Synthesis and Cytotoxic Evaluation of Monocarbonyl Analogs of Curcumin as Potential Anti-Tumor Agents

Article abstract of DOI:10.1002/ddr.21291

(Table Presented) A series of mono-carbonyl curcumin analogs with different substituents at the 4/4′-position of the phenyl group were synthesized and screened for in vitro cytotoxicity against a panel of human cancer cell lines using a methyl thiazolyl tetrazolium assay. Several of the curcumin analogs, especially B114, exhibited a wide-spectrum of anti-tumor properties in all tested cell lines, indicating their potential in as anti-cancer lead compounds. Further toxicity testing in the NRK-52E kidney cell line revealed that the analogs A111, A113, and B114 had comparable or higher safety than curcumin. These data suggested that the introduction of appropriate substituents in the 4/4′-positions could be a promising approach for curcumin-based drug design.

Full text of DOI:10.1002/ddr.21291

DRUG DEVELOPMENT RESEARCH 77 : 43–49 (2016)
Research Article
Synthesis and Cytotoxic Evaluation of Monocarbonyl
Analogs of Curcumin as Potential Anti-Tumor Agents
Zheer Pan,1,2 Chengwei Chen,2 Yeli Zhou,2 Feng Xu,2 and Yaozeng Xu1*
1The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu,
People’s Republic of China
2Department of Orthopedic Surgery, The First Affiliated Hospital, Wenzhou Medical University,
Wenzhou, Zhejiang, People’s Republic of China
Strategy, Management and Health Policy
Enabling
Preclinical Development
Toxicology, Formulation
Drug Delivery,
Clinical Development
Phases I-III
Preclinical
Research
Postmarketing
Phase IV
Technology,
Genomics,
Proteomics
Regulatory, Quality,
Manufacturing
Pharmacokinetics
ABSTRACT
A series of mono-carbonyl curcumin analogs with different substituents at the 4/4’-posi-
tion of the phenyl group were synthesized and screened for in vitro cytotoxicity against a panel of
human cancer cell lines using a methyl thiazolyl tetrazolium assay. Several of the curcumin analogs,
especially B114, exhibited a wide-spectrum of anti-tumor properties in all tested cell lines, indicating
their potential in as anti-cancer lead compounds. Further toxicity testing in the NRK-52E kidney cell
line revealed that the analogs A111, A113, and B114 had comparable or higher safety than curcumin.
These data suggested that the introduction of appropriate substituents in the 4/4’-positions could be a
C
V
promising approach for curcumin-based drug design. Drug Dev Res 77 : 43–49, 2016.
2016 Wiley
Periodicals, Inc.
Key words: monocarbonyl curcumin analogs; synthesis; anti-tumor activity
INTRODUCTION
modification and analog design of curcumin to identify
more stable entities that may improve the in vivo meta-
bolic profile and enhance anti-proliferative activity
against cancer cells.
Curcumin is a polyphenolic natural product iso-
lated from the rhizome of Curcuma longa Linn
(Turmeric). It has multiple biological properties includ-
ing antioxidant, anti-inflammatory, anti-infective, anti-
cancer, and wound healing activities [Wilken et al.,
2011; Prasad et al., 2014; Rainey et al., 2015]. In
regard to its anti-cancer properties, curcumin can
inhibit cell growth and induce apoptosis in a variety of
cancer cell lines by modulating the activities of numer-
ous transcription factors, growth regulators, adhesion
molecules, apoptotic genes, and cellular signaling path-
ways [Chen et al., 2014; Wang et al., 2015]. Due to its
anti-tumor properties and extremely low toxicity, cur-
cumin is regarded as an ideal candidate for cancer
therapy [Hatcher et al., 2008]. However, its clinical
utility is limited by its chemical instability in vitro and
poor metabolic properties in vivo [Pan et al., 1999;
This is an open access article under the terms of the Creative
Commons Attribution-NonCommercial-NoDerivs License, which
permits use and distribution in any medium, provided the original
work is properly cited, the use is non-commercial and no modifica-
tions or adaptations are made.
*Correspondence to: Yaozeng Xu, The First Affiliated
Hospital of Soochow University, Suzhou, Jiangsu, People’s
Republic of China. E-mail: xyzsz2001@126.com
Contract grant sponsor: Zhejiang Natural Science Funding; con-
tract grant number: LY13H060007
Received 30 December 2015; Accepted 5 January 2016
Published online in Wiley Online Library (wileyonlinelibrary.
Rosemond et al., 2004]. This prompted the chemical com). DOI: 10.1002/ddr.21291
C
V
2016 The Authors Drug Development Research Published by Wiley Periodicals, Inc.

44
PAN ET AL.
The instability and metabolic defects of curcumin
may result from the high reactivity of the b-diketone
group in the curcumin structure. Deletion of the b-
diketone moiety increases the stability and improves the
bioavailability of curcumin analogs in rat [Liang et al.,
2009; Zhang et al., 2014]. Mono-carbonyl analogs of
curcumin (MACs) with better pharmacokinetic proper-
ties and bioactivities than curcumin may represent
novel therapeutic entities for the treatment of tumor
and inflammatory diseases [Zhao et al., 2013].
Substituents on the 4/40-position of curcumin
may represent an important pharmacophore for bio-
logical activity [Ohtsu et al., 2002; Lin et al., 2006a,b;
Quincoces Suarez et al., 2010]. The curcumin analog
ASC-J9, which has a methoxy group at the 4/40-posi-
tion had enhanced anti-androgenic activity and cyto-
toxicity against prostate cancer cell lines [Lin et al.,
2006a,b; Shi et al., 2009].
Fig. 1. Design of mono-carbonyl curcumin analogs. [Color figure
can be viewed in the online issue, which is available at wileyonli-
nelibrary.com.]
General Procedure (A)
In the present study, a variety of functional
groups was used to replace the 4/40-OH groups in
MACs to develop potent and selective anticancer
agents\(Figure.1). Three series of compounds with
different 5-carbon linkers (series A: cyclopentanone,
series B: acetone and series C: cyclopentanone) and
various substituents on the 4/40-position of benzene
rings were designed and synthesized. The cytotoxicity
of these MACs was screened in the non-small cell
lung cancer cell line H460 using a methyl thiazolyl
tetrazolium (MTT) assay. Further, an anti-tumor
evaluation in a panel of tumor cell lines showed that
some analogs may possess improved anti-cancer activ-
ities as compared to curcumin.
Compounds 4a, 4b, or 4c (5 mmol) were dis-
solved in dry acetone (10 mL) and anhydrous K2CO3
(7.5 mmol) and various alkyl halide (10 mmol) were
added. The mixture was refluxed until TLC analysis
indicated the reaction was complete; water was added
and the mixture extracted with ethyl acetate (50
mL33). The combined organic phase was washed
with H2O, dried over anhydrous MgSO4 and the sol-
vent removed under vacuum. The products were sep-
arated by column chromatography using petroleum
ether and ethyl acetate as eluent to yield compounds
A111, A112, A115, A116, B111, B115, B116,
C111, C112, C115, and C116, respectively.
METHODS AND MATERIALS
Chemistry
General Procedure (B)
Ac2O or other anhydride (10 mmol) was added
to a solution of 4a, 4b, or 4c (5 mmol) in THF
(10 mL) in the presence of triethylamine (2–3 drops).
The reaction mixture was stirred at room
Melting points were determined on a SGW X-4
melting point apparatus and are uncorrected. Electron-
spray ionization-mass spectra in positive mode (ESI-MS)
data were obtained with a Bruker Esquire 30001 spec-
1
trometer. H-NMR spectra were recorded on Bruker
600 MHz instrument, and chemical shifts presented as
parts per million with TMS as the internal reference. Sol-
vents were distilled and dried by standard methods. Tet-
˚
rahydrofuran (THF) was prepared by drying over 4A
molecular sieves overnight. Various alkyl halide and anhy-
dride reagent were purchased from Aladdin and Sigma-
Aldrich. Other chemicals were obtained from local sup-
pliers and were used without further purification.
(2E,5E)22,5-bis(4-hydroxy-3-methoxybenzylidene)cyclo-
pentanone (4a), (1E,4E)21,5-bis (4-hydroxy-3-methoxy-
phenyl)penta-1,4-dien-3-one (4b) and (2E,5E)22,5-
bis(4-hydroxy-3-methoxybenzylidene) cyclohexanone (4c)
were prepared as described in the literature. [Liang
et al., 2008].
Fig. 2. Synthesis of target compounds. Reagents and conditions: (a)
pyridine-PTSA, CH2Cl2, rt.; (b) acetone or cyclopentanone, NaOH,
EtOH, rt; (c) HCl, CH2Cl2, rt. (d) K2CO3, acetone, reflux; (e) TEA,
THF, rt.
Drug Dev. Res.

ANTI-TUMOR ACTIVITY OF MONOCARBONYL CURCUMIN ANALOGS
45
temperature and the mixture extracted with ethyl
A-115 - (2E,5E)-2,5-bis(3-Methoxy-4-((3-
methylbut-2-en-1-yl)oxy)benzylidene)
cyclopentanone
acetate (50 mL33) and the organic phase was
washed with H2O, dried over anhydrous MgSO4 and
the solvent evaporated to dryness. The crude product
was purified by column chromatography eluting with
petroleum ether and ethyl acetate to afford A110,
A113, B110, B113, and B114.
Orange powder, 98.9% yield, mp 148.2-148.68C.
1H-NMR (CDCl3) d:1.754-1.785(12H, m, ACH334),
3.113 (4H, s, CH2ACH2), 3.917 (6H, s, AOCH332),
4.645 (4H, d, J 5 6.6 HZ, AOCH2 32), 5.522 (2H, t,
ACH@C 32), 6.935 (2H, d, J 5 8.4 HZ, ArAH532),
7.135 (2H, s, ArAH232), 7.205 (2H, d, J 5 8.4 HZ,
ArAH632), 7.542(2H, s, ArACH@32). ESI-MS m/z:
489.1(M1H)1, calculated for C31H36O5: 488.26.
A110 - ((1E,1’E)-(2-Oxocyclopentane-1,3-
diylidene)bis(methanylylidene))bis(2-methoxy-4,1-
phenylene) dibenzoate
Yellow powder, 64.5% yield, mp 193.7–194.98C.
1H-NMR (CDCl3) d: 3.163 (4H, s, CH2ACH2), 3.855
(6H, s, AOCH3), 7.243 (2H, d, J 5 9.0 HZ, ArAH5),
A-116 - (2E,5E)-2,5-bis(3-Methoxy-4-
propoxybenzylidene)cyclopentanone
7.25 (2H, s, ACH@C), 7.282 (2H, 0d, J 5 8.4 HZ, Ar-
Yellow powder, 68.5% yield, mp 112.2-114.28C.
1H-NMR (CDCl3) d: 1.043 (6H, t, ACH332), 1.877-
1.913 (4H, m, ACH2A32), 3.112 (4H, s,
CH2ACH2), 3.865 (6H, s, AOCH332), 4.038 (4H, t,
AOCH232), 6.933 (2H, d, J 5 8.4 HZ, ArAH532),
7.140 (2H, s, ArAH232), 7.211(2H, d, J 5 8.4 HZ,
ArAH632), 7.541 (2H, s, ArACH@ 32). ESI-MS m/
z: 437.1(M1H)1, calculated for C27H32O5: 436.22.
0
H6), 7.483–7.540 (4H, m, ArAH3 ,5 ), 7.609 (2H, s,
0
ArAH2), 7.659(2H, d, J 5 7.2 HZ, ArAH4 ), 8.212-
0
0
8.236
(4H,
m,
ArAH2 ,6 ).
ESI-MS
m/z:
561.1(M1H)1, calculated for C35H28O7: 560.18.
A-111 - (2E,5E)-2,5-bis(4-(2-Hydroxyethoxy)-3-
methoxybenzylidene)cyclopentanone
Yellow powder, 73.6% yield, mp 185.3–187.88C.
1H-NMR (CDCl3) d: 3.108 (4H, s, CH2ACH2), 3.917
(6H, s, AOCH332), 3.991 (4H, t, AOCH2 32),
4.185 (4H, t, AOCH2 32), 6.974 (2H, d, J 5 7.8 HZ,
ArAH532), 7.133(2H, s, ArAH2 32), 7.208(2H, d,
J 5 7.8 HZ, ArAH632), 7.535 (2H, s, ArACH@32).
ESI-MS m/z: 441.0(M1H)1, calculated for
C25H28O7: 440.18.
B-110 - ((1E,4E)-3-Oxopenta-1,4-diene-1,5-
diyl)bis(2-methoxy-4,1-phenylene)dibenzoate
Yellow powder, 56.2% yield, mp 234.0–235.78C.
1H-NMR (CDCl3) d: 3.88 (6H, s, AOCH332), 7.02
(2H, d, J 5 15.6 Hz, COACH@), 7.23 (2H, d,
ArAH5), 7.280 (2H, d, ArAH6), 7.53 (4H, t, J 5 7.8
0
HZ, ArAH3 ,5 ), 7.61 (2H, s, ArAH2), 7.65 (2H, t,
0
J 5 7.8 HZ, ArAH4 ), 7.71 (2H, d, J 5 15.6 HZ,
0
0
ArACH@), 8.23 (4H, d, ArAH2 ,6 ). ESI-MS m/z:
535.0(M1H)1, calculated for C33H26O7: 534.17.
A-112 - (2E,5E)-2,5-bis(4-(Cyclopentyloxy)-3-
methoxybenzylidene)cyclopentanone
Yellow powder, 45.3% yield, mp 114.0–116.28C.
0
0
1H-NMR (CDCl3) d: 1.58 (8H, m, H3 ,4 ), 2.23 (8H,
B-111 - (1E,4E)-1,5-bis(4-(2-Hydroxyethoxy)-3-
methoxyphenyl)penta-1,4-dien-3-on
0
0
0
m, H2 ,5 ), 3.81 (2H, s, H1 ), 3.91 (6H, s, AOCH332),
3.10 (4H, s, CH2ACH2), 6.96 (2H, d, J 5 7.8 HZ,
ArAH5), 7.20 (2H, d, J 5 7.8 HZ, ArAH6), 7.27 (2H,
s, ArAH2), 7.53 (2H, s, ArACH@]). ESI-MS m/z:
489.1 (M1H)1, calculated for C31H36O5: 488.26.
Yellow powder, 75.4% yield, mp 91.2–93.68C.
1H-NMR (DMSO) d: 3.344 (6H, s, AOCH332),
3.387 (2H, s, AOH), 3.73 (4H, t, J 5 5.4 HZ,
CH2ACH2), 4.03 (4H, t, J 5 4.8 HZ, AOCH2), 7.03
(2H, d, J 5 15.6 HZ, COACH@), 7.17 (2H, d,
ArAH5), 7.29 (2H, d, ArAH6), 7.49 (2H, s, ArAH2),
7.71 (2H, d, J 5 15.6 HZ, ArACH@). ESI-MS m/z:
437.0(M1Na)1, calculated for C23H26O7: 414.17.
A-113- (1E,1’E)-(2-Oxocyclopentane-1,3-
diylidene)bis(methanylylidene))bis (2-methoxy-4,1-
phenylene) diacetate
Yellow powder, 92.0% yield, mp 200.6-201.88C.
1H-NMR (CDCl3) d: 2.337 (6H, s, COACH332),
3.110 (4H, s, CH2ACH2), 3.884 (6H, s, AOCH332),
7.110 (2H, d, J 5 7.8 HZ, ArAH532), 7.173(2H, s,
ArAH232), 7.214 (2H, d, J 5 8.4 HZ, ArAH632),
7.557 (2H, s, ArACH@ 32). ESI-MS m/z: 437.2
B-113 (1E,4E)-3-Oxopenta-1,4-diene-1,5-diyl)bis(2-
methoxy-4,1-phenylene)diacetate
Yellow powder, 60.0% yield, mp 72.8–75.28C .
1H-NMR (CDCl3) d: 2.33 (6H, s, ACOCH332), 3.88
(6H, s, AOCH3 32), 7.03 (2H, d, J 5 15.6 HZ,
(M1H)1, 459.2 (M1Na)1, calculated for C25H24O7: ACOCH@ 32), 7.11 (2H, d, J 5 7.8 HZ, ArAH532),
436.15.
7.22 (2H, d, J 5 8.4 HZ, ArAH6 32), 7.27 (2H, s,
Drug Dev. Res.

46
PAN ET AL.
TABLE 1. The Structures of Synthesized Compounds
Compd.
n
R
Compd.
N
R
Compd.
n
R
A110
A111
A112
A113
A115
A116
2
2
2
2
2
2
-acetophenone
-CH2CH2OH
-cyclopentane
-OCOCH3
-CH2CH5C(CH3)2
-(CH2)2CH3
B110
B111
B113
B114
B115
B116
0
0
0
0
0
0
-acetophenone
-CH2CH2OH
-OCOCH3
-COCH2CH3
-CH2CH5C(CH3)2
-(CH2)2CH3
C111
C112
C115
C116
3
3
3
3
-CH2CH2OH
-cyclopentane
-CH2CH5C(CH3)2
-(CH2)2CH3
ArAH2 32), 7.71 (2H, d, J 5 15.6 HZ, ArACH@ ArACH@32). ESI-MS m/z: 411.1(M1H)1, calcu-
32). ESI-MS m/z: 432.1 (M1Na)1, calculated for lated for C25H30O5: 410.42.
C23H22O7: 410.14.
C-111-(2E,6E)-2,6-bis(4-(2-Hydroxyethoxy)-3-
B-114 - ((1E,4E)-3-Oxopenta-1,4-diene-1,5-
diyl)bis(2-methoxy-4,1-phenylene)dipropionate
methoxybenzylidene)cyclohexanone
Yellow powder, 72.1% yield, mp 136.3–138.48C
1
Yellow powder, 47.9% yield, mp 147.4–148.78C
.1H-NMR (CDCl3) d: 1.247–1.298 (6H, m,
ACH332), 2.614–2.652 (4H, m, ACH232), 3.885
(6H, s, AOCH332), 7.010(2H, d, J 5 15.6 Hz,
COACH@32), 7.078 (2H, d, J 5 7.8Hz, ArAH532),
7.181 (2H, s, ArAH2 32), 7.219 (2H, d, J 5 7.8Hz,
. H-NMR (CDCl3) d: 1.23–1.25 (2H, m, ACH2A),
2.93 (4H, d, J 5 6.0 HZ, ACH2A), 3.81 (4H, t,
ACH2A32), 3.91 (6H, s, AOCH332), 4.19 (4H, t,
AOCH232), 6.96 (2H, d, J 5 7.8 HZ, ArAH532),
7.20 (2H, d, J 5 7.8 HZ, ArAH632), 7.27 (2H, s,
ArAH232), 7.53 (2H, s, ArACH@32). ESI-MS m/z:
455.2(M11)1, 477.1(M1Na)1, calculated for
C26H30O7: 454.20.
ArAH6
32),
7.810(2H,
d,
J 5 15.6
HZ,
ArACH@32). ESI-MS m/z: 438.9(M1H)1, calcu-
lated for C25H26O7: 438.17.
C-112 (2E,6E)22,6-bis(4-(Cyclopentyloxy)-3-
B-115 -(1E,4E)-1,5-bis(3-Methoxy-4-((3-methylbut-
2-en-1-yl)oxy)phenyl)penta-1,4-dien-3-one
methoxybenzylidene)cyclohexanone
Yellow powder, 42.3% yield, mp 96.2–99.68C .
1H-NMR (CDCl3) d: 1.24–1.25 (2H, m, ACH2A),
1.57–1.59 (8H, m, ACH2A), 2.22–2.23 (8H, m,
ACH2A), 2.93 (4H, t, ACH2A), 3.81 (2H, s,
Brick red powder, 58.6% yield, mp 93.9–95.28C.
1H-NMR (CDCl3) d: 1.755–1.780 (12H, m,
ACH334), 3.910 (6H, s, AOCH332), 4.65 (4H, d,
J 5 6.6 HZ, AOCH232), 5.520 (2H, t, ACH@C32),
6.9400 (2H, d, J 5 8.4 HZ, ArAH532), 7.02 (2H, d,
J 5 15.6 Hz, ACH@32), 7.190 (2H, d, J 5 8.4 HZ,
ArAH632), 7.27 (2H, s, ArAH232), 7.71(2H, d,
150
100
J 5 15.6
HZ,
ArACH@32).
ESI-MS
m/z:
50
**
463.1(M1H)1, calculated for C29H34O5: 462.24.
**
**
**
0
n
0
1
5
0
i
11
B116 C1
12
13
11
14
15
B1
B-116 - (1E,4E)-1,5-bis(3-Methoxy-4-
propoxyphenyl)penta-1,4-dien-3-one
1
115
C116
1
1
1
B
MSO
A11
A11
A112
A
A11
A116 B11
B
B113
C
C
cum
r
D
Cu
Yellow powder, 42.2% yield, mp 134.5–136.98C.
1H-NMR (CDCl3) d: 1.02 (6H, t, ACH332), 1.83
(4H, m, ACH232), 3.86 (6H, s, AOCH332), 4.05
(4H, t, AOCH232), 6.94 (2H, d, ArAH532), 7.14
(2H, d, ArAH632), 7.21 (2H, s, ArAH232), 7.54
Fig. 3. Evaluation of the anti-proliferative activities of synthesized
compounds. U2-OS cells were seeded in 96 well plate and incu-
bated with different compounds with the concentration of 20 lM
for 24 hours. Cell viability was assessed using an MTT assay. Data
are expressed as fold change relative to control values (samples
treated with DMSO alone), mean 6 SEM. n ꢀ 3. **P < 0.01 vs.
(2H, s, ArACH@32), 7.71 (2H, d, J 5 15.6 HZ, DMSO group.
Drug Dev. Res.

ANTI-TUMOR ACTIVITY OF MONOCARBONYL CURCUMIN ANALOGS
47
5000 cells per well in 1640 medium, supplemented
with 5% heat-inactivated serum, 100 U/ml penicillin,
and 100 lg/mL streptomycin. Cells were maintained
at 378C in a humidified atmosphere containing 5%
CO2. All experiments were carried out 24 h after
cells were seeded. Tested compounds were dissolved
in DMSO, and diluted with 1640 medium to the dif-
ferent concentrations of each compound. The tumor
cells were incubated with test compounds for 72 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. Plates
were then incubated in a CO2 incubator for 3 h, cells
dissolved with 150 lL DMSO, and then analyzed in
a multi-well-plate reader at 490 nm. All results were
representative from three or more independent
experiments.
TABLE 2. Cytotoxic Test of A111, A113, B114 (IC50) Against
Different Cancer Cell Lines
IC50 (M)
Comp.
cell line
A111
A113
B114
Curcumin
U2-OS
OS-732
A549
HepG2
P815
2.84
8.17
>20
>20
3.26
2.23
>20
6.91
8.15
>20
>20
>20
>20
>20
7.24
9.23
10.86
15.32
0.84
9.94
11.6
13.97
>20
10.33
15
PC 3
Hela
4.12
3.23
17.5
ACHA), 3.91 (6H, s, AOCH332), 6.96 (2H, d,
J 5 7.8 HZ, ArAH532), 7.20 (2H, d, J 5 7.8 HZ,
ArAH632), 7.27 (2H, s, ArAH232), 7.53 (2H, s,
ArACH@32). ESI-MS m/z: 503.2(M11)1, calcu-
lated for C32H38O5: 502.27.
RESULTS AND DISCUSSION
Chemistry
C-115 (2E,6E)-2,6-bis(3-Methoxy-4-((3-methylbut-
2-en-1-yl)oxy)benzylidene)cyclohexanone
The synthesis of the target MACs is shown in Fig-
ure 2. As the method reported in literature, the synthesis
of compounds 4a, 4b and 4c started from the protection
of commercially available vanillin with tetrahydropyran-
2-yl to afford protected compound 2. 3-methoxy-4-(tetra-
hydro-2H-pyran-2-yloxy)benzaldehyde 2 was obtained
by reacting vanillin 1 with 3,4-2H-dihydropyran in the
presence of pyridinium p-toluenesulfonate. The aldol
condensation of 2 with cyclopentanone or acetone
afforded compound 3a, 3b and 3c, respectively. Hydroxy-
lated analogs 4a, 4b and 4c were then obtained by
deprotection with diluted hydrochloric acid as catalyst.
Brick red powder, 56.7% yield, mp 89.2–91.68C
. H-NMR (CDCl3) d: 1.25-1.26 (2H, m, ACH2A),
1
2.93 (4H, t, ACH2A), 3.81 (4H, t, ACH2A32), 3.91
(6H, s, AOCH332), 4.19 (4H, t, AOCH232), 6.96
(2H, d, J 5 7.8 HZ, ArAH532), 7.12 (2H, d, J 5 15.6
Hz, ACHA32), 7.20 (2H, d, J 5 7.8 HZ, ArAH632),
7.27 (2H, s, ArAH232), 7.53 (2H, s, ArACH@32).
ESI-MS m/z: 455.2(M11)1, 477.1(M1Na)1, calcu-
lated for C26H30O7: 454.20.
C-116 - (2E,6E)-2,6-bis(3-Methoxy-4-
propoxybenzylidene)cyclohexanone
150
Yellow powder, 45.3% yield, mp 136.3–138.48C.
1H-NMR (CDCl3) d: 1.039 (6H, t, ACH332), 1.24–
1.26 (2H, m, ACH2A), 1.81 (4H, m, ACH2A32),
2.93 (4H, t, ACH2A), 3.89 (6H, s, AOCH332), 4.03
(4H, t, AOCH232), 6.91 (2H, d, J 5 8.4 HZ,
ArAH532), 7.09 (2H, d, J 5 8.4 HZ, ArAH632),
7.27 (2H, s, ArAH232), 7.74 (2H, s, ArACH@32).
ESI-MS m/z: 451.1(M11)1, calculated for C28H34O5:
450.57.
100
*
50
0
Cell Culture and Methyl Thiazolyl Tetrazolium
Assay
n
4
1
3
i
1
A1
1
A1
1
B1
SO
DM
um
c
r
Cu
U2-OS, OS-732, HepG2, A549, P815, PC3,
HeLa and normal renal NRK-52E cell lines were
obtained from the American Type Culture Collection
(ATCC, USA). All cell lines were cultured according
to ATCC recommendations. Antiproliferative activity
was determined using a MTT assay. Briefly, cells
Fig. 4. Evaluation of the cytotoxicity of active compounds in NRK-
52E cells. NRK-52E cells were seeded in 96 well plate and incu-
bated with active compounds at a concentration of 20 lM for 24
hours. Cell viability was assessed using MTT assay. Data are
expressed as fold change relative to control values (samples treated
were seeded into 96-well plates at a density of 3000– with DMSO alone), mean 6 SEM. n ꢀ 3. *P < 0.05 vs. DMSO group.
Drug Dev. Res.

48
PAN ET AL.
Finally, the intermediate compounds 4a, 4b and 4c which inhibited tumor cell proliferation. In particular,
were reacted with different alkyl halides in the presence
of anhydrous potassium carbonate to yield the target
compounds A111, 112, A115, A116, B111, B115,
B116, C111, C112, C115, and C116. The 40-OH of
4a, 4b, and 4c was also substituted by various alkyl esters
through reacted with anhydride in the presence of TEA
as catalyst. In this way, A110, A113, B110, B113, and
B114 were obtained in good yield. The structures of all
compounds are listed in Table 1.
B114 had higher activity than curcumin against the
tested tumor cell lines. Meanwhile, A111 showed
potent cytotoxicity against human osteosarcoma cell
line U2-OS, prostate cancer cell line PC-3, human
osteosarcoma cell line OS-732, and mouse mastocy-
toma cell line P815, implying their specific potential
in the chemotherapy of cancer. Toxicity testing in
vitro showed that A111, A113, and B114 did not
affect growth of normal renal cells. This study
presents a series of novel curcumin derivatives as
potential anti-tumor candidates.
Antiproliferative Activity
The in vitro anti-tumor activities of synthesized
compounds were first evaluated in the human osteo-
sarcoma cell line U2-OS using MTT assay at a con-
centration of 20 lM. The results presented in Figure
3 showed that these synthetic curcumin analogues
exhibited different cytotoxic activities against U2-OS
cells. Most compounds displayed lower activity com-
pared with curcumin except compounds A111,
A113, and B114. These showed more pronounced
anti-tumor activity than curcumin in U2-OS cells. As
curcumin has a wide-spectrum of anti-tumor proper-
ties, the IC50 values for A111, A113, and B114 were
determined in other cancer cell lines. The anti-
proliferative activity of active compounds A111,
A113, and B114 on human osteosarcoma OS-732,
human hepatic cancer HepG2, lung cancer A549,
prostate cancer PC-3, HeLa and mouse leukemia
P815 cell lines were evaluated in the MTT assay. The
results shown in Table 2 suggest that B114 had
wide-spectrum anti-proliferative properties, especially
against P815 (IC50 5 840 nM). A111 had selective,
potent cytotoxicity against U2-OS (IC50 5 2.84 mM),
OS-732 (IC50 5 8.17 mM), P815 (IC50 5 3.26 mM)
and PC-3 (IC50 5 2.23 mM) cell lines, while A113
was only cytotoxic in U2-OS (IC50 5 7.24 mM) and
OS-732 (IC50 5 9.23 mM) cells. Further studies were
carried out to observe the preliminary safety of these
compounds in the normal rat renal epithelial cell line
NRK-52E by MTT assay. The results shown in Fig-
ure 4 indicated that the cell viability was reduced by
approximately 50% when cells were incubated with
20 lM curcumin. However, growth of NRK-52E cells
was only minimally affected by A111, while A113
and B114 had no inhibitory effect on renal cell
growth suggesting that these three compounds have
comparable or improved safety over curcumin at the
same concentration.
ACKNOWLEDGMENT
Financial support was provided by the Zhejiang
Natural Science Funding (Grants LY13H060007).
REFERENCES
Chen B, Zhang Y, Wang Y, Rao J, Jiang X, Xu Z. 2014. Curcumin
inhibits proliferation of breast cancer cells through Nrf2-
mediated down-regulation of Fen1 expression. J Steroid Bio-
chem Mol Biol 143:11–18.
Hatcher H, Planalp R, Cho J, Torti FM, Torti SV. 2008. Curcu-
min: from ancient medicine to current clinical trials. Cell Mol
Life Sci 65:1631–1652.
Liang G, Shao L, Wang Y, Zhao C, Chu Y, Xiao J, Zhao Y, Li X,
Yang S. 2009. Exploration and synthesis of curcumin analogues
with improved structural stability both in vitro and in vivo as
cytotoxic agents. Bioorg Med Chem 17:2623–2631.
Liang G, Yang S, Jiang L, Zhao Y, Shao L, Xiao J, Ye F, Li Y, Li
X. 2008. Synthesis and anti-bacterial properties of mono-car-
bonyl analogues of curcumin. Chem Pharm Bull 56(2):162–167.
Lin L, Shi Q, Su CY, Shih CC, Lee KH. 2006a. Antitumor agents
247. New 4-ethoxycarbonylethyl curcumin analogs as potential
antiandrogenic agents. Bioorg Med Chem 14:2527–2534.
Lin L, Shi Q, Nyarko AK, Bastow KF, Wu CC, Su CY, Shih CY,
Lee KH. 2006b. Antitumor agents. 250. Design and synthesis
of new curcumin analogues as potential anti-prostate cancer
agents. J Med Chem 49:3963–3972.
Ohtsu H, Xiao Z, Ishida J, Nagai M, Wang HK, Itokawa H, Su
CY, Shih C, Chiang T, Chang E, et al. 2002. Antitumor agents.
217. Curcumin analogues as novel androgen receptor antago-
nists with potential as anti-prostate cancer agents. J Med Chem
45:5037–5042.
Pan MH, Huang TM, Lin JK. 1999. Biotransformation of curcu-
min through reduction and glucuronidation in mice. Drug
Metab Dispos 27:486–494.
Prasad S, Gupta SC, Tyagi AK, Aggarwal BB. 2014. Curcumin, a
component of golden spice: from bedside to bench and back.
Biotechnol Adv 32:1053–1064.
Quincoces Suarez JA, Rando DG, Santos RP, Goncalves CP,
Ferreira E, de Carvalho JE, et al. 2010. New antitumoral
agents I: in vitro anticancer activity and in vivo acute toxicity of
synthetic 1,5-bis(4-hydroxy-3-methoxyphenyl)21,4-pentadien-3-
one and derivatives. Bioorg Med Chem 18:6275–6281.
CONCLUSIONS
In summary, we designed and synthesized a
series of monocarbonyl curcumin analogs, some of
Drug Dev. Res.

ANTI-TUMOR ACTIVITY OF MONOCARBONYL CURCUMIN ANALOGS
49
Rainey N, Motte L, Aggarwal BB, Petit PX 2015. Curcumin
hormesis mediates a cross-talk between autophagy and cell
death. Cell Death Dis 6:e2003.
mitochondrial-mediated apoptosis through hexokinase II in
human colorectal cancer cells in vitro. Anti-Cancer Drugs 26:
15–24.
Rosemond MJ, St John-Williams L, Yamaguchi T, Fujishita T,
Walsh JS. 2004. Enzymology of a carbonyl reduction clearance
pathway for the HIV integrase inhibitor, S-1360: role of human
liver cytosolic aldo-keto reductases. Chem Biol Interact 147:
129–139.
Wilken R, Veena MS, Wang MB, Srivatsan ES. 2011. Curcumin:
a review of anti-cancer properties and therapeutic activity in
head and neck squamous cell carcinoma. Mol Cancer 10:12.
Zhang Y, Zhao C, He W, Wang Z, Fang Q, Xiao B, et al. 2014.
Discovery and evaluation of asymmetrical monocarbonyl ana-
logs of curcumin as anti-inflammatory agents. Drug Des Dev
Ther 8:373–382.
Shi Q, Shih CC, Lee KH. 2009. Novel anti-prostate cancer curcu-
min analogues that enhance androgen receptor degradation
activity. anti-cancer agents in medicinal chemistry 9:904–912.
Zhao C, Liu Z, Liang G 2013. Promising curcumin-based drug
design: mono-carbonyl analogues of curcumin (MACs). Curr
Pharm Des 19:2114–2135.
Wang K, Fan H, Chen Q, Ma G, Zhu M, Zhang X, et al. 2015.
Curcumin
inhibits
aerobic
glycolysis
and
induces
Drug Dev. Res.