Synthesis and anti-inflammatory evaluation of novel mono-carbonyl analogues of curcumin in LPS-stimulated RAW 264.7 macrophages

Article abstract of DOI:10.1016/j.ejmech.2010.09.037

Curcumin is a multifunctional natural product with regulatory effects on inflammation. However, a major limitation for the application of curcumin is its poor bioavailability. We previously demonstrated that the mono-carbonyl analogues of curcumin possess

Full text of DOI:10.1016/j.ejmech.2010.09.037

European Journal of Medicinal Chemistry 45 (2010) 5773e5780
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anti-inflammatory evaluation of novel mono-carbonyl analogues of
curcumin in LPS-stimulated RAW 264.7 macrophages
,
,
,
Chengguang Zhao a 1, Yuepiao Cai a 1, Xuzhi He a, Jianling Li a, Li Zhang a, Jianzhang Wu a b, Yunjie Zhao a,
**
Shulin Yang b, Xiaokun Li a, Wulan Li c , Guang Liang
,
a,b,
*
a School of Pharmacy, Wenzhou Medical College, 1210 College Town, Wenzhou, Zhejiang 325035, China
b College of Chemical Engineering, Nanjing University of Science and Technology, 200 Xiaolingwei St., Nanjing, Jiangsu 210094, China
c College of Mathematics and Computer Science, Hunan Normal University, Changsha, Hunan 410081, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Curcumin is a multifunctional natural product with regulatory effects on inflammation. However, a major
limitation for the application of curcumin is its poor bioavailability. We previously demonstrated that the
mono-carbonyl analogues of curcumin possessed improved pharmacokinetic profiles. In this study, 33
Received 13 August 2010
Received in revised form
11 September 2010
Accepted 16 September 2010
Available online 8 October 2010
novel mono-carbonyl analogues of curcumin were synthesized and their inhibition against TNF-a and IL-6
release was evaluated in LPS-stimulated RAW 264.7 macrophages. Based on the screening data, quanti-
tative structureeactivity relationship was conducted, indicating that electron-withdrawing groups in
benzene ring are favourable to anti-inflammatory activities of B-class compounds. Furthermore,
compounds AN1 and B82 demonstrated anti-inflammatory abilities in a dose-dependent manner. These
raise the possibility that these compounds might serve as potential agents for the treatment of inflam-
matory diseases.
Keywords:
Curcumin
Mono-carbonyl analogues
QSAR
Ó 2010 Elsevier Masson SAS. All rights reserved.
TNF-
IL-6
a
Anti-inflammation
1. Introduction
chronic diseases, its efficacy in clinic has been limited by its poor
bioavailability and fast metabolism in vivo [15,16]. For example,
Curcumin (diferuloylmethane) is an orange-yellow and dietary
polyphenolic phytochemical in turmeric (Curcuma longa). In the
past decades, curcumin has been shown to exhibit antioxidant,
anti-inflammatory, antiviral, antibacterial, and thus has a potential
against various malignant cancers, diabetes, allergies, arthritis and
other chronic illnesses [1e6]. These effects have been proved to be
mediated through the regulation of various transcriptional factors,
inflammatory cytokines and other enzymes [7e10]. This molecule
is under clinical trials for cancer preventive drug development and
for the treatment of rheumatoid arthritis and infectious diseases
[11e14].
with oral administration at the dose of 450e3600 mg/day in
a phase I trial, the blood concentration of curcumin in plasma and
target tissues is under the detection limit [17]. Curcumin seems to
be metabolized and degraded through reduction. It is believed from
recent studies that the b-diketone moiety in the structure of cur-
cumin appears to be a specific substrate of a series of aldoeketo
reductases and can be decomposed rapidly in vivo [18,19]. Some of
analogues without the b-diketone moiety have been designed and
yielded in our previous study, and as expected, they have been
demonstrated to possess enhanced stability in vitro and improved
pharmacokinetic profiles in vivo [20].
Although curcumin has been shown to be safe and modulates
several targets that have been linked with cancer and various other
Proinflammation is involved in many different pathophysio-
logical disease processes, and the inflammatory conditions may
serve as an important and common pattern in various diseases
including cardiovascular diseases and cancer [21e23]. Numerous
cytokines are present with proinflammation. For example, TNF-
* Corresponding author. Bioorganic
& Medicinal Chemistry Research Center,
a
and IL-6 are the two proinflammatory cytokines involved in the
School of Pharmacy, Wenzhou Medical College, 1210 College Town, Wenzhou,
Zhejiang 325035, China. Tel.: þ86 577 86699892; fax: þ86 577 86689983.
** Corresponding author.
pathogenesis of hemodialysis-related (cardiovascular) diseases
[24,25]. Therapeutical candidates targeting proinflammatory cyto-
kines or inhibiting the over-expressions of cytokines become
a focus of novel drug development for inflammatory disease,
E-mail addresses: wulanli2009@163.com (W. Li), cuiliang1234@163.com
(G. Liang).
1
These authors contribute equally to this work.
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.09.037

5774
C. Zhao et al. / European Journal of Medicinal Chemistry 45 (2010) 5773e5780
cancer, diabetes and cardiovascular disease [26e28]. With respect
to inflammatory cytokines in vitro, curcumin and its derivatives
have been shown to down-regulate expression of several cytokines,
Therefore, three series of 5-carbon linker-containing mono-carbonyl
analogues of curcumin, 1,5-diaryl-1,4-pentadiene-3-ones (B),
together with cyclopentanone (A) and cyclohexanone (C) analogues,
including TNF-
a
, IL-1, IL-6, and IL-12 by inhibiting the activation of
weredesignedbydisplacingb-diketonemoietywithasinglecarbonyl
free radical-activated transcription factors, such as nuclear factor
-kappaB and AP-1 [29e31].
group. The same methodology as previously reported was used to
carry out the synthesis of new compounds here [20,33]. Different
substituents withopposingelectronic properties in the benzene rings
were designed to investigate and discuss the structureeactivity
relationship. The synthesis and structures of compounds N1e4 and
76e88 were shown in Fig. 1. The synthesis essentially involved an
aldol condensation of the appropriate aromatic aldehyde with
cyclohexanone, cyclopentanone or acetone in an alkaline medium at
room temperature, respectively. Generally, the coupling reaction
yielded the respective products in high yields (50e91%). The diaryl
structurewasconfirmedby the absence ofmethyl protons adjacent to
the carbonyl group in the 1H NMR spectra of A-class compounds and
the absence of two methylene protons near to the central carbonyl in
the spectra of B and C-class compounds.
Previously, we have evaluated
a part of mono-carbonyl
analogues of curcumin for anti-inflammatory properties using
lipopolysaccharide (LPS)-stimulated mouse macrophages and dis-
cussed the primary structureeactivity relationship (SAR) [32,33].
As part of our ongoing research for potential anti-inflammatory
drug candidates, in this study, we further presented 33 newly
synthesized mono-carbonyl analogues of curcumin and their
quantitative SAR analysis for their anti-inflammatory activities in
mouse RAW 264.7 macrophages. Following initial examination, we
then showed that the best two compounds can prevent LPS-
induced inflammation in a dose-dependent manner.
2. Result and discussion
2.2. Inhibitory screening against LPS-induced TNF-
release
a and IL-6
2.1. Chemistry
As reported previously [20], the deletion of the central
b-diketone
TNF-
a and IL-6 are two of well characterized cytokines that play
may improve pharmacokinetic profiles of curcumin derivatives.
an important role in many inflammatory diseases caused by
O
O
H3CO
HO
OCH3
OH
Curcumin
Delete the beta-diketone moiety
to obtain the structurally stable
mono-carbonyl analogues with
improved pharmacokinetic profiles
O
O
B: n = 0
A: n = 2
C: n = 3
CHO
alkali / alcohol
R
R
R
n
n
N1
N2
N3
N
N
N
N
O
CH3
OH
F
F
H3CO
F3C
N4
77
76
Cl
F
F
Cl
R
78
80
84
81
F3C
CF3
OCH3
OCH3
OCH2CH3
F
82
87
86
H3CO
H3CO
Br
CF3
OCH3
88
H3CH2CO
F3C
OCH2CH3
Fig. 1. Chemical structure of curcumin and the design of its mono-carbonyl analogues as well as general synthesis and chemical structures of mono-carbonyl analogues of curcumin.

C. Zhao et al. / European Journal of Medicinal Chemistry 45 (2010) 5773e5780
5775
endotoxins produced by Gram-negative bacteria. Over-expression
of TNF- and IL-6 contributes significantly to the pathological
complications observed in various diseases [24,25]. TNF- and IL-6
are produced mainly in cells of reticuloendothelial origin (e.g.,
macrophages), especially when induced by many external stimuli,
such as lipopolysaccharide (LPS), a component of the cell wall of
Gram-negative bacteria.
6 release and B84 also showed 51.1% inhibition of IL-6. Further-
more, compounds AN4, BN4, A81, and B78 showed more than 30%
inhibitory effects against IL-6 expression compared to LPS control.
Compounds AN1 and B82, especially, were more potent than cur-
cumin at the same concentration in inhibiting LPS-induced TNF-
a
a
a
and IL-6 expression. Among the tested compounds, AN1 showed
the strongest inhibitory effect on LPS-induced TNF- and IL-6
a
Curcumin and its 33 synthetic analogues were evaluated for
release and its inhibitory rates reached 67.5 and 92.7%, respectively,
compared to the LPS control.
their ability to inhibit the TNF-a and IL-6 release in RAW 264.7
mouse macrophages stimulated by LPS. Macrophages were stimu-
lated with LPS in the presence or absence of the compounds at
Chemical modifications as well as the synthesis of the curcumin
analogues have been tried by many research groups to find out the
SAR conclusion and better leads for the treatment of inflammatory
diseases. However, few significantly potent analogues were found
to be under final considerations as a drug. Concurrently, 33 mono-
carbonyl analogues were synthesized and their inhibitory effects on
a concentration of 10
showed a significant inhibition against TNF-
m
M, where the leading compound curcumin
and IL-6 production.
a
The cells were pre-incubated for 60 min with curcumin analogues
and DMSO as a control. After that, cells were treated with LPS
(0.5
m
g/ml) for 22 min at 37 ꢀC. The amount of TNF-
a
and IL-6 in
TNF-a and IL-6 release were evaluated. As reported previously, it is
media was detected through enzyme-linked immunosorbant assay
(ELISA) and normalized by protein concentration of cells harvested
in homologous culture plates.
Fig. 2 gave the results of the anti-inflammatory evaluation of
curcumin and three analogue classes. Curcumin reduced LPS-
observed here that acetone-derived B-class analogues are more
effective, especially against LPS-induced IL-6 expression, than
cyclopentanone-derived A-class and cyclohexanone-derived
C-class analogues, indicating that the structure of a 5-canbonyl
linker may play a role on such activities. However, AN1 was claimed
the strongest inhibition against LPS-induced IL-6 release. The
detailed rationale is unclear yet. Some special conformation, not
molecular structure, of AN1 may contribute to its strong bioactivity.
mediated TNF-
tively. Among these 33 compounds, the majority of the tested
compounds inhibited LPS-induced TNF- and IL-6 expression to
a and IL-6 expression at 35.6% and 75.7%, respec-
a
various degrees. Compounds AN1, AN4, CN3, B78, and B82
exhibited higher inhibitory ability than the leading curcumin
2.3. Quantitative structureeactivity relationship
(35.6%) against LPS-induced TNF-
BN3, BN4, A78, A84, B84, B86, C77, C81 and C87 showed inhibition
of TNF- production in a range of 20e35.6%. With regard to IL-6,
substituted amino-containing analogues BN1e3 showed inhibition
of IL-6 over 50% compared to the LPS-treated control. Compounds
B82 and B86 exhibited comparable activity to curcumin against IL-
a expression. Compounds AN2,
To further demonstrate the SAR of B-class compounds and to
evaluate the effects of various substituents on the activity, QSARs
were calculated on the nine B-class compounds. The relative
a
amounts of TNF-
a and IL-6 expression in screening section were
used as the factors of biological activity, named as RACTNF- and
a
TNF-α
200
150
100
*
*
*
*
*
*
**
**
50
0
**
**
IL-6
200
150
100
50
*
*
*
*
*
**
**
**
**
**
*
**
**
0
Fig. 2. Curcumin and its analogues inhibited LPS-induced TNF-
a
and IL-6 secretion in RAW 264.7 macrophages. Macrophages were plated at a density of 4.0 ꢁ 105/plate for
overnight in 37 ꢀC and 5% CO2. Cells were pre-treated with curcumin or its analogues (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 was indicated, *p < 0.05, **p < 0.02.

5776
C. Zhao et al. / European Journal of Medicinal Chemistry 45 (2010) 5773e5780
RACIL-6, respectively. Log (1/RACTNF-a) and log (1/RACIL-6) were used
as the dependent variables in the linearization procedure. To
construct QSAR model, various types of parameters are operated,
including the quantum-chemical descriptors calculated by density
functional theory (DFT) method, molar refractivity and the coeffi-
cient of molecular partition octanolewater (log P) which reflects
the molecular properties, and topological descriptors including
kappa index, Zagreb index and Wiener index. A good correlation
was observed with some quantum chemical descriptors for B-class
compounds. However, most of the parameters have to be dis-
regarded due to the low correlation to the biological activity, such
as electrostatic potential (EP), HOMO and LUMO energies, molec-
ular dipole moment (MDP) and molecular polarizability (MP),
molar refractivity, log P. The calculated values of the quantum
chemical descriptors are shown in Table S1 (see Table S1 in Sup-
porting material).
correlation between the biological activity and different substituted
groups. The model involves the DFT-calculated qp and SDN-sub,
which mean the sum of
p-electron densities of the whole mole-
cules and the sum of nucleophilic superdelocalizabilities of the
substituent moiety, respectively. These parameters are frequently
employed to characterize molecular interactions and can be used to
compare corresponding atoms in different molecules. This equation
indicates that the electronic properties play an important role in
the anti-TNF-
a properties of B-class compounds. The correlation
analysis shows that qp correlates Log (1/RACTNF-a) at the signifi-
cance level of 0.0004, with correlation coefficient of ꢃ0.744 in this
model. Negative coefficient of qp indicates that electron-with-
drawing substituents would benefit the activity.
The correlation analysis between anti-IL-6 activities of B-class
compounds and their quantum chemical descriptors is illustrated
in Fig. 3B. Eq. (2) suggests that net atomic charge on the carbon
atom numbered 3 and the oxygen atom numbered 8 in B-class
compounds are important parameters influencing the RACIL-6
activity. It has been proven that local electron densities or charges
are important in many chemical reactions and physico-chemical
properties of compounds. Mulliken atomic charges of the C-3 and
O-8 atom were calculated at B3LYP/6-31G level of theory. In most
cases the presence of donor and acceptor groups in aromatic ring
has a direct influence on the charge of this C atom and O atom.
Although this equation gives a relatively modest correlation with R2
Multiple linear regressions between the different activities of
B-class derivatives and the quantum chemical descriptors were
studied, and the QSAR equations (shown as Eq. (1) and Eq. (2) in
Fig. 3) with the largest correlation coefficient were obtained. As
illustrated in Fig. 3A, the Eq. (1) has high quality and the parameters
used in this equation can explain the variance in the RACTNF-
activity of B-class derivatives. Eq. (1) satisfactorily describes the
a
of 0.71, the QSAR conclusion is coincident to RACTNF- analysis of
a
these compounds. The calculated correlation coefficient between
QC3 and log (1/RACIL-6) is ꢃ0.736, which indicates that electron-
withdrawing group is favourable because it will decrease the
electrophilicity of C-3 and increase the biological activity.
2.4. AN1 and B82 inhibit TNF-a and IL-6 release in a dose-
dependent manner
Among the active analogues above, two compounds, AN1 and
B82, which demonstrated the highest activities and low cytotox-
icity (data not shown) in macrophages, were chosen for further
evaluation of their dose-dependent inhibitory effects against LPS-
induced TNF-
treated with AN1 or B82 in a series of concentrations (1, 2.5, 5.0, 10
and 20 M) for 2 h and were subsequently incubated with LPS
(0.5 g/ml) for 22 h. As shown in Fig. 4, our data demonstrated
a dose-dependent inhibition of LPS-induced TNF- and IL-6 release
a and IL-6 release. RAW 264.7 macrophages were pre-
m
m
a
by AN1 and B82. Accordingly, the IC50 values of these two
compounds are determined and listed in Fig. 4. AN1 exhibited the
lowest IC50 value (1.07
mM) when inhibiting IL-6 release. The
inhibition of TNF- and IL-6 release by AN1 and B82 in a dose-
a
dependent manner, suggests the potential of these compounds as
anti-inflammatory agents.
Combined with our previous studies [20], it is further confirmed
that these mono-carbonyl analogues without
b-diketone may
render themselves favorably to the development of promising
curcumin-based anti-inflammatory drugs from both pharmacoki-
netic and pharmacological standpoints. Further studies including
analysis of the design of novel B-class compounds with electron-
withdrawing substituents and investigation of the underlying
molecular mechanisms of these active compounds at the tran-
scriptional or posttranscriptional level are necessary to further
establish this investigational pathway.
3. Conclusion
Fig. 3. Plot of predicted activity against the corresponding experimental activity. N, the
number of compounds taken into account in the regression; R2, the multiple corre-
lation coefficient; R2adj, adjusted multiple correlation coefficient; s, residual standard
error; F value is related to the F-statistic analysis (Fischer test). The numbers in
parentheses mean the standard deviation of the coefficients.
In summary, we presented a series of novel 5-carbon linker-
containing mono-carbonyl analogues of curcumin and evaluated
their potent inhibitory activities against LPS-induced TNF-
a and

C. Zhao et al. / European Journal of Medicinal Chemistry 45 (2010) 5773e5780
5777
TNF-α
IC50
AN1 16.4 μM
B82 6.28 μM
IL-6
IC50
100
50
0
100
50
0
AN1 1.07 μM
B82 3.64 μM
*
*
**
*
*
*
**
*
*
*
**
*
**
**
**
LPS
AN1(μM)
B82(μM)
-
+
-
-
+
+
2.5
-
+
5
-
+
+
+
-
1
+
-
2.5
+
-
5
+
-
+
LPS
AN1(μM)
B82(μM)
-
+
-
-
+
1
-
+
2.5
-
+
5
-
+
+
+
-
1
+
+
-
+
-
+
-
-
1
10 20
-
-
10 20
-
-
-
-
-
10 20
-
-
-
2.5 5 10 20
Fig. 4. AN1 and B82 inhibited LPS-induced TNF-
a
and IL-6 release in a dose-dependent manner in RAW 264.7 macrophages. Macrophages were plated at a density of 4.0 ꢁ 106/plate
overnight in 37 ꢀC and 5% CO2. Cells were pre-treated with specific compounds as indicated for 2 h, followed by LPS (0.5
mg/ml) treatment for 22 h. TNF-a and IL-6 levels in the
culture media were measured by ELISA and were normalized to the total protein amount. The results are expressed as percent of LPS control. Each bar represents mean ꢂ SE of 3e5
independent experiments. Statistical significance relative to LPS is indicated, *p < 0.05; **p < 0.01.
IL-6 release in RAW 264.7 macrophages. It was concluded that
acetone-derived B-class analogues are more effective than cyclo-
pentanone-derived A-class and cyclohexanone-derived C-class
analogues. Combined with the QSAR analysis, electron-with-
drawing groups in benzene ring are favourable to anti-inflamma-
tory activities of B-class compounds. Compounds AN1 and B82
were the most potent analogues and exhibited anti-inflammatory
abilities in a dose-dependent manner in macrophages. This pres-
ents the possibility that mono-carbonyl analogues of curcumin
might serve as potential agents for the treatment of various
inflammatory diseases.
4.1.2. (2E,5E)-2,5-Bis(2-fluoro-3-(trifluoromethyl)benzylidene)
cyclopentanone (A77)
Yellow powder, 58.1% yield, mp 221.8e222.4 ꢀC [223 ꢀC, lit. [34]].
ESI-MS m/z: 433.1 (M þ H)þ, calcd for C21H12F8O: 432.31.
4.1.3. (2E,5E)-2,5-Bis(2-fluoro-6-(trifluoromethyl)benzylidene)
cyclopentanone (A78)
Yellow powder, 50.4% yield, mp 95.4e97.9 ꢀC. 1H NMR (CDCl3)
d:
7.64 (2H, s, AreCH]C ꢁ2), 7.47 (2H, d, J ¼ 8.4 Hz, AreH5 ꢁ2),
7.44e7.47 (2H, d, J ¼ 8.4 Hz, AreH4 ꢁ2), 7.33e7.35 (2H, t, J ¼ 9.0 Hz,
AreH3 ꢁ2), 2.60 (4H, s, CH2eCH2). ESI-MS m/z: 433.3 (M þ H)þ,
455.1 (M þ Na)þ, calcd for C21H12F8O: 432.31.
4.1.4. (2E,5E)-2,5-Bis(2-fluoro-4-(trifluoromethyl)benzylidene)
cyclopentanone (A80)
4. Experimental section
Yellow powder, 86.4% yield, mp 164.7e169.5 ꢀC. 1H NMR (CDCl3)
d:
4.1. General procedure for the synthesis of compounds
7.77 (2H, s, AreCH]C ꢁ2), 7.68 (2H, t, J ¼ 7.5 Hz, AreH5 ꢁ2), 7.48 (2H,
d, J ¼ 8.4 Hz, AreH6 ꢁ2), 7.41 (2H, d, J ¼ 8.4 Hz, AreH3 ꢁ2), 3.07 (2H, s,
CH2eCH2). ESI-MS m/z: 432.8 (M þ H)þ, calcd for C21H12F8O: 432.31.
All reagents for syntheses were obtained from SigmaeAldrich
and Fluka, and were used without purification. Thin-layer chro-
matography (TLC) was performed on Kieselgel 60 F254 plates.
Melting points were determined on a Fisher-Johns melting appa-
ratus and were uncorrected. 1H NMR spectra were recorded on
a Bruker 600 MHz instruments. The chemical shifts were presented
in terms of parts per million with TMS as the internal reference.
Electron-spray ionization mass spectra in positive mode (ESI-MS)
data were recorded on a Bruker Esquire 3000þ spectrometer.
Column chromatography purifications were carried out on Silica
Gel 60 (E. Merck, 70e230 mesh).
Briefly, an amount of 7.5 mmol acetone (B-class), cyclo-
pentanone (A-class), or cyclohexanone (C-class) was added to
a solution of 15 mmol arylaldehyde in MeOH (10 ml). The solution
was stirred at room temperature for 20 min, followed by dropwise
addition of NaOCH3/CH3OH (1.5 ml, 7.5 mmol). The mixture was
stirred at room temperature and monitored with TLC. When the
reaction was finished, the residue was poured into saturated NH4Cl
solution and filtered. The precipitate was washed with water and
cold ethanol, and dried in vacuum. The solid was purified by
chromatography over silica gel using CH2Cl2/CH3OH as the eluent to
yield compounds [34].
4.1.5. (2E,5E)-2,5-Bis(2-fluoro-5- methoxybenzylidene)
cyclopentanone (A81)
Yellow powder, 69.6% yield, mp 175.6e176.7 ꢀC. 1H NMR (CDCl3)
d
: 7.76 (2H, s, AreCH]C ꢁ2), 7.04e7.07 (4H, m, AreH3,4 ꢁ2),
6.88e6.90 (2H, m, AreH6 ꢁ2), 3.82 (6H, s, AreOCH3 ꢁ2), 3.05 (4H, s,
CH2eCH2). ESI-MS m/z: 357.2 (M þ H)þ, calcd for C21H18F2O3:356.36.
4.1.6. (2E,5E)-2,5-Bis(5-bromo-2 ethoxybenzylidene)
cyclopentanone (A82)
Yellow powder, 94.5% yield, mp 202.1e203.5 ꢀC. 1H NMR (CDCl3)
d
: 7.90 (2H, s, AreCH]C ꢁ2), 7.59 (2H, d, J ¼ 2.4 Hz, AreH6 ꢁ2), 7.41
(2H, dd, J¼ 2.4 Hz, J ¼ 9.0Hz,AreH4 ꢁ2), 6.80 (2H, d, J¼ 9.0 Hz, AreH3
ꢁ2), 4.07 (4H, m, OeCH2 ꢁ2), 3.02 (4H, s, CH2eCH2),1.45(6H, m, CH3
ꢁ2). ESI-MS m/z: 507.1 (M þ H)þ, calcd for C23H22Br2O3: 506.23.
4.1.7. (2E,5E)-2,5-Bis(2-fluoro-4-methoxybenzylidene)
cyclopentanone (A84)
Yellow powder, 41.7% yield, mp 179.1e181.9 ꢀC. 1H NMR (CDCl3)
d
: 7.76 (2H, s, AreCH]C ꢁ2), 7.53 (2H, t, J ¼ 8.4 Hz, AreH6 ꢁ2), 6.75
(2H, d, J ¼ 8.4 Hz AreH5 ꢁ2), 6.67 (2H, d, J ¼ 12 Hz, AreH3 ꢁ2), 3.84
(6H, s, AreOCH3 ꢁ2), 3.02 (4H, s, CH2eCH2). ESI-MS m/z: 357.2
(M þ H)þ, calcd for C21H18F2O3: 356.36.
4.1.1. (2E,5E)-2,5-Bis(2-hydroxy-3-methoxylbenzylidene)
cyclopentanone (A76)
Green powder, 61.2% yield, mp 120.1e122.7 ꢀC. 1H NMR (CDCl3)
d
: 7.74 (2H, s, AreCH]C ꢁ2), 7.19 (2H, m, AreH6 ꢁ2), 7.13 (2H, m,
4.1.8. (2E,5E)-2,5-Bis(2,4,5-trimethoxybenzylidene)cyclopentanone
(A86)
AreH5 ꢁ2), 6.98 (2H, m, AreH4 ꢁ2), 5.35 (2H, s, AreOH ꢁ2), 3.96
(6H, s, AreOeCH3 ꢁ2), 2.78 (4H, s, CH2eCH2). ESI-MS m/z: 351.2
(M ꢃ H)ꢃ, calcd for C22H14F8O: 352.38.
Orange powder, 92.6% yield, mp 225.8e227.1 ꢀC. 1H NMR
(CDCl3)
d
: 7.97 (2H, s, AreCH]C ꢁ2), 7.13 (2H, s, AreH6 ꢁ2), 6.54

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C. Zhao et al. / European Journal of Medicinal Chemistry 45 (2010) 5773e5780
(2H, s, AreH3 ꢁ2), 3.94 (6H, s, Ar2eOCH3 ꢁ2), 3.88 (12H, s,
Ar4,5eOCH3 ꢁ2), 3.06 (4H, s, CH2eCH2). ESI-MS m/z: 441.8
(M þ H)þ, calcd for C25H28O7: 440.49.
AreH6 ꢁ2), 7.05 (2H d, J ¼ 16.2 Hz, COeCH]C ꢁ2), 6.75 (2H, dd,
J ¼ 8.4 Hz, J ¼ 8.4 Hz, AreH3 ꢁ2), 6.66 (2H dd, J ¼ 1.8 Hz, J ¼ 8.4 Hz,
AreH5 ꢁ2), 3.85 (6H, s, AreOCH3 ꢁ2). ESI-MS m/z: 331.6 (M þ H)þ,
calcd for C19H16F2O3: 330.33.
4.1.9. (2E,5E)-2,5-Bis(3-(diethoxymethyl) benzylidene)
cyclopentanone (A88)
4.1.18. (1E,4E)-1,5-Bis(2,4,5-trimethoxyphenyl)penta-1,4-dien-3-
Yellow powder, 95.5% yield, mp 84.4e85.1 ꢀC. 1H NMR (CDCl3)
d:
one (B86) [38]
8.10 (2H, s, AreCH]C ꢁ2), 7.91 (2H, d, J ¼ 7.8 Hz, AreH4 ꢁ2), 7.85
(2H, d, J ¼ 7.8 Hz, AreH5 ꢁ2), 7.62e7.66 (4H, m, AreH2,6 ꢁ2), 5.56
(2H, s, AreCHeO ꢁ2), 3.72 (8H, m, OeCH2 ꢁ4), 3.18 (4H, s,
CH2eCH2), 1.26 (12H, m, CH3 ꢁ4). ESI-MS m/z: 466.1 (M þ H)þ,
calcd for C29H36O5: 464.59.
Orange powder, 58% yield, mp 173.1e174.8 ꢀC. 1H NMR (CDCl3)
d:
8.03 (2H, d, J ¼ 15.6 Hz, AreCH]C ꢁ2), 7.13 (2H, s, AreH6 ꢁ2), 7.02
(2H, d, J ¼ 16.2 Hz, COeCH]C ꢁ2), 6.52 (2H, s, AreH3 ꢁ2), 3.94 (6H,
s, AreOCH3 ꢁ2), 3.90 (12H, s, Ar4,5eOCH3 ꢁ4). ESI-MS m/z: 415.9
(M þ H)þ, calcd for C23H26O7: 414.45.
4.1.10. (2E,5E)-2,5-Bis(4-(diethylamino)benzylidene)
cyclopentanone (AN1) [35]
4.1.19. (1E,4E)-1,5-Bis(4-(diethylamino)phenyl)penta-1,4-dien-3-
one (BN1) [35]
Red powder, 89.3% yield, mp 168.5e170.9 ꢀC. ESI-MS m/z: 403.4
(M þ H)þ, calcd for C27H34N2O: 402.57.
Brick red powder, 50.9% yield, mp 155.5e157.8 ꢀC. ESI-MS m/z:
377.2 (M þ H)þ, calcd for C25H32N2O: 376.53.
4.1.11. (2E,5E)-2,5-Bis(4-(piperidin-1-yl)benzylidene)
cyclopentanone (AN2)
4.1.20. (1E,4E)-1,5-Bis(4-(piperidin-1-yl)phenyl)penta-1,4-dien-3-
one (BN2)
Brick red powder, 92.9% yield, mp 256.7e258.2 ꢀC [260e262 ꢀC,
lit. [36]]. ESI-MS m/z: 427.3(M þ H)þ, calcd for C29H34N2O: 426.59.
Orange powder, 41.2% yield, mp 181.2e184.9 ꢀC. 1H NMR (CDCl3)
d
: 7.75 (2H, d, J ¼ 16.2 Hz, AreCH]C ꢁ2), 7.53 (4H, d, J ¼ 9.0 Hz,
AreH2,6 ꢁ2), 6.95 (2H, d, J ¼ 16.2 Hz, COeCH] ꢁ2), 6.92 (4H, d,
J ¼ 4.8 Hz, AreH3,5 ꢁ2), 3.33 (8H, t, J ¼ 5.4 Hz, NeCH2 ꢁ4), 1.72 (4H,
s, pCH2 ꢁ2), 1.67 (8H, d, J ¼ 4.8 Hz, CH2eCeCH2e ꢁ6). ESI-MS m/z:
401.3 (M þ H)þ, calcd for C27H32N2O: 400.56.
4.1.12. (2E,5E)-2,5-Bis(4-morpholinobenzylidene)cyclopentanone
(AN3)
Orange powder, 94.6% yield, mp 270e272 ꢀC [275e277 ꢀC, lit.
[36]]. ESI-MS m/z: 431.3 (M þ H)þ, calcd for C27H30N2O3: 430.5.
4.1.21. (1E,4E)-1,5-Bis(4-morpholinophenyl)penta-1,4-dien-3-one
(BN3)
4.1.13. (2E,6E)-2,6-Bis(4-(bis(2-chloroethyl)amino)benzylidene)
cyclohexanone (AN4)
Orange powder, 92.5% yield, mp 215.1e217.0 ꢀC. 1H NMR (CDCl3)
Yellow powder, 95.5% yield, mp 181.4e185.1 ꢀC. 1H NMR (CDCl3)
d
: 7.70 (2H, d, J ¼ 15.6 Hz, AreCH]C ꢁ2), 7.49 (4H, d, J ¼ 8.4 Hz,
d
: 7.58 (4H, d, J ¼ 9.0 Hz, AreH2,6 ꢁ2), 7.54 (2H, s, AreCH]C ꢁ2),
AreH2,6 ꢁ2), 6.95 (2H, d, J ¼ 15.6 Hz, COeCH]C ꢁ2), 6.92 (4H, t,
J ¼ 9.0 Hz, AreH3,5 ꢁ2), 3.89 (8H, t, J ¼ 4.8 Hz, OeCH2 ꢁ4), 3.29 (8H,
t, J ¼ 4.8 Hz, NeCH2 ꢁ4). ESI-MS m/z: 405.3 (M þ H)þ, calcd for
C25H28N2O3: 404.5.
6.76 (4H, d, J ¼ 9.0 Hz, AreH3,5 ꢁ2), 3.83 (8H, t, J ¼ 7.2 Hz, NeCH2
ꢁ4), 3.71 (8H, t, J ¼ 7.2 Hz, CleCH2 ꢁ4), 3.10 (4H, s, CH2eCH2). ESI-
MS m/z: 541.1 (M þ H)þ, calcd for C27H30Cl4N2O: 540.35.
4.1.14. (1E,4E)-1,5-Bis(2-hydroxy-3-methylphenyl)penta-1,4-dien-
3-one (B76)
4.1.22. (1E,4E)-1,5-Bis(4-(bis(2-chloroethyl)amino)phenyl)penta-
1,4-dien-3-one (BN4)
Green powder, 78.9% yield, mp 109.3e112.7 ꢀC. 1H NMR (CDCl3)
Yellow powder, 74.6% yield, mp 197.4e203.0 ꢀC. 1H NMR (CDCl3)
d
: 7.43 (2H, d, J ¼ 16.2 Hz, AreCH]C ꢁ2), 7.19 (2H, d, J ¼ 7.8 Hz,
d
: 7.67 (2H, d, J ¼ 15.6 Hz, AreCH]C ꢁ2), 7.54 (4H, d, J ¼ 8.4 Hz,
AreH6 ꢁ2), 7.12 (2H, d, J ¼ 7.8 Hz, AreH5 ꢁ2), 7.08 (2H, d,
J ¼ 16.2 Hz, COeCH]C ꢁ2), 6.98 (2H, t, J ¼ 8.4 Hz, AreH4 ꢁ2), 5.35
(2H, s, eOH ꢁ2), 3.93 (6H, s, AreOCH3 ꢁ2). ESI-MS m/z: 324.9
(M ꢃ H)ꢃ, calcd for C19H18O5: 326.34.
AreH2,6 ꢁ2), 6.90 (2H, d, J ¼ 15.6 Hz, COeCH]C ꢁ2), 6.57 (4H, d,
J ¼ 8.4 Hz, AreH3,5 ꢁ2), 3.81 (8H, m, NeCH2 ꢁ4), 3.65 (8H, m,
CleCH2 ꢁ4). ESI-MS m/z: 515.1 (M þ H)þ, calcd for C27H30Cl4N2O:
514.31.
4.1.15. (1E,4E)-1,5-Bis(2-fluoro-6-(trifluoromethyl)phenyl)penta-
1,4-dien-3-one (B78)
4.1.23. (2E,6E)-2,6-Bis(2-fluoro-3-(trifluoromethyl)benzylidene)
cyclohexanone (C77)
Yellow powder, 65.2% yield, mp 89.4e92.1 ꢀC. 1H NMR (CDCl3)
d:
Yellow powder, 62.5% yield, mp 119.7e122.3 ꢀC. 1H NMR (CDCl3)
8.11 (2H, d, J ¼ 16.2 Hz, AreCH]C ꢁ2), 7.56 (2H, m, AreH5 ꢁ2),
7.25e7.29 (4H, t, AreH3,4 ꢁ2), 6.89 (2H, d, J ¼ 16.2 Hz, COeCH]C
ꢁ2). ESI-MS m/z:429.4 (M þ Na)þ, calcd for C19H10F8O: 406.27.
d
: 7.78 (2H, s, AreCH]C ꢁ2), 7.60 (2H, t, J ¼ 7.2 Hz, AreH4 ꢁ2), 7.55
(2H, t, J ¼ 7.2 Hz, AreH6 ꢁ2), 7.27 (2H, t, J ¼ 7.2 Hz, AreH5 ꢁ2), 2.78
(4H, t, J ¼ 5.6 Hz, CH2eCeCH2), 1.79 (2H, m, pCH2). ESI-MS m/z:
447.2 (M þ H)þ, calcd for C22H14F8O: 446.33.
4.1.16. (1E,4E)-1,5-Bis(5-bromo-2-ethoxyphenyl)penta-1,4-dien-3-
one (B82)
4.1.24. (2E,6E)-2,6-Bis(2-fluoro-6-(trifluoromethyl)benzylidene)
cyclohexanone (C78)
Yellow powder, 73.1% yield, mp 132.45e134.55 ꢀC. 1H NMR
(CDCl3)
d
: 7.95 (2H, d, 16.2 Hz, AreCH]C ꢁ2), 7.69 (2H, d, J ¼ 1.8 Hz,
Yellow powder, 56.5% yield, mp 114.9e116.7 ꢀC. 1H NMR (CDCl3)
AreH6 ꢁ2), 7.41 (2H, dd, J ¼ 1.8 Hz, J ¼ 8.4 Hz, AreH4 ꢁ2), 7.14 (2H,
d, J ¼ 16.2 Hz, COeCH]C ꢁ2), 6.80 (2H, d, J ¼ 8.4 Hz, AreH3 ꢁ2),
4.11 (4H, m, eOCH2 ꢁ2), 1.45 (6H, s, eCH3 ꢁ2). ESI-MS m/z: 480.8
(M þ H)þ, calcd for C21H20Br2O3: 480.19.
d
: 7.62 (2H, s, AreCH]C ꢁ2), 7.53 (2H, d, J ¼ 8.4 Hz, AreH5 ꢁ2), 7.44
(2H, dd, J ¼ 8.4 Hz, J ¼ 8.4 Hz, AreH4 ꢁ2), 7.31 (2H, t, J ¼ 8.4 Hz, AreH3
ꢁ2), 2.45 (4H t, J ¼ 6.3 Hz, CH2eCeCH2), 1.69 (2H, m, pCH2). ESI-MS
m/z: 447.2 (M þ H)þ, 469.0 (M þ Na)þ, calcd for C22H14F8O: 446.33.
4.1.17. (1E,4E)-1,5-Bis(2-fluoro-4-methoxyphenyl)penta-1,4-dien-
3-one (B84) [37]
4.1.25. (2E,6E)-2,6-Bis(2-fluoro-4-(trifluoromethyl)benzylidene)
cyclohexanone (C80)
Yellow powder, 52% yield, mp 123.2e124.5 ꢀC. 1H NMR (CDCl3)
Yellow powder, 88.5% yield, mp 145.4e147.7 ꢀC. 1H NMR (CDCl3)
d: 7.79 (2H, d, J ¼ 16.2 Hz, AreCH]C ꢁ2), 7.55 (2H, t, J ¼ 8.7 Hz,
d
: 7.78 (2H, s, AreCH]C ꢁ2), 7.48 (2H, t, J ¼ 7.8 Hz, AreH5 ꢁ2), 7.45

C. Zhao et al. / European Journal of Medicinal Chemistry 45 (2010) 5773e5780
5779
(2H, d, J ¼ 8.4 Hz, AreH6 ꢁ2), 7.39 (2H, d, J ¼ 9.6 Hz, AreH3 ꢁ2), 2.81
4.2. Cell line and reagents
(4H, t, J ¼ 6.0 Hz, CH2eCeCH2), 1.80 (2H, m, pCH2). ESI-MS m/z:
445.7 (M ꢃ H)ꢃ, calcd for C22H14F8O: 446.33.
Mouse RAW 264.7 macrophages were obtained from the
American Type Culture Collection (ATCC, USA). Cell culture reagents
were obtained from Gibco. Fetal bovine serum was from HyClone
and was heat-inactivated for 30 min at 65 ꢀC. LPS purchased from
Sigma was dissolved in PBS. Curcumin and its analogues were
dissolved in DMSO before use.
4.1.26. (2E,6E)-2,6-Bis(2-fluoro-5-methoxy benzylidene)
cyclohexanone (C81)
Yellow powder, 58.3% yield, mp 91.3e93.4 ꢀC. 1H NMR (CDCl3)
d:
7.77 (2H, s, AreCH]C ꢁ2), 7.03 (2H, t, J ¼ 9.0 Hz, AreH3 ꢁ2),
6.83e6.87 (4H, m, AreH4,6 ꢁ2), 3.80 (6H, s, AreOCH3 ꢁ2), 2.81 (4H,
t, J ¼ 5.4 Hz, CH2eCeCH2), 1.78 (2H, m, pCH2). ESI-MS m/z: 371.0
(M þ H)þ, calcd for C22H20F2O3: 370.39.
4.3. Cell treatment and ELISA assay [32,33]
Mouse RAW 264.7 macrophages were incubated in DMEM
media (Gibco) supplemented with 10% FBS,100 U/ml penicillin, and
4.1.27. (2E,6E)-2,6-Bis(5-bromo-2-ethoxy benzylidene)
cyclohexanone (C82)
100
treated with 10
2 h, then treated with LPS (0.5 m
m
g/ml streptomycin at 37 ꢀC with 5% CO2. Cells were pre-
Yellow powder, 91.1% yield, mp 136.5e138.14 ꢀC. 1H NMR
mM of curcumin, analogues or vehicle control for
(CDCl3)
d
: 7.89 (2H, s, AreCH]C ꢁ2), 7.4 (2H, d, J ¼ 2.4 Hz,
g/ml) for 22 h. After treatment, the
AreH6 ꢁ2), 7.38 (2H, d, J ¼ 2.4 Hz, J ¼ 9.0 Hz, AreH4 ꢁ2), 6.78
(2H, d, J ¼ 9.0 Hz, AreH3 ꢁ2), 4.05 (4H, m, eOCH2 ꢁ2), 2.83
(4H, t, J ¼ 5.4 Hz, CH2eCeCH2), 1.78 (2H, m, pCH2), 1.42 (6H, m,
CH3 ꢁ2). ESI-MS m/z: 520.8 (M þ H)þ, calcd for C24H24Br2O3:
520.25.
culture media and cells were collected separately. The culture
media collected were centrifuged at 1000 rpm for 10 min. The
levels of TNF-
using mouse TNF-
a
and IL-6 in the media were determined by ELISA
and mouse IL-6 ELISA Kits (BOSTER, USA). After
a
centrifugation, the supernatant was separated and stored at ꢃ70 ꢀC
until use. Cells were washed with PBS and harvested with cell lysis
buffer (Tris-HCl 20 mM, NP40 1%, NaCl 150 mM, EDTA 2 mM,
Na3VO4 200 mM, SDS 0.1%, NaF 20 mM). The mixed liquor was
shaken vigorously for 10 min in lysis buffer at 0 ꢀC. After being
centrifuged at 12,000 rpm for 5 min at 4 ꢀC, the total protein was
collected and the concentrations were determined using Bio-Rad
protein assay reagents. The total amount of the inflammatory factor
in the media was normalized to the total protein amount of the
viable cell pellets.
4.1.28. (2E,6E)-2,6-Bis(2,4,5-trimethoxybenzylidene)cyclohexanone
(C86)
Yellow powder, 62% yield, mp 121.2e123.6 ꢀC. 1H NMR (CDCl3)
d:
7.97 (2H, s, AreCH]C ꢁ2), 6.91 (2H, s, AreH6 ꢁ2), 6.53 (2H, s,
AreH3 ꢁ2), 3.84 (18H, s, AreOCH3 ꢁ6), 2.85 (4H, t, J ¼ 4.8 Hz,
CH2eCeCH2),1.78 (2H, m, pCH2). ESI-MS m/z: 455.1 (M þ H)þ, calcd
for C26H30O7: 454.51.
4.1.29. (2E,6E)-2,6-Bis(2,4,-bis(trifluoromethyl)benzylidene)
cyclohexanone (C87)
4.4. Conventional quantum chemical descriptors
Yellow powder, 91% yield, mp 129.1e130.8 ꢀC. 1H NMR (CDCl3)
d:
7.98 (2H, s, AreH2 ꢁ2), 7.91 (2H, s, AreCH]C ꢁ2), 7.83 (2H, d,
J ¼ 7.8 Hz, AreH5 ꢁ2), 7.47 (2H, d, J ¼ 8.4 Hz, AreH6 ꢁ2), 2.60 (2H, t,
J ¼ 5.1 Hz, CH2eCeCH2), 1.73(2H, m, pCH2). ESI-MS m/z: 547.9
(M þ H)þ, calcd for C24H14F12O: 546.35.
EHOMO/ELUMO: energy of the highest occupied molecular orbital
(EHOMO) and energy of the lowest unoccupied molecular orbital
(ELUMO);
DE: orbital energy gap between HOMO and LUMO
(ELUMO ꢃ EHOMO); QA: net atomic charge on atom A; FrE=FrN: indices
of frontier electron density, FrE ¼ frE=EHOMO,FrN ¼ frN=ELUMO, where
frE and frN are the electrophilic and nucleophilic atomic frontier
4.1.30. (2E,6E)-2,6-Bis(4-(diethylamino)benzylidene)
cyclohexanone (CN1) [35]
electron densities, respectively; qp: the sum of
p electron densities
Brick red powder, 21.4% yield, mp 143.7e145.2 ꢀC. ESI-MS m/z:
417.4 (M þ H)þ, calcd for C28H36N2O: 416.6.
of all atoms; SDE/SDN: the sum of electrophilic and nucleophilic
superdelocalizabilities. In order to better represent quantitatively
the contributions of these orbitals in two distinct parts of the
molecules, SDE-BS/SDN-BS and SDE-sub/SDN-sub were calculated
over the basic structure moiety and the variable radical moiety [39].
4.1.31. (2E,6E)-2,6-Bis(4-(piperidin-1-yl)benzylidene)
cyclohexanone (CN2)
Yellow powder, 72.4% yield, mp 230.4e233.6 ꢀC [230e232 ꢀC, lit.
[36]]. ESI-MS m/z: 441.4 (M þ H)þ, calcd for C28H36N2O: 440.62.
4.5. Calculation method and models
4.1.32. (2E,6E)-2,6-Bis(4-morpholinobenzylidene)cyclohexanone
(CN3)
Initially, full geometric optimization of the 33 molecules was
performed using DFT/B3LYP methods with the 6-31G(d) basis sets.
All molecular orbitals are calculated by the Firefly QC package,
which is partially based on the GAMESS (US) source code. Based on
these precise quantum chemical descriptors, a quantitative struc-
tureeactivity relationships study has been carried out. The QSAR
equations and statistics were calculated with the open-source R
framework, a powerful tool for statistical computing and graphics
[39,40].
Yellow powder, 22% yield, mp 234.3e237.1 ꢀC. 1H NMR (CDCl3)
d
: 7.77 (2H, s, AreCH] ꢁ2), 7.47 (4H, d, J ¼ 9.0 Hz, AreH2,6 ꢁ2), 6.94
(4H, d, J ¼ 8.4 Hz, AreH3,5 ꢁ2), 3.90 (8H, d, J ¼ 4.2 Hz, OeCH2 ꢁ4),
3.27 (8H, t, J ¼ 4.2 Hz, NeCH2 ꢁ4), 2.95 (4H, s, eCH2eCH2), 1.60 (2H,
m, pCH2). ESI-MS m/z: 445.3 (M þ H)þ, calcd for C28H32N2O3:
444.57.
4.1.33. (2E,6E)-2,6-Bis(4-(bis(2-chloroethyl)amino)benzylidene)
cyclohexanone (CN4)
Acknowledgment
Yellow powder, 87.2% yield, mp 201.4e204.2 ꢀC. 1H NMR
(CDCl3)
d
: 7.77 (4H, m, AreH2,6 ꢁ2), 7.45 (2H, m, AreCH]C ꢁ2),
This work was supported by the National Natural Science
Funding of China (20802054), Young Talent Funding of Zhejiang
Department of Health (2009QN020), Key Project of Wenzhou
Sci&Tech Bureau (Y20090009 & Y20100006) and Zhejiang Natural
Science Funding (Y20101108).
6.68e6.76 (4H, m, AreH3,5 ꢁ2), 3.77e3.86 (8H, m, NeCH2 ꢁ4),
3.64e3.69 (8H, m, CleCH2 ꢁ4), 2.92 (4H, m, CH2eCH2), 1.81 (2H, s,
pCH2). ESI-MS m/z: 555.7 (M þ H)þ, calcd for C27H30Cl4N2O:
554.38.

5780
C. Zhao et al. / European Journal of Medicinal Chemistry 45 (2010) 5773e5780
[20] G. Liang, L. Shao, Y. Wang, C. Zhao, Y. Chu, J. Xiao, Y. Zhao, X. Li, S. Yang, Bioorg.
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