Discovery of novel anti-tumor curcumin analogues from the optimization of curcumin scaffold

Article abstract of DOI:10.1007/s00044-017-1946-2

A series of novel curcumin analogues were synthesized by optimization of its aromatic ring. The antiproliferative activities of these analogues were screened against four human cancer cell lines by MTT assay and some displayed significant improvement in a

Full text of DOI:10.1007/s00044-017-1946-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1946-2
ORIGINAL RESEARCH
Discovery of novel anti-tumor curcumin analogues from the
optimization of curcumin scaffold
1 ●
2 ●
3 ●
3 ●
3
Laiyin Zhang Haiyang Zong Huiping Lu Jingru Gong Fenfen Ma
Received: 7 March 2017 / Accepted: 3 June 2017
©
Springer Science+Business Media, LLC 2017
Abstract A series of novel curcumin analogues were syn-
thesized by optimization of its aromatic ring. The anti-
proliferative activities of these analogues were screened
against four human cancer cell lines by MTT assay and
some displayed significant improvement in antiproliferative
activity compared with curcumin. The most potent com-
pound 10 exhibited antiproliferative activity against Caco-2,
Bel-7402, MDA-MB-231, and DU145 cancer cells with
IC50 values of 2.04, 1.54, 4.99, and 3.22 μM, respectively.
Annexin V/propidium iodide double staining and flow
cytometry cell cycle analysis using propidium iodide DNA
staining revealed that compound 10 induced apoptosis and
cell cycle arrest at G1 phase in Bel-7402 cells in a dose-
dependent manner. Moreover, in an in vivo mouse model of
liver cancer xenograft, compound 10 substantially inhibited
liver tumor growth at a dose of 20 mg/kg/day without
observable toxic effect.
Intorduction
Natural products (NPs) proved to be an excellent source of
lead compounds for novel drug discovery, especially for the
discovery of antitumor agents (Ganesan 2008; Cragg and
Newman 2013). Although naturally active substances are
good lead compounds for the discovery of new drugs, most
of them suffer from various deficiencies or shortcomings.
Therefore, structural modification of NPs is highly neces-
sary to develop novel compounds with specific properties
(Newman 2008).
The polyphenol curcumin (1, Fig. 1), the primary
bioactive compound found in the rhizomes of Curcuma
longa L. (turmeric), is one of the most thoroughly investi-
gated natural product, documented by numerous studies
claiming its efficacy and safety as an antioxidative, anti-
inflammatory, and antitumor agent (Lelli et al. 2016; Lin
and Wu 2016; Yu et al. 2016). Recently, researches have
demonstrated the remarkable cancer preventive properties
of curcumin, it exhibits growth-suppressive activity against
a broad range of cancers including skin, stomach, breast,
and colon cancers (Goldhahn et al. 2015; Liu et al. 2016;
Sahu et al. 2016; Singh et al. 2016; Yu et al. 2016). Its’
antitumor properties include growth inhibition and apopto-
sis induction in a veriety of cancer cell lines in vitro, as well
as the ability to inhibit tumorigenesis in vivo (Cheng et al.
●
●
●
Keywords Curcumin Optimization Antitumor
Apoptosis
Electronic supplementary material The online version of this article
doi:10.1007/s00044-017-1946-2) contains supplementary material,
which is available to authorized users.
(
2
016; Zhu et al. 2016). However, its clinical application
*
Fenfen Ma
3211030048@fudan.edu.cn
was largely hampered by its low bioavailability and mod-
erate potency (Mahran et al. 2016; Liu et al. 2016).
Therefore, it is highly desirable to synthesize novel curcu-
min analogues through structural modification and develop
potential anticancer drugs (Momtazi and Sahebkar 2016).
There are some naturally existing curcuminoids featured
extremely similar structures to curcumin (Figs. 1–3), and
slight variations of structures between these curcuminoids
1
1
2
3
Department of Pharmacy, Linyi people’s Hospital, Linyi 276000,
China
Department of Orthopedic Surgery, Changzheng Hospital, The
Second Military Medical University, Shanghai 200003, China
Department of Pharmacy, Pudong Hospital, Fudan University,
Shanghai 201399, China

Med Chem Res
Fig. 1 Structures of curcumin (1), desmethoxycurcumin (2), and bisdesmethoxycurcumin (3)
focus on the aromatic ring. It is noteworthy that these
analogues display better antiproliferative activities when
compared with curcumin, which indicates that structural
modifications on the aromatic ring might be an optimal way
to get novel curcumin analogues possessing better anti-
tumor profiles (Wang and Qiu 2013; Makarov et al. 2016;
Teymouri et al. 2016).
In this research, in order develop potent and selective
antitumor curcuminoids, a series of curcumin analogues
bearing various substituents on the aromatic ring were
prepared and their antiproliferative activity against four
human cancer cell lines were evaluated. Besides, the pri-
mary anticancer mechanism and in vivo antitumor activity
of the most potent compound were also studied.
Scheme 1 Synthesis of substituted dione 5–8
Antiproliferative effects against cancer cells
Ten curcumin analogues (9–18) were evaluated for anti-
proliferative activities against a panel of human cancer cell
lines, and curcumin was also tested as the positive control.
The results are summarized in Table 1; curcumin showed a
broad and moderate cytotoxicity against cancer cells as
reported. Most of the synthesized compounds were more
active against human epithelial colorectal adenocarcinoma
cells, and human breast cancer cells were less sensitive to
these compounds. By comparing the cytotoxicity results in
Table 1, we can draw the primary structure–activity rela-
tionships. At least one side of the aromatic ring should be
substituted with hydroxy or methoxy group, for example,
compound 12 bearing dimethylamino groups at the both
sides of aromatic ring was less active than other com-
pounds. The substituented groups on the aromatic ring has
no great affect on the antiproliferation of target compounds
by comparing the data of compounds 13, 16, 17. The
diketone moiety was important to the activity, which existed
in the enol form from the NMR analysis. The most potent
compound 10 exhibited potent anticancer activity against
Caco-2, Bel-7402, MDA-MB-231, and DU145 cancer cell
lines with IC50 values of 2.04, 1.54, 4.99, and 3.22 μM,
respectively. This results might be attributed to the intro-
duction of dimethylamino group, which increased the
solubility and bioavailability of compound 10. Therefore,
compound 10 was chosen for further investigation of
mechanism and in vivo antitumor activity.
Results and discussion
Chemistry
These newly designed curcuminoids were synthesized as
described in Schemes 1 and 2. To prevent Knovenagel
condensation at C-3 methylene group, boric anhydride was
first reacted with acetylacetone (4) to generate an acetone-
boric oxide complex. One of the less active methyl moiety
was then reacted further with an appropriate ratio of dif-
ferent kinds of substituented benzaldehyde to ensure single
aldol condensation. The reaction was performed in ethyl
acetate, and tributyl borate was used as a desiccant to pre-
vent the reaction from moist. After decomplexation in the
presence of hydrochloric acid, compounds 5–8 were
obtained in good yields. Subsequently, these intermediates
were subjected to a second aldol condensation with various
benzaldehydes to afford target compounds 9–18 after col-
umn chromatography (Wichitnithad et al. 2011). The
structures of synthesized curcuminoids were fully char-
acterized by nuclear magnetic resonance (NMR) and mass
spectroscopy (MS). The β-diketone functionality is
responsible for the intramolecular hydrogen atom transfer
leading to the keto and enol tautomeric conformations, and
these tautomeric forms exist in different cis and trans forms,
which depends on factors such, as temperature, polarity of
the solvent, and substitution on the aromatic rings. From the
analysis of NMR, it was observed that these newly syn-
thesized compounds existed in the enol form (Supporting
Information).
Analysis of cell cycle effects
Although antitumor agents may initiate cell damage or
stress, the cellular proteins that are involved in control of

Med Chem Res
Scheme 2 Synthesis of
curcumin analogues 9–18
Table 1 Antiproliferative
effects of Curcumin (1) and its
analogues (9–18) against four
human cancer cell lines
a
Compd
IC50 (μM)
Caco-2
Bel-7402
MDA-MB-231
DU145
Cisplatin
4.18 ± 0.29
6.09 ± 0.74
5.61 ± 0.27
2.04 ± 0.09
3.10 ± 0.27
18.58 ± 1.67
4.42 ± 0.22
3.43 ± 0.48
2.94 ± 0.28
6.74 ± 0.64
6.67 ± 0.32
5.87 ± 0.74
5.82 ± 0.72
9.05 ± 1.34
6.66 ± 1.03
1.54 ± 0.12
4.49 ± 0.68
23.43 ± 3.13
8.02 ± 0.94
4.07 ± 0.39
9.69 ± 0.88
10.47 ± 1.46
7.21 ± 0.37
4.28 ± 0.66
6.18 ± 0.79
18.77 ± 1.97
21.39 ± 2.07
4.99 ± 1.02
8.01 ± 0.99
>30
4.25 ± 0.42
15.53 ± 1.55
17.28 ± 1.89
3.22 ± 0.32
11.02 ± 0.79
22.79 ± 3.04
11.26 ± 1.18
11.88 ± 1.19
5.30 ± 0.47
12.09 ± 1.12
10.23 ± 1.43
16.82 ± 1.99
1
9
10
11
12
13
14
15
16
17
18
19.58 ± 1.16
14.17 ± 1.32
13.48 ± 1.20
15.32 ± 1.54
24.32 ± 2.61
28.19 ± 3.04
Caco-2, human epithelial colorectal adenocarcinoma cells; Bel-7402, human hepatocellular carcinoma cells;
MDA-MB-231, human breast cancer cells; DU145, human prostate cancer cells
a
MTT methods, cells were incubated with indicated compounds for 72 h, (means ± SD, n = 3)
cell cycle and apoptosis are the final arbiters of cell fate
Pietenpol and Stewart 2002). To determine whether the
Compound 10-induced apoptosis
(
effects of these analogues on the proliferation of cancer
cells were mediated by inhibition of cell cycle progression,
DNA-based cell cycle analysis was performed by flow
cytometry to understand how the cell growth was inhibited
by compound 10. As illustrated in Fig. 2, treatment of 10
caused blockage of the cell cycle at G1 phase. Compared
with the control cells treated with dimethyl sulfoxide
To further investigate whether compound 10 could induce
apoptosis in Bel-7402 cells, vehicle-treated or 10-treated
Bel-7402 cells were stained with Annexin V and PI.
Externalization of phosphatidylserine (PS) from the inner
leaflet to the outer leaflet of the plasma membrane is a
distinct phenomenon of early apoptosis. As Annexin V
possesses high affinity for PS, apoptotic cells can easily be
detected by fluorescently labeling with Annexin V. In
contrast, PI can detect necrotic cells due to its ability to
permeate damaged cell membranes (Segawa and Nagata
2015). Thus, flow cytometry analysis of the cells identified
four groups: viable cells (Annexin V−, PI−), early apop-
totic cells (Annexin V+, PI−), later apoptotic cells
(Annexin V+, PI+) and necrotic cells (Annexin V−, PI+)
(
DMSO), when Bel-7402 cells were treated with increasing
concentrations of 10 (0.5, 1.0, and 2.0 μM), the percentage
of cells in the G1 phase increased from 42.91 to 56.44%,
and the percentages of cells in S and G2/M phases
decreased concomitantly. The above results suggested that
these derivatives may induce cancer cell cycle arrest at G1
phase.

Med Chem Res
Fig. 2 Compound 10 caused a G1 arrest of Bel-7402 cancer cells. Bel-
402 cells were treated with DMSO and varying concentrations of 10
0.5, 1.0, 2.0 μM) for 48 h, cells were harvested and stained with
propidium iodide (PI), then analyzed by flow cytometry. The per-
centage of cells of different phases of cell cycle was analyzed by
ModFit 4.1
7
(
cells. As shown in Fig. 3, 10 treatment induced a dose-
dependent increase in both early and late stage apoptosis of
Bel-7402 cells, treatment with four different concentrations
of 10 (0.5, 1.0, and 2.0 μM) resulted in increased amounts
of total apoptotic cells in a dose-dependent manner from
10 was efficacious in inhibiting the growth of liver cancer
growth in vivo and deserved further evaluations.
Conclusions
2
.8% up to 21.0%. Together, these results demonstated that
1
0 can induce apoptosis of Bel-7402 cancer cells.
The structural modification of curcumin around the aro-
matic ring led to the discovery of a series of novel curcumin
analogues, and part of the analogues displayed improved
antiproliferative activities vs. curcumin against a panel of
human cancer cell lines. We extended the structure–activity
data reported for natural product curcumin in this study and
demonstrated that the aromatic ring of curcumin was sui-
table for modification to improve its antitumor activity. The
most potent compound 10 exhibited about six-fold
improved anti-proliferative activity compared to the parent
molecule curcumin against Bel-702 cancer cell line.
Besides, compound 10 was shown to arrest the cell cycle at
the G1 phase, and caused a marked increase in apoptosis,
which was determined by externalization of PS. More
importantly, the antitumor efficacy of 10 was demonstrated
in xenograft-bearing mice. In this model, there was
significant inhibition of tumor growth with a dosage of
Evaluate the antitumor effect of compound 10 in vivo
To evaluate the in vivo antitumor activity of compound 10,
human liver cancer xenograft was established by intrave-
nous injection of H22 cells into mice. The tumor size and
body weight changes of the mice were monitored and
recorded every 2 days. The results showed that compound
1
0 significantly suppressed the tumor volume and reduced
tumor weight by 62.1% with a dose of 20 mg/kg/day (iv.),
which was similar to that of positive control cyclopho-
sphamide, which caused a 65.1% of reduction. Of note,
compound 10 had little effect on mice body weight and had
no overt toxicity under these experimental conditions
(
Fig. 4). Taken together, these data indicated that compound

Med Chem Res
Fig. 3 Compound 10 induced apoptosis on Bel-7402 cells. Compound
treatment, cells were collected and stained with Annexin V/PI, fol-
lowed by flow cytometric analysis. Histograms display the percentage
of cell distribution
1
0 induced apoptosis on Bel-7402 cells. Bel-7402 cells were treated
with varying concentrations of 10 (0, 0.5, 1.0, 2.0 μM). After 24 h of
2
0 mg/kg 10, and the treated animals had no significant
coupling constants (J) in Hz. Low-resolution mass spectrum
and high-resolution mass spectrum were measured on Fin-
nigan MAT 95 spectrometer (Finnigan, Germany).
A mixture of 2,4-pentanedione (1.03 mL, 10 mmol) and
boric anhydride (0.17 g, 2.5 mmol) was dissolved in ethyl
acetate (15 mL). The mixture was stirred at 80 °C for 30
min, and a mixture of vanillin (0.38 g, 2.5 mmol) and tri-
butyl borate (1.36 mL, 5 mmol) in ethyl acetate (3 mL) was
added to the reaction solution. The solution was stirred for
15 min at 50 °C with dropwise addition of n-butylamine
weight loss. In summary, this newly developed curcumin
analogues showed marked biological activity in vitro and
in vivo and has potential for further development as a pro-
mising antitumor lead.
Experimental section
Chemistry
(0.2 mL, 2.7 mmol) in 2 mL ethyl acetate. The reaction
All commercially available reagents were used without
further purification. Anhydrous solvents were dried through
routine protocols. Flash column chromatography was car-
ried out on 200–300 mesh silica gel (Qingdao Haiyang
Chemical, China). Reactions were monitored by thin-layer
chromatography (TLC) on 0.25 mm silicagel plates
mixture was stirred overnight at room temperature. 1 N HCl
(8 mL) was added and the mixture was stirred for another
30 min. The organic layers were separated and the aqueous
fraction was extracted with ethyl acetate (30 mL). The
extract was washed with saturated NaHCO
brine, dried over anhydrous Na SO , filtered, and evapo-
solution and
3
2
4
1
13
(
GF254) and visualized under UV light. H NMR and
C
rated to afford compound 7 as a pale yellowish solid. A
complex of 7 (0.4 g, 1.71 mmol) and boric anhydride (0.6 g,
0.85 mmol) was formed in ethyl acetate (10 mL) and then
stirred at 80 °C for 30 min. Then, a mixture of p-dimethy-
laminobenzaldehyde (0.25 g, 1.71 mmol) and tributyl borate
NMR spectra were recorded with a Bruker AV-300 spec-
trometer (Bruker Company, Germany) in the indicated
solvents (CDCl , TMS as internal standard): the values of
3
the chemical shifts are expressed in δ values (ppm) and the

Med Chem Res
Fig. 4 Compound 10 inhibits
liver cancer xenograft growth
in vivo. a H22 cells were
subcutaneously inoculated into
the right flank of mice. The mice
were randomly divided into four
groups with six mice in each
group. Mice were treated
intravenously with 10 (20 mg/
kg), 10 (10 mg/kg), curcumin
(
(
(
20 mg/kg), cyclophosphamide
15 mg/kg) and DMSO
dissolved in sodium chloride,
control) every day for 2 weeks.
The resulting tumors were
excised from the animals after
treatment. b Body weight
changes of mice during
treatment
1
(
0.46 mL, 1.71 mmol) was added and stirred at 50 °C for 30
53%, mp 128–130 °C. H NMR (CDCl , 300 MHz): δ
3
min. A mixture of n-butylamine (0.17 mL) in 2 mL ethyl
acetate was added dropwisely over 10 min and the solution
was stirred overnight at room temperature. 1N HCl (10 mL)
was added and the mixture was further stirred for 30 min.
The organic layers were separated and the aqueous fraction
was extracted with ethyl acetate (30 mL). The extract was
(ppm) 16.17 (br, 1H, OHC=CH), 7.62, 6.41 (dd, J = 15.9
Hz, each 2H, CH=CH), 7.42 (d, J = 8.4 Hz, 2H, ArH), 7.18
(d, J = 8.4 Hz, 2H, ArH), 6.99 (d, J = 8.4 Hz, 2H, ArH),
6.69 (d, J = 8.7 Hz, 2H, ArH), 5.78 (s, 1H, OHC=CH),
3.92 (s, 3H, OCH ), 3.47 (q, J = 6.9 Hz, 4H, N(CH CH ) ),
3
2
3 2
1
3
1.25 (t, J = 6.9 Hz, 6H, N(CH CH ) ); C NMR (CDCl3,
2
3 2
washed with saturated NaHCO solution and brine, dried
75 MHz): δ (ppm) 178.4 (C=O), 162.4, 147.8, 146.6,
141.2, 139.4, 130.1, 124.3, 122.7, 121.9, 114.5, 111.2,
109.1, 101.4 (OHC=CH), 60.1 (OCH3), 44.4 (N
(CH CH ) ), 12.7 (N(CH CH ) ); MS (ESI) m/z: 378.3 [M
3
over anhydrous Na SO . After removal of the solvent under
2
4
vacuum, the crude products were purified by flash column
chromatography to afford compound 9 as a yellowish solid.
2
+
3 2
2
3 2
1
Yield 62%, mp 114–116 °C. H NMR (CDCl , 300 MHz): δ
+ H] , HR-MS (ESI) m/z: calcd for C H NNaO [M +
3
24 27 3
+
(
ppm) 16.23 (br, 1H, OHC=CH), 7.65, 6.45 (dd, J = 15.6
Hz, each 2H, ArH), 7.42 (d, J = 8.7 Hz, 2H, ArH), 7.13 (m,
H, ArH), 6.93 (d, J = 8.4 Hz, 1H, ArH), 6.67 (d, J = 8.7
Hz, 2H, ArH), 5.93 (br, 1H, OH), 5.78 (s, 1H, OHC=CH),
Na] 400.1883, found 400.1872.
Following the procedure described for preparation of
2
compound 9, compound 11 was prepared with yield of
1
61%, mp 132–133 °C. H NMR (CDCl , 300 MHz): δ
3
3
1
7
1
1
.96 (s, 3H, OCH ), 3.43 (q, J = 6.9 Hz, 4H, N(CH CH ) ),
(ppm) 16.19 (br, 1H, OHC=CH), 7.61, 6.41 (dd, J = 15.9
Hz, each 2H, CH=CH), 7.41 (d, J = 8.4 Hz, 2H, ArH), 7.21
(d, J = 8.4 Hz, 2H, ArH), 6.97 (d, J = 8.4 Hz, 2H, ArH),
6.71 (d, J = 8.7 Hz, 2H, ArH), 6.01 (br, 1H, OH), 5.77 (s,
1H, OHC=CH), 3.47 (q, J = 6.9 Hz, 4H, N(CH CH ) ),
3
2
3 2
1
3
.21 (t, J = 6.9 Hz, 6H, N(CH CH ) ); C NMR (CDCl3,
2
3 2
5 MHz): δ (ppm) 177.2 (C=O), 162.8, 147.6, 146.7,
41.5, 139.6, 130.3, 127.9, 122.7, 121.9, 114.8, 111.3,
09.5, 101.6 (OHC=CH), 60.0 (OCH3), 44.6 (N
2
3 2
1
3
(
CH CH ) ), 12.6 (N(CH CH ) ); MS (ESI) m/z: 394.2 [M
1.24 (t, J = 6.9 Hz, 6H, N(CH CH ) ); C NMR (CDCl3,
2
3 2
2
3 2
2 3 2
+
+
H] , HR-MS (ESI) m/z: calcd for C H NNaO [M +
75 MHz): δ (ppm) 178.2 (C=O), 162.9, 148.1, 146.9,
143.1, 138.9, 132.0, 124.3, 122.6, 121.4, 114.1, 111.6,
109.0, 101.2 (OHC=CH), 44.7 (N(CH CH ) ), 12.6 (N
2
4
27
4
+
Na] 416.1832, found 416.1830.
Following the procedure described for preparation of
2
3 2
+
compound 9, compound 10 was prepared with yield of
(CH CH ) ); MS (ESI) m/z: 364.1 [M + H] , HR-MS (ESI)
2 3 2

Med Chem Res
+
m/z: calcd for C H NNaO [M + Na] 386.1727, found
Following the procedure described for preparation of
2
3
25
3
3
86.1623.
Following the procedure described for preparation of
compound 9, compound 12 was prepared with yield of
compound 9, compound 16 was prepared with yield of
1
68%. H NMR (CDCl , 300 MHz): δ (ppm) 15.95 (br, 1H,
3
OHC=CH), 7.65, 6.45 (dd, J = 15.6 Hz, each 2H,
CH=CH), 7.43 (d, J = 8.7 Hz, 2H, ArH), 7.05 (dd, J = 8.4,
2.1 Hz, 1H, ArH), 6.96 (d, J = 2.1 Hz, 2H, ArH), 6.83 (m,
1H, ArH), 6.82 (d, J = 8.7 Hz, 2H, ArH), 5.86 (br, 1H, OH),
1
6
8%. H NMR (CDCl , 300 MHz): δ (ppm) 16.18 (br, 1H,
3
OHC=CH), 7.70, 6.51 (dd, J = 15.6 Hz, each 2H,
CH=CH), 7.62 (d, J = 8.7 Hz, 4H, ArH), 6.90 (d, J = 8.7
Hz, 4H, ArH), 5.81 (s, 1H, OHC=CH), 3.45 (q, J = 6.9 Hz,
5.71 (s, 1H, OHC=CH), 3.86 (s, 3H, OCH ), 3.76 (s, 3H,
3
1
3
8
H, N(CH CH ) ), 1.23 (t, J = 6.9 Hz, 12H, N(CH CH ) );
OCH3); C NMR (CDCl , 75 MHz): δ (ppm) 182.2
2
3 2
2
3 2
3
1
3
C NMR (CDCl , 75 MHz): δ (ppm) 182.0 (C=O), 162.6,
(C=O), 160.8, 147.4, 146.3, 140.0, 139.7, 129.3, 127.3,
122.4, 121.3, 114.3, 111.9, 109.1, 100.8 (OHC=CH), 55.4
3
1
46.9, 141.1, 130.3, 124.7, 121.4, 116.9, 101.2
+
(
OHC=CH), 46.3 (N(CH CH ) ), 12.6 (N(CH CH ) ); MS
(OCH ), 54.9 (OCH ); MS (ESI) m/z: 353.2 [M + H] , HR-
2
3 2
2
3 2
3
3
+
+
(
ESI) m/z: 419.3 [M + H] , HR-MS (ESI) m/z: calcd for
MS (ESI) m/z: calcd for C H NaO [M + Na] 375.1203,
21 20 5
+
C H N NaO [M + Na] 441.2512, found 441.2517.
found 375.1200.
2
7
34
2
2
Following the procedure described for preparation of
Following the procedure described for preparation of
compound 9, compound 13 was prepared with yield of
compound 9, compound 17 was prepared with yield of
1
1
6
6%, mp 146–148 °C. H NMR (CDCl , 300 MHz): δ
61%, mp 108–109 °C. H NMR (CDCl , 300 MHz): δ
3
3
(
ppm) 16.05 (br, 1H, OHC=CH), 7.66, 6.48 (dd, J = 15.9
Hz, each 2H, CH=CH), 8.31 (d, J = 8.7 Hz, 2H, ArH), 8.03
d, J = 8.7 Hz, 2H, ArH), 7.16 (m, 2H, ArH), 6.97 (d, J =
.4 Hz, 1H, ArH), 5.95 (br, 1H, OH), 5.74 (s, 1H,
(ppm) 15.81 (br, 1H, OHC=CH), 7.65, 6.44 (dd, J = 15.6
Hz, each 2H, CH=CH), 7.42 (m, 2H, ArH), 7.29 (m, 3H,
ArH), 7.05 (dd, J = 8.4, 2.1 Hz, 1H, ArH), 6.82 (d, J = 8.7
(
8
Hz, 2H, ArH), 5.86 (br, 1H, OH), 5.71 (s, 1H, OHC=CH),
1
3
13
OHC=CH), 3.91 (s, 3H, OCH3); C NMR (CDCl , 75
3.85 (s, 3H, OCH3); C NMR (CDCl , 75 MHz): δ (ppm)
3
3
MHz): δ (ppm) 178.4 (C=O), 161.9, 148.1, 146.7, 140.9,
182.2 (C=O), 160.8, 149.1, 147.3, 140.0, 138.7, 129.3,
1
1
+
39.6, 130.1, 127.5, 122.8, 121.2, 114.8, 110.9, 109.8,
127.1, 122.5, 121.3, 114.4, 111.3, 109.2, 101.1
+
01.3 (OHC=CH), 59.4 (OCH ); MS (ESI) m/z: 368.1 [M
(OHC=CH), 55.1 (OCH ); MS (ESI) m/z: 323.1 [M + H] ,
3
3
+
+
H] , HR-MS (ESI) m/z: calcd for C H NNaO [M +
HR-MS (ESI) m/z: calcd for C H NaO [M + Na]
2
0
17
6
20 18
4
+
Na] 390.0948, found 390.0935.
345.1097, found 345.1093.
Following the procedure described for preparation of
Following the procedure described for preparation of
compound 9, compound 14 was prepared with yield of
compound 9, compound 18 was prepared with yield of
1
1
5
7%, mp 134–135 °C. H NMR (CDCl , 300 MHz): δ
71%, mp 104–105 °C. H NMR (CDCl , 300 MHz): δ
3
3
(
ppm) 16.17 (br, 1H, OHC=CH), 7.63, 6.45 (dd, J = 15.6
Hz, each 2H, CH=CH), 8.30 (d, J = 8.7 Hz, 2H, ArH), 8.01
d, J = 8.7 Hz, 2H, ArH), 7.19 (d, J = 8.7 Hz, 2H, ArH),
(ppm) 16.10 (br, 1H, OHC=CH), 7.67, 6.54 (dd, J = 15.9
Hz, each 2H, CH=CH), 7.53 (d, J = 8.7 Hz, 4H, ArH), 6.91
(d, J = 8.7 Hz, 4H, ArH), 5.80 (s, 1H, OHC=CH), 3.87 (s,
(
1
3
7
3
1
1
.00 (d, J = 8.4 Hz, 2H, ArH), 5.71 (s, 1H, OHC=CH),
6H, OCH3); C NMR (DMSO-d , 75 MHz): δ (ppm) 182.2
6
1
3
.87 (s, 3H, OCH3); C NMR (CDCl , 75 MHz): δ (ppm)
(C=O), 162.9, 148.1, 141.1, 129.0, 123.1, 121.4, 114.9,
3
79.2 (C=O), 162.1, 147.2, 145.7, 140.4, 139.5, 130.2,
111.6, 101.2 (OHC=CH), 56.3 (OCH ); MS (ESI) m/z:
3
+
28.4, 121.9, 121.2, 114.8, 110.5, 109.6, 101.2
337.2 [M + H] , HR-MS (ESI) m/z: calcd for C H NaO
2
1
20
4
+
+
(
OHC=CH), 59.6 (OCH ); MS (ESI) m/z: 352.1 [M + H] ,
[M + Na] 359.1254, found 359.1251.
3
+
HR-MS (ESI) m/z: calcd for C H NNaO [M + Na]
2
0
17
5
3
74.0999, found 390.0935.
Following the procedure described for preparation of
compound 9, compound 15 was prepared with yield of
Determination of in vitro anticancer activity
The overall growth level of human cancer cell lines was
determined using the colorimetric MTT (3-[4,5-dimethyl-
thiazol-2-yl]diphenyl tetrazolium bromide, Sigma) assay. The
MTT colorimetric assay is based on the capability of living
cells to reduce the yellow product MTT to a blue product,
formazan, by a reduction reaction occurring in the mito-
chondria. Briefly, the cell lines were incubated at 37 °C in a
1
5
9%, mp 136–138 °C. H NMR (CDCl , 300 MHz): δ
3
(
ppm) 16.18 (br, 1H, OHC=CH), 7.63, 6.41 (dd, J = 15.6
Hz, each 2H, CH=CH), 8.33 (d, J = 8.7 Hz, 2H, ArH), 8.02
d, J = 8.7 Hz, 2H, ArH), 7.21 (d, J = 8.7 Hz, 2H, ArH),
(
6
1
1
1
.98 (d, J = 8.7 Hz, 2H, ArH), 5.90 (br, 1H, OH) 5.72 (s,
1
3
H, OHC=CH); C NMR (CDCl , 75 MHz): δ (ppm)
3
78.1 (C=O), 161.4, 148.1, 144.3, 140.7, 139.5, 130.2,
humidified 5% CO incubator for 24 h in 96-microwell plates
2
28.2, 121.8, 121.1, 114.7, 110.5, 109.6, 101.1
prior to the experiments. After medium removal, 100 μL of
fresh medium containing the test compound at different
concentrations was added to each well and incubated at 37 °C
for 72 h, the percentage of DMSO in the medium never
+
(
OHC=CH); MS (ESI) m/z: 338.1 [M + H] , HR-MS (ESI)
+
m/z: calcd for C H NNaO [M + Na] 360.0842, found
3
1
9
15
5
60.0831.

Med Chem Res
exceeded 0.25%. The number of living cells after 72 h of
culture in the presence (or absence: control) of the various
compounds is directly proportional to the intensity of the
blue, which is quantitatively measured by spectrophotometry
where L is the length and W the width. Ratio of inhibition of
tumor (%) = (1 – average tumor weight of treated group/
average tumor weight of control group) × 100%. All proce-
dures were performed following institutional approval in
accordance with the NIH Guide for the Care and Use of
Laboratory Animals. All animal handling and procedures
were approved by the Ethical Committee of the Fudan
University.
(
Biorad, Nazareth, Belgium) at a 570 nm wavelength.
The experiment was performed in quadruplicate and repeated
times.
3
Cell cycle analysis
Statistical analysis of the data
4
A total of 5 × 10 cells were seeded into six-well plates and
incubated overnight. Cells were then treated with various
concentrations of compounds for 48 h. The cells were har-
vested, washed with cold phosphate-buffered saline (PBS)
and then fixed with 70% ethanol in PBS at −20 °C for 12 h.
Subsequently, the cells were resuspended in PBS containing
All results were confirmed in at least three separate
experiments. The data are expressed as means ± S.D.
Statistical comparisons were made by Student’s t-test.
P < 0.05 was considered significant.
Acknowledgements This work was supported by grants from the
National Program on Key Basic Research Project (973 Program, No.
1
00 ug/mL RNase and 50 ug/mL PI and incubated at 37 °C
for 30 min. Cell cycle distribution of nuclear DNA was
determined by flow cytometry on a FC500 cytometer
2
011CB504405), the National Natural Scientific Foundation of China
(No. 81372121, 81572146), and the Key Project of Shanghai Science
(
Beckman Colter).
and Technology Commission (No. 13411951002).
Compliance with ethical standards
Flow cytometry analysis of apoptotic cells
Conflict of interest The authors declare that they have no competing
interests.
5
MCF-7 cells (1 × 10 /well) were cultured in complete
medium in six-well plates for 24 h and treated in triplicate
with different concentrations of compound for 24 h. The
control cells wre treated with vehicle (1‰ DMSO in
complete medium). The cells were harvested, washed and
stained with PI, and FITC-Annexin-V in the dark for 15 min
using the FITC-AnnexinV Kit (BD Parmingen, San Diego,
CA, USA). The percentages of apoptotic cells were deter-
mined by flow cytometry on an FC500 cytometer (Beckman
Colter).
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