Synthesis and biological evaluation of allylated and prenylated mono-carbonyl analogs of curcumin as anti-inflammatory agents

Article abstract of DOI:10.1016/j.ejmech.2013.10.061

Curcumin has been shown to possess anti-inflammatory activities but has been limited for its low stability and poor bioavailability. We have previously reported four series of 5-carbon linker-containing mono-carbonyl analogs of curcumin (MACs). In continuation of our ongoing research, we designed and synthesized 33 novel allylated or prenylated MACs here, and evaluated their anti-inflammatory effects in RAW 264.7 macrophages. A majority of them effectively inhibited the LPS-induced expression of TNF-α and IL-6, especially IL-6. The preliminary SAR and quantitative SAR analysis were conducted. Compound 14q is the most potent analog among them, and exhibits significant protection against LPS-induced death in septic mice. Together, these data present a series of new analogs of curcumin as promising anti-inflammatory agents.

Full text of DOI:10.1016/j.ejmech.2013.10.061

European Journal of Medicinal Chemistry 74 (2014) 671e682
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of allylated and prenylated
mono-carbonyl analogs of curcumin as anti-inflammatory agents
,
d
,
,
,
Zhiguo Liu a 1, Longguang Tang a 1, Peng Zou b 1, Yali Zhang b, Zhe Wang a, Qilu Fang a,
**
Lili Jiang c, Gaozhi Chen a, Zheng Xu a, Huajie Zhang a , Guang Liang
a Chemical Biology Research Center at School of Pharmaceutical Sciences, Wenzhou Medical University, 1210 University Town, Wenzhou, Zhejiang 325035,
,
a,
*
China
b School of Environmental and Biological Engineering, Nanjing University of Science and Technology, No. 200 Xiaolingwei Road, Xuanwu District, Nanjing,
Jiangsu 210094, China
c Department of Pediatrics, The Second Affiliated Hospital, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China
d Wenzhou Undersun Biotechnology Co., Ltd., Wenzhou, Zhejiang, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Curcumin has been shown to possess anti-inflammatory activities but has been limited for its low sta-
bility and poor bioavailability. We have previously reported four series of 5-carbon linker-containing
mono-carbonyl analogs of curcumin (MACs). In continuation of our ongoing research, we designed
and synthesized 33 novel allylated or prenylated MACs here, and evaluated their anti-inflammatory
effects in RAW 264.7 macrophages. A majority of them effectively inhibited the LPS-induced expres-
Received 19 August 2013
Received in revised form
18 October 2013
Accepted 24 October 2013
Available online 1 November 2013
sion of TNF-a and IL-6, especially IL-6. The preliminary SAR and quantitative SAR analysis were con-
ducted. Compound 14q is the most potent analog among them, and exhibits significant protection
against LPS-induced death in septic mice. Together, these data present a series of new analogs of cur-
cumin as promising anti-inflammatory agents.
Keywords:
Curcumin
Mono-carbonyl analogs
Anti-inflammation
QSAR
Ó 2013 Elsevier Masson SAS. All rights reserved.
TNF-
IL-6
a
1. Introduction
pathways. Inhibition of these cytokines has been successful in
preclinical and clinical studies for the treatment of sepsis, cancer,
Macrophages play important roles in host immune defense
systems during infection and disease development. Activation of
macrophages by stimuli such as the bacterial lipopolysaccharide
(LPS) endotoxins, increases the production of numerous inflam-
matory mediators and cytokines [1,2]. Two representative cyto-
diabetic complications, and rheumatoid arthritis [6,7]. Therefore,
inhibition of the release of cytokines from activated macrophages is
an important mode of action for anti-inflammatory drugs and has
therefore become the focus of much current drug discovery and
development research.
kines, Interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-
a
),
Curcumin [7-bis(4-hydroxy-3-methoxypentyl)-1,6-hepadiene-
3,5-dione, 1], a yellow component in the root of the plant, Curcuma
longa., has been shown to exert multiple pharmacological activities,
such as anti-inflammation [8], anti-cancer, anti-oxidation, and car-
diovascular protection [9,10]. It has been demonstrated that cur-
cumin can attenuate inflammatory diseases by inhibiting the
have been well recognized as pro-inflammatory agents. These
multifunctional cytokines, produced mainly by cells of the mono-
cyte/macrophage lineage, have been shown to play important roles
in acute and chronic inflammatory diseases, such as sepsis, rheu-
matoid arthritis [3], cancer [4], atherosclerosis, and inflammatory
bowel disease [5], by amplifying inflammatory signals via multiple
release of cytokines such as TNF-a and IL-6 both in vitro and in vivo
[11e13]. The natural safety and pharmacological efficacy of curcu-
min make it a potential agent for treatment and prevention of awide
variety of human diseases, however, its high metabolic instability
and low bioavailability have dramatically limited its practical
application. Evidence suggests that the active methylene group and
* Corresponding author. School of Pharmaceutical Sciences, Wenzhou Medical
University, China. Tel./fax: þ86 577 86699396.
** Corresponding author. Tel./fax: þ86 577 86689736.
E-mail addresses: zhj@wzmc.net (H. Zhang), cuiliang1234@163.com,
wzmcliangguang@163.com (G. Liang).
b-diketone moiety in the structure of curcumin contribute to its
1
instability under physiological conditions and induce its rapid
These authors contribute equally to this work.
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.10.061

672
Z. Liu et al. / European Journal of Medicinal Chemistry 74 (2014) 671e682
Fig. 1. Structural design of allylated and prenylated mono-carbonyl analogs of curcumin.
degradation and metabolism [14,15]. Recently, medicinal chemists
have carried out several studies on curcumin derivatives and ana-
logs and have found that mono-carbonyl analogs of curcumin
We report here the design, synthesis and anti-inflammatory
evaluation of a series of novel allylated or prenylated MACs.
Furthermore, preliminary and quantitative structureeactivity
relationship analyses were carried out to outline the effects of the
structures of MACs on their pharmacological activity. Active com-
pounds exhibited significant therapeutic effects in a septic mouse
model, suggesting the potential for MACs to be developed as new
anti-inflammatory agents.
(MACs), which have no b-diketone moiety, show greater metabolic
stability and bioactivity than that of curcumin [16e18]. Over several
years, our research group has synthesized several different MACs
and evaluated their anti-inflammatory properties (Fig. 1) [19e21].
Recently, we found that a large number of natural products, such as
4-hydroxycordoin (2), piperkadsin A (3), cudraflavone B (4) and
erythro-honokitriol (5), showed effectively inhibitory activity
against inflammation and related diseases [22e25]. Interestingly,
each of these compounds contains either an allyl or prenyl group.
Covalent modification by prenylation has recently been recognized
as a mechanism of promoting membrane interactions and biological
activity of a variety of cellular proteins [26]. Thus, we proposed that
allyl or prenyl groups in compounds may play important roles in
their bioactivity. As shown in Fig. 1, it is further hypothesized that
linking allyl or prenyl groups may enhance the anti-inflammatory
ability of MACs (Fig. 1).
2. Chemistry
The synthetic routes for the production of the allylated and
prenylated mono-carbonyl analogs of curcumin (8aei, 10aec and
14aeu) used in this study are shown in Schemes 1 and 2; and their
structures are shown in Table 1. The key O-allylated or O-prenylated
benzaldehydes 7 were prepared from commercially available or
previously synthesized 4-hydroxybenzaldehydes 6 with allyl bro-
mide or 1-chloro-3-methylbut-2-ene in the presence of a sufficient
amount of potassium carbonate. Aldol condensation of 7 with
Scheme 1. General synthetic routes for 8aed, 8gei, and 10aec. Reagents and conditions: (a) allyl bromide or 1-chloro-3-methylbut-2-ene, K2CO3, acetone, rt, 1e5 h, 50e95%; (b)
aq. NaOH, EtOH, rt, 3e5 h, 47e85%; (c) acetone, 20% (w/v) NaOH, rt, 4 h, 70e90%; (d) different benzaldehydes, EtOHeH2O (2:1), rt, overnight, 30e80%.

Z. Liu et al. / European Journal of Medicinal Chemistry 74 (2014) 671e682
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Scheme 2. General synthetic routes for 8eef and 14aeu. Reagents and conditions: (e) N,N-diethylaniline, 190 ꢀC, 20 h, 65%; (f) 3,4-dihydro-2H-pyran, PPTS, CH2Cl2, 4 h, 95%; (g)
methyl iodide, K2CO3, acetone, rt, 3 h, 100%; (h) 1 M HCl, EtOH, rt, 1 h, 90e95%.
acetone or different cyclones with sodium hydroxide as a catalyst,
yield the symmetric MACs; 8aed and 8gei, respectively. Alterna-
tively, asymmetric MACs 10aec are obtained by coupling benzal-
dehydes with different a,b-unsaturated ketones 9 derived from a
unilateral aldol condensation between the intermediates 7 and
acetone in a catalyst of 20% NaOH.
N,N-dimethylaniline solvent to afford allylbenzene 11. The product
was subsequently methylated, or protected by 3,4-dihydro-2H-
pyran to afford 12a or 12b, respectively. 12a was used to synthesize
the symmetric MACs, 8e, which was subsequently deprotected to
produce 8f. 12a and 12b were converted to intermediates 13a and
13b by the same procedure used for the synthesis of 9. Finally, after
removal of the THP protecting group, 13a and 13b were reacted in
situ with equimolar amounts of benzaldehydes to produce the
desired MACs (14aeu). All of the new products were isolated by
conventional work-up with satisfactory yields. Analytical and
spectral data of all synthesized compounds are in full agreement
with the proposed structures (Table 1).
To produce the compound 4-[(3-methylbut-2-en-1-yl)oxy]
benzaldehyde, a Claisen Rearrangement was carried out in boiling
Table 1
Chemical structures of all synthetic compounds.
3. Results and discussion
3.1. Anti-inflammatory and cytotoxic evaluation of synthetic MACs
The stimulation of macrophages with LPS induces a pro-
inflammatory process through the activation of cytokine expres-
sion and release [27]. 33 newly synthetic analogs were evaluated
Comp.
R(A-ring)
R0(B-ring)
n
8a
8b
8c
8d
8e
8f
8g
8h
4-O-allyl
4-O-prenyl
40-O-allyl
0
0
0
0
2
2
2
2
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
40-O-prenyl
40-OH, 50-allyl
40-O-prenyl, 50-OMe
40-OTHP, 50-allyl
40-OH, 50-allyl
40-O-prenyl, 50-OMe
40-O-prenyl, 60-OH
40-O-prenyl
30, 40-OMe
30, 40-OMe
40-F
for their ability to inhibit TNF-a and IL-6 release in RAW 264.7
4-OH, 5-allyl
4-O-prenyl, 5-OMe
4-OTHP, 5-allyl
4-OH, 5-allyl
4-O-prenyl, 5-OMe
4-O-prenyl, 6-OH
4-O-prenyl
mouse macrophages, and the results were compared to those for
curcumin. Macrophages were stimulated by LPS for 22 h in the
presence or absence of the compounds at a concentration of 10
mM.
The amount of TNF- and IL-6 subsequently found in the culture
a
medium was detected through enzyme-linked immunosorbant
assay (ELISA) and the results were normalized using the protein
concentration of cells harvested in homologous culture plates. The
results of the anti-inflammation assay when MACs were used are
shown in Fig. 2A and B. The initial screening showed that the ma-
jority of these derivatives inhibited the LPS-induced expression of
8i
10a
10b
10c
14a
14b
14c
14d
14e
14f
14g
14h
14i
14j
14k
14l
14m
14n
14o
14p
14q
14r
14s
14t
14u
4-O-allyl
4-O-prenyl
4-O-allyl, 6-OH
4-OH, 5-allyl
4-OH, 5-allyl
4-OMe, 5-allyl
4-OMe, 5-allyl
4-OMe, 5-allyl
4-OMe, 5-allyl
4-OMe, 5-allyl
4-OMe, 5-allyl
4-OMe, 5-allyl
4-OMe, 5-allyl
4-OMe, 5-allyl
4-OMe, 5-allyl
4-OMe, 5-allyl
4-OMe, 5-allyl
4-OMe, 5-allyl
4-OMe, 5-allyl
4-OMe, 5-allyl
4-OMe, 5-allyl
4-OMe, 5-allyl
4-OMe, 5-allyl
4-OMe, 5-allyl
30, 40-OMe
40-NO2
30, 40-OMe
40-CF3
TNF-
expression. 20 of these compounds exhibited 50e95% inhibition
rates at a dosage of 10 M. Their inhibitory abilities were compa-
a and IL-6, and had a particularly pronounced effect on IL-6
40-N(Me)2
40-O-C(Me)3
50-Br
m
rable to, or more pronounced than, that of curcumin at the same
concentration. Among these compounds, 5 were found to be more
potent than curcumin in inhibiting LPS-induced TNF-a expression
(39e76% inhibition), and 7 compounds showed a greater inhibitory
effect on IL-6 expression (67e90% inhibition). Compounds 8c, 14q,
14r, and 14u exhibited a stronger inhibition of both TNF-a and IL-6
than curcumin. Importantly, 14q, (1E,4E)-1-(3-allyl-4-methoxy
phenyl)-5-(2,5-dibromophenyl)penta-1,4-dien-3-one, showed the
most potent effect on LPS-induced TNF-a and IL-6 production with
its inhibitory rates reaching 67.5% and 91.1%, respectively.
40-Br
40-Morpholine
40-Piperidine
40-O-allyl
30-Ph-40
20,50-F
20-F, 60-Br
20,30-Cl
20-Cl
20,50-Br
20-Br, 50-F
20-CF3
Before the further evaluation, synthetic MACs were tested for
their cytotoxicity and safety in the human normal hepatic cell line,
HL-7702, by MTT after 24-h treatment of the cells with compounds
20-OMe, 50-F
20,40,50-OMe

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Z. Liu et al. / European Journal of Medicinal Chemistry 74 (2014) 671e682
150
100
50
A
IL-6
*
*
*
*
*
*
**
**
**
**
**
**
**
**
**
0
i
j
l
f
f
14
t
r
c
10
c
k
s
a
b
a
b
d
e
g
h
n
o
p
q
u
8i
8
m
8c
8a 8b
8d 8e
8g 8h
14 14
14
14
SO
14
14
14
14
10 10
14 14
14 14
14 14
14 14 14 14
14
LPS
14
CUR
DM
LPS (0.5 g/ml)
150
100
50
B
TNF-
*
*
**
**
**
**
0
i
j
l
14
f
14
t
r
c
10
c
k
14
s
e
h
8
a
b
a
b
d
e
g
h
n
o
p
q
u
8i
8f
m
8c
8a 8b
8d
8
8g
14 14
14
14
SO
14
14
14
10 10
14 14
14 14
14 14
14 14 14 14
LPS
14
CUR
DM
LPS (0.5 g/ml)
150
C
HL-7702 Cells
100
50
0
i
j
l
14
f
f
t
r
c
c
s
4
a
a
b
d
e
g
h
n
o
p
u
8i
8
8c
4k
8a 8b
8d 8e
8g 8h
0b
1
4q
14
1
14 14
14
4m
14
14
10
14
1
1
10
14 14
14 14
14 14
14 14 14
LPS
1
CUR
DMSO
Fig. 2. Curcumin and its analogs inhibited LPS-induced IL-6 (A) and TNF-a (B) secretion in RAW 264.7 macrophages as well as the cytotoxic evaluation (C) in HL-7702 cells.
Macrophages were plated at a density of 4.0 ꢁ 105/plate at 37 ꢀC and 5% CO2 overnight. Cells were pre-treated with curcumin or its analogs (10
mM) for 2 h, then treated with LPS
(0.5 mg/ml) for 22 h. TNF-a and IL-6 levels in the culture media were measured by ELISA and were normalized by the total protein. The results were expressed as the percent of LPS
control. Each bar represents mean ꢂ SE of 3e5 independent experiments. Statistical significance relative to LPS group was indicated, *p < 0.05, **p < 0.01.
at a concentration of 10
compounds displayed no toxicity in hepatic cells, indicating that
they are relatively safe.
m
M. As shown in Fig. 2C, all of these
analogs (series 8). In series 8, the cyclopenta- or cyclohexa-
containing analogs, such as 8f, 8g and 8i, exhibited lower potency
than the corresponding acetone derivatives, 8b and 8c. Compound
8c, which has a 4-hydroxyl group at the ortho position of the 5-allyl
moiety, displayed the greatest activity, with an inhibition ratio of
96.4%. However, etherification at the 4-hydroxy group with THP
produced the compound 8e with the weakest inhibitory activity
compound 8e. This indicates that the hydroxyl group at this posi-
tion is favorable for anti-inflammatory activity. A similar conclusion
was drawn when the asymmetric MACs, 14a and 14c, were
compared. To examine the SAR of ring B, the effects of group size,
electronic properties, and position were tested by modifying the
substituents of analogs 14ceu. Preliminary SAR conclusions can be
3.2. Preliminary and quantitative evaluation of structureeactivity
relationship (SAR)
All synthetic compounds, except 8e and 10a, inhibited IL-6 with
inhibition ratios ranging from 20% to 95%, and the majority showed
greater inhibitory activity than curcumin. However, it is difficult to
analyze the SAR of MACs related to TNF-a inhibition due to their
relatively low activity. The asymmetric MACs (series 14) generally
showed higher activity against IL-6 production than symmetric

Z. Liu et al. / European Journal of Medicinal Chemistry 74 (2014) 671e682
675
drawn: (1) the electronic properties of the substituents seem to
have no influence on bioactivity; and (2) the di- or tri-substituted
compounds are much more active than mono-substituted
compounds.
dose-dependent inhibitory effects against LPS-induced IL-6 release.
RAW 264.7 macrophages were pretreated with analogs at a series
of concentrations (1.1, 3.3, 10 mM) for 2 h and were subsequently
incubated with LPS (0.5 mg/ml) for 22 h. Fig. 4A shows the dose-
dependent inhibition of LPS-induced IL-6 release by these six an-
alogs, further suggesting the potential of these compounds as anti-
inflammatory agents. We next examined the inhibitory effects of
these six compounds on inflammatory gene transcription in LPS-
stimulated macrophages. It can be seen from Fig. 4B that the
To further demonstrate the SAR of these compounds in the in-
hibition of IL-6 expression, a quantitative SAR (QSAR) was calcu-
lated for 33 synthetic MACs. The materials and methods for this
computer-assistant QSAR study are described in our previous
publications [20,28]. To construct the QSAR model, multiple pa-
rameters were operated, including the quantum-chemical de-
scriptors calculated by the density functional theory (DFT) method,
molar refractivity and the coefficient of molecular partition octa-
nolewater (log P) which reflects the molecular properties and to-
pological descriptors, such as kappa index, Zagreb index and
Wiener index. Statistically significant models with three variables
were derived for anti-IL-6 activity. The scatter plot of predicted
values versus experimental values is illustrated in Fig. 3. It can be
seen that an excellent QSAR model was obtained with the
GETAWAY descriptors [MTA and RDF], which exhibited a high
adjusted squared regression coefficient (R2adj ¼ 0.80) and was able
to describe more than 82% variance in the experimental activity. R-
GETAWAY descriptors can be calculated with different atomic
weightings, including van der Waals volume, electronegativity,
atomic mass, and polarizability. In this model, the MTA means the
multiple-template approach and RDF seems to relate to the pres-
ence of electronegative atoms at the inner atmosphere of the
structure. The QSAR results indicate that the electro-negativity and
molecular polarizability may play an important role in the anti-IL-6
activity. However, no statistically significant correlation between
mRNA levels of IL-1
incubation. Pretreatment with six compounds significantly reduced
the mRNA transcription of IL-6, TNF- and in particular, IL-1 to
b, IL-6, and TNF-a were all up-regulated by LPS
a
b
different extents, indicating that they may exert anti-inflammatory
properties at the transcriptional level.
3.4. Assessment of chemical stability of representative compounds
The clinical application of curcumin has been significantly
limited by its instability and poor metabolism. MACs were designed
to improve the chemical stability of curcumin, therefore, we tested
the chemical stability of representative compounds, 8c, 14q, and
14r, in phosphate buffer (pH 7.4) using an absorption spectrum
assay. As shown in Fig. 5A, the UVevisible absorption spectrum of
curcumin displayed an intense peak with an absorption maximum
close to 425 nm, and the absorption intensity of the curcumin
spectrum decreases significantly in phosphate buffer (pH 7.4) with
time. Within 25 min of its incubation in phosphate buffer, curcumin
lost more than 45% of its original intensity, while 8c, 14q, and 14r
degraded much less than curcumin (Fig. 5BeD). These three ana-
logs showed almost complete stability in phosphate buffer within
the 25-min incubation. This result indicates that these allylated and
prenylated MACs are much more stable than curcumin in vitro.
anti-TNF-
a
activity and the molecular descriptors was obtained,
in-
which is consistent with the preliminary SAR results on TNF-
a
hibition. Taken together, the SAR and QSAR results on the IL-6-
inhibitory activities of the allylated and prenylated MACs may
provide valuable information for the further design of novel anti-
inflammatory agents.
3.5. Compound 14q attenuates LPS-induced inflammatory
mortality in C57BL/6 mice and inhibits cytokine release in MPMs
We further determined whether the representative analogs
were able to attenuate endotoxin shock through the inhibition of
inflammatory cytokine release in vivo. Compound 14q was selected
for this study. The C57BL/6 mice were injected intraperitoneally
with 20 mg/kg of LPS in the presence or absence of 14q, and their
survival rates were monitored for seven days. As shown in Fig. 6A,
all animals treated with LPS alone died within 48 h as a result of the
septic shock. In animals receiving 14q at 10 mg/kg prior to LPS
treatment, the survival rates were significantly increased (45%
survival in 14q-treated group, v.s. LPS alone group, p < 0.01). Thus,
pre-treatment with 14q can prolong the survival of LPS-induced
acute inflammatory models. The inhibitory effect of 14q on LPS-
3.3. Active compounds inhibited LPS-induced inflammatory gene
expression
The most promising compounds from those tested; 8c, 14a, 14i,
14q, 14r, and 14u, were selected for further assessment of their
induced IL-6 and TNF-a release was then tested in mouse pri-
mary peritoneal macrophages (MPMs). As shown in Figs. 6B, 14q
dose-dependently inhibited the expression of these two cytokines.
These data further suggest that 14q could attenuate sepsis via in-
hibition of inflammatory mediators in vivo.
4. Conclusion
In summary, we have synthesized a series allylated or preny-
lated mono-carbonyl analogs of curcumin and evaluated their
inhibitory activities against LPS-induced TNF-a and IL-6 release in
RAW 264.7 macrophages. The MACs used for this study showed
excellent chemical stability in vitro. The majority of derivatives
effectively inhibited the LPS-induced expression of TNF-a and IL-6,
especially IL-6. The preliminary and quantitative SAR analyses
suggested that the asymmetric MACs possessed higher anti-
inflammatory activity than symmetric analogs, and the electro-
Fig. 3. Scatter plot of predicted activity against the corresponding experimental ac-
tivity. N is the number of compounds taken into account in the regression, R2 is the
multiple correlation coefficient, R2adj is adjusted multiple correlation coefficient, s is
residual standard error and F value is related to the F-statistic analysis (Fischer test).
The numbers in parentheses are the standard deviation of the coefficients. p is the
significance of the variables in the model.

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Z. Liu et al. / European Journal of Medicinal Chemistry 74 (2014) 671e682
A
150
100
50
IL-6
*
*
**
**
**
**
**
**
**
**
**
**
**
**
**
**
0
1
3
1
( M)
10
10
10
10
10
10
1.1 3.3
1.
3.3
1.1 3.
1.1 3.3
1.
3.3
1.1 3.3
LPS
DMSO
LPS (0.5 g/ml)
TNF-
150
100
50
B
IL-1
IL-6
*
*
*
*
*
**
**
**
**
**
**
**
**
**
**
**
** **
**
0
LPS (0.5 g/ml)
LPS (0.5 g/ml)
LPS (0.5 g/ml)
Fig. 4. Six active MACs inhibited LPS-induced IL-6 release in a dose-dependent manner. (A) RAW 264.7 macrophages were plated at a density of 4.0 ꢁ 105/plate at 37 ꢀC and 5% CO2
overnight. Cells were pre-treated with compounds at indicated concentrations (1.1, 3.3, and 10
m
M) for 2 h, then treated with LPS (0.5
m
g/ml) for 22 h. IL-6 levels in the culture media
g/ml) for 6 h. The mRNA
-actin mRNA and was expressed as a ratio to DMSO. Each bar
represents mean ꢂ SD of 3e5 independent experiments. Statistical significance relative to LPS group was indicated, *p < 0.05, **p < 0.01.
were measured by ELISA and were normalized by the total protein. (B) Cells were pre-treated with compounds at 10
levels of IL-6, TNF- , and IL-1 were determined by RT-qPCR. The mRNA value for each gene was normalized to b
mM for 2 h, and treated with LPS (0.5 m
a
b
negativity and molecular polarizability of the MAC structure was
important for the inhibition of LPS-induced IL-6 expression. Com-
pound 14q was the most potent analog and was found to exhibit
anti-inflammatory ability in LPS-induced septic mouse models and
primary peritoneal macrophages. Taken together, these data pre-
sent the possibility that allylated or prenylated MACs might serve
as potential agents for the treatment of cytokine-mediated acute
inflammatory diseases.
were presented in terms of parts per million with TMS as the in-
ternal reference. The 1H and 13C NMR spectra of six active anti-
inflammatory compounds were provided in Supplementary data.
Electron-spray ionization mass spectra in positive mode (ESI-MS)
data were recorded on a Bruker Esquire 3000t spectrometer. All
general chemicals were the highest available grade.
5.1.2. Synthesis of O-allylated and O-prenylated benzaldehydes 7
A
solution of substituted 4-hydroxybenzaldehyde (6,
40.9 mmol) in dry acetone (20 mL) was added K2CO3 (9.73 g,
70.1 mmol) portionwise, followed by allyl bromide or 1-chloro-3-
methylbut-2-ene (40.9 mmol) at room temperature. After 1e5 h,
the reaction mixture was then filtered and the filtercake was
washed with acetone (20 mL). The filtrate was concentrated under
reduced pressure and the residue was dissolved in EtOAc (50 mL),
the organic layer was then dried over anhydrous MgSO4, filtered,
and concentrated under reduced pressure. The residue was further
purified by chromatography to afford target benzaldehydes 7.
5. Experimental section
5.1. Chemistry
5.1.1. General
In general, reagents, solvents, and other chemicals were used as
purchased without further purification. All reagents for synthesis
were obtained from Sigma Aldrich and Fluka. Thin-layer chroma-
tography (TLC) was performed on Kieselgel 60 F254 plates
and flash column chromatography (medium pressure liquid chro-
matography) purifications were carried out using Merck silica gel
60 (230e400 mesh ASTM) (Merck KGaA, Darmstadt, Germany).
Melting points were determined on a Fisher-Johns melting appa-
ratus and were uncorrected. 1H NMR and 13C NMR spectra were
recorded on a Bruker 600 MHz instruments. The chemical shifts
5.1.3. Synthesis of a,b-unsaturated ketones 9
To a solution of different benzaldehydes 7 (11.3 mmol) in
acetone (20 mL) was added 20% NaOH dropwise, and the mixture
was stirred at room temperature for 4 h. The resulting solution was
then quenched with water (20 mL), the acetone was removed

Z. Liu et al. / European Journal of Medicinal Chemistry 74 (2014) 671e682
677
B
A
Curcumin
8c
0min
5min
10min
15min
20min
25min
0.12
0.10
0.08
0.06
0.04
0min
5min
10min
15min
20min
25min
0.12
0.10
0.08
0.06
0.04
300
400
500
600
300
400
500
600
nm
nm
D
C
14r
0min
5min
10min
15min
20min
25min
14q
0min
5min
10min
15min
20min
25min
0.12
0.10
0.08
0.06
0.04
0.12
0.10
0.08
0.06
0.04
300
400
500
600
300
400
500
600
nm
nm
Fig. 5. UVevisible absorption spectra of curcumin (A), 8c (B), 14q (C), and 14r (D) in phosphate buffer (pH 7.4).
under reduced pressure and extracted with EtOAc. The combined
organic layers were washed with brine (100 mL) and dried over
anhydrous MgSO4, filtered, and concentrated under reduced pres-
sure. The residue was purified by flash chromatography on silica gel
chromatography on silica gel to afford target Asymmetric MACs
10aec.
5.1.4.1. (1E,4E)-1-[4-(Allyloxy)phenyl]-5-(3,4-dimethoxyphenyl)
to furnish various
yields.
a
,b
-unsaturated ketones 9 with satisfactory
penta-1,4-dien-3-one (10a). Yellow oil, 60.5% yield. 1H NMR
(600 MHz, CDCl3):
d
(ppm) 7.710 (1H, d, J ¼ 16.2 Hz, H-
a
0), 7.682
(1H, J ¼ 16.2 Hz, H-
a
), 7.563 (2H, d, J ¼ 9.0 Hz, H-20, H-60), 7.201 (1H,
5.1.4. General procedure for synthesis of asymmetric MACs 10aec
To a solution of various -unsaturated ketones 9 (0.28 mmol)
d, J ¼ 7.8 Hz, H-6), 7.147 (1H, s, H-2), 6.979 (1H, d, J ¼ 16.2 Hz, H-
b),
a,
b
6.949 (1H, J ¼ 16.2 Hz, H-
b
0), 6.935 (2H, d, J ¼ 9.0 Hz, H-30, H-50),
and different benzaldehydes (0.3 mmol) in EtOH (5 mL) at room
temperature was added dropwise a solution of 20% NaOH. The re-
action mixture was stirred at room temperature overnight and the
resulting mixture was diluted with H2O (15 mL) and extracted with
EtOAc. The combined organic layers were washed with brine
(15 mL) and dried over anhydrous MgSO4, filtered, and concen-
trated under reduced pressure. The residue was further purified by
6.895 (1H, d, J ¼ 7.8 Hz, H-5), 5.510e5.486 (1H, m, ArOCH2CH]
CH2), 4.560 (2H, d, J ¼ 6.6 Hz, ArOCH2CH]CH2), 3.945 (3H, s,
ArOCH3), 3.931 (3H, s, ArOCH3). ESI-MS m/z: 350.1 (M)þ.
5.1.4.2. (1E,4E)-1-(3,4-Dimethoxyphenyl)-5-{4-[(3-methylbut-2-en-
1-yl)oxy]phenyl}penta-1,4- dien-3-one (10b). Yellow oil, 61.7% yield.
1H NMR (600 MHz, CDCl3):
d
(ppm) 7.708 (1H, d, J ¼ 16.2 Hz, H-
a
0),
B
A
150
100
IL-6
TNF-α
80
100
60
**
**
**
**
**
50
40
Vehicle
LPS + 14q
LPS
20
0
0
(
10
M)
.1
.1
.3
3
10
1
3.3
1
SO
LPS
LPS
0
50
100
150
DMSO
DM
LPS (0.5 g/ml)
LPS (0.5 g/ml)
Hours
Fig. 6. 14q attenuates septic death in mice. (A) Mice were treated with 10 mg/kg 14q (i.v.) 15 min before injection of 20 mg/kg of LPS (i.p.). Survival was recorded for 7 days after the
LPS injection at the interval of 12 h n ¼ 9 animals in each group (**p < 0.01 vs LPS group). (B) MPMs were harvested and plated at a density of 4.0 ꢁ 105/plate at 37 ꢀC and 5% CO2
overnight. Cells were pre-treated with 14q for 2 h, and then treated with LPS (0.5 mg/ml) for 22 h. IL-6 and TNF-a levels in the culture media were measured by ELISA and were
normalized by the total protein. Statistical significance for 3 independent experiments relative to LPS group was indicated, *p < 0.05, **p < 0.01.

678
Z. Liu et al. / European Journal of Medicinal Chemistry 74 (2014) 671e682
7.680 (1H, J ¼ 16.2 Hz, H-
a
), 7.565 (2H, d, J ¼ 9.0 Hz, H-20, H-60),
flash chromatography on silica gel to furnish symmetric MACs 8ae
k with the yields of 47e85%.
7.202 (1H, d, J ¼ 7.8 Hz, H-6), 7.143 (1H, s, H-2), 6.977 (1H, d,
J ¼ 16.2 Hz, H-
b
), 6.951 (1H, J ¼ 16.2 Hz, H-
b
0), 6.937 (2H, d,
J ¼ 9.0 Hz, H-30, H-50), 6.897 (1H, d, J ¼ 7.8 Hz, H-5), 5.506e5.484
[1H, m, ArOCH2CH]C(CH3)2], 4.558 [2H, d, J ¼ 6.6 Hz, ArOCH2CH]
C(CH3)2], 3.947 (3H, s, ArOCH3), 3.931 (3H, s, ArOCH3), 1.810 [3H, s,
ArOCH2CH]C(CH3)2], 1.760 [3H, s, ArOCH2CH]C(CH3)2]. ESI-MS
m/z: 378.9 (M)þ.
5.1.8.1. (1E,4E)-1,5-bis[4-(Allyloxy)phenyl]penta-1,4-dien-3-one (8a).
Yellow powder, 81.7% yield, mp: 119.2e121.5 ꢀC. 1H NMR (600 MHz,
CDCl3):
d
(ppm) 7.691 (2H, d, J ¼ 15.6 Hz, H-
b, H-b
0), 7.560 (4H, d,
J ¼ 9.0 Hz, H-2, H-20, H-6, H-60), 6.957 (4H, d, J ¼ 9.0 Hz, H-3, H5,
H30, H50), 6.951 (2H, d, J ¼ 15.6 Hz, H-
a, H-a
0), 6.091e6.027 (2H, m,
A,B-ArOCH2CHCH2), 5.432 (2H, d, J ¼ 17.4 Hz, A,B-ArOCH2CHCH2),
5.318 (2H, d, J ¼ 10.2 Hz, A,B-ArOCH2CHCH2), 4.566 (4H, d,
J ¼ 5.4 Hz, A,B-ArOCH2CHCH2). ESI-MS m/z: 346.4 (M)þ.
5.1.4.3. (1E,4E)-1-(4-(Allyloxy)-2-hydroxyphenyl)-5-(4-
fluorophenyl)penta-1,4-dien-3-one (10c). Yellow oil, 54.6% yield. 1H
NMR (600 MHz, CDCl3):
d
(ppm) 8.091 (1H, d, J ¼ 15.6 Hz, H-
a
),
7.972 (1H, d, J ¼ 8.4 Hz, H-6), 7.821 (1H, d, J ¼ 15.6 Hz, H-
a
0), 7.720
5.1.8.2. (1E,4E)-1,5-bis{4-[(3-Methylbut-2-en-1-yl)oxy]phenyl}
(2H, d, J ¼ 8.4 Hz, H-20, H-60), 7.19 (1H, d, J ¼ 8.4 Hz, H-30, H-50),
penta-1,4-dien-3-one (8b). Yellow oil, 80.5% yield. 1H NMR
7.031 (1H, d, J ¼ 15.6 Hz, H-
b
0), 6.862 (1H, d, J ¼ 15.6 Hz, H-
b
), 6.545
(600 MHz, CDCl3):
d
(ppm) 7.694 (2H, d, J ¼ 16.2 Hz, H-
b, H-b
0),
(1H, d, J ¼ 8.4 Hz, H-5), 6.531 (1H, s, H-3), 6.081e5.936 (1H, m,
ArCH2CH]CH2), 5.405e5.381 (2H, m, A-ArCH2CH]CH2), 5.323
(1H, s, OH), 3.311 (2H, s, ArCH2CH]CH2). ESI-MS m/z: 324.0 (M)þ,
347.0 (M þ Na)þ.
7.565e7.461 (4H, m, H-2, H-20, H-6, H-60), 6.963e6.914 (4H, m, H-3,
H5, H30, H50), 6.606 (2H, d, J ¼ 16.2 Hz, H-
a, H-a
0), 5.06e5.474 [2H,
m, A,B-ArOCH2CH]C(CH3)2], 4.552 [4H, m, A,B-ArOCH2CH]
C(CH3)2], 1.808 [6H, s, A-ArOCH2CH]C(CH3)2], 1.759 [6H, s, B-
ArOCH2CH]C(CH3)2]. ESI-MS m/z: 402.5 (M)þ.
5.1.5. Synthesis of 3-allyl-4-hydroxybenzaldehyde 11
The solution of 4-[(3-methylbut-2-en-1-yl)oxy]-benzaldehyde
(4.0 g, 24.6 mmol) in N,N-diethylaniline (20 mL) was heated at
190 ꢀC for 20 h. After cooling to room temperature, the mixture was
diluted with H2O (20 mL) and 6 N-HCl (10 mL) was added slowly
then extracted with EtOAC. The combined organic layers were
washed with brine (50 mL) and dried over anhydrous MgSO4,
filtered, and concentrated under reduced pressure. The residue was
further purified by chromatography on silica gel to give 3-allyl-4-
hydroxybenzaldehyde (11, 2.50 g) as white solid with 65% yield.
5.1.8.3. (1E,4E)-1,5-bis(3-Allyl-4-hydroxyphenyl)penta-1,4-dien-3-
one (8c). Yellow oil, 60.3% yield. 1H NMR (600 MHz, CDCl3):
d (ppm)
7.680 (2H, d, J ¼ 16.2 Hz, H-
b
, H-
b
0), 7.394 (2H, d, J ¼ 6.6 Hz, H-2, H-
20), 7.400 (2H, s, H-6, H-60), 6.946 (2H, d, J ¼ 15.6 Hz, H-
a, H-a
0),
6.850 (2H, d, J ¼ 9 Hz, H-3, H-30), 6.065e5.999 (2H, m, A,B-
ArCH2CH]CH2), 5.903 (2H, s, -OH), 5.198e5.170 (4H, m, A,B-
ArCH2CH]CH2), 3.442 (2H, d,
J
¼
6.6 Hz, A,B-ArCH2CH]
(ppm) 156.6, 143.1,
CH2) (Fig. S1). 13C NMR (600 MHz, CDCl3):
d
135.9, 131.0, 128.8, 126.1, 123.5, 117.2, 116.5, 35.1 (Fig. S2). ESI-MS m/
z: 346.4 (M)þ.
5.1.6. Synthesis of 3-allyl-4-[(tetrahydro-2H-pyran-2-yl)oxy]
benzaldehyde 12a
5.1.8.4. (1E,4E)-1,5-bis{3-Methoxy-4-[(3-methylbut-2-en-1-yl)oxy]
phenyl}penta-1,4-dien-3-one (8d). Brick red powder, 58.6% yield,
mp: 93.9e95.2 ꢀC. 1H NMR (600 MHz, CDCl3): 7.71 (2H, d,
To a solution of 5.0 mL (4.63 g, 55.3 mol) of dihydropyran in
30 mL CH2Cl2 was added with stirring 7.48 g (46.2 mmol) of 3-allyl-
4-hydroxybenzaldehyde 11 and 50 mg (0.198 mmol) of p-TsOH. The
resulting solution was stirred at room temperature for 5 h, and then
quenched with 50 mL H2O and extracted with CH2Cl2 (100 mL). The
combined organic layers were washed with brine, dried over
anhydrous MgSO4, filtered, and concentrated in vacuo. The residue
was purified by flash chromatography on silica gel to give 3-allyl-4-
[(tetrahydro-2H-pyran-2-yl)oxy]benzaldehyde as a colorless solid
with 95% yield.
J ¼ 15.6 Hz, H-
a
, H-
a
0), 7.27 (2H, s, H-2, H-20), 7.19 (2H, d, J ¼ 8.4 Hz,
H-6, H-60), 7.02 (2H, d, J ¼ 15.6 Hz, H-
b
, H-b
0), 6.94 (2H, d, J ¼ 8.4 Hz,
H-5, H-50), 5.52e5.36 [2H, m, A,B-ArOCH2CH]C(CH3)2], 4.65 [4H,
d, J ¼ 6.6 Hz, A,B-ArOCH2CH]C(CH3)2], 3.91 (6H, s, A,B-OCH3), 1.78
[12H, d, A,B-ArOCH2CH]C(CH3)2]. ESI-MS m/z: 463.1 (M þ H)þ.
5.1.8.5. (2E,5E)-2,5-bis{3-Allyl-4-[(tetrahydro-2H-pyran-2-yl)oxy]
benzylidene}cyclopenta- none (8e). Yellow powder, 50.3% yield, mp:
108.7e110.8 ꢀC. 1H NMR (600 MHz, CDCl3):
d (ppm) 7.531 (2H, s, H-
5.1.7. Synthesis of 3-allyl-4-methoxybenzaldehyde 12b
a
, H-
a
0), 7.401 (2H, d, J ¼ 17.4 Hz, H-2, H-20), 7.170 (2H, s, H-6, H-60),
A
solution of 3-allyl-4-hydroxybenzaldehyde 11 (6.10 g,
6.890 (2H, d, J ¼ 17.4 Hz, H-3, H-30), 6.071e5.976 (2H, m, A,B-
ArCH2CH]CH2), 5.720 (2H, t, J ¼ 6.6 Hz A,B-THP), 5.203e5.084 (4H,
m, A,B-ArCH2CH]CH2), 4.036e3.513 (4H, m, A,B-THP), 3.454 (4H,
d, J ¼ 6.6 Hz, A,B-ArCH2CH]CH2), 3.069 (4H, s, Cyclopentanonee
CH2eCH2e), 1.904e1.534 (12H, m, A,B-THP). ESI-MS m/z: 541.1
(M þ H)þ.
37.6 mmol) in dry acetone (70 mL) was added K2CO3 (9.73 g,
70.1 mmol) portionwise, followed by MeI (1.8 mL, 38.0 mmol) at
room temperature. After 3 h, the reaction mixture was then filtered
and the filtercake was washed with acetone (100 mL). The filtrate
was concentrated under reduced pressure and the residue was
dissolved in EtOAc (50 mL), washed with water and brine. The
organic layer was dried over MgSO4, and filtered and the filtrate
was concentrated in vacuo to give 3-allyl-4-methoxybenzaldehyde
12b as a white crystalline solid.
5.1.8.6. (2E,5E)-2,5-bis(3-Allyl-4-hydroxybenzylidene)cyclo-
pentanone (8f). Yellow powder, 60.5% yield, mp: 109.3e111.2 ꢀC. 1H
NMR (600 MHz, CDCl3):
d (ppm) 7.532 (2H, s, H-a, H-a
0), 7.417 (2H,
d, J ¼ 8.4, H-2, H-20), 7.368 (2H, s, H-6, H-60), 6.883 (2H, d, J ¼ 8.4, H-
3, H-30), 6.070e6.014 (2H, m, A,B-ArCH2CH]CH2), 5.211 (2H, s, e
OH), 5.192e5.182 (4H, m, A,B-ArCH2CH]CH2), 3.448 (4H, d,
J ¼ 6.0 Hz, A,B-ArCH2CH]CH2), 3.072 (4H, s, Cyclopentanonee
CH2eCH2e). ESI-MS m/z: 372.4 (M)þ.
5.1.8. General procedure for synthesis of symmetric MACs 8aek
To a solution of different benzaldehydes 7 or 12a (0.15 mmol) in
EtOH (10 mL) was added saturated NaOH dropwise, and the
mixture was stirred at room temperature for 3e5 h. The resulting
solution was then quenched with water (20 mL), the EtOH was then
removed under reduced pressure and the resulting solution was
extracted with EtOAc. The combined organic layers were washed
with brine (100 mL) and dried over anhydrous MgSO4, filtered, and
concentrated under reduced pressure. The residue was purified by
5.1.8.7. (2E,5E)-2,5-bis{3-Methoxy-4-[(3-methylbut-2-en-1-yl)oxy]
benzylidenecyclopenta-none (8g). Orange powder, 98.9% yield, mp:
148.2e148.6 ꢀC. 1H NMR (600 MHz, CDCl3):
d (ppm) 7.54 (2H, s, H-a,
H-a
0), 7.27 (2H, s, H-2, H-20), 7.19 (2H, d, J ¼ 8.4 Hz, H-6, H-60), 6.94

Z. Liu et al. / European Journal of Medicinal Chemistry 74 (2014) 671e682
679
(2H, d, J ¼ 8.4 Hz, H-5, H-50), 5.478e5.065 [2H, m, A,B-ArOCH2CH]
C(CH3)2], 4.65 [4H, d, J ¼ 6.6 Hz, A,B-ArOCH2CH]C(CH3)2], 3.91
(6H, s, A,B-OCH3), 3.11 (4H, s, CyclopentanoneeCH2eCH2e), 1.78
[12H, d, A,B-ArOCH2CH]C(CH3)2]. ESI-MS m/z: 489.1 (M þ H)þ.
(1H, s, H-6), 6.608 (1H, d, J ¼ 16.2 Hz, H-4), 6.036e6.969 (1H, m,
ArCH2CH]CH2), 5.102e5.037 (2H, m, ArCH2CH]CH2), 3.921 (9H,
s, OCH3), 3.402 (2H, d, J ¼ 6.6 Hz, ArCH2CH]CH2). ESI-MS m/z:
364.9 (M)þ.
5.1.8.8. (2E,5E)-2,5-bis{2-Hydroxy-4-[(3-methylbut-2-en-1-yl)oxy]
benzylidene}cyclopenta-none (8h). Yellow powder, 75.6% yield, mp:
5.1.10.4. (1E,4E)-1-(3-Allyl-4-methoxyphenyl)-5-(4-(trifluoromethyl)
phenyl)penta-1,4-dien-3-one (14d). Yellow oil, 65.7% yield. 1H NMR
89.2e91.3 ꢀC. 1H NMR (600 MHz, CDCl3):
d
(ppm) 7.957 (2H, s, H-
a
,
(600 MHz, CDCl3):
d
(ppm) 7.719 (1H, d, J ¼ 15.6 Hz, H-
a
0), 7.719 (1H,
H-a
0), 7.481 (2H, d, J ¼ 8.4 Hz, H-2, H-20), 6.532 (2H, d, J ¼ 17.4 Hz, H-
d, J ¼ 15.6 Hz, H-
a
), 7.720e6.658 (4H, m, H-20, H-30, H-50, H-60),
3, H-30), 6.505 (2H, s, H-5, H50), 5.525e5.488 [(4H, m, A,B-
ArOCH2CH]C(CH3)2, A,B-OH], 4.545 [4H, d, J ¼ 6.6 Hz, A,B-
ArOCH2CH]C(CH3)2], 2.982 (4H, s, CyclopentanoneeCH2eCH2e),
1.810 [6H, s, A-ArOCH2CH]C(CH3)2], 1.786 [6H, s, B-ArOCH2CH]
C(CH3)2]. ESI-MS m/z: 540.6 (M)þ.
7.479 (1H, d, J ¼ 8.4 Hz, H-2), 7.453 (1H, s, H-6), 7.154 (1H, d,
J ¼ 16.2 Hz, H-
b
0), 6.943 (1H, d, J ¼ 16.2 Hz, H-
b), 6.891 (1H, d,
J ¼ 8.4 Hz, H-3), 6.035e5.955 (1H, m, ArCH2CH]CH2), 5.108e5.081
(2H, m, ArCH2CH]CH2), 3.687 (3H, s, OCH3), 3.405 (2H, d,
J ¼ 6.6 Hz, ArCH2CH]CH2). ESI-MS m/z: 373.3 (M þ H)þ.
5.1.8.9. (2E,6E)-2,6-bis{4-[(3-Methylbut-2-en-1-yl)oxy]benzylidene}
5.1.10.5. (1E,4E)-1-(3-Allyl-4-methoxyphenyl)-5-(4-(dimethylamino)
cyclohexanone (8i). Yellow powder, 75.6% yield, mp: 111.7e
phenyl)penta-1,4-dien-3-one (14e). Yellow oil, 70.3% yield. 1H NMR
113.7 ꢀC. 1H NMR (600 MHz, CDCl3):
d
(ppm) 7.759 (2H, s, H-
b, H-
b
0),
(600 MHz, CDCl3):
d
(ppm) 7.702 (1H, d, J ¼ 15.6 Hz, H-
a
0), 7.664
7.443 (4H, d, J ¼ 9.0 Hz, H-2, H-20, H-6, H-60), 6.941 (4H, d, J ¼ 8.4 Hz,
H-3, H-5, H-30, H-50), 5.500 [2H, t, J ¼ 6.6 Hz, A,B-ArOCH2CH]
C(CH3)2], 4.550 [4H, d, J ¼ 6.6 Hz, A,B-ArOCH2CH]C(CH3)2], 2.923
(4H, t, J ¼ 5.4 Hz, CyclohexanoneeCH2CHCH2e), 1.808 [6H, s, A-
ArOCH2CH]C(CH3)2], 1.758 [6H, s, B-ArOCH2CH]C(CH3)2], 1.808e
1.758 (2H, m, CyclohexanoneeCH2CHCH2e). ESI-MS m/z: 442.5
(M)þ.
(1H, d, J ¼ 16.2 Hz, H-
a
), 7.520 (2H, d, J ¼ 8.4 Hz, H-20, H-60), 7.453
(1H, d, J ¼ 8.4 Hz, H-2), 7.439 (1H, s, H-6), 6.954 (1H, d, J ¼ 16.2 Hz,
H-b
), 6.888 (1H, d, J ¼ 15.6 Hz, H-
b
0), 6.871 (1H, d, J ¼ 8.4 Hz, H-3),
6.694 (2H, d, J ¼ 8.4 Hz, H-30, H-50), 6.037e5.970 (1H, m, ArCH2CH]
CH2), 5.104e5.078 (2H, m, ArCH2CH]CH2), 3.873 (3H, s, OCH3),
3.401 (2H, d, J ¼ 6.6 Hz, ArCH2CH]CH2), 3.041 [6H, s, N(CH3)2]. ESI-
MS m/z: 347.8 (M)þ.
5.1.9. Synthesis of
a
,
b
-unsaturated ketones 13a and 13b
5.1.10.6. (1E,4E)-1-(3-Allyl-4-methoxyphenyl)-5-[4-(tert-butoxy)
These compounds were synthesized using the same method
described for synthesis of compounds 9 with the yields of 72.5%
and 76%.
phenyl]penta-1,4-dien-3-one (14f). Yellow oil, 65.4% yield. 1H NMR
(600 MHz, CDCl3):
d
(ppm) 7.706 (1H, d, J ¼ 16.2 Hz, H-
a
0), 7.680
(1H, d, J ¼ 15.6 Hz, H-
a
), 7.535 (2H, d, J ¼ 8.4 Hz, H-20, H-60), 7.463
(1H, d, J ¼ 16.2 Hz, H-2), 7.364 (1H, s, H-6), 7.017 (1H, d, J ¼ 8.4 Hz,
5.1.10. General procedure for synthesis of MACs 14aeu
These compounds were synthesized using the same method
described for synthesis of asymmetric MACs 10aec.
H-30, H-50), 6.968 (1H, d, J ¼ 16.2 Hz, H-
b
0), 6.941 (1H, d, J ¼ 15.6 Hz,
H-b), 6.024e5.945 (1H, m, ArCH2CH]CH2), 5.106e5.091 (2H, m,
ArCH2CH]CH2), 3.869 (3H, s, OCH3), 3.410e3.379 (2H, m,
ArCH2CH]CH2), 1.399 [9H, s, ArOC(CH3)3]. ESI-MS m/z: 376.9 (M)þ,
398.9 (M þ Na)þ.
5.1.10.1. (1E, 4E)-1-(3-Allyl-4-hydroxyphenyl)-5-(3, 4-
dimethoxyphenyl)penta-1,4-dien-3-one (14a). Yellow oil, 48.3%
yield. 1H NMR (600 MHz, CDCl3):
d
(ppm) 7.682 (1H, J ¼ 15.6 Hz,
5.1.10.7. (1E,4E)-1-(3-Allyl-4-methoxyphenyl)-5-(3-bromophenyl)
H-
a
0), 7.667 (1H, J ¼ 15.6 Hz, H-
a
), 7.510 (2H, d, J ¼ 7.2 Hz, H-2, H-
penta-1,4-dien-3-one (14g). Yellow oil, 64.7% yield. 1H NMR
3), 7.456 (1H, d, J ¼ 8.4 Hz, H-60), 7.438 (1H, s, H-20), 6.951 (2H, d,
(600 MHz, CDCl3): d
(ppm) 7.771 (1H, s, H-60), 7.705 (1H, d,
J ¼ 15.6 Hz, H-
b
, H-
b
0), 6.894 (1H, s, H-6), 6.875 (1H, d, J ¼ 8.4 Hz,
J ¼ 16.2 Hz, H-
a
0), 7.630 (1H, d, J ¼ 16.2 Hz, H-
a), 7.515 (2H, d,
H-50), 6.036e5.973 (1H, m, ArCH2CH]CH2), 5.092 (2H, d,
J ¼ 13.8 Hz, ArCH2CH]CH2), 3.876 (3H, s, OCH3), 3.404 (2H, d,
J ¼ 13.8 Hz, ArCH2CH]CH2), 3.311 (3H, s, OCH3) (Fig. S3). 13C
J ¼ 7.8 Hz, H-20, H-40), 7.474 (1H, dd, J ¼ 8.4 Hz, H-2), 7.448 (1H, s, H-
6), 7.284 (1H, t, J ¼ 7.8 Hz, H-30), 7.085 (1H, d, J ¼ 16.2 Hz, H-
b
0),
6.923 (1H, d, J ¼ 16.2 Hz, H- ), 6.888 (1H, d, J ¼ 8.4 Hz, H-3), 6.035e
b
NMR (600 MHz, CDCl3):
d
(ppm) 188.8, 156.6, 149.0, 143.1, 142.8,
5.964 (1H, m, ArCH2CH]CH2), 5.110e5.081 (2H, m, ArCH2CH]
CH2), 3.885 (3H, s, OCH3), 3.405 (2H, d, J ¼ 6.6 Hz, ArCH2CH]CH2).
ESI-MS m/z: 382.7 (M)þ.
135.7, 130.5, 128.3, 127.6, 127.1, 126.2, 123.5, 122.8, 122.7,
116.4, 115.9, 110.9, 109.6, 55.75, 55.70, 34.4 (Fig. S4). ESI-MS m/z:
350.1 (M)þ.
5.1.10.8. (1E,4E)-1-(3-Allyl-4-methoxyphenyl)-5-(4-bromophenyl)
5.1.10.2. (1E,4E)-1-(3-Allyl-4-hydroxyphenyl)-5-(4-nitrophenyl)
penta-1,4-dien-3-one (14h). Yellow oil, 70.6% yield. 1H NMR
penta-1,4-dien-3-one (14b). Yellow oil, 58.6% yield. 1H NMR
(600 MHz, CDCl3):
d
(ppm) 7.699 (1H, d, J ¼ 16.2 Hz, H-
a
0), 7.648 (1H,
(600 MHz, CDCl3):
d
(ppm) 10.140 (1H, s, OH), 8.289 (2H, d,
d, J ¼ 16.2 Hz, H-
a
), 7.542 (2H, d, J ¼ 8.4 Hz, H-20, H-60), 7.475 (1H, d,
J ¼ 8.4 Hz, H-30, H-50), 8.065 (1H, d, J ¼ 8.4 Hz, H-20, H-60), 7.794 (1H,
J ¼ 8.4 Hz, H-30, H-50), 7.468 (2H, d, J ¼ 8.4 Hz, H-2), (7.443 (1H, s, H-
d, J ¼ 15.6 Hz, H-
a
0), 7.664 (1H, d, J ¼ 15.6 Hz, H-
a
), 7.631 (1H, d,
6), 7.076 (1H, d, J ¼ 16.2 Hz, H-
b
), 6.927 (1H, d, J ¼ 16.2 Hz, H-
b
0),
J ¼ 8.4 Hz, H-2), 7.602 (1H, d, J ¼ 15.6 Hz, H-
b
0), 7.515 (1H, s, H-6),
6.883 (1H, d, J ¼ 8.4 Hz, H-3), 6.021e5.965 (1H, m, ArCH2CH]CH2),
5.105e5.080 (2H, m, ArCH2CH]CH2), 3.882 (3H, s, OCH3), 3.404
(2H, d, J ¼ 6.6 Hz, ArCH2CH]CH2). ESI-MS m/z: 384.8 (M þ H)þ.
7.065 (1H, d, J ¼ 15.6 Hz, H- ), 6.973 (1H, d, J ¼ 8.4 Hz, H-3), 6.031e
b
5.939 (1H, m, ArCH2CH]CH2), 5.090e5.040 (2H, m, ArCH2CH]
CH2), 3.321 (2H, s, ArCH2CH]CH2). ESI-MS m/z: 335.9 (M)þ.
5.1.10.9. (1E,4E)-1-(3-Allyl-4-methoxyphenyl)-5-(4-morpholino
5.1.10.3. (1E,4E)-1-(3-Allyl-4-methoxyphenyl)-5-(3,4-dimethoxy
phenyl)penta-1,4-dien-3-one (14i). Yellow powder, 72.8% yield, mp:
phenyl)penta-1,4-dien-3-one (14c). Yellow oil, 63.5% yield. 1H NMR
141.3e143.5 ꢀC. 1H NMR (600 MHz, CDCl3):
d (ppm) 7.684 (1H, d,
(600 MHz, CDCl3):
d
(ppm) 7.695 (1H, d, J ¼ 15.6 Hz, H-
a
0), 7.683
J ¼ 15.6 Hz, H-
a
0), 7.675 (1H, d, J ¼ 16.2 Hz, H-
a), 7.543 (2H, d,
(1H, d, J ¼ 15.6 Hz, H-
a
), 7.464 (1H, d, J ¼ 16.2 Hz, H-3), 7.205 (1H, d,
J ¼ 5.4 Hz, H-20, H-60), 7.455 (1H, d, J ¼ 8.4 Hz, H-2), 7.260 (1H, s, H-
J ¼ 8.4 Hz, H-60), 7.127 (1H, d, J ¼ 8.4 Hz, H-50), 7.076 (1H, s, H-20),
6), 6.945 (2H, d, J ¼ 15.6, H-
b, H-b
0), 6.899e6.866 (3H, m, H-3, H-30,
6.964 (1H, d, J ¼ 15.6 Hz, H-
b
0), 6.695 (1H, d, J ¼ 15.6 Hz, H-
b), 6.889
H-50), 6.035e5.968 (1H, m, ArCH2CH]CH2), 5.090 (2H, d,

680
Z. Liu et al. / European Journal of Medicinal Chemistry 74 (2014) 671e682
J ¼ 12.0 Hz, ArCH2CH]CH2), 3.874 (3H, s, OCH3), 3.864e3.856 [4H,
m, AreN(CH2eCH2)2O], 3.402 (2H, d, J ¼ 6.6 Hz, ArCH2CH]CH2),
3.272e3.256 [4H, m, AreN(CH2eCH2)2O] (Fig. S5). 13C NMR
yield, mp: 110.3e112.5 ꢀC. 1H NMR (600 MHz, CDCl3):
d
(ppm) 8.086
(1H, d, J ¼ 15.6 Hz, H-
a
0), 7.710 (d, J ¼ 16.2 Hz, H-
a), 7.609 (1H, d,
J ¼ 7.2 Hz, H-40), 7.495 (1H, d, J ¼ 9.0 Hz, H-2), 7.467e7.485 (1H, m,
H-50), 7.443 (1H, s, H-6), 7.243 (1H, d, J ¼ 7.8 Hz, H-60), 7.023 (1H, d,
(600 MHz, CDCl3):
d (ppm) 188.6, 159.0, 152.2, 142.5, 142.4, 136.0,
129.7, 129.2, 129.0, 128.4, 127.2, 123.3, 122.3, 115.7, 114.5, 110.2, 66.3,
J ¼ 13.8 Hz, H-
b
), 6.960 (1H, d, J ¼ 13.8 Hz, H-
b
0), 6.888 (1H, d,
55.3, 47.8, 33.8 (Fig. S6). ESI-MS m/z: 390.2 (M)þ.
J ¼ 8.4 Hz, H-3), 5.963e6.030 (1H, m, ArCH2CH]CH2), 5.079e5.105
(2H, m, ArCH2CH]CH2), 3.884 (3H, s, OCH3), 3.403 (2H, d,
J ¼ 6.6 Hz, ArCH2CH]CH2). ESI-MS m/z: 372.8 (M)þ, 394.7
(M þ Na)þ.
5.1.10.10. (1E,4E)-1-(3-Allyl-4-methoxyphenyl)-5-[4-(piperidin-1-yl)
phenyl]penta-1,4-dien-3- one (14j). Yellow oil, 67.7% yield. 1H NMR
(600 MHz, CDCl3):
J ¼ 15.6 Hz, H-
d
(ppm) 7.681 (1H, d, J ¼ 15.6 Hz, H-
a
0), 7.666 (d,
a
), 7.507 (2H, d, J ¼ 8.4 Hz, H20, H-60), 7.452 (1H, d,
5.1.10.16. (1E,4E)-1-(3-Allyl-4-methoxyphenyl)-5-(2-chlorophenyl)
J ¼ 8.4 Hz, H-2), 7.437 (1H, s, H-6), 6.948 (1H, d, J ¼ 15.6 Hz, H-
b
),
penta-1,4-dien-3- one (14p). Yellow oil, 67.7% yield. 1H NMR
6.927 (1H, d, J ¼ 9.6 Hz, H-3), 6906 (1H, J ¼ 15.6 Hz, H-
b
0), 6.885 (2H,
(600 MHz, CDCl3):
d
(ppm) 8.103 (1H, d, J ¼ 16.2 Hz, H-
a
0), 7.709
d, J ¼ 9.0 Hz, H-30, H-50), 5.968e6.036 (1H, m, ArCH2CH]CH2),
5.079e5.103 (2H, m, ArCH2CH]CH2), 3.873 (3H, s, OCH3), 3.400
(2H, d, J ¼ 6.6 Hz, ArCH2CH]CH2), 3.299e3.317 [4H, m, AreN(CH2e
CH2)2CH2], 1.683e1.691 [4H, m, AreN(CH2eCH2)2CH2], 1.243e1.255
[2H, m, AreN(CH2eCH2)2CH2]. ESI-MS m/z: 388.1 (M þ H)þ.
(1H, d, J ¼ 15.6 Hz, H-
a
), 7.497e7.444 (2H, m, H-2, H-30), 7.396e
7.357 (1H, m, H-60), 7.326e7.301 (1H, m, H-50), 7.241e7.192 (1H, m,
H-40), 7.171 (1H, s, H-6), 7.049 (1H, d, J ¼ 15.6 Hz, H-
b
0), 6.979 (1H, d,
J ¼ 15.6 Hz, H- ), 6.886 (1H, d, J ¼ 8.4 Hz, H-3), 6.033e5.897 (1H, m,
b
ArCH2CH]CH2), 5.106e5.031 (2H, m, ArCH2CH]CH2), 3.881 (3H, s,
OCH3), 3.410e3.323 (2H, m, ArCH2CH]CH2). ESI-MS m/z: 339.8
(M þ H)þ.
5.1.10.11. (1E,4E)-1-(3-Allyl-4-methoxyphenyl)-5-[4-(allyloxy)
phenyl]penta-1,4-dien-3-one (14k). Yellow oil, 62.7% yield. 1H NMR
(600 MHz, CDCl3):
d
(ppm) 7.693 (1H, d, J ¼ 16.2 Hz, H-
a
0), 7.683
5.1.10.17. (1E,4E)-1-(3-Allyl-4-methoxyphenyl)-5-(2,5-
(1H, d, J ¼ 15.6 Hz, H-
a
), 7.564 (2H, d, J ¼ 9.0 Hz, H-20, H-60), 7.459
dibromophenyl)penta-1,4-dien-3- one (14q). Yellow powder, 67.1%
(1H, d, J ¼ 8.4 Hz, H-2), 7.441 (1H, s, H-6), 6.972 (1H, d, J ¼ 16.2 Hz,
yield, mp: 83.4e85.2 ꢀC. 1H NMR (600 MHz, CDCl3):
d (ppm) 7.946
H-
b
0), 6.943 (1H, d, J ¼ 9 Hz, H-30, H-50), 6.939 (1H, d, J ¼ 15.6 Hz, H-
(1H, d, J ¼ 16.2 Hz, H-
a
0), 7.814 (1H, d, J ¼ 2.4 Hz, H-30), 7.715 (1H, d,
b), 6.879 (1H, d, J ¼ 8.4 Hz, H-3), 5.969e6.094 (2H, m, ArCH2CH]
J ¼ 15.6 Hz, H-
a
), 7.485 (1H, s, H-60), 7.472 (1H, d, J ¼ 2.4 Hz, H-40),
CH2, ArOCH2CH]CH2), 5.436 (1H, d, J ¼ 15.6 Hz, ArOCH2CH]CH2),
5.320 (1H, d, J ¼ 10.2 Hz, ArOCH2CH]CH2), 5.080e5.104 (2H, m,
ArCH2CH]CH2), 4.589 (2H, d, J ¼ 5.4 Hz, ArOCH2CH]CH2), 3.878
(3H, s, OCH3), 3.404 (2H, d, J ¼ 6.6 Hz, ArCH2CH]CH2). ESI-MS m/z:
361.1 (M þ H)þ.
7.449 (1H, s, H-6), 7.353 (1H, d, J ¼ 8.4 Hz, H-2), 7.006 (1H, d,
J ¼ 15.6 Hz, H-
b
), 6.937 (1H, d, J ¼ 16.2 Hz, H-
b
0), 6.890 (1H, d,
J ¼ 8.4 Hz, H-3), 6.032e5.957 (1H, m, ArCH2CH]CH2), 5.110e5.081
(2H, m, ArCH2CH]CH2), 3.885 (3H, s, OCH3), 3.405 (2H, d,
J ¼ 6.6 Hz, ArCH2CH]CH2) (Fig. S7). 13C NMR (600 MHz, CDCl3):
d
(ppm) 187.9, 159.4, 144.0, 139.2, 136.8, 135.9, 134.5, 133.5, 130.2,
5.1.10.12. (1E,4E)-1-(3-allyl-4-methoxyphenyl)-5-(naphthalen-2-yl)
129.5, 129.2, 128.7, 128.6, 126.7, 123.9, 122.8, 121.3, 115.8, 110.2, 55.3,
penta-1,4-dien-3-one (14l). Yellow oil, 71.6% yield. 1H NMR
33.8 (Fig. S8). ESI-MS m/z: 462.6 (M)þ.
(600 MHz, CDCl3):
10, H-
0, H-
), 7.773 (1H, d, J ¼ 8.4 Hz, H-2), 7.738 (1H, d, J ¼ 15.6 Hz,
H-
60), 7.471 (1H, s, H-6), 7.211 (1H, d, H-
d
(ppm) 8.023e7.839 (6H, m, H-20, H-50, H-7, H-
a
a
5.1.10.18. (1E,4E)-1-(3-Allyl-4-methoxyphenyl)-5-(2-bromo-5-
b
), 7.526 (2H, t, J ¼ 4.8 Hz, H-8, H-9), 7.488 (1H, d, J ¼ 7.8 Hz, H-
fluorophenyl)penta-1,4-dien-3- one (14r). Yellow powder, 65.1%
b
0), 6.894 (1H, d, J ¼ 8.4 Hz, H-
yield, mp: 47.9e49.1 ꢀC. 1H NMR (600 MHz, CDCl3):
d (ppm) 7.975
3), 6.049e5.981 (1H, m, ArCH2CH]CH2), 5.120e5.098 (2H, m,
ArCH2CH]CH2), 3.888 (3H, s, OCH3), 3.417 (2H, d, J ¼ 6.6 Hz,
ArCH2CH]CH2). ESI-MS m/z: 355.3 (M þ H)þ.
(1H, d, J ¼ 15.6 Hz, H-
a
0), 7.863 (1H, d, J ¼ 1.8 Hz, H-30), 7.807 (1H, d,
J ¼ 15.6 Hz, H- ), 7.751 (1H, d, J ¼ 8.4 Hz, H-2), 7.693 (1H, d,
a
J ¼ 1.8 Hz, H-40), 7.503 (1H, s, H-6), 7.295 (1H, d, J ¼ 8.4 Hz, H-3),
7.241 (1H, s, H-60), 6.527 (1H, d, J ¼ 15.6 Hz, H-
b), 6.490 (1H, d,
5.1.10.13. (1E,4E)-1-(3-allyl-4-methoxyphenyl)-5-(2,5-
difluorophenyl)penta-1,4-dien-3-one (14m). Yellow oil, 60.7% yield.
J ¼ 16.2 Hz, H-
b
0), 6.017e5.922 (1H, m, ArCH2CH]CH2), 5.096e
5.042 (2H, m, ArCH2CH]CH2), 3.850 (3H, s, OCH3), 3.345 (2H, d,
1H NMR (600 MHz, CDCl3):
d
(ppm) 7.974 (1H, d, J ¼ 15.6 Hz, H-
a
0),
), 7.750
J ¼ 6.6 Hz, ArCH2CH]CH2) (Fig. S9). 13C NMR (600 MHz, CDCl3):
7.853 (1H, d, J ¼ 1.8 Hz, H-30), 7.806 (1H, d, J ¼ 15.6 Hz, H-
a
d (ppm) 188.4, 161.3, 159.8, 144.4, 140.0, 136.3, 134.8, 131.0, 129.9,
(1H, d, J ¼ 8.4 Hz, H-2), 7.691 (1H, d, J ¼ 1.8 Hz, H-40), 7.502 (1H, s, H-
129.1, 127.1, 123.1, 118.5, 116.2, 114.5, 110.6, 55.7, 34.2 (Fig. S10). ESI-
6), 7.294 (1H, d, J ¼ 8.4 Hz, H-3), 7.240 (1H, s, H-60), 6.525 (1H, d,
MS m/z: 402.8 (M)þ, 422.7 (M þ Na)þ.
J ¼ 15.6 Hz, H-
b
), 6.488 (1H, d, J ¼ 16.2 Hz, H-
b
0), 6.016e5.921 (1H,
m, ArCH2CH]CH2), 5.095e5.041 (2H, m, ArCH2CH]CH2), 3.849
(3H, s, OCH3), 3.344 (2H, d, J ¼ 6.6 Hz, ArCH2CH]CH2). ESI-MS m/z:
340.8 (M)þ, 362.8 (M þ Na)þ
5.1.10.19. (1E,4E)-1-(3-Allyl-4-methoxyphenyl)-5-[2-(tri-
fluoromethyl)phenyl]penta-1,4-dien-3-one (14s). Yellow powder,
66.1% yield, mp: 92.4e94.5 ꢀC. 1H NMR (600 MHz, CDCl3):
d (ppm)
8.048 (1H, d, J ¼ 15.6 Hz, H-
a
0), 7.794 (1H, d, J ¼ 7.8 Hz, H-30), 7.725
5.1.10.14. (1E,4E)-1-(3-Allyl-4-methoxyphenyl)-5-(2-bromo-6-
(1H, d, J ¼ 8.4 Hz, H-2), 7.705 (1H, d, J ¼ 16.2 Hz, H-
a), 7.594 (1H, t,
fluorophenyl)penta-1,4-dien-3- one (14n). Yellow oil, 67.7% yield. 1H
J ¼ 7.8 Hz, H-50), 7.492 (1H, t, J ¼ 7.8 Hz, H-40), 7.438 (1H, s, H-6),
NMR (600 MHz, CDCl3):
(1H, d, J ¼ 15.6 Hz, H-
), 7.534e7.463 (2H, m, H-2, H-50), 7.441 (1H,
0), 7.204e7.168 (2H, m, H-30, H-
d
(ppm) 7.85 (1H, d, J ¼ 16.2 Hz, H-
a
0), 7.698
7.003 (1H, d, J ¼ 16.2 Hz, H-
b
), 6.954 (1H, d, J ¼ 15.6 Hz, H-
b
0), 6.888
a
(1H, d, J ¼ 8.4 Hz, H-3), 6.029e5.951 (1H, m, ArCH2CH]CH2),
5.106e5.081 (2H, m, ArCH2CH]CH2), 3.883 (3H, s, OCH3), 3.403
(2H, d, J ¼ 6.6 Hz, ArCH2CH]CH2). ESI-MS m/z: 372.9 (M)þ.
s, H-6), 7.310 (1H, d, J ¼ 16.2 Hz, H-
b
40), 6.921 (1H, d, J ¼ 16.2 Hz, H-
), 6.882 (1H, d, J ¼ 8.4 Hz, H-3),
b
6.029e5.957 (1H, m, ArCH2CH]CH2), 5.103e5.074 (2H, m,
ArCH2CH]CH2), 3.880 (3H, s, OCH3), 3.406e3.344 (2H, m,
ArCH2CH]CH2). ESI-MS m/z: 401.5 (M)þ.
5.1.10.20. (1E,4E)-1-(3-Allyl-4-methoxyphenyl)-5-(5-fluoro-2-
methoxyphenyl)penta-1,4-dien-3-one (14t). Yellow oil, 79.6% yield.
1H NMR (600 MHz, CDCl3):
d
(ppm) 7.775 (1H, d, J ¼ 15.6 Hz, H-
a
0),
a), 7.551
5.1.10.15. (1E,4E)-1-(3-Allyl-4-methoxyphenyl)-5-(2,3-
dichlorophenyl)penta-1,4-dien-3- one (14o). Yellow powder, 73.1%
7.663 (1H, d, J ¼ 1.8 Hz, H-30), 7.607 (1H, d, J ¼ 15.6 Hz, H-
(1H, d, J ¼ 8.4 Hz, H-2), 7.493 (1H, d, J ¼ 1.8 Hz, H-40), 7.403 (1H, s, H-

Z. Liu et al. / European Journal of Medicinal Chemistry 74 (2014) 671e682
681
6), 7.195 (1H, d, J ¼ 8.4 Hz, H-3), 7.041 (1H, s, H-60), 6.327 (1H, d,
The levels of TNF-a and IL-6 in the media were determined by
J ¼ 15.6 Hz, H-
b
), 6.290 (1H, d, J ¼ 16.2 Hz, H-
b
0), 6.024e5.917 (1H,
ELISA. The total protein in cultural plates was collected and the
concentrations were determined using Bio-Rad protein assay re-
agents. The total amount of the inflammatory factor in the media
was normalized to the total protein amount of the viable cell
pellets.
m, ArCH2CH]CH2), 5.104e5.044 (2H, m, ArCH2CH]CH2), 3.922
(3H, s, OCH3), 3.360 (2H, d, J ¼ 6.6 Hz, ArCH2CH]CH2). ESI-MS m/z:
352.1 (M)þ.
5.1.10.21. (1E,4E)-1-(3-Allyl-4-methoxyphenyl)-5-(2,4,5-
trimethoxyphenyl)penta-1,4-dien-3-one (14u). Yellow oil, 75.3%
5.5. MTT (methyl thiazolyl tetrazolium) assay
yield. 1H NMR (600 MHz, CDCl3):
d
(ppm) 8.041 (1H, d, J ¼ 15.6 Hz,
H-
a
0),7.680 (1H, d, J ¼ 15.6 Hz, H-
a
), 7.469 (1H, d, J ¼ 8.4 Hz, H-2),
HL7702 cells were seeded into 96-well plates at a density of
5000 cells per well in 1640 medium, supplemented with 5% heat-
7.446 (1H, s, H-6), 7.118 (1H, s, H-60), 7.006 (1H, d, J ¼ 15.6 Hz, H-
b),
6.974 (1H, d, J ¼ 15.6 Hz, H-
b
0), 6.874 (1H, d, J ¼ 8.4 Hz, H-3), 6.520
inactivated serum, 100 U/ml penicillin, and 100 mg/ml strepto-
(1H, s, H-30), 6.038e5.971 (1H, m, ArCH2CH]CH2), 5.101e5.077
(2H, m, ArCH2CH]CH2), 3.944 (3H, s, B-OCH3), 3.911 (3H, s, B-
OCH3), 3.898 (3H, s, A-OCH3), 3.873 (3H, s, B-OCH3), 3.403 (2H, d,
J ¼ 6.6 Hz, ArCH2CH]CH2). (Fig. S11). 13C NMR (600 MHz, CDCl3):
mycin. The cells were maintained at 37 ꢀC in 5% CO2. Tested com-
pounds were dissolved in DMSO and diluted with 1640 medium to
the final concentrations of 10 mM. Cells were incubated with test
compounds for 24 h before the MTT assay. A fresh solution of MTT
(5 mg/ml) prepared in NaCl solution (0.9%) was added to each
single well of the 96-well plate. The plate was then incubated in a
CO2 incubator for 3 h, cells dissolved with 150
analyzed in a multi-well-plate reader at 570 nM.
d
(ppm) 189.0, 159.0, 154.0, 152.1, 143.1, 142.3, 137.5, 136.1, 129.3,
129.0, 128, 127.3, 123.9, 122.9, 115.6, 115.2, 110.6, 110.2, 96.6, 56.2,
56.1, 55.8, 55.3, 33.9 (Fig. S12). ESI-MS m/z: 395.1 (M þ H)þ.
mL DMSO, and then
5.2. Animals
5.6. UVevisible absorption spectra of curcumin and its analogs
Male ICR mice and C57BL/6 mice weighing 18e22 g were ob-
tained from the Animal Center of Wenzhou Medical College
(Wenzhou, China). Animals were housed at a constant room tem-
perature with a 12:12 h lightedark cycle, and fed with a standard
rodent diet and water. The animals were acclimatized to the labo-
ratory 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: 2012/APWC/0110).
Absorbance readings were taken from 250 to 600 nm using a
spectrum Max M5 (Molecular Devices, USA). A stock solution of
1 mM curcumin or its analogs was prepared and diluted by phos-
phate buffer (pH 7.4) to a final concentration of 20 mM. In the ex-
periments where degradation of curcumin was recorded, the
absorption spectra were collected for over 25 min at 5 min in-
tervals. The UVevisible absorbance spectrum was measured at
25 ꢀC at varying time interval in a 1 cm path-length quartz cuvette.
5.3. Cells and reagents
5.7. Real-time quantitative PCR
RAW 264.7 macrophages were incubated in DMEM medium
(Gibco, Eggenstein, Germany) supplemented with 10% FBS
(Hyclone, Logan, UT), 100 U/ml penicillin, and 100 mg/ml strepto-
mycin at 37 ꢀC with 5% CO2. LPS purchased from Sigma was dis-
solved in PBS. Curcumin and its analogs were dissolved in DMSO
before use. For peritoneal macrophage preparation, ICR mice were
stimulated by intraperitoneal (i.p.) injection of 3 mL thioglycollate
solution (0.3 g beef extract, 1 g tryptone, 0.5 g sodium chloride, and
6 g soluble starch were dissolved and boiled in 100 ml water. Before
Cells were homogenized in TRIZOL kit (Invitrogen, Carlsbad, CA)
for extraction of RNA according to each manufacturer’s protocol.
Both reverse transcription and quantitative PCR were carried out
using a two-step M-MLV Platinum SYBR Green qPCR SuperMix-
UDG kit (Invitrogen, Carlsbad, CA). Eppendorf Mastercycler ep
realplex detection system (Eppendorf, Hamburg, Germany) was
used for q-PCR analysis. The primers of genes including TNF-
IL-1 , and -actin were synthesized by Invitrogen. The primer se-
quences of mouse genes used are shown as follows:
a, IL-6,
b
b
used, the solution was filtrated with 0.22
mm filter.) per mouse and
TNF-a
sense: 50-TGGAACTGGCAGAAGAGG-30;
kept in pathogen-free conditions for 3 days before peritoneal
macrophage isolation. Total peritoneal macrophages were har-
vested by washing the peritoneal cavity with PBS containing 30 mM
of EDTA (8 mL per mouse), centrifuged, then the pellet was re-
suspended in RPMI-1640 medium (Gibco, Eggenstein, Germany)
antisense: 50-AGACAGAAGAGCGTGGTG-30;
IL-6 sense: 50-GAGGATACCACTCCCAACAGACC-30;
antisense: 50-AAGTGCATCATCGTTGTTCATACA-30;
IL-1
antisense: 50-CCATCAGAGGCAAGGAGGAA-30;
-actin sense: 50-TGGAATCCTGTGGCATCCATGAAAC-30;
antisense: 50-TAAAACGCAGCTCAGTAACAGTCCG-30.
The amount of each gene was determined and normalized by
the amount of -actin.
b
sense: 50-ACTCCTTAGTCCTCGGCCA-30;
with 10% FBS (Hyclone, Logan, UT), 100 U/ml penicillin, and 100
mg/
b
ml streptomycin. Nonadherent cells were removed by washing
with medium at 3 h after seeding. Experiments were undertaken
after the cells adhered firmly to the culture plates. Before used,
peritoneal macrophages were cultured in RPMI-1640 medium on
35 mm plates at a density of 4.0 ꢁ 105/plate and maintained at 37 ꢀC
in a 5% CO2-humidified air.
b
5.8. LPS-induced inflammatory mortality in C57BL/6 mice
Compound 14q were firstly dissolved with macrogol 15
hydroxystearate (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 14q was 2 mg/ml. The
concentration of solubilizer was ranged 5e10%, and MCT 0.5e2% in
final solution. For the vehicle, the mixture of solubilizer and MCT
was prepared at 10% and 2% respectively. Male B6 mice weighing
18e22 g were pretreated with compounds in a water solution
5.4. Determination of TNF-a and IL-6
After treatment of cells with indicated compounds and LPS, the
TNF- and IL-6 levels in medium were determined with an ELISA kit
(eBioScience, San Diego, CA) according to the manufacturer’s in-
structions. Briefly, cells were pretreated with 10 M of curcumin or
analogs for 2 h, then treated with LPS (0.5 g/ml) for 22 h. After
treatment, the culture media and cells were collected separately.
a
m
m
(200 mL, 15 mg/kg) by i.v. injection 15 min before the i.p. injection of

682
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The results are presented as means ꢂ SD. Student’s t-test was
employed to analyze the differences between sets of data. Statistics
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Acknowledgment
Financial support was provided by the National Natural Science
Funding of China (21272179 and 21202124), High-level Innovative
Talent Funding of Zhejiang Department of Health (G.L.), Project of
Wenzhou Sci & Tech Bureau (Y20120061), Zhejiang Natural Science
Funding (Q12H300009), Project of Zhejiang Provincial Key
Constructive Subject (Traditional Chinese Medicine, 2012-XK-A28),
Qianjiang Talent Project of Zhejiang Province (2013R10020),
Zhejiang Medical
& Health Science and Technology Project
(2013KYB168), and Zhejiang Key Group Project in Scientific Inno-
vation (2010R50042).
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.10.061.
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