Synthesis of mono-carbonyl analogues of curcumin and their effects on inhibition of cytokine release in LPS-stimulated RAW 264.7 macrophages

Article abstract of DOI:10.1016/j.bmc.2010.03.001

Curcumin has been reported to possess multifunctional bioactivities, especially the ability to inhibit proinflammatory induction. We previously demonstrated that the mono-carbonyl analogues of curcumin possessed improved pharmacokinetic profiles both in v

Full text of DOI:10.1016/j.bmc.2010.03.001

Bioorganic & Medicinal Chemistry 18 (2010) 2388–2393
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of mono-carbonyl analogues of curcumin and their effects on
inhibition of cytokine release in LPS-stimulated RAW 264.7 macrophages
Chengguang Zhao a, , Ju Yang a, , Yi Wang a, Donglou Liang a, Xuyi Yang a, Xiaoxia Li a, Jianzhang Wu a,
Xiaoping Wu a, Shulin Yang b, Xiaokun Li a, Guang Liang a,b,
*
a Bioorganic & Medicinal Chemistry Research Center, 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
a r t i c l e i n f o
a b s t r a c t
Article history:
Curcumin has been reported to possess multifunctional bioactivities, especially the ability to inhibit pro-
inflammatory induction. We previously demonstrated that the mono-carbonyl analogues of curcumin
possessed improved pharmacokinetic profiles both in vitro and in vivo. In this study, we synthesized
and examined a series of 5-carbon linker-containing mono-carbonyl analogues of curcumin with potent
Received 21 December 2009
Revised 1 March 2010
Accepted 2 March 2010
Available online 6 March 2010
inhibitory activities against TNF-a and IL-6 release in LPS-stimulated RAW 264.7 macrophages. Discus-
sion and conclusions are given regarding structure-activity relationships (SAR). The two most potent
analogues among the tested compounds, B75 and C12, exhibited anti-inflammatory abilities in a dose-
dependent manner in macrophages. This raises the possibility that mono-carbonyl analogues of curcumin
might serve as potential agents for the treatment of various inflammatory diseases.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Curcumin
Mono-carbonyl analogues
SAR
TNF-a
IL-6
Macrophage
1. Introduction
cytokines by curcumin is thought to be mediated by the inhibition
of the activation of NF-j
B and AP-1.16–18
Proinflammation is a widespread phenomenon that is associ-
ated with various diseases including cardiovascular diseases and
cancer.1–3 Numerous cytokines are present with proinflammation.
In spite of the favorable biological properties of curcumin, there
are drawbacks that limit the development of curcumin as a poten-
tial therapeutic agent, including low bioavailability and instability
at neutral to basic conditions.19,20 For example, with oral adminis-
tration of 450–3600 mg/day in a phase I trial, the curcumin blood
concentration in plasma and target tissues is under the detection
limit.21 Thus, the weakness of a pharmacokinetic profile in vivo
of curcumin significantly inhibits its clinical application.
For example, TNF-a and IL-6 are two inflammatory cytokines in-
volved in the pathogenesis of cardiovascular diseases, cancer and
diabetes.4–6 Anti-inflammatory candidates targeting proinflamma-
tory cytokines or inhibiting the over-expressions of cytokines are a
major focus of current drug development.7–9
Curcumin, 1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadi-
ene-3,5-dione, is a key active component in the traditional herb
Curcuma Longa and has been extensively investigated for its poten-
tial biological benefits, including anti-tumor, anti-inflammation,
cardio-protection and anti-virus.10–13 Presently, curcumin has been
evaluated in clinic trials for the treatment of many diseases such as
liver disease, rheumatoid arthritis, infectious diseases and can-
cers.14 The therapeutic effects of curcumin are attributed to its
activity on a wide range of molecular targets, particularly a series
of inflammatory factors and cytokines.15 Curcumin is able to inhi-
bit the production of proinflammatory cytokines, both at the
mRNA and protein levels. The decrease in the expression of
It is believed that the presence of an active methylene group
and b-diketone moiety contributes to the instability of curcumin
under physiological conditions, and induces rapid degradation
and metabolism of curcumin.22,23 In our previous study, we de-
signed and synthesized a series of mono-carbonyl analogues of
curcumin by deleting the b-diketone moiety.24 We have also eval-
uated a total of 87 analogues for anti-inflammatory properties
using lipopolysaccharide (LPS)-stimulated mouse macrophages
and discuss the primary structure–activity relationship (SAR).25,26
The evidence from a degradation degree assay in pH 7.4 buffer
in vitro and a pharmacokinetic study in vivo demonstrated that
mono-carbonyl analogues exhibit enhanced stability and improved
pharmacokinetic profiles.24 In this study, we further present 23
newly designed mono-carbonyl analogues of curcumin and their
SAR results for their anti-inflammatory activities in mouse RAW
264.7 macrophages. Following initial examination, we further
* Corresponding author. Tel.: +86 577 86699524; fax: +86 577 86699527.
E-mail address: cuiliang1234@163.com (G. Liang).
These authors contribute equally to this work.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.03.001

C. Zhao et al. / Bioorg. Med. Chem. 18 (2010) 2388–2393
2389
show that compounds B75 and C12 prevented LPS-induced inflam-
mation in a dose-dependent manner.
release in mouse RAW 267 macrophages. The macrophages were
pre-treated with 10 M compounds for 2 h and then incubated
with 0.5 g/ml LPS for 22 h. The amount of TNF- and IL-6 in med-
l
l
a
ia were detected thru enzyme-linked immunosorbant assays (ELI-
SA) and normalized by protein concentration of cells harvested in
homologous culture plates.
The results of the anti-inflammatory assay of three analog clas-
ses are shown in Fig. 2A and B, respectively. Among these 23 com-
2. Result and discussion
2.1. Chemistry
Three series of mono-carbonyl analogues of curcumin, 1,5-dia-
ryl-1,4-pentadiene-3-ones (B), together with cyclopentanone (A)
and cyclohexanone (C) analogues, were designed by displacing
beta-diketone moiety with a single carbonyl group based on expla-
nations we reported previously.24 Different substituents with
opposing electronic properties in the benzene rings were designed
to investigate and discuss the structure–activity relationship. As
shown in Figure 1, compounds 12 and 67–75 were synthesized
by coupling the appropriate aromatic aldehyde with cyclohexa-
none, cyclopentanone or acetone in an alkaline medium, respec-
tively. The general process for synthesis of these compounds was
previously reported.27 The synthetic yields, melting points, 1H
NMR and ESI-MS analysis of unpublished compounds are described
in the Section 4. The diaryl structure is confirmed by the absence of
methyl protons adjacent to the carbonyl group in the 1H NMR spec-
tra of A-class compounds and the absence of two methylene pro-
tons near to the central carbonyl in the spectra of B- and C-class
compounds.
pounds, the majority inhibited LPS-induced TNF-
expression to various degrees and compounds B68, A69, B69,
B72, C72, B75 and C12 exhibited the highest inhibitory abilities
a and IL-6
against LPS-induced TNF-
a expression (Fig. 2A), and compounds
A68, B68, B69, B72, C72, A73, B75 and C12 showed inhibitory ef-
fects within 60% against IL-6 release compared to the LPS-control.
Compounds B75 and C12, especially, were more potent than the
leading curcumin at the same concentration in inhibiting LPS-in-
duced TNF-
oxyl-substituted compound, showed the strongest inhibitory effect
on LPS-induced TNF- and IL-6 release among the tested analogues
a and IL-6 expression. C12, a 3-(dimethylamino) prop-
a
and its inhibitory rates reached 96.9% and 97.1%, respectively,
compared to the LPS-control.
2.3. Structure–activity relationship analysis of curcumin
analogues
As an excellent leading compound, curcumin has been investi-
gated in depth in the field of medicinal chemistry. A number of
analogues of curcumin have also been designed and synthesized
for the development of new anti-inflammatory and anti-cancer
drugs.29–31 Our previous publications demonstrated that the struc-
tural replacement of C7 ‘ene-[1,3-dioxo]-ene’ linker in curcumin by
C5 ‘ene-oxoene’ linker forms mono-carbonyl analogues and leads
to the enhancement of stability in vitro and improvement of phar-
macokinetic profiles in vivo.24,31 In our previous studies,26 a series
of mono-carbonyl derivatives of curcumin containing 1,4-pentadi-
ene-3-one linker and their cyclopenta- and cyclohexa-analogues,
2.2. Inhibitory effects on LPS-induced TNF-a and IL-6 release by
curcumin analogues
Lipopolysaccharide (LPS) is an important structural component
of the outer membrane of gram-negative bacteria, and it is a well-
studied immunostimulator that induces a systemic inflammation
response,28 and especially, the expression of proinflammatory
cytokines, such as TNF-
thetic analogues in the 5-carbon linker series were evaluated for
their inhibitory abilities against LPS-induced TNF- and IL-6
a and IL-6. Here, curcumin and its 23 syn-
a
Figure 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.

2390
C. Zhao et al. / Bioorg. Med. Chem. 18 (2010) 2388–2393
Figure 2. Curcumin and its analogues inhibited LPS-induced TNF-
for overnight in 37 °C and 5% CO2. Cells were pre-treated with curcumin or its analogues (10 lM) for 2 h, then treated with LPS (0.5 lg/ml) for 22 h. TNF-a and IL-6 levels in
a
and IL-6 secretion in RAW 264.7 macrophages. Macrophages were plated at a density of 4.0 ꢀ 105/plate
the culture media were measured by ELISA and were normalized to the total protein. The results are expressed as percent of LPS control. Each bar represents mean SE of 3–5
independent experiments. Statistical significance relative to LPS is indicated, *p <0.05.
exhibited an ability to inhibit LPS-induced TNF-
sion in mouse macrophages. Further, our studies found that five ac-
tive compounds exhibited inhibitory effects on LPS-induced TNF-
a
and IL-6 expres-
These results indicate that the bioactivity of analogues against
inflammation induced by LPS is associated with electronegativity
of the 40-substituent: the electron-donating ability of the 40-
substituent may increase the anti-inflammatory abilities of the
mono-carbonyl analogues whereas electric neutrality and elec-
tron-withdrawing moiety may reduce or remove such bioactivity.
Among all 23 compounds, C12 showed the highest potential as
an anti-inflammatory agent. In our previous publication25, we re-
ported that a structurally similar compound, A12, 2,6-bis(4-(3-
(dimethylamino)propoxy)benzylidene)cyclohexanone, possessed
intensive inhibitory effects against LPS-induced expression of
inflammatory factors. Here, our data demonstrated again that
N,N-dimethyl-propoxy containing long-chain analogues may be
considered as promising compounds for developing anti-inflam-
matory candidates. However, further studies including analysis of
the SAR of N-containing long-chain substituents and examination
of the underlying molecular mechanisms of these kinds of
compounds at the transcriptional or posttranscriptional level are
necessary to further establish this investigational pathway.
a,
IL-1b, IL-6, MCP-1, COX-2, PGES, iNOS and p65 NF-jB mRNA pro-
duction.25 The previous results indicated that these mono-carbonyl
analogues may possess anti-inflammatory activities comparable to
curcumin despite the absence of the b-diketone.
Concurrently, 23 mono-carbonyl analogues were synthesized
and their inhibitory effects on TNF-
ated (Fig. 1). Among 16 compounds which exhibited high inhibi-
tory abilities against LPS-induced TNF- and/or IL-6 expression,
a and IL-6 release were evalu-
a
the percentage of B-class compounds was 53.3%. As reported pre-
viously, it is observed here that acetone-derived B-class analogues
are more effective than cyclopentanone-derived A-class and cyclo-
hexanone-derived C-class analogues, indicating that the structure
of a 5-canbonyl linker may have a role on such activities.
Previous reports have found that the electric property of a sub-
stituent in the 40-position plays an important role in anti-inflam-
matory activities.25 In this study, compounds 70, 73 and 74
without substituents at the para position of the phenyl rings
showed little inhibitory activity; and an electron-withdrawing
chloro substituent at the 40-position removes the anti-inflamma-
tory activities of compound 67. In comparison, methoxyl-contain-
ing 68 and 72 and methyl-containing 69 showed significant
2.4. B75 and C12 inhibit TNF-a and IL-6 release in a dose-
dependent manner
Among the active analogues above, two compounds, B75 and
C12, demonstrated the highest activities and low cytotoxicity (data
not shown) in macrophages. They were chosen for further evalua-
tion of their dose-dependent inhibitory effects against LPS-induced
inhibitory activities against LPS-induced TNF-a and IL-6. The N,N-
dimethyl-propyl-alkalized C12 exhibited the strongest bioactivity
among the tested compounds.

C. Zhao et al. / Bioorg. Med. Chem. 18 (2010) 2388–2393
2391
TNF-
with B75 or C12 in a series of concentrations (1, 2.5, 5.0 and
10 M) for 2 h and were subsequently incubated with LPS
(0.5 g/ml) for 22 h. As shown in Figure 3, our data indicated a
dose-dependent inhibition of LPS-induced TNF- and IL-6 release
by B75 and C12. The inhibition of TNF- and IL-6 release by B75
a
and IL-6 release. RAW 264.7 macrophages were pre-treated
might serve as potential agents for the treatment of various inflam-
matory diseases.
l
l
4. Experimental section
4.1. Chemical synthesis
a
a
and C12 in a dose-dependent manner demonstrated their potential
for development as anti-inflammatory agents.
Melting points were determined on a Fisher-Johns melting
apparatus and were uncorrected.1 H NMR spectra were recorded
on a Varian INOVA-400 spectrometer. 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 3000+ spectrom-
eter. Column chromatography purifications were carried out on Sil-
ica Gel 60 (E. Merck, 70–230 mesh). The general procedure for
synthesis of these compounds was the same as reported in our pre-
vious papers.27 Briefly, an amount of 7.5 mmol acetone (B-class),
cyclopentanone (A class), or cyclohexanone (C-class) was added
to a solution of 15 mmol arylaldehyde in MeOH (10 ml). The solu-
tion was stirred at room temperature for 20 min, followed by drop-
wise 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
Combined with our previous studies24 regarding to the stability
and pharmacokinetics of these mono-carbonyl analogues, we con-
sidered that these mono-carbonyl analogues without b-diketone
may lend themselves favorably to the development of curcumin-
based anti-inflammatory drugs from both pharmacokinetic and
pharmacological standpoints.
3. Conclusions
In conclusion, we examined a series of 5-carbon linker-contain-
ing mono-carbonyl analogues of curcumin with potent inhibitory
activities against TNF-a and IL-6 release in LPS-stimulated RAW
264.7 macrophages. Discussion and conclusions were made
regarding structure-activity relationships. Compounds B75 and
C12 were the most potent analogues and exhibited anti-inflamma-
tory abilities in a dose-dependent manner in macrophages. This
presents the possibility that mono-carbonyl analogues of curcumin
Figure 3. B75 and C12 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
lg/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 SE of 3–5 independent experiments. Statistical significance relative to LPS is indicated, * p <0.05; ** p <0.01.

2392
C. Zhao et al. / Bioorg. Med. Chem. 18 (2010) 2388–2393
purified by chromatography over silica gel using CH2Cl2/CH3OH as
the eluent to yield compounds.
4.1.10. (2E,5E)-2,5-Bis(2,3-dichlorobenzylidene)cyclopentanone
(A70)
Yellow powder, 82.6% yield, mp 204.6–206.8 °C [215–216 °C,
lit.35]. 1H NMR (CDCl3) d: 2.95 (4H, s, CH2–CH2), 7.25 (2H, d,
J = 8.0 Hz, Ar-H6 ꢀ 2), 7.41 (2H, d, J = 8.0 Hz, Ar-H5 ꢀ 2), 7.47 (2H,
d, J = 8.0 Hz, Ar-H4 ꢀ 2), 7.88 (2H, s, Ar-CH@C ꢀ 2). ESI-MS m/z:
399.89 (M+1)+, calcd for C19H12Cl4O: 398.11.
4.1.1. (2E,5E)-2,5-Bis(2,4-dichlorobenzylidene)cyclopentanone
(A67)
Yellow powder, 82.7% yield, mp 209.4–212 °C [206–208 °C,
lit.32]. ESI-MS m/z: 399.16 (M+1)+, calcd for C19H12Cl4O: 398.11.
4.1.2. (1E,4E)-1,5-Bis(2,4-dichlorophenyl)penta-1,4-dien-3-
one(B67)
4.1.11. (2E,6E)-2,6-Bis(2,3-dichlorobenzylidene)cyclohexanone
(C70)
Yellow powder, 78.8% yield, mp 176.4–179.6 °C. 1H NMR
(CDCl3) d: 1.75 (2H, m, ˜CH2), 2.72 (4H, t, J = 4.0 Hz, CH2–C–CH2),
7.21 (4H, d, J = 8.0 Hz, Ar-H5,6 ꢀ 2), 7.44 (2H, t, J = 8.0 Hz, Ar-
H4 ꢀ 2), 7.85 (2H, s, Ar-CH@C ꢀ 2). ESI-MS m/z: 413.2 (M+1)+, calcd
for C20H14Cl4O: 412.14.
Yellow powder, 67.7% yield, mp 160.4–163.8 °C [168–169 °C,
lit.33]. ESI-MS m/z: 373.32 (M+1)+, calcd for C17H10Cl4O: 372.07.
4.1.3. (2E,6E)-2,6-Bis(2,4-dichlorobenzylidene)cyclohexanone
(C67)
Yellow powder, 73.9% yield, mp 160.1–163 °C [163–164 °C,
lit.32].
4.1.12. (2E,5E)-2,5-Bis(2,4,6-trimethoxybenzylidene)
cyclopentanone(A72)
4.1.4. (2E,5E)-2,5-Bis(2,4-dimethoxybenzylidene)
cyclopentanone(A68)
Yellow powder, 24.0% yield, mp 195–197.8 °C. 1H NMR (CDCl3)
d: 2.51 (4H, s, CH2–CH2), 3.78 (12H, s, Ar2,6-O–CH3 ꢀ 2), 3.82 (6H, s,
Ar4-O–CH3 ꢀ 2), 6.12 (4H, s, Ar-H3,5 ꢀ 2), 7.55 (2H, s, Ar-
CH@C ꢀ 2). ESI-MS m/z: 441.6 (M+1)+, calcd for C25H28O7: 440.49.
Yellow powder, 34.1% yield, mp 176.1–180.3 °C. 1H NMR
(CDCl3) d: 2.98 (4H, s, CH2–CH2), 3.84 (6H, s, Ar4-O–CH3 ꢀ 2),
3.86 (6H, s, Ar2-O–CH3 ꢀ 2), 6.46 (2H, s, Ar-H3 ꢀ 2), 6.52 (2H, d,
J = 8.0 Hz, Ar-H5 ꢀ 2), 7.49 (2H, d, J = 8.0 Hz, Ar-H6 ꢀ 2), 7.94 (2H,
s, Ar-CH@C ꢀ 2). ESI-MS m/z: 381.3 (M+1)+, calcd for C23H24O5:
380.43.
4.1.13. (1E,4E)-1,5-Bis(2,4,6-trimethoxyphenyl)penta-1,4-dien-
3-one(B72)30
Yellow powder, 17.0% yield, mp 210.4–215.4 °C. 1H-NMR
(CDCl3) d: 3.84 (6H, s, Ar4-O–CH3 ꢀ 2), 3.88 (12H, s, Ar2,6-O–
CH3 ꢀ 2), 6.12 (4H, s, Ar-H3,5 ꢀ 2), 7.44 (2H, d, J = 18.0 Hz, –CH–
C@O ꢀ 2), 8.11 (2H, d, J = 18.0 Hz, Ar-CH@C ꢀ 2). ESI-MS m/z:
415.3 (M+1)+, calcd for C23H26O7: 414.45.
4.1.5. (1E,4E)-1,5-Bis(2,4-dimethoxyphenyl)penta-1,4-dien-3-
one(B68)
Yellow powder, 35.7% yield, mp 132.7–136.5 °C [138–139 °C,
lit.34].
4.1.14. (2E,6E)-2,6-Bis(2,4,6-
4.1.6. (2E,6E)-2,6-Bis(2,4-dimethoxybenzylidene)cyclohexanone
(C68)
trimethoxybenzylidene)cyclohexanone(C72)29
Yellow powder, 39.7% yield, mp 201.8–203.4 °C. 1H NMR
(CDCl3) d: 1.62 (2H, t, J = 6.0 Hz, CH2), 2.43 (4H, t, J = 6.0 Hz, CH2–
C–CH2), 3.79 (12H, s, Ar2,6-O–CH3 ꢀ 2), 3.82 (6H, s, Ar4-O–
CH3 ꢀ 2), 6.13 (4H, s, Ar-H3,5 ꢀ 2), 7.61 (2H, s, Ar-CH@C ꢀ 2). ESI-
MS m/z: 456.1 (M+1)+, calcd for C26H30O7: 454.51.
Yellow powder, 36.2% yield, mp 155.8–160.7 °C. 1H NMR
(CDCl3) d: 1.74 (2H, t, J = 6.0 Hz, ˜CH2), 2.82 (4H, t, J = 6.0 Hz,
CH2–C–CH2), 3.82 (12H, s, –O–CH3 ꢀ 4), 6.46 (2H, s, Ar-H3 ꢀ 2),
6.49 (2H, d, J = 6.0 Hz, Ar-H5 ꢀ 2), 7.26 (2H, d, J = 6.0 Hz, Ar-
H6 ꢀ 2), 7.95 (2H, s, Ar-CH@C ꢀ 2). ESI-MS m/z: 395.5 (M+1)+, calcd
for C24H26O5: 394.46.
4.1.15. Bis(2-carboxylbenzylidene)cyclopentanone(A73)36
Green powder, 87.9% yield, mp >260 °C. 1H-NMR (HDO) d: 2.73
(4H, s, CH2–CH2), 7.32–7.44 (4H, m, Ar-H4,6 ꢀ 2), 7.52–7.63 (4H, m,
Ar-H5 ꢀ 2, Ar-CH@C ꢀ 2), 7.80 (2H, s, Ar-H3 ꢀ 2). ESI-MS m/z: 347.3
(M-1)ꢁ, calcd for C21H26O5: 348.35.
4.1.7. (2E,5E)-2,5-Bis(2,4-dimethylbenzylidene)cyclopentanone
(A69)
Yellow powder, 72.6% yield, mp 128.8–131.8 °C. 1H NMR
(CDCl3) d:2.35 (6H, s, Ar4-CH3 ꢀ 2), 2.43 (6H, s, Ar2-CH3 ꢀ 2), 2.99
(4H, s, CH2-CH2), 7.04 (2H, s, Ar-H5 ꢀ 2), 7.07 (2H, s, Ar-H3 ꢀ 2),
7.40 (2H, d, J = 8.0 Hz, Ar-H6 ꢀ 2), 7.79 (2H, s, Ar-CH@C ꢀ 2). ESI-
MS m/z: 317.4 (M+1)+, 339.3 (M+Na), calcd for C23H24O: 316.44.
4.1.16. Bis(2-carboxylbenzylidene)acetone(B73)
Green powder, 52.2% yield, mp >260 °C. 1H-NMR (HDO) d: 7.11
(2H, d, J = 18.0 Hz, @CH–C@O ꢀ 2), 7.29–7.34 (4H, m, Ar-H4,6 ꢀ 2),
7.51 (4H, m, Ar-H5 ꢀ 2, Ar-CH@C ꢀ 2), 7.61 (2H, d, J = 18.0 Hz, Ar-
CH@C ꢀ 2), 7.87 (2H, s, Ar-H3 ꢀ 2). ESI-MS m/z: 321.2 (Mꢁ1)ꢁ,
calcd for C19H14O5: 322.31.
4.1.8. (1E,4E)-1,5-Bis(2,4-dimethylphenyl)penta-1,4-dien-3-
one(B69)
Yellow powder, 38.2% yield, mp 95.5–98 °C. 1H-NMR (CDCl3) d:
2.34 (6H, s, Ar4-CH3 ꢀ 2), 2.44 (6H, s, Ar2-CH3 ꢀ 2), 6.96 (2H, d,
J = 16.0 Hz, @CH–C@O ꢀ 2), 7.03 (2H, d, J = 8.0 Hz, Ar-H5 ꢀ 2),
7.05 (2H, s, Ar-H3 ꢀ 2), 7.56 (2H, d, J = 8.0 Hz, Ar-H6 ꢀ 2), 8.00
(2H, d, J = 16.0 Hz, Ar-CH@C ꢀ 2). ESI-MS m/z: 292.4 (M+1)+,
603.9 (2 M+Na)+, calcd for C21H22O: 290.4.
4.1.17. Bis(2-carboxylbenzylidene)cyclohexanone(C73)
Green powder, 77.7% yield, mp >260 °C. 1H NMR (HDO) d: 1.65
(2H, m, ˜CH2), 2.66 (4H, t, J = 8.0 Hz, CH2–C–CH2), 7.36 (2H, d,
J = 8.0 Hz, Ar-H4 ꢀ 2), 7.41 (2H, t, J = 8.0 Hz, Ar-H6 ꢀ 2), 7.44 (2H,
s, Ar-CH@C ꢀ 2), 7.51 (2H, d, J = 8.0 Hz, Ar-H5 ꢀ 2,), 7.84 (2H, s,
Ar-H3 ꢀ 2). ESI-MS m/z: 361.3 (Mꢁ1)ꢁ, calcd for C22H18O5: 362.38.
4.1.9. (2E,6E)-2,6-Bis(2,4-dimethylbenzylidene)cyclohexanone
(C69)
Yellow powder, 36.8% yield, mp 118.6–121.5 °C. 1H NMR
(CDCl3) d: 1.70 (2H, m, ˜CH2), 2.32 (6H, s, Ar4-CH3 ꢀ 2), 2.34 (6H,
s, Ar2-CH3 ꢀ 2), 2.77 (4H, t, J = 4.0 Hz, CH2–C–CH2), 7.01 (2H, d,
J = 8.0 Hz, Ar-H5 ꢀ 2), 7.05 (2H, s, Ar-H3 ꢀ 2), 7.16 (2H, d,
J = 8.0 Hz, Ar-H6 ꢀ 2), 7.88 (2H, s, Ar-CH@C ꢀ 2). ESI-MS m/z:
331.6 (M+1)+, calcd for C24H26O: 330.46.
4.1.18. (2E,5E)-2,5-Bis(2,5-difluorobenzylidene)cyclopentanone
(A74)
Yellow powder, 29.4% yield, mp 208.1–209.8 °C. 1H NMR
(CDCl3) d: 3.05 (4H, s, CH2–CH2), 7.06–7.10 (6H, m, Ar-H), 7.73
(2H, s, Ar-CH@C ꢀ 2). ESI-MS m/z: 334.5 (M+1)+, 687.3 (2 M+Na)+,
calcd for C19H12F4O: 332.29.

C. Zhao et al. / Bioorg. Med. Chem. 18 (2010) 2388–2393
2393
4.1.19. (1E,4E)-1,5-bis(2,5-difluorophenyl)penta-1,4-dien-3-
one(B74)
TNF-
mouse TNF-
a
and IL-6 in the media were determined by ELISA using
and mouse IL-6 ELISA Kits (BOSTER, USA). After cen-
a
Yellow powder, 22.0% yield, mp 84.4–90.5 °C. 1H NMR (CDCl3)
d: 7.08 (2H, d, J = 18.0 Hz, @CH–C@O ꢀ 2), 7.09–7.13 (2H, m, Ar-
H6 ꢀ 2), 7.29–7.32 (4H, m, Ar-H3,4 ꢀ 2), 7.79 (2H, d, J = 18.0 Hz,
Ar-CH@C ꢀ 2). ESI-MS m/z: 306.2 (M+1)+, calcd for C17H10F4O:
306.25.
trifugation, the supernatant was separated and stored at ꢁ70 °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 cen-
trifuged at 12,000 rpm for 30 min at 4 °C, the total protein was col-
lected and the concentrations were determined using Bio-Rad
protein assay reagents. The total amount of the inflammatory fac-
tor in the media was normalized to the total protein amount of the
viable cell pellets.
4.1.20. (2E,6E)-2,6-Bis(2,5-difluorobenzylidene)cyclohexanone
(C74)
Yellow powder, 45.3% yield, mp 132–135.4 °C. 1H NMR (CDCl3)
d: 1.80 (2H, m, ˜CH2), 2.80 (4H, t, J = 6.0 Hz, CH2–C–CH2), 6.99–7.08
(6H, m, Ar-H), 7.74 (2H, s, Ar-CH@C ꢀ 2). ESI-MS m/z: 348.1 (M+1)+,
715.4 (2M+Na), calcd for C20H14F4O: 346.32.
Acknowledgments
4.1.21. (2E,5E)-2,5-Bis(2,6-difluorobenzylidene)cyclopentanone
(A75)
This work was supported by the National Natural Science Fund-
ing of China (20802054), Young Talent Funding of Zhejiang Depart-
ment of Health (2009QN020), China Postdoctroal Science
Foundation (20090461121) and Zhejiang Extremely Key Subject
of Pharmacology and Biochemical Pharmaceutics.
Yellow powder, 79.2% yield, mp 146.8–149.6 °C. 1H NMR
(CDCl3) d: 2.73 (4H, s, CH2–CH2), 6.92–6.95 (4H, m, Ar-H3,5 ꢀ 2),
7.29–7.34 (2H, m, Ar-H4 ꢀ 2), 7.50 (2H, s, Ar-CH@C ꢀ 2). ESI-MS
m/z: 334.4 (M+1)+, 687.4 (M+Na), calcd for C19H12F4O: 332.29.
Reference and notes
4.1.22. (1E,4E)-1,5-Bis(2,6-difluorophenyl)penta-1,4-dien-3-
one(B75)
1. Yang, H. S.; Gu, J.; Lin, X.; Grossman, H. B.; Ye, Y. Q.; Dinney, C. P.; Wu, X. F. Clin.
Cancer Res. 2008, 14, 2236.
2. McInnes, I. B.; Schett, G. Nat. Rev. Immunol. 2007, 7, 429.
3. Esch, T.; Stefano, G. Med. Sci. Monit. 2002, 8, HY1.
4. Lee, A. S.; Jung, Y. J.; Kim, D. H.; Lee, T. H.; Kang, K. P.; Lee, S.; Lee, N. H.; Sung, M.
J.; Kwon, D. Y.; Park, S. K.; Kim, W. Cell Physiol. Biochem. 2009, 24, 503.
5. Frazier, W. J.; Wang, X.; Wancket, L. M.; Li, X. A.; Meng, X.; Nelin, L. D.; Cato, A.
C.; Liu, Y. J. Immunol. 2009, 183, 7411.
Yellow powder, 49.3% yield, mp 135–138.5 °C. 1H NMR (CDCl3)
d: 6.95 (4H, t, Ar-H3,5 ꢀ 2), 7.29 (2H, d, J = 18.0 Hz, @CH–C@O ꢀ 2),
7.31–7.36 (2H, m, Ar-H4 ꢀ 2), 7.81 (2H, d, J = 18.0 Hz, Ar-
CH@C ꢀ 2). ESI-MS m/z: 307.7 (M+1)+ 329.6 (M+Na) 635.3
(2 M+Na), calcd for C17H10F4O: 306.25.
6. Waldner, M. J.; Neurath, M. F. Inflamm. Allergy-Drug Targets 2008, 7, 187.
7. Kim, K. J.; Lee, J. S.; Kwak, M. K.; Choi, H. G.; Yong, C. S.; Kim, J. A.; Lee, Y. R.;
Lyoo, W. S.; Park, Y. J. Eur. J. Pharmacol. 2009, 622, 52.
8. Esposito, E.; Cuzzocrea, S. Curr. Med. Chem. 2009, 16, 3152.
9. Mendis, E.; Kim, M. M.; Rajapakse, N.; Kim, S. K. Bioorg. Med. Chem. 2008, 16,
8390.
10. Reddy, B. V.; Sundari, J. S.; Balamurugan, E.; Menon, V. P. Mol. Cell Biochem.
2009, 331, 127.
11. O’Sullivan-Coyne, G.; O’Sullivan, G. C.; O’Donovan, T. R.; Piwocka, K.; McKenna,
S. L. Br. J. Cancer 2009, 101, 1585.
12. Aggarwal, B. B.; Kumar, A.; Bharti, A. C. Anticancer Res. 2003, 23, 363.
13. Kelloff, G. J.; Crowell, J. A.; Hawk, E. T. J. Cell Biochem. Suppl. 1996, 26, 72.
14. Hsu, C. H.; Cheng, A. L. Adv. Exp. Med. Biol. 2007, 595, 471.
15. Cho, J. W.; Lee, K. S.; Kim, C. W. Int. J. Mol. Med. 2007, 19, 469.
16. Reuter, S.; Charlet, J.; Juncker, T.; Teiten, M. H.; Dicato, M.; Diederich, M. Ann.
N.Y. Acad. Sci. 2009, 1171, 436.
4.1.23. (2E,6E)-2,6-Bis(2,6-difluorobenzylidene)cyclohexanone
(C75)
Yellow powder, 42.5% yield, mp 127.4–130.8 °C. 1H NMR
(CDCl3) d: 1.73 (2H, m, ˜CH2), 2.59 (4H, s, CH2–C–CH2), 6.91–6.94
(4H, m, Ar-H3,5 ꢀ 2), 7.27–7.32 (2H, m, Ar-H4 ꢀ 2), 7.55 (2H, s,
Ar-CH@C ꢀ 2). ESI-MS m/z: 348.7 (M+1)+, 715.2 (2M+Na), calcd
for C20H14F4O: 346.32.
4.1.24. (2E,6E)-2,6-Bis(4-(3-(dimethylamino)propoxy)
benzylidene)cyclohexanone (C12)
Green powder, 64.7% yield, mp 89.7–91.5 °C. 1H NMR (CDCl3) d:
1.85 (2H, m, ˜CH2), 1.97 (4H, m, –CH2– ꢀ 2), 2.25 (12H, s, N–
CH3 ꢀ 4), 2.45 (4H, t, J = 6.9 Hz, N–CH2– ꢀ 2), 2.91 (4H, t,
J = 4.8 Hz, CH2–CH2), 4.02 (4H, m, O–CH2 ꢀ 2), 6.92 (4H, d,
J = 8.4 Hz, Ar-H3,5 ꢀ 2), 7.26 (2H, s, Ar-CH@C ꢀ 2), 7.43 (4H, d,
J = 8.4 Hz, Ar-H2,6 ꢀ 2). ESI-MS m/z: 477.3 (M+1)+, calcd for
C30H40N2O3: 476.65.
17. Ghosh, A. K.; Kay, N. E.; Secreto, C. R.; Shanafelt, T. D. Clin. Cancer Res. 2009, 15,
1250.
18. Gautam, S. C.; Gao, X.; Dulchavsky, S. Adv. Exp. Med. Biol. 2007, 595, 321.
19. Sharma, R. A.; Steward, W. P.; Gescher, A. J. Adv. Exp. Med. Biol. 2007, 595, 453.
20. Pan, M. H.; Huang, T. M.; Lin, J. K. Drug Metab. Dispos. 1999, 27, 486.
21. Garcea, G.; Jones, D. J.; Singh, R.; Dennison, A. R.; Farmer, P. B.; Sharma, R. A.;
Steward, W. P.; Gescher, A. J.; Berry, D. P. Br. J. Cancer 2004, 90, 1011.
22. Rosemond, M. J.; St. John-Williams, L.; Yamaguchi, T.; Fujishita, T.; Walsh, J. S.
Chem. Biol. Interact. 2004, 147, 129.
23. Wang, Y. J.; Pan, M. H.; Cheng, A. L.; Lin, L. I.; Ho, Y. S.; Hsieh, C. Y.; Lin, J. K. J.
Pharm. Biomed. Anal. 1997, 15, 1867.
4.2. Cell line and reagents
24. Liang, G.; Shao, L. L.; Wang, Y.; Zhao, C. G.; Chu, Y. H.; Xiao, J.; Zhao, Y.; Li, X. K.;
Yang, S. L. Bioorg. Med. Chem. 2009, 17, 2623.
25. Liang, G.; Zhou, H. P.; Wang, Y.; Gurley, E. C.; Feng, B.; Chen, L.; Xiao, J.; Yang, S.
L.; Li, X. K. J. Cell Mol. Med. 2009.
26. Liang, G.; Li, X. K.; Chen, L.; Yang, S. L.; Wu, X. P.; Studer, E.; Gurley, E.;
Hylemon, P. B.; Ye, F. Q.; Li, Y.; Zhou, H. P. Bioorg. Med. Chem. Lett. 2008, 18,
1525.
27. Liang, G.; Yang, S. L.; Jiang, L. J.; Zhao, Y.; Shao, L. L.; Xiao, J.; Ye, F. Q.; Li, Y.; Li, X.
K. Chem. Pharm. Bull. (Tokyo) 2008, 56, 162.
Mouse RAW 264.7 macrophages were obtained from the Amer-
ican 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 dis-
solved in DMSO.
28. Stoll, L. L.; Denning, G. M.; Weintraub, N. L. Curr. Pharm. Des. 2006, 12, 4229.
29. Lee, K. H.; Ab Aziz, F. H.; Syahida, A.; Abas, F.; Shaari, K.; Israf, D. A.; Lajis, N. H.
Eur. J. Med. Chem. 2009, 44, 3195.
4.3. Cell treatment and ELISA assay
30. Fuchs, J. R.; Pandit, B.; Bhasin, D.; Etter, J. P.; Regan, N.; Abdelhamid, D.; Li, C.;
Lin, J.; Li, P. K. Bioorg. Med. Chem. Lett. 2009, 19, 2065.
Mouse RAW 264.7 macrophages were incubated in DMEM med-
ia (Gibco) supplemented with 10% FBS, 100 U/ml penicillin, and
31. Amolins, M. W.; Peterson, L. B.; Blagg, B. S. Bioorg. Med. Chem. 2009, 17, 360.
32. Yang, L.; Lu, J.; Bai, Y. J. Chin. J. Org. Chem. 2003, 23, 659.
33. Chen, G. F.; Li, J. T.; Duan, H. Y.; Li, T. S. Chem. J. Int. 2004, 6, 7.
34. Weber, W. M.; Hunsaker, L. A.; Abcouwer, S. F.; Deck, L. M.; Vander Jagt, D. L.
Bioorg. Med. Chem. 2005, 13, 3811.
100
ted with 10
then treated with LPS (0.5
l
g/ml streptomycin at 37 °C with 5% CO2. Cells were pre-trea-
M of curcumin, analogues or vehicle control for 2 h,
g/ml) for 22 h. After treatment, the cul-
l
l
35. Tereshko, A. B.; Kozlov, N. G.; Gusak, K. N. Rus. J. Gen. Chem. 2003, 73, 1619.
36. Liu, H.; Dinkova-Kostova, A.; Talalay, P. PNAS 2008, 105, 15926.
ture media and cells were collected separately. The culture media
collected were centrifuged at 1000 rpm for 10 min. The levels of