious inhibition only on JNK phosphor-
jB signaling, I
j
B degradation frees NF-
j
B p65 subunit and allows
ylation in LPS-stimulated macrophages (Fig. 3C).
it to translocate to the nucleus, followed by turning on transcrip-
tion of inflammatory genes.14,15 MAPKs include three kinases: the
extracellular-signal-regulated kinase (ERK), the JUN N-terminal ki-
nase (JNK) and the p38 kinase.14,15 Among them, p38 has also been
identified to regulate the cytokine expression at the post-transcrip-
tional level.
To gain insight into involved signaling, we determined whether
MAPKs and NF-jB might be involved in anti-inflammatory actions
of active curcumin analogues. In order to closely imitate the path-
ological practice, the primary mouse peritoneal macrophages
(MPMs) were employed here instead of RAW264.7 cell line. MPMs
were treated with compounds before LPS stimulation. LPS induced
compound F35 seems to exert anti-inflammatory actions partly
via inhibiting NF-
inflammatory cytokines is accompanied with inactivation of NF-
ERK, and JNK. However, the bioactivities of F8 and F29 may be NF-
B/MAPKs-independent. Curcumin has been identified as a multi-tar-
get compound and there is recent evidence that curcumin can inhibit
MAPKs and NF- B at relatively high concentrations. We have previ-
ously demonstrated that several curcumin analogues affected NF-
B and ERK/JNK pathways.16,17 These results indicated a mechanistic
jB and ERK pathways; the inhibition of F36 on
jB,
j
j
j
difference among active anti-inflammatory curcumin analogues,
although they are derived from the same structural lead. This sug-
gests that the structural modification reduces and even changes the
possible molecular targets of curcumin. It is really worthy to be fur-
ther investigated in the future for the chemical and structural fea-
tures influencing the biological mechanism of curcumin analogs.
the I
as shown in Figure 3. A pretreatment with active F35 and F36 sig-
nificantly reversed LPS-induced degradation of I B, while F8 and
F29 had no effect on it (Fig. 3A). F35 and F36 further showed
dose-dependent inhibition against LPS-induced I B degradation
jB degradation and the phosphorylation of ERK, p38, and JNK
j
j
2.5. F35 and F36 attenuated the LPS-induced septic
death in mice
(Fig. 3B). We also tested the effects of four compounds on LPS-in-
duced MAPK phosphorylation. Figure 3C revealed that F36 could
significantly block the phosphorylation of ERK and JNK, F35 only
suppressed ERK activation, while F8 and F29 showed no effect on
all three MAP kinases. Subsequently, the inhibitory activity of
F35 and F36 against LPS-induced ERK activation was confirmed
by dose-dependent experiments (Fig. 3D). A similar result was also
As a major endotoxin, LPS has been implicated as a major cause
of sepsis.18 A number of different approaches have been investi-
gated to treat and/or prevent the septic shock associated with
infections caused by Gram-negative bacteria, including blockage
of one or more of the cytokines induced by LPS signaling.15,18

J. Wu et al. / Bioorg. Med. Chem. 21 (2013) 3058–3065
3061
A
B
Cur
10
+
F35
5
F36
5
+
Comps (uM)
-
-
2.5
+
10
+
Comps (10uM)
-
-
Cur F8 F29 F35 F36
-
-
-
+
2.5
+
10
+
LPS (10ng/ml)
-
+
+
LPS (10ng/ml) -
IκB
+
+
+
+
+
+
IκB
GAPDH
150
GAPDH
150
**
**
150
100
50
**
**
**
**
**
100
100
50
0
50
0
0
F36
C
D
E
F35
5
+
F36
5
+
Comps (uM)
-
-
2.5
+
5
+
10
+
Cur F8 F29 F35 F36
Comps (10uM)
-
-
-
+
Comps (uM)
-
-
-
+
2.5
+
10 2.5
10
+
LPS (10ng/ml)
-
+
LPS (10ng/ml)
+
+
+
+
+
LPS (10ng/ml)
+
+
p-JNK
p-ERK
p-ERK
ERK
JNK
ERK
p-JNK
JNK
100
50
0
100
50
0
**
**
p-P38
P38
**
**
**
**
**
**
**
**
Figure 3. Active compounds F8, F19, F35, and F36 affected LPS-induced NF-
jB and MAPK signaling activation in MPMs. (A) Four compounds affected IjB degradation; (B) F35
and F36 dose-dependently reversed LPS-induced I B degradation; (C) Four compounds inhibited the phosphorylation of JNK, ERK, and p38; (D) F35 and F36 dose-
j
dependently inhibited ERK activation; (E) F36 dose-dependently decreased JNK phosphorylation (For clarity, lanes 3rd–5th showing the effects of F35 at three concentrations
have been cut. The full unmodified gels are not shown.). MPMs were plated with a density of 1.2 Â 106/plate for overnight. Cells were pretreated with DMSO or curcumin
(Cur) or indicated compounds at indicated concentrations for 2 h, then treated with LPS (10 ng/mL) for 1 h. The cells were collected and lysated. The protein level of IjB, p-
JNK, p-ERK, or p-p38 was detected by western blot with GAPDH or corresponding total protein as loading control. Representative blots of 4–6 dependent experiments in each
study are shown. The column figures show the normalized optical density as a percentage of control. Bars represent the mean SEM of 3–5 independent experiments: (⁄⁄
)
p <0.01.
A
B
28
21
14
7
vehicle
LPS
100
80
60
40
20
0
**
**
F35+LPS
F36+LPS
vehicle
Vehicle+LPS
F35+LPS
F36+LPS
0
0
1
2
3
4
5
6
7
8
0
1
2
3
5
6
7
Days
Days4
Figure 4. F35 and F36 attenuated LPS-induced septic shock in vivo. Male C57BL/6 mice were pretreated with F35 or F36 (ip, 20 mg/kg) or vehicle, followed by injection of LPS
⁄⁄
(iv, 20 mg/kg). Survival (A) and body weight (B) were recorded for 7 days at an interval of 24 h after the LPS injection. n = 10 animals in each group. p <0.01 versus LPS
group.
Our data have demonstrated the inhibitory effects of these analogs
on LPS-induced signaling activation and IL-6 release. For the poten-
tial clinical application, we further determine whether F35 and F36
are able to attenuate LPS-induced septic death in mice. Compounds
were used in a water-soluble preparation for intraperitoneal (ip)
administration. Mice were injected with LPS at the dosage of
20 mg/kg intravenously (iv) 15 min after the ip injection of F35
or F36, and their survival rates were monitored for seven days,
respectively. As shown in Figure 4A, 80% of animals treated with
LPS alone died within 4 days as a result of the septic shock. In ani-
mals receiving F35 or F36 at 15 mg/kg 15 min prior to LPS injec-
tion, the survival rates were significantly increased compared to
that of the control group (80% survival in F35-treated group and
60% survivals in F36-treated group, p <0.01 in both groups vs LPS
group). Meanwhile, the body weight of compound-treated mice
decreased during day 0–3, but regained slowly 3 days after LPS
treatment (Fig. 4B). Thus, our data provide the in vivo evidence
for the anti-inflammatory effects of these piperid-4-one-contain-
ing MCACs.
3. Conclusion
In summary, we synthesized and screening several piperid-4-
one-containing analogues of curcumin, which exhibited strong

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J. Wu et al. / Bioorg. Med. Chem. 21 (2013) 3058–3065
inhibition on LPS-induced IL-6 release in RAW264.7 macrophage
cell. Generally, this kind of MCAC is more effective against LPS-in-
duced IL-6 expression than previous 3 series of 5-carbon linker-
containing MCACs. Their SAR profiles were discussed and active
compounds were selected for further anti-inflammatory investiga-
tion. Compounds F8, F29, F33, F35 and F36 showed dose-depen-
dent inhibition on LPS-induced IL-6 expression, and pretreatment
of F35 and F36 prolonged survival in LPS-induced acute inflamma-
tory mouse model. We also test the effects of active compounds on
(d, J = 1.8 Hz, 2H, Ar-H2 Â 2), 6.873 (dd, J1 = 1.8 Hz, J2 = 8.4 Hz, 2H,
Ar-H6 Â 2), 6.799 (d, J = 8.4 Hz, 2H, Ar-H5 Â 2), 2.845 (t, J = 4.8 Hz,
4H, CH2–C–CH2), 1.725 (t, J = 4.8 Hz, 2H, C–CH2–C). ESI-MS m/z:
339.1(M+1)+, calcd for C20H18O5: 338.35.
4.1.3. (1E,4E)-1,5-Bis(3,4-dihydroxyphenyl)penta-1,4-dien-3-
one (F3)
15% Yield, mp 149–151.7 °C [150–154 °C, lit.19]. 1H NMR
(DMSO-d6) d: 9.697 (s, 2H, Ar–CH@C Â 2), 9.524 (s, 2H, Ar–
C@CH Â 2), 7.265 (m, 4H, Ar-H2 Â 2, Ar-H5 Â 2), 6.904 (d, 2H, Ar-
H6 Â 2). ESI-MS m/z: 296.9(MÀ1)À, calcd for C17H14O5: 298.29.
ESI-MS m/z: 296.9(MÀ1)À, calcd for C17H14O5: 298.29.
NF-jB and MAPK signaling pathways. The results indicate that
they exert cytokine-inhibitory activity via different signaling
mechanism, although they are derived from the same lead. These
findings suggest that piperid-4-one-containing MCACs may be
promising anti-inflammatory agents and have potential in the
therapy of sepsis and other inflammatory diseases. In the next step,
we may examine the introduction of the other heteroatom into the
central linker of MCACs. Further studies are also necessary to
investigate the underlying molecular mechanisms and different
targets of these analogues at the transcriptional or post-transcrip-
tional level.
4.1.4. (3E,5E)-3,5-Bis(4-hydroxy-3-methoxybenzylidene)-1-
propylpiperid-4-one (F4)
36.5% Yield, mp 213.8–214.7 °C. 1H NMR (DMSO-d6) d: 7.833 (S,
2H, Ar–CH@C Â 2), 7.139 (s, 2H, Ar-H2 Â 2), 7.033 (d, J = 8.4 Hz, 2H,
Ar-H6 Â 2), 6.946 (d, J = 8.4 Hz, 2H, Ar-H5 Â 2), 3.842 (s, 6H, 3-
OCH3 Â 2), 3.356 (S, 4H, –CH2–N–CH2–), 2.500 (s, 2H, N–CH2),
1.678-1.716 (m, 2H, –CH2–), 0.889 (t, J = 7.2 Hz, 3H, –CH3). ESI-
MS m/z: 410.1 (M+1)+, calcd for C24H27NO5: 409.47.
4. Experimental section
4.1. Chemical synthesis
4.1.5. (3E,5E)-3,5-Bis(2-chlorobenzylidene)-1-propylpiperid-4-
one (F5)
76.6% Yield, mp 126.2–127.9 °C. 1H NMR (CDCl3), d: 8.003 (s, 2H,
Ar–CH@C Â 2), 7.452–7.468 (m, 2H, Ar-H6 Â 2), 7.281–7.320 (m,
4H, Ar-H3 Â 2, Ar-H4 Â 2), 7.235–7.251 (m,2H, Ar-H5 Â 2), 3.692
(s, 4H, N–CH2–C Â 2), 2.420 (t, J = 7.8 Hz, 2H, N–CH2), 1.343–
1.384 (m, 2H, N–C–CH2), 0.809 (t, J = 7.8 Hz, 3H, CH3). ESI-MS m/
z: 386.2, 388.1 (M+1)+, calcd for C22H21Cl2NO: 386.31.
All chemical reagents were obtained from Sigma–Aldrich, Fluka,
and Aladdin (Beijing, China). Silica gel (GF254) for thin-layer chro-
matography and column chromatography (100–200 and 200–300
mesh) were obtained from Aladdin. Melting points were tested
on a Fisher-Johns melting apparatus. Electron-spray ionization
mass spectra (ESI-MS) data were determined on a Bruker Esquire
HCT spectrometer. The 1H NMR spectra data was recorded on a
600 MHz spectrometer (Bruker Corporation, Switzerland).
All compounds were synthesized by aldol condensation
between aryl aldehydes and ketones (acetone, cyclohexanone,
cyclopentanone, piperid-4-one, 1-methylpiperid-4-one, 1-ethyl
piperid-4-one, or 1-propylpiperid-4-one). The general procedure
for synthesis of these compounds is briefly described as following.
The piperid-4-one (2 mmol) and corresponding aryl (4 mmol) alde-
hydes are dissolved in the mixture solvent of ethanol and water
(10:1), and other ketones (2 mmol) and corresponding aryl alde-
hydes (4 mmol) are dissolved in ethanol absolute. The compounds
containing –OH group in the benzene ring were catalyzed by HCl.
Other compounds were catalyzed by NaOH at 5–8 °C. All reactions
were monitored by the silica gel TLC. At the end of the reaction,
water is added into the reaction mixture to precipitate the product.
Compound F3–F6, F9, F11–F16, F19–F21, F25, F27–F36 were puri-
fied by column chromatography using PE/EA or CHCl3/CH3OH. The
crude of other compounds were recrystallized in the ethanol or the
mixture of trichloromethane and ethanol. Their structures were
determined by spectral data from ESI-MS and 1H NMR. The spectral
data of new or unreported compounds are shown below.
4.1.6. (3E,5E)-3,5-Bis(3,4-dihydroxybenzylidene)-1-
propylpiperid-4-one (F6)
43.15% Yield, mp 232.2–233.8 °C. 1H NMR (DMSO-d6), d: 7.700
(s, 2H, Ar–CH@C Â 2), 7.221 (d, J = 1.8 Hz, 2H, Ar-H2 Â 2), 6.900
(d, J = 7.8 Hz, 2H, Ar-H6 Â 2), 6.871 (d, J = 7.8 Hz, 2H, Ar-H5 Â 2),
4.524 (s, 4H, CH2–N–CH2), 3.146–3.151 (m, 2H, N–CH2),1.655–
1.694 (m, 2H, N–C–CH2), 0.912–0.925 (m, 3H, CH3). ESI-MS m/z:
380.1 (MÀ1)À, calcd for C22H23NO5: 381.42.
4.1.7. (3E,5E)-3,5-Bis(3,4-dimethoxybenzylidene)-1-
propylpiperid-4-one (F8)
61.1% Yield, mp 162.7–165.2 °C. 1H NMR (CDCl3), d: 7.769 (s,
2H, Ar–CH@C Â 2), 7.021 (dd, J1 = 1.8 Hz, J2 = 8.4 Hz, 2H, Ar-
H6 Â 2), 6.955 (d, J = 1.2 Hz, 2H, Ar-H2 Â 2), 6.928 (d, J = 8.4 Hz,
2H, Ar-H5 Â 2), 3.952 (s, 6H, 3-OCH3 Â 2), 3.924 (s, 6H, 4-
OCH3 Â 2), 3.854 (s, 4H, CH2–N–CH2), 2.520 (t, 2H, N–CH2), 1.483
(m, 2H, CH3CH2), 0.884 (t, J = 7.2 Hz, 3H, CH3). ESI-MS m/z:
438.3(M+1)+, calcd for C26H31NO5: 437.53.
4.1.8. (3E,5E)-3,5-Bis(2,4-dichlorobenzylidene)-1-
methylpiperid-4-one (F9)
88.6% Yield, mp 145.9–147.6 °C. 1H NMR (CDCl3), d: 7.913 (s,
1H, Ar–CH@C), 7.481 (d, J = 1.8 Hz, 2H, Ar-H3 Â 2), 7.305 (dd,
J1 = 1.8 Hz, J2 = 7.8 Hz, 2H, Ar-H6 Â 2), 7.187 (d, J = 7.8 Hz, 2H, Ar-
H5 Â 2), 3.735 (s, 4H, CH2–N–CH2), 2.453 (s, 3H, N–CH3). ESI-MS
m/z: 427.8, 425.9, 429.8(M+1)+, calcd for C20H15Cl4NO: 427.15.
4.1.1. (2E,5E)-2,5-Bis(3,4-dihydroxybenzylidene)
cyclopentanone (F1)
13.7% Yield, mp 260.7 °C. 1H NMR (DMSO-d6), d: 9.563 (br s, 2H,
OH-4), 9.211 (br s, 2H, OH-3), 7.238 (s, 2H, Ar–CH@C Â 2), 7.113 (d,
J = 1.8 Hz, 2H, Ar-H2 Â 2), 7.006 (dd, J1 = 1.8 Hz, J2 = 8.4 Hz, 2H, Ar-
H6 Â 2), 6.834 (d, J = 8.4 Hz, 2H, Ar-H5 Â 2), 3.001 (s, 4H, CH2–O–
CH2). ESI-MS m/z: 323.1(MÀ1)À, calcd for C19H16O5: 324.33.
4.1.9. (3E,5E)-3,5-Bis(4-(dimethylamino)benzylidene)-1-
methylpiperid-4-one (F10)
16.8% Yield, mp 225.8–227.3 °C [223–225 °C, lit.21]; 1H NMR
(CDCl3), d: 7.785 (s, 2H, Ar–CH@C Â 2), 7.345 (d, J = 9.0 Hz, 4H,
Ar-H2 Â 2, Ar-H6 Â 2), 6.716 (d, J = 8.4 Hz, 4H, Ar-H3 Â 2, Ar-
H5 Â 2), 3.845 (s, 4H, CH2–N–CH2), 3.023 (s, 12H, 4-N(CH3) Â 2),
2.510 (s, 3H, N–CH3). ESI-MS m/z: 376.1(M+1)+, calcd for
4.1.2. (2E,6E)-2,6-Bis(3,4-dihydroxybenzylidene)cyclohexanone
(F2)
69% Yield, mp 234.4 °C. 1H NMR (DMSO-d6), d: 9.438 (br s, 2H,
OH-4), 9.131 (br s, 2H, OH-3), 7.446 (s, 2H, Ar–CH@C Â 2), 6.980
C24H29N3O: 375.51.

J. Wu et al. / Bioorg. Med. Chem. 21 (2013) 3058–3065
3063
4.1.10. (3E,5E)-3,5-Bis(4-hydroxy-3-methoxybenzylidene)-1-
methylpiperid-4-one (F11)
2H, Ar-H5 Â 2), 3.937 (s, 6H, 3-OCH3 Â 2), 3.915 (s, 6H, 4-
OCH3 Â 2), 3.833 (s, 4H, CH2–N–CH2), 2.497 (s, 3H, N–CH3). ESI-
MS m/z: 410.1(M+1)+, calcd for C24H27NO5: 409.47.
17.1% Yield, mp 190.2–192.7 °C [195–197 °C, lit.20]. 1H NMR
(DMSO-d6) d: 7.821 (S, 2H, Ar–CH@C Â 2), 7.418 (dd, J1 = 1.8 Hz,
J2 = 7.8 Hz, 1H, Ar-H6 Â 2), 7.131 (d, J = 1.2 Hz, 2H, Ar-H2Â2),
6.969 (d, J = 8.4 Hz, 2H, Ar-H6 Â 2), 3.841 (s, 6H, 3-OCH3 Â 2),
3.345 (S, 4H, –CH2–N–CH2–), 2.500 (s, 3H, N–CH3). ESI-MS m/z:
382.1(M+1)+, calcd for C22H23NO5: 381.42
4.1.18. (3E,5E)-3,5-Bis(2,4-dichlorobenzylidene)-1-ethylpiperid-
4-one (F19)
92.21% Yield, mp 100.4–103.7 °C. 1H NMR (CDCl3), d: 7.925 (s,
1H, Ar–CH@C), 7.487 (d, J = 1.8 Hz, 2H, Ar-H3 Â 2), 7.294 (dd,
J1 = 1.8 Hz, J2 = 7.8 Hz, 2H, Ar-H6 Â 2), 7.175 (d, J = 7.8 Hz, 2H, Ar-
H5 Â 2), 3.662 (s, 4H, N–CH2 Â 2), 2.546 (q, J = 7.2 Hz, 2H, N–
CH2), 0.996 (t, J = 7.2 Hz, 3H, CH3). ESI-MS m/z: 442.1, 440.2,
444.0(M+1)+, calcd for C21H17Cl4NO: 441.18.
4.1.11. (3E,5E)-3,5-Bis(2-chlorobenzylidene)-1-methylpiperid-
4-one (F12)
78.5% Yield, mp 143.9–145.2 °C [150–151 °C, lit.21]. 1H NMR
(CDCl3), d: 8.010 (s, 2H, Ar–CH@C Â 2), 7.458 (d, J = 9.0 Hz, 2H,
Ar-H6 Â 2), 7.285–7.322 (m, 4H, Ar-H3 Â 2, Ar-H4 Â 2), 7.231–
7.262 (m, 2H, Ar-H5 Â 2), 3.659 (s, 4H, CH2–N–CH2), 2.379 (s, 3H,
N–CH3). ESI-MS m/z: 358.3, 360.1, 361.1(M+1)+, calcd for
C20H17Cl2NO: 358.26.
4.1.19. (3E,5E)-3,5-Bis(4-(dimethylamino)benzylidene)-1-
ethylpiperid-4-one (F20)
34.2% Yield, mp 188.6–191.1 °C. 1H NMR (CDCl3), d: 7.797 (s,
2H, Ar–CH@C Â 2), 7.355 (d, 4H, Ar-H2 Â 2, Ar-H6 Â 2), 6.720 (d,
4H, Ar-H3 Â 2, Ar-H5 Â 2), 3.909 (s, 4H, N–CH2 Â 2), 2.670 (q,
J = 7.2 Hz, 2H, N–CH2), 1.115 (t, J = 7.2 Hz, 3H, CH3). ESI-MS m/z:
390.3(M+1)+, calcd for C25H31N3O: 389.53.
4.1.12. (3E,5E)-3,5-Bis(2-fluorobenzylidene)-1-methylpiperid-4-
one (F13)
95.2% Yield, mp 138.5–141.8 °C. 1H NMR (CDCl3), d: d: 7.902 (s,
2H, Ar–CH@C Â 2), 7.355–7.371 (m, 2H, Ar-H6 Â 2), 7.294 (dt,
J = 1.2 Hz, 7.2 Hz, 2H, Ar-H3 Â 2), 7.187 (dt, J1 = 0.6 Hz, J2 = 7.2 Hz,
2H, Ar-H4 Â 2), 7.128 (dt, J1 = 0.6 Hz, J2 = 8.4 Hz, 2H, Ar-H5 Â 2),
3.653 (s, 4H, N–CH2 Â 2), 2.409 (s, 3H, N–CH3). ESI-MS m/z:
326.2(M+1)+, calcd for C20H17F2NO: 325.35.
4.1.20. 2-{[(3E,5E)-5-[(2-Carboxyphenyl)methylidene]-1-ethyl-
4-oxopiperidin-3-ylidene]methyl}benzoic acid (F21)
25.0% Yield, mp 64.8–66.9 °C. 1H NMR (CDCl3), d: 7.889 (d, 2H,
Ar-H3 Â 2), 7.709 (t, 2H, Ar–CH@C Â 2), 7.592 (m, 4H, Ar-H5 Â 2,
Ar-H6 Â 2), 3.932 (m, 4H, CH2–N–CH2). ESI-MS m/z:
390.1(MÀ1)À, calcd for C23H21NO5: 391.42.
4.1.13. (3E,5E)-3,5-Bis(4-hydroxybenzylidene)-1-methylpiperid-
4-one (F14)
4.1.21. (3E,5E)-3,5-Bis(2,4,6-trimethoxybenzylidene)-1-
ethylpiperid-4-one (F22)
36.7% yield, mp 116.5–117.8 °C. 1H NMR (DMSO-d6) d: 7.758 (d,
J = 8.4 Hz, 4H, Ar–CH@C Â 2, Ar-H2 Â 2, Ar-H6 Â 2), 6.927 (d,
J = 8.4 Hz, 4H, Ar-H3 Â 2, Ar-H5 Â 2), 3.324 (S, 4H, –CH2–N–CH2–),
2.498 (s, 3H, N–CH3). ESI-MS m/z: 319.7(MÀ1)À, calcd for
18.66% Yield, mp 193.7–196.2 °C. 1H NMR (CDCl3), d: 7.782 (s,
2H, Ar–CH@C Â 2), 6.118 (s, 4H, Ar-H3 Â 2, Ar-H5 Â 2), 3.876 (s,
6H, 4-OCH3 Â 2), 3.800 (s, 12H, 2-OCH3 Â 2, 6-OCH3 Â 2), 3.482
(s, 4H, CH2–N–CH2), 2.467 (m, 2H, NCH2), 0.896 (t, J = 7.2 Hz, 3H,
CH3). ESI-MS m/z: 484.4(M+1)+, calcd for C27H33NO7: 483.55.
C20H19NO3: 321.37.
4.1.14. (3E,5E)-3,5-Bis(4-fluorobenzylidene)-1-methylpiperid-4-
one (F15)
4.1.22. (3E,5E)-3,5-Bis(2,5-dimethoxybenzylidene)-1-
ethylpiperid-4-one (F23)
67.8% Yield, mp 173.8–175.8 °C. 1H NMR (DMSO-d6) d: 7.559–
7.595 (m, 6H, Ar–CH@C Â 2, Ar-H6 Â 2, Ar-H2 Â 2), 7.311 (d,
J = 8.4 Hz, 4H, Ar-H3 Â 2, Ar-H5 Â 2), 3.316 (S, 4H, –CH2–N–CH2–),
2.500 (s, 3H, N–CH3). ESI-MS m/z: 324.36(MÀ1)À, calcd for
69.01% Yield, mp 88.9–92.1 °C. 1H NMR (CDCl3), d: 8.010 (s, 2H,
Ar–CH@C Â 2), 6.875 (d, J = 3.0 Hz, 2H, Ar-H3 Â 2), 6.855 (s, 2H, Ar-
H6 Â 2), 6.775 (d, J = 3.0 Hz, 2H, Ar-H4 Â 2), 3.751–3.807 (m, 12H,
OCH3 Â 4), 3.761 (s, 4H, N–CH2 Â 2), 2.541 (q, J = 7.2 Hz, 2H, N–
CH2), 0.996 (t, J = 7.2 Hz, 3H, CH3). ESI-MS m/z: 424.2(M+1)+, calcd
for C25H29NO5: 423.5.
C20H17F2NO: 325.35.
4.1.15. (3E,5E)-3,5-Bis(2-methoxybenzylidene)-1-
methylpiperid-4-one (F16)
35.92% Yield, mp 108.2–110.9 °C [117–118 °C, lit.21]. 1H NMR
(CDCl3), d: 8.093 (s, 2H, Ar–CH@C Â 2), 7.335–7.363 (m, 2H, Ar-
H6 Â 2), 7.183 (dd, J1 = 1.2 Hz, J2 = 7.8 Hz, Ar-H4 Â 2), 6.974 (t,
J = 7.8 Hz, Ar-H5 Â 2), 6.924 (d, J = 8.4 Hz, Ar-H3 Â 2), 3.857 (s, 6H,
2-OCH3 Â 2), 3.735 (s, 4H, CH2–N–CH2), 2.404 (s, 3H, N–CH3).
ESI-MS m/z: 350.1 (M+1)+, calcd for C22H23NO3: 349.42.
4.1.23. (3E,5E)-3,5-bis(2-bromobenzylidene)-1-ethylpiperid-4-
one (F24)
84.5% Yield, mp 133.4–136 °C. 1H NMR (CDCl3), d: 7.946 (s, 2H,
Ar–CH@C Â 2), 7.655 (d, J = 7.8 Hz, 2H, Ar-H3 Â 2), 7.346 (t,
J = 7.2 Hz, 2H, Ar-H5 Â 2), 7.187–7.236 (m, 4H, Ar-H4 Â 2, Ar-
H6 Â 2), 3.667 (s, 4H, N–CH2 Â 2), 7.525 (q, J = 7.2 Hz, 2H, N–
CH2), 0.972 (t, J = 7.2 Hz, 3H, CH3). ESI-MS m/z: 462.1, 463.9,
460.3, 462.9, 464.9(M+1)+, calcd for C21H19Br2NO: 461.19.
4.1.16. (3E,5E)-3,5-Bis(2,3-dimethoxybenzylidene)-1-
methylpiperid-4-one (F17)
38.1% Yield, mp 126.6–128.4 °C. 1H NMR (CDCl3), d: 7.173 (s,
2H, Ar–CH@C Â 2), 7.052 (d, J = 7.8 Hz, 2H, Ar-H6 Â 2), 7.027 (t,
J = 7.8 Hz, 2H, Ar-H4 Â 2), 7.003 (d, J = 7.8 Hz, 2H, Ar-H5 Â 2),
3.887 (s, 4H, CH2–N–CH2), 3.851–3.876 (m, 12H, 2-OCH3 Â 2, 3-
OCH3 Â 2), 3.838 (s, 3H, N-CH3). ESI-MS m/z: 410.2(M+1)+, calcd
for C24H27NO5: 409.47.
4.1.24. (3E,5E)-3,5-Bis(3,4-difluorobenzylidene)-1-ethylpiperid-
4-one (F25)
68.69% Yield, mp 122.2–124.3 °C. 1H NMR (CDCl3), d: 7.688 (s,
2H, Ar–CH@C Â 2), 7.191–7.232 (m, 4H, Ar-H2 Â 2, Ar-H6 Â 2),
7.133–7.151 (m, 1H, Ar-H5 Â 2), 3.766 (s, 4H, N–CH2 Â 2), 2.635
(q, J = 7.2 Hz, 2H, N–CH2), 1.082 (t, J = 7.2 Hz, 3H, CH3). ESI-MS m/
z: 376.0, calcd for C21H17F4NO: 375.36.
4.1.17. (3E,5E)-3,5-bis(3,4-dimethoxybenzylidene)-1-
methylpiperid-4-one (F18)
4.1.25. (3E,5E)-3,5-Bis(3,4-dimethoxybenzylidene)-1-
ethylpiperid-4-one (F26)
52.3% Yield, mp 157.3–159.4 °C. 1H NMR (CDCl3), d: 7.785 (s,
2H, Ar–CH@C Â 2), 6.999 (dd, J1 = 1.2 Hz, J2 = 8.4 Hz, 2H, Ar-
H6 Â 2), 6.936 (d, J = 1.8 Hz, 2H, Ar-H2 Â 2), 6.916 (d, J = 8.4 Hz,
66.32% Yield, mp 188.7–190.8 °C. 1H NMR (CDCl3), d: 7.783
(s, 2H, Ar–CH@C Â 2), 7.017 (dd, J1 = 1.8 Hz, J2 = 8.4 Hz, 2H,

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J. Wu et al. / Bioorg. Med. Chem. 21 (2013) 3058–3065
Ar-H6 Â 2), 6.950 (d, J = 1.8 Hz, 2H, Ar-H2 Â 2), 6.922 (d, J = 8.4 Hz,
2H, Ar-H3 Â 2), 3.920 (m, 12H, OCH3 Â 4), 3.888 (s, 4H, N–CH2–
C Â 2), 1.085 (t, J = 7.2 Hz, 3H, CH3), 2.641 (d, J = 7.2 Hz, 2H, N–
CH2). ESI-MS m/z: 424.5(M+1)+, calcd for C25H29NO5: 423.5.
6.933 (m, 4H, Ar-H5 Â 2, Ar-H6 Â 2), 4.191 (s, 4H, N–CH2–C Â 2),
3.895–3.947 (m, 12H, OCH3 Â 4), 1.780 (br s, 1H, N–H). ESI-MS
m/z: 396.2(M+1)+, calcd for C23H25NO5: 395.45.
4.2. Animals
4.1.26. (3E,5E)-3,5-Bis(4-methoxybenzylidene)piperid-4-one
(F27)
Male C57BL/6 and ICR mice weighing 18–22 g were obtained
from the Animal Center of Wenzhou Medical College (Wenzhou,
China). Animals were housed at a constant room temperature with
a 12:12 hour light-dark cycle, and fed with a standard rodent diet
and water. The animals were acclimatized to the laboratory for at
least 7 days before used in experiments. Protocols involving the
use of animals were approved by the Wenzhou Medical College
Animal Policy and Welfare Committee (Approval documents:
2009/APWC/0031).
11.1% Yield, mp 182.0–184.6 °C [180–182 °C, lit.22] ESI-MS m/z:
336.1(M+1)+, calcd for C21H21NO3: 335.4.
4.1.27. (3E,5E)-3,5-Bis(2-fluorobenzylidene)piperid-4-one (F28)
44.68% Yield, mp 139.6–142.5 °C. 1H NMR (CDCl3), d: 7.884 (s,
2H, Ar–CH@C Â 2), 7.285–7.389 (m, 4H, Ar-H3 Â 2, Ar-H6 Â 2),
7.204 (t, J = 7.2 Hz, 2H, Ar-H4 Â 2), 7.148 (t, J = 9.0 Hz, 2H, Ar-
H5 Â 2), 4.049 (s, 4H, CH2–N–CH2). ESI-MS m/z: 312.3(M+1)+, calcd
for C19H15F2NO: 311.33.
4.3. Reagents and cells
4.1.28. (3E,5E)-3,5-Bis(4-hydroxybenzylidene)piperid-4-one
(F29)
Chemical reagents and lipopolysaccharide (LPS) were pur-
chased from Sigma (Louis, MO). Saline was prepared as 0.9% NaCl
solution. Mouse RAW 264.7 macrophages were obtained from
the American Type Culture Collection (ATCC, USA). RAW 264.7
macrophages were incubated in DMEM medium (Gibco, Eggen-
stein, Germany) supplemented with 10% FBS (Hyclone, Logan,
UT), 100 U/mL penicillin, and 100 mg/mL streptomycin at 37 °C
with 5% CO2. The mouse primary peritoneal macrophage (MPMs)
were obtained from ICR mice according to the method described
in our previous paper.22
7.6% Yield, mp 298.5 °C decompose [>300 °C, lit.23]. 1H NMR
(DMSO-d6), d: 10.270 (s, 2H, Ar–CH@C Â 2), 7.392 (d, J Â 8.4 Hz,
4H, Ar-H2 Â 2, Ar-H6 Â 2), 6.920 (d, J Â 8.4 Hz, 4H, Ar-H3 Â 2, Ar-
H5 Â 2), 4.451 (s, 4H, CH2–N–CH2). ESI-MS m/z: 308.1 (M+1)+;
306.0 (MÀ1)À, calcd for C19H17NO3: 307.34.
4.1.29. (3E,5E)-3,5-Bis(2,4,6-trimethoxybenzylidene)piperid-4-
one (F30)
30.61% Yield, mp 152.3–156.1 °C. 1H NMR (CDCl3), d: 7.662 (s,
2H, Ar–CH@C Â 2), 6.124 (s, 4H, Ar-H3 Â 2, Ar-H5 Â 2), 3.843 (s,
6H, 4-OCH3 Â 2), 3.799 (s, 12H, 2-OCH3 Â 2, 6-OCH3 Â 2), 3.686
(s, 4H, CH2-N-CH2). ESI-MS m/z: 456.2(M+1)+, calcd for
4.4. Determination of IL-6 by ELISA method
After treatment of cells with indicated compounds and LPS, the
IL-6 levels in medium were determined with an ELISA kit (eBio-
Science, San Diego, CA) according to the manufacturer’s instruc-
tions and the method described in our previous paper.16 The
total amount of the inflammatory factor in the medium was nor-
malized to the total protein quantity of the viable cell pellets.
C25H29NO7: 455.19.
4.1.30. (3E,5E)-3,5-Bis(2,4-dimethoxybenzylidene)piperid-4-one
(F31)
41.61% Yield, mp 150.7–154.1 °C. 1H NMR (CDCl3), d: 7.993 (s,
2H, Ar–CH@C Â 2), 7.108 (d, J = 8.4 Hz, 2H, Ar-H6 Â 2), 6.500 (d,
J = 7.8 Hz, 2H, Ar-H5 Â 2), 6,464 (s, 2H, Ar-H3 Â 2), 4.046 (s, 4H,
N–CH2–C Â 2), 3.843 (s, 6H, 2-OCH3 Â 2), 3.834 (s, 6H, 4-
OCH3 Â 2), 1.975 (br s, 1H, N–H). ESI-MS m/z: 396.1 (M+1)+, calcd
for C23H25NO5: 395.45.
4.5. Western blot analysis
The treated cells were collected and lysated, then 40–60 lg of
the whole cell lysates were separated by 10% sodium dodecyl sul-
fate-polyacrylamide gel electrophoresis and electrotransferred to a
nitrocellulose membrane. Each membrane was preincubated for
1 h at room temperature in tris-buffered saline, pH 7.6, containing
0.05% tween 20 and 5% non-fat milk. The nitrocellulose membrane
4.1.31. (3E,5E)-3,5-Bis(3,4-dihydroxybenzylidene)piperid-4-one
(F32)
31% Yield, mp 81.6–83.5 °C. ESI-MS m/z: 337.8 (MÀ1)À, calcd
for C19H13F4NO: 339.34.
was incubated with specific antibodies against p-JNK, JNK, IjB, p-
p38, p38, p-ERK, ERK, or Actin (Santa Cruz Biotech Co. LTD, CA),
respectively. Immunoreactive bands were then detected by incu-
bating with secondary antibody conjugated with horseradish per-
oxidase and visualized using enhanced chemiluminescence
reagents (Bio-Rad, Hercules, CA).
4.1.32. (3E,5E)-3,5-Bis(3-hydroxybenzylidene)piperid-4-one
(F33)
41.2% Yield, mp >300 °C. 1H NMR (CDCl3), d: 8.955 (s, 2H, Ar–
CH@C Â 2), 7.764 (s, 2H, Ar-H2 Â 2), 7.405 (t, 2H, Ar-H6 Â 2),
6.967–6.996 (m, 4H, Ar-H4 Â 2, Ar-H5 Â 2), 3.905 (s, 4H, CH2–N–
CH2). ESI-MS m/z: 308.7(M+1)+, calcd for C19H16O4: 307.34.
4.6. LPS-induced inflammatory mortality in mice
Compounds were firstly dissolved with macrogol 15 hydroxy-
stearate (a nonionic solubilizer for injection from BASF) with or
without medium chain triglycerides (MCT, from BASF) in water
bath at 37 °C. The concentration of compounds was 2 mg/mL. The
concentration of solubilizer was ranged 5–10%, and MCT 0.5–2%
in final solution. For the vehicle, the mixture of solubilizer and
MCT was prepared at 10% and 2% respectively. Male C57BL/6 mice
weighing 18–22 g were pretreated with compounds (20 mg/kg) in
a water solution by ip injection 15 min before the iv injection of
4.1.33. (3E,5E)-3,5-Bis(3,4,5-trimethoxybenzylidene)piperid-4-
one (F35)
29.73% Yield, mp 193.7–194.0 °C. 1H NMR (CDCl3), d: 7.734 (s,
2H, Ar–CH@C Â 2), 6.615 (s, 4H, Ar-H2 Â 2, Ar-H6 Â 2), 4.210 (s,
4H, CH2–N–CH2), 3.930 (s, 6H, 4-OCH3 Â 2), 3.880 (s, 12H, 3-
OCH3 Â 2, 5-OCH3 Â 2). ESI-MS m/z: 456.2(M+1)+, calcd for
C25H29NO7: 455.5.
4.1.34. (3E,5E)-3,5-Bis(3,4-dimethoxybenzylidene)piperid-4-one
(F36)
LPS (20 mg/kg). Control animals received
(200 L) of vehicle. Body weight change and mortality were re-
corded for 7 days.
a similar volume
8.39% Yield, mp 162.2–165.4 °C. 1H NMR (CDCl3), d: 7.758 (s,
2H, Ar–CH@C Â 2), 7.008 (d, J = 8.4 Hz, 2H, Ar-H2 Â 2), 6.908–
l

J. Wu et al. / Bioorg. Med. Chem. 21 (2013) 3058–3065
3065
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Financial support was provided by the National Natural Science
Funding of China (Grants 21272179, 81102310, and 81272462),
High-Level Innovative Talent Funding of Zhejiang Department of
Health (G.L.), Project of Wenzhou Sci-Tech Bureau (Y20120061),
Zhejiang Natural Science Funding (Grant LY12H16003), Grant from
Zhejiang Department of Health (2012KYA129), Zhejiang Zhejiang
Key Group Project in Scientific Innovation (2010R50042), and Chi-
na Postdoctoral Science Foundation (Grants 20090461121 and
201003591).
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A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.03.057.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
References and notes
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