Synthesis and antitumor properties of novel curcumin analogs

Article abstract of DOI:10.1007/s00044-013-0854-3

A series of novel curcumin (CC) analogs were synthesized by reacting substituted aldehydes with inter-mediates 4a and 4b. The inhibitory activities of these CC analogs were investigated on human cancer cells PC3, Bcap-37, and MGC-803 in vitro by MTT assay. The results showed that most of the title compounds displayed moderate to high levels of antitumor activities. Compound 5f, the most active CC analogs, has the IC₅₀ values of 1.34 ± 0.28, 3.90 ± 0.36, and 0.86 ± 0.44 μM against the three human cancer cells assayed, respectively. Furthermore, subsequent fluorescence staining and flow cytometry analysis indicated compound 5f could induce apoptosis in PC3, Bcap-37, and MGC-803 cells, and the apoptosis ratio reachs the peak (27.1%) in MGC-803 cells at 24 h after treatment at 10 μM. Springer Science+Business Media 2013.

Full text of DOI:10.1007/s00044-013-0854-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:2584–2595
DOI 10.1007/s00044-013-0854-3
ORIGINAL RESEARCH
Synthesis and antitumor properties of novel curcumin analogs
•
•
•
Hui Luo Shengjie Yang Qi Zhao
Hongmei Xiang
Received: 22 July 2013 / Accepted: 15 October 2013 / Published online: 26 October 2013
Ó Springer Science+Business Media New York 2013
Abstract A series of novel curcumin (CC) analogs were
synthesized by reacting substituted aldehydes with inter-
mediates 4a and 4b. The inhibitory activities of these CC
analogs were investigated on human cancer cells PC3,
Bcap-37, and MGC-803 in vitro by MTT assay. The results
showed that most of the title compounds displayed mod-
erate to high levels of antitumor activities. Compound 5f,
the most active CC analogs, has the IC50 values of
1.34 0.28, 3.90 0.36, and 0.86 0.44 lM against the
three human cancer cells assayed, respectively. Further-
more, subsequent fluorescence staining and flow cytometry
analysis indicated compound 5f could induce apoptosis in
PC3, Bcap-37, and MGC-803 cells, and the apoptosis ratio
reachs the peak (27.1 %) in MGC-803 cells at 24 h after
treatment at 10 lM.
Keywords Curcumin analogs Á Antitumor Á Apoptosis Á
Flow cytometry analysis
Abbreviations
ADM
AO/EB
CC
Adriamycin
Acridine orange/ethidium bromide
Curcumin
13C NMR 13C nuclear magnetic resonance
DMSO
FCM
Dimethyl sulfoxide
Flow cytometry
HCPT
10-Hydroxyl camptothecine
1H NMR Proton nuclear magnetic resonance
IR
Infra-red
MTT
3-(4,5-Dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide
Hui Luo and Shengjie Yang have contributed equally to this study.
H. Luo
Guizhou Fruit Institute, Guizhou Academy of Agricultural
Sciences, Guiyang 550006, People’s Republic of China
Introduction
Curcumin (CC) is the principal curcuminoid of the popular
Indian spice turmeric, which is a member of the ginger
family (Zingiberaceae) (Archana et al., 2011; Khanna
et al., 2009). CC and its derivatives have been revealed
having antitumor (Wilken et al., 2011; Sa and Das, 2008),
antioxidant (Menon and Sudheer, 2007; Jayaprakasha
et al., 2006), antiarthritic (Zahidah et al., 2012), antiamy-
loid (Ringman et al. 2005), anti-ischemic (Shukla et al.,
2008), and anti-inflammatory (Menon and Sudheer, 2007;
Chainani-Wu, 2003) properties in both in vitro and animal
studies. They are particularly known for their ability to
resist mutation and raise antitumor activity (Lee et al.,
2011). Their potential anticancer effects stem from an
ability to induce apoptosis in cancer cells without cytotoxic
S. Yang
Research Institute of Traditional Chinese Medicine, Yangtze
River Pharmaceutical Group Co., Ltd, Taizhou 225321,
People’s Republic of China
H. Luo Á S. Yang Á Q. Zhao Á H. Xiang
State Key Laboratory Breeding Base of Green Pesticide and
Agricultural Bioengineering, Key Laboratory of Green Pesticide
and Agricultural Bioengineering, Ministry of Education,
Guizhou University, Guiyang 550025,
People’s Republic of China
H. Luo (&) Á S. Yang Á Q. Zhao Á H. Xiang
Ctr for R&D of Fine Chemicals, Guizhou University, Huaxi St,
Guiyang 550025, People’s Republic of China
e-mail: luohui8732@163.com
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Fig. 1 Design of the target
compounds
effects on healthy cells (Ravindran et al., 2009; Almanaa
et al., 2012). Researchers have demonstrated that CC
interfered with activity of the transcription factor NF-jB,
which had been linked to a number of diseases such as
cancer (Lin and Lin, 2008; Kohli et al., 2005). In Senft
et al. (2010) reported that CC could inhibit tumor cell
proliferation, migration, and invasion which were mediated
through interference with a signaling pathway (Senft et al.,
2010). In addition, some studies indicated that CC had
phytoestrogenic activity that might affect onset of breast
cancer (Bachmeier et al., 2010). However, the potential use
of CC as a therapeutic agent was severely affected by its
low water solubility, poor ex vivo bioavailability, and rapid
metabolism. Thus, great interest has been devoted to the
synthesis of novel CC analogs exhibiting enhanced bio-
logic properties, e.g., the novel ferrocenyl curcuminoids
were synthesized by covalent anchorage of three different
ferrocenyl ligands. Shibata et al. reported some newly
synthesized CC analogs containing methoxymethyl group
showed unexpected improvements in the anticolorectal
cancer activities in vivo (Shibata et al., 2009), whereas
Yamakoshi et al. obtained similar class of compounds by
studying structure–activity relationship of C5-curcumi-
noids Ai–ii (Yamakoshi et al., 2010). Our group reported
several newly synthesized CC analogs showed obvious
antitumor activities (Yuan et al., 2011). In present work,
quinazoline groups were introduced into CC analogs, and
sixteen CC analogs were synthesized (Fig. 1). These CC
analogs were evaluated their antitumor activities against
PC3, Bcap-37, and MGC-803 human cancer cell lines by
MTT assay, and were studied their preliminary mechanism
by AO/EB staining, Hoechst 33258 staining, DNA Ladder,
and flow cytometry analysis.
obtain intermediates 4-(4-hydroxy-phenyl)-3-butylene-2-
one (3). o-Aminobenzoic acid was reacted with formamide,
and the reaction was maintained for 4.5 h at 140–145 °C to
obtain intermediate (1). Intermediate (1), SOCl2 was added
in DMF, and the mixture was refluxed to obtain 4-chloro-
quine quinazolin (2) (reactive intermediate). Intermediates
(2), and 4-(4-hydroxy-phenyl)-3-butylene-2-one (3) were
reacted with K2CO3 for 3.5 h at 30–40 °C. The crude
products were recrystallized from anhydrous alcohol to get
intermediates 4a and 4b with 76.8 and 80.6 % yields,
respectively. The structures of these compounds were
confirmed by elements analysis, IR, 1H NMR, and 13C
NMR.
Biologic activity
The in vitro antitumor activities of these newly synthesized
compounds were evaluated against human prostate cancer
cell line PC3, breast cancer cell line Bcap-37, and gastric
cancer cell line MGC-803 by 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl-tetrazolium bromide (MTT) cell proliferation
assay. Adriamycin (ADM) and the compounds were dis-
solved in DMSO. The negative control cells were treated
with culture medium containing 0.1 % DMSO, while ADM
was used as positive control. Each experiment was repeated
at least three times. The results were reported in terms of
IC50 values and summarized in Table 1. From the IC50
values, all the tested compounds had good activities on all
the selected cell lines, with IC50 values ranging from 0.86
to 30.29 lM. Compounds 5a–5n showed significant anti-
tumor activities, but intermediates 4a and 4b exhibited
only moderate activities. The variation among the different
substitutes on R1 greatly affected the activities. For
example, intermediate 4a (R1 was H) presented stronger
inhibition than 4b (R1 was 8-CH3). Interestingly, when
aromatic aldehydes were introduced to the intermediate 4a
or 4b, an apparent increase in antitumor activity was found.
Compounds 5a–5n (IC50, \15 lM) showed more promis-
ing activity as compared to the intermediates 4a and 4b.
Compound 5f, the most active compound, had shown IC50
values of 1.34 0.28, 3.90 0.36, and 0.86 0.44 lM
on PC3, Bcap-37, and MGC-803 cell lines, respectively.
Further experiments indicated that proliferation of these
three cancer cells was significantly inhibited by compound
Results and discussion
Chemistry
Target compounds 5a–5n were synthesized by reacting the
substituted aldehydes with intermediates 4a, 4b in EtOH at
room temperature, as shown in Fig. 2. Starting from ace-
tone and salicylaldehyde, acetone was excessive and was
stirred in sodium hydroxide (1 %) at room temperature to
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Med Chem Res (2014) 23:2584–2595
Fig. 2 Synthetic route of 5a–5n
5f in a concentration-dependent manner, as shown in
Fig. 3.
analyze the mechanism of growth inhibition of cancer
cells. In order to study the preliminary mechanism of
action, changes in the morphological character of PC3,
Bcap-37, and MGC-803 cells were investigated using
acridine orange (AO)/ethidium bromide (EB) staining,
Hoechst 33258 staining, and apoptosis-DNA ladder assay
to determine compound 5f whether the growth inhibitory
activity was related to the induction of apoptosis. HCPT,
which was isolated from a Chinese tree Camptotheca
acuminate, has a broad spectrum of anticancer activity
in vitro and in vivo (Fei et al., 2013). It was reported that
HCPT could induce apoptosis in human cancer cells (Zhai
et al., 2010). Therefore, HCPT was used as positive control
to study the action mechanism of compound 5f.
In addition, ADM and the active compounds (5c, 5e, and
5f) were also tested on normal cell line NIH3T3. As shown
in Table 2, compound 5f was less toxic on normal cell line
than on cancer cell lines and more selective to cancer cell
lines than compounds 5c, 5e, and ADM. Thus, compounds
5c, 5e, and ADM showed less selective to cancer cells and
had some cytotoxic effect on normal cells.
Apoptosis is a physiologic pattern of cell death char-
acterized by morphological features and extensive DNA
fragmentation, the frequency, and time of appearance of
which depend on the cell line and the apoptosis inducing
signal (Wu et al., 2012). Thus compound 5f was selected to
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Table 1 The in vitro antitumor activities of compounds 4a, 4b, and
5a–5n against PC3, Bcap-37, and MGC-803 cancer cell lines
Table 2 Selectivity index of 5c, 5e, and 5f toward cancer cells
Compound
PC3
Bcap-37
MGC-803
Compound
IC50 (lM)a
5c
3.40
4.46
[8
\2
\2
5.72
\2
6.13
6.57
[8
PC3
Bcap-37
MGC-803
5e
5f
4a
4b
5a
5b
5c
21.31 0.46
25.79 0.51
5.62 0.34
7.35 0.38
3.66 0.29
4.57 0.43
2.96 0.32
1.34 0.28
11.52 0.51
7.36 0.32
8.79 0.46
5.17 0.45
15.24 0.53
4.71 0.39
2.79 0.24
16.42 0.49
1.01 0.21
28.91 0.52
30.29 0.46
8.53 0.46
11.35 0.42
7.82 0.26
10.81 0.58
7.81 0.29
3.90 0.36
18.9 0.44
9.25 0.50
12.26 0.32
9.26 0.41
32.75 0.56
8.17 0.38
7.89 0.53
21.33 0.41
1.90 0.24
18.45 0.48
19.87 0.45
3.84 0.19
4.84 0.27
2.03 0.21
2.77 0.34
2.01 0.36
0.86 0.44
9.31 0.38
4.36 0.38
8.10 0.50
3.76 0.37
5.13 0.49
2.32 0.32
1.86 0.28
11.56 0.32
0.74 0.18
ADM
\2
\2
Selectivity index (IC50 on mouse embryo fibroblast cell line/IC50 on
corresponding cancer cell line)
5d
5e
condensed or fragmented chromatin; late apoptotic cells
display condensed and fragmented orange chromatin,
whereas necrotic cells showed structurally normal orange
nucleus (Kasibhatla et al., 2006; Sukardiman et al., 2007).
The cytotoxicity of compound 5f at 5 lM against PC3,
Bcap-37, and MGC-803 cells for 12 h was detected by AO/
EB staining, with HCPT used as a positive control at
10 lM for 12 h.
As can be seen in Fig. 4, green yellow or orange dots
were detected in the HCPT after 12 h. Yellow and orange
dots in PC3, Bcap-37, and MGC-803 cells showed early
and late apoptotic cells, and the appearance of little red
cells indicated that compound 5f was associated with low
cytotoxicity. The results suggested that compound 5f was
able to induce apoptosis in tumor cells.
5f
5g
5h
5i
5j
5k
5l
5m
5n
ADM
The data were the mean SD obtained from three independent
experiments, with ADM used as the positive control
a
Agent concentration (micromolar) that inhibited cell growth by
50 % 72 h after treatment
Hoechst 33258 staining was also performed to investi-
gate the apoptosis-inducing activities of compound 5f in
PC3, Bcap-37, and MGC-803 cells. Live cells with
homogeneous light blue nuclei could be observed under
fluorescence microscopy after staining with Hoechst
33258, and the apoptotic cells displayed bright blue due to
karyopyknosis and chromatin condensation after treating
with Hoechst 33258, and the nuclei of dead cells could not
be stained (Yang et al., 2012). PC3, Bcap-37, and MGC-
803 cells treated with compound 5f at 5 lM for 12 h were
stained with Hoechst 33258. HCPT was used as a positive
control at 10 lM for 12 h. The results are shown in Fig. 5.
It can been seen from Fig. 5 that a part of cells with
smaller nuclei and condensed staining appeared in the
positive control compared with the negative control. After
treated with compound 5f, some cell nuclei became pyk-
notic (shrunken and dark) or appeared to be highly con-
densed (brightly stained). These findings once again
indicated that compound 5f could induce apoptosis in
tumor cells.
Fig. 3 Effect of 5f on proliferation of tumor cells. After PC3, Bcap-
37, and MGC-803 cells were treated with 5f for 72 h in the
concentration varied from 0.5 to 8 lM. Data are presented as
mean SD, n = 4
Endonucleases were activated when cells exhibited
apoptosis, then DNA was degraded selectively, forming
50–300 kb big fragments, then cleaved in the vicinity of
nucleosome and formed DNA fragments of or multiples of
180–200 bp. These DNA fragments could be extracted
from cells. The DNA appeared by agarose gel electro-
phoresis and EB staining (Yuan et al., 2011). With HCPT
The morphologic changes in cells after treatment with
compound 5f were assessed by fluorescence microscopy
after staining with AO/EB. Live cells exhibit normal green
nuclei early apoptotic cells show bright green nuclei with
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Fig. 4 Nuclei morphological
changes during 5f induced
apoptosis in tumor cells
detected by AO/EB staining
Fig. 5 Nuclei morphological
changes during 5f induced
apoptosis in tumor cells
detected by Hechst 33258
staining
Therefore, the results indicated that compound 5f could
induce apoptosis in MGC-803 cells.
The apoptosis ratios induced by compound 5f in tumor
cells were quantitatively assessed by FCM. In the early
stages of apoptosis, phosphatidylserine was translocated
from within the cell membrane to the cell exterior. Annexin V,
a calcium-dependent phospholipid-binding protein associ-
ated with a high affinity for phosphatidylserine, was used
to detect early apoptotic cells. Propidine iodide (PI) is a
red fluorescent dye that stains cells with lost their mem-
brane integrity. Cells stained with Annexin V-fluores-
cein isothiocyanate and PI were classified as necrotic
(Q1, Annexin-/PI?), late apoptotic (Q2, Annexin?/PI?),
Fig. 6 The DNA ladder of 5f on MGC-803 cells
used as positive control at 10 lM for 12 h, the compound
5f induced apoptosis in MGC-803 cells at 5 lM for 12 h,
as detected by agarose gel electrophoresis. As shown in
Fig. 6, the HCPT and compound 5f groups appeared as
typical DNA Ladder compared with negative group.
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Table 3 The apoptosis ratios of MGC-803 cells treated with 5f assessed by flow cytometry
Compound
6 ha
12 h
24 h
1 lLb
5 lL
10 lL
1 lL
5 lL
10 lL
1 lL
5 lL
10 lL
5f (%)
2.4
1.2
4.1
1.8
6.4
3.0
2.8
2.0
6.2
5.8
10.5
8.6
5.3
4.7
8.9
7.6
27.1
24.5
HCPT (%)
a
Treating time
b
Agent concentration
Fig. 7 Flow cytometry analysis
for apoptosis inducing activities
of 5f on MGC-803 cells. In the
figure, the MGC-803 cells were
treated with HCPT and
compound 5f (10 lM) for 6, 12,
and 24 h
intact (Q3, Annexin-/PI-), and early apoptotic (Q4,
Annexin?/PI-) cells (Yang et al., 2013). As shown in
Table 3, with HCPT (1, 5, and 10 lM) as positive control,
compound 5f (1, 5, and 10 lM) could induce apoptosis in
MGC-803 cells. The highest apoptosis ratio (including the
early and late apoptosis ratios) of 27.1 % was obtained
after 24 h of treatment at a concentration of 10 lM, and it
was markedly higher than that of the positive control
HCPT (24.5 % at 10 lM after 24 h of treatment). Fur-
thermore, as shown in Fig. 7, the apoptosis of MGC-803
cells which is treated with compound 5f increased gradu-
ally in a time-dependent manner.
and MGC-803 cells. And DNA ladder and flow cytometry
analysis also revealed that it inhibited the growth of MGC-
803 cells via inducing apoptosis, with apoptosis ratio of
27.1 % at 24 h after treatment at 10 lM, higher than the
ratio observed for the positive control HCPT (24.5 %).
Further studies of the specific mechanisms of these com-
pounds on human malignant tumors are currently
underway.
Experimental
General procedures and reagents
Conclusion
1H NMR and 13C NMR were recorded using a JEOL-
ECX500 spectrometer at 22 °C, with tetramethylsilane as
the internal standard and CDCl3 as the solvent. Elemental
analysis was performed with an Elementar Vario-III CHN
analyzer. Infrared spectra were recorded on a Bruker
VECTOR22 spectrometer in KBr disks. The melting points
were determined using an XT-4 digital microscope (melt-
ing point apparatus) (Beijing Tech Instrument Co.).
Reagents of analytical grade were obtained from Yuda
Chemistry Co., Ltd., and used without further purification
unless otherwise noted. Column chromatography was per-
formed using silica gel (200–300 mesh) (Qingdao Marine
In summary, we have synthesized sixteen novel CC ana-
logs and evaluated for their antitumor activities. Among
these new compounds, most of them exhibited good anti-
proliferative activities against PC3, Bcap-37, and MGC-
803 cells. Compound 5f was found to have the best anti-
tumor activities, with the IC50 values of 1.34 0.28,
3.90 0.36, and 0.86 0.44 lM on these three cells,
respectively. In addition, subsequent fluorescence staining
(AO/EB staining and Hoechst 33258 staining) indicated
that the compound could induce apoptosis in PC3, Bcap-37,
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1
1385, 1385, 1211, 972, 777; H NMR (500 MHz, CDCl3)
Chemistry Co., Qingdao, China). Analytical thin-layer
chromatography (TLC) was performed on silica gel GF254
(400 mesh).
d: 8.81 (s, 1H, Qu-2-H), 8.31(d, J = 8.5 Hz, 1H, Qu-5-H),
8.09 (d, J = 8.6 Hz, 1H, Qu-7-H), 7.78 (d, J = 8.6 Hz,
1H, Ar–CH=), 7.54–7.71 (m, 3H, Ar-2,3,5-H), 7.34 (d,
J = 8.6 Hz, 1H, Ar-6-H), 6.73 (d, J = 16.5 Hz, 1H,
ArC=CH), 2.66 (s, 3H, Qu-CH3), 2.41(s, 3H, –CH3); 13C
NMR (125 MHz, CDCl3) d: 198.4 (C=O), 162.7 (C-4),
153.1 (C-2), 152.7 (C-12), 150.9 (C-9), 142.4 (C-18), 136.5
(C-7), 134.9 (C-8, 15), 129.8 (C-14, 16), 128.6 (C-6), 127.3
(C-19), 123.6 (C-13, 17), 122.7 (C-5), 116.1 (C-10), 27.4
(CH3), 17.6 (CH3).
Synthetic procedures
Quinazolin-4(3H)-one,
8-methyl-quinazolin-4(3H)-one,
8-methyl-4-chloroquiazoline, and 4-chloroquiazoline were
prepared according to a previously described method (Liu
et al., 2004). Intermediate 4-chloroquiazoline was prepared
following standard synthetic protocols (Liu et al., 2006).
(E)-4-(2-Hydroxyphenyl)-3-butylene-2-one was prepared
according to a previously reported (McGookin and Heil-
bron, 1924).
(1E,4E)-1-(4-fluorophenyl)-5-(4-(quinazolin-4-
yloxy)phenyl)penta-1,4-dien-3-one (5a)
General synthetic procedures for title compounds
Yellow powder; mp 149–151 °C; yield: 56.9 %; ESI–MS:
397 [M?H]?; IR (KBr, cm-1) m: 1653, 1620, 1568, 1493,
1335, 1220, 986; H NMR (500 MHz, CDCl3) d: 8.78 (s,
Compounds 5a–5n were synthesized according to Fig. 2.
The starting materials were substituted 2-aminobenzoic
acid, formamide, salicylaldehyde, and acetone. Substituted
o-aminobenzoic acid was reacted with formamide for 4.5 h
at 140–145 °C to obtain the intermediate 4-quinazolinone
1. Substituted 4-chloroquinazoline 2 was synthesized via
efficient chlorination with use of SOCl2. Intermediates 4a
and 4b were synthesized starting from the (E)-4-(4-
hydroxyphenyl)-3-butene-2-one with K2CO3 in acetonitrile
for 3.5 h at 30–40 °C. Finally, compounds 5a–5n were
obtained via condensation of intermediate 4a or 4b and
1
1H, Qu-2-H), 8.42 (d, J = 8.5 Hz, 1H, Qu-8-H), 8.30 (d,
J = 8.0 Hz, 1H, Qu-5-H), 8.22–8.26 (m, 2H, Qu-6,7-H),
8.07 (d, J = 8.6 Hz, 1H, Ar–CH=), 7.98 (d, J = 8.5 Hz,
1H, F–Ar–CH=), 7.72–7.83 (m, 6H, F–Ar-2,6-H, F–Ar–
C=CH, Ar–C=CH, Ar-3,5-H), 7.32 (d, J = 8.6 Hz, 2H, F–
Ar-3,5-H), 7.20 (d, J = 16.5 Hz, 2H, Ar-2-H), 7.06 (d,
J = 16.0 Hz, 1H, Ar-6-H); 13C NMR (125 MHz, CDCl3)
d: 188.8 (C=O), 166.8 (C-4), 162.6 (C-25), 160.6 (C-2),
154.2 (C-11), 151.9 (C-9), 151.6 (C-21), 136.8 (C-17),
136.1 (C-7), 134.5 (C-14), 131.7 (C-13, 15), 129.4 (C-22),
128.3 (C-23, 27), 128.2 (C-8), 128.1 (C-6), 127.1 (C-20),
126.7 (C-18), 123.6 (C-12, 16), 123.5 (C-5), 116.2 (C-10),
111.5 (C-24, 26); Anal. Calcd for C25H17FN2O2: C, 75.75;
H, 4.32; N, 7.07; Found: C, 75.79; H, 4.71; N, 7.23.
1
substituted aldehydes. IR, H NMR, 13C NMR, and ele-
mental analysis data for all the synthesized compounds are
reported in the experimental protocols.
(E)-4-(4-(Quinazolin-4-yloxy)phenyl)but-3-en-2-one (4a)
(1E,4E)-1-(4-chlorophenyl)-5-(4-(quinazolin-4-
Yellow powder; mp 97–99 °C; yield: 76.8 %; ESI–MS: 291
[M?H]?; IR (KBr, cm-1) m: 1690, 1609, 1578, 1478, 1400,
1354, 1219, 936, 768; 1H NMR (500 MHz, CDCl3) d: 8.74
(s, 1H, Qu-2-H), 8.33 (d, J = 2.0 Hz, 1H, Qu(Quinazolin)-
8-H), 7.98 (d, J = 8.6 Hz, 1H, Qu-5-H), 7.90 (t,
J = 8.4 Hz, 1H, Qu-7-H), 7.63–7.65 (m, 3H, Qu-6-H, Ar–
CH=, Ar-3-H), 7.51 (d, J = 16.5 Hz, 1H,Ar-5-H), 7.29 (d,
J = 8.6 Hz, 1H, Ar-2,6-H), 6.68 (d, J = 16.0 Hz, 1H, Ar–
C=CH), 2.37 (s, 3H, –CH3); 13C NMR (125 MHz, CDCl3)
d: 198.3 (C=O), 166.7 (C-4), 154.1 (C-2), 154.0 (C-11),
151.8 (C-9), 142.3 (C-17), 134.4 (C-14), 132.4 (C-13,
C-15), 129.8 (C-7), 128.0 (C-8), 127.9 (C-6), 127.4 (C-18),
123.6 (C-12, 16), 122.7(C-5), 116.4 (C-10), 27.8 (CH3).
yloxy)phenyl)penta-1,4-dien-3-one (5b)
Yellow powder; mp 220–222 °C; yield: 59.4 %; ESI–MS:
413 [M?H]?; IR (KBr, cm-1) m: 1654, 1595, 1522, 1489,
1
1385, 1336, 1275, 964; H NMR (500 MHz, CDCl3) d:
8.78 (s, 1H, Qu-2-H), 8.37 (d, J = 7.5 Hz, 1H, Qu-8-H),
8.14 (d, J = 16.5 Hz, 1H, Cl–Ar–CH=), 8.02 (d,
J = 8.0 Hz, 1H, Qu-5-H), 7.93 (t, J = 7.6 Hz, 1H, Qu-7-
H), 7.69–7.79 (m, 5H, Qu-6-H, Ar–CH=, Cl–Ar-3,6-H, Ar-
3-H), 7.43 (d, J = 7.5 Hz, 1H, Ar-5-H), 7.31–7.35 (m, 4H,
Cl–Ar-4,5-H, Cl–Ar–C=CH, Ar–C=CH), 7.12 (d,
J = 16.0 Hz, 1H, Ar-2-H), 7.03 (d, J = 16.5 Hz, 1H, Ar-
6-H); 13C NMR (125 MHz, CDCl3) d: 188.8 (C=O), 166.8
(C-4), 154.2 (C-2), 153.2 (C-11), 151.9 (C-9), 142.7 (C-
17), 139.2 (C-21), 134.4 (C-22), 133.1 (C-25), 132.7 (C-
14), 131.3 (C-7), 130.0 (C-13, 15), 128.2 (C-23, 27), 128.1
(C-8), 127.9 (C-24, 26), 127.8 (C-6), 127.2 (C-20), 125.1
(C-18), 123.6 (C-12, 16), 122.7 (C-5), 116.3 (C-10); Anal.
(E)-4-(4-((8-methylquinazolin-4-yl)oxy)phenyl)but-3-en-2-
one (4b)
Yellow powder; mp 145–147 °C; yield: 80.6 %; ESI–MS:
305 [M?H]?; IR (KBr, cm-1) m: 1665, 1601, 1566, 1487,
123

Med Chem Res (2014) 23:2584–2595
2591
Calcd for C25H17ClN2O2: C, 72.73; H, 4.15; N, 6.97;
Found: C, 72.36; H, 4.44; N, 6.56.
Ar–CH=, Ar-3,5-H), 7.20–7.34 (m, 5H, Ar–C=CH, CH3
O-Ar–C=CH, Ar-2,6-H, CH3O-Ar-6-H), 6.93 (d, J =
15.0 Hz, 1H, CH3O-Ar-3-H), 6.79 (d, J = 16.5 Hz, 1H,
CH3O-Ar-4-H), 3.92 (s, 3H, CH3O-2-Ar), 3.97 (s, 3H,
CH3O-6-Ar); 13C NMR (125 MHz, CDCl3) d: 188.7
(C=O), 166.7 (C-4), 155.3 (C-2), 154.2 (C-11, 26), 153.9
(C-21), 151.8 (C-23), 151.0 (C-9), 141.7 (C-17), 134.4 (C-7),
129.8 (C-14), 128.1(C-13, 15), 127.9 (C-8), 126.7 (C-6),
125.5 (C-18), 123.7 (C-20), 122.6 (C-12, 16), 122.5 (C-5),
116.3 (C-22), 116.2 (C-10), 116.1 (C-27), 113.2 (C-24),
112.6 (C-25), 56.2 (OCH3), 55.9 (OCH3); Anal. Calcd
for C27H22N2O4: C, 73.96; H, 5.06; N, 6.39; Found: C, 73.65;
H, 5.34; N, 6.15.
(1E,4E)-1-(2-methoxyphenyl)-5-(4-(quinazolin-4-
yloxy)phenyl)penta-1,4-dien-3-one (5c)
Yellow powder; mp 161–163 °C; yield: 45.9 %; ESI–MS:
409 [M?H]?; IR (KBr, cm-1) m: 1647, 1618, 1570, 1489,
1
1337, 1213, 983; H NMR (500 MHz, CDCl3) d: 8.77 (s,
1H, Qu-2-H), 8.38 (d, J = 2.5 Hz, 1H, Qu-8-H), 8.02–8.10
(m, 2H, Qu-5,7-H), 7. 94 (d, J = 3.6 Hz, 1H, CH3O-Ar–
CH=), 7.68–7.77 (m, 4H, Qu-6-H, Ar–CH=, CH3O-Ar-6-
H, Ar–C=CH), 7.61 (t, J = 7.6 Hz, 1H, CH3O-Ar-4-H),
7.33–7.38 (m, 3H, Ar-3,5-H,CH3O-Ar–C=CH), 7.12–7.15
(m, 2H, CH3O-Ar-3,5-H), 6.93–6.99 (m, 2H, Ar-2,6-H);
13C NMR (125 MHz, CDCl3) d: 189.5 (C=O), 166.8 (C-4),
158.7 (C-23), 154.2 (C-2), 153.9 (C-11), 151.9 (C-9, 21),
141.8 (C-17), 138.9 (C-27), 134.4 (C-14), 133.1 (C-7),
131.9 (C-13, 15), 128.9 (C-25), 128.1 (C-8), 127.9 (C-6),
126.5 (C-22), 125.6 (C-20), 123.7 (C-18), 123.6 (C-12, 16),
122.6 (C-26), 120.9 (C-5), 115.8 (C-10), 111.3 (C-24), 55.6
(OCH3);
(1E,4E)-1-(2-chloro-5-nitrophenyl)-5-(4-(quinazolin-4-
yloxy)phenyl)penta-1,4-dien-3-one (5f)
Yellow powder; mp 235–237 °C; yield: 64.7 %; ESI–MS:
458 [M?H]?; IR (KBr, cm-1) m: 1653, 1624, 1570, 1522,
1
1489, 1346, 1217, 906; H NMR (500 MHz, CDCl3) d:
8.79 (s, 1H, Qu-2-H), 8.59 (s, 1H, Cl–Ar-6-H), 8.38 (d,
J = 8.0 Hz, 1H, Qu-8-H), 8.18 (d, J = 6.3 Hz, 1H, Cl–Ar-
4-H), 8.02–8.10 (m, 2H, Qu-5,7-H), 7.94 (t, J = 7.9 Hz,
1H, Qu-6-H), 7.62–7.85 (m, 6H, Cl–Ar–CH=Cl–Ar–
C=CH, Ar–CH=, Cl–Ar-3-H, Ar-3,5-H), 7.37 (d,
J = 8.6 Hz, 1H, Ar–C=CH), 7.23 (d, J = 16.0 Hz, 1H, Ar-
2-H), 7.09 (d, J = 16.5 Hz, 1H, Ar-6-H); 13C NMR
(125 MHz, CDCl3) d: 187.8 (C=O), 166.7 (C-4), 154.4 (C-
2), 154.1 (C-11), 151.6 (C-9, 21), 146.6 (C-26), 143.6 (C-
17), 141.7 (C-23), 134.5 (C-22), 134.0 (C-7), 132.4 (C-13,
15), 131.4 (C-14), 130.2 (C-24), 129.8 (C-8), 128.1 (C-6),
127.9 (C-25), 125.6 (C-20), 125.2 (C-18), 123.6 (C-27),
122.8 (C-12, 16), 122.6 (C-5), 116.4 (C-10); Anal. Calcd
for C25H16ClN3O4: C, 65.58; H, 3.52; N, 9.18; Found: C,
65.55; H, 3.21; N, 9.17.
Anal. Calcd for C26H20N2O3: C, 76.45; H, 4.94; N, 6.86;
Found: C, 76.21; H, 5.24; N, 7.02.
(1E,4E)-1-(2,4-dichlorophenyl)-5-(4-(quinazolin-4-
yloxy)phenyl)penta-1,4-dien-3-one (5d)
Yellow powder; mp 154–156 °C; yield: 65.4 %; ESI–MS:
447 [M?H]?; IR (KBr, cm-1) m: 1653, 1616, 1489, 1338,
1217, 906; 1H NMR (500 MHz, CDCl3) d: 8.78 (s, 1H, Qu-
2-H), 8.38–8.39 (d, J = 5.0 Hz, 1H, Qu-8-H), 8.03–8.07
(m, 5H, Qu-5,6,7-H, Cl–Ar–CH=, Ar–CH=), 7.65–7.76 (m,
5H, Cl–Ar-3,5,6-H,Ar-3,5-H), 7.34–7.36 (m, 2H, Cl–Ar–
C=CH, Ar–C=CH), 7.10 (d, J = 15.0 Hz, 1H, Ar-2-H),
7.03 (d, J = 15.0 Hz, 1H, Ar-6-H); 13C NMR (125 MHz,
CDCl3) d: 188.5 (C=O), 166.8 (C-4), 157.1 (C-2), 154.2
(C-11), 153.1 (C-21), 151.9 (C-9), 142.9 (C-17), 137.9 (C-
23), 136.6 (C-7), 136.1 (C-14), 134.5 (C-22), 132.7 (C-27),
131.7 (C-13, 15), 130.2 (C-24), 130.0 (C-8), 128.5 (C-6),
128.4 (C-26), 127.9 (C-25), 127.7 (C-20), 125.2 (C-18),
123.6 (C-12, 16), 122.7 (C-5), 116.4 (C-10); Anal. Calcd
for C25H16Cl2N2O2: C, 67.13; H, 3.61; N, 6.26; Found: C,
67.49; H, 3.45; N, 5.97.
(1E,4E)-1-(5-chloro-1,3-dimethyl-1H-pyrazol-4-yl)-5-(4-
(quinazolin-4-yloxy)phenyl)penta-1,4-dien-3-one (5g)
Yellow powder; mp 143–145 °C; yield: 57.6 %; ESI–MS:
431 [M?H]?; IR (KBr, cm-1) m: 1655, 1618,1587, 1481,
1316, 1219, 987; 1H NMR (500 MHz, CDCl3, ppm) d: 8.74
(s, 1H, Qu-2-H), 8.43 (d, J = 8.0 Hz, 1H, Qu-8-H), 8.27
(d, J = 8.5 Hz, 1H, Qu-5-H), 7.87 (t, J = 7.4 Hz, 1H, Qu-
7-H), 7.80 (d, J = 8.5 Hz, 1H, Ar–CH=), 7.61–7.74 (m,
2H, pyrazole-CH=, Qu-6-H), 7.28–7.51 (m, 3H, Ar-2,3,5-
H), 7.28 (d, J = 8.0 Hz, 1H, pyrazole-C=CH), 7.09 (d,
J = 16.0 Hz, 1H, Ar–C=CH), 6.68 (d, J = 16.5 Hz, 1H,
Ar-6-H), 3.75 (s, 3H, N–CH3), 2.18 (s, 3H, CH3); 13C NMR
(125 MHz, CDCl3) d: 187.6 (C=O), 165.8 (C-4), 152.2 (C-
2), 150.9 (C-11), 150.4 (C-9, 21), 148.7 (C-26), 141.6 (C-
17), 135.9 (C-23), 134.3 (C-7), 132.7 (C-14), 129.2 (C-13,
15), 128.2 (C-20), 128.1 (C-8), 127.9 (C-6), 123.8 (C-8),
(1E,4E)-1-(2,5-dimethoxyphenyl)-5-(4-(quinazolin-4-
yloxy)phenyl)penta-1,4-dien-3-one (5e)
Yellow powder; mp 146–148 °C; yield: 46.3 %; ESI–MS:
439 [M?H]?; IR (KBr, cm-1) m: 1627, 1566, 1520, 1487,
1
1341, 1217, 983; H NMR (500 MHz, CDCl3) d: 8.77 (s,
1H, Qu-2-H), 8.38 (d, J = 8.5 Hz, 1H, Qu-8-H), 7.95–8.07
(m, 3H, Qu-5,6,7-H), 7.68–7.79 (m, 4H, CH3O-Ar–CH=,
123

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Med Chem Res (2014) 23:2584–2595
123.4 (C-12, 16), 121.9 (C-5), 116.2 (C-10), 112.5 (C-22),
36.3 (CH3), 14.1 (CH3); Anal. Calcd for C24H19ClN4O2: C,
66.90; H, 4.44; N, 13.00; Found: C, 66.71; H, 4.23; N,
13.28.
Qu-5,6,7-H, CH3O-Ar-6-H), 7.55–7.61 (m, 2H, Ar-3,5-H),
7.31–7.37 (m, 3H, CH3O-Ar-4-H, CH3O-Ar–C=CH, Ar–
C=CH), 7.12–7.19 (m, 2H, CH3O-Ar-3,5-H), 6.95 (d,
J = 16.5 Hz, 2H, Ar-2,6-H), 3.86 (s, 3H. OCH3), 2.77 (s,
3H. CH3); 13C NMR (125 MHz, CDCl3) d: 189.5 (C=O),
167.1 (C-4), 158.7 (C-24), 154.2 (C-2), 154.1 (C-12), 153.2
(C-22), 150.9 (C-9), 141.9 (C-18), 138.9 (C-7), 136.5 (C-
28), 134.47 (C-8), 131.9 (C-15), 129.9 (C-14, 16), 129.7
(C-26), 128.9 (C-6), 127.4 (C-23), 126.5 (C-19), 125.6 (C-
21), 123.8 (C-13, 17), 122.6 (C-27), 121.2 (C-5), 120.9 (C-
10), 111.3 (C-25), 55.6 (OCH3), 17.7 (CH3); Anal. Calcd
for C27H22N2O3: C, 76.76; H, 5.25; N, 6.63; Found: C,
76.60; H, 5.16; N, 6.52.
(1E,4E)-1-(4-fluorophenyl)-5-(4-((8-methylquinazolin-4-
yl)oxy)phenyl)penta-1,4-dien-3-one (5h)
Yellow powder; mp 159–161 °C; yield: 60.5 %; ESI–MS:
411 [M?H]?; IR (KBr, cm-1) m: 1668, 1616, 1584, 1479,
1
1404, 1360, 1220, 984; H NMR (500 MHz, CDCl3) d:
8.82 (s, 1H, Qu-2-H), 8.22 (d, J = 8.0 Hz, 1H, Qu-5-H),
7.69–7.78 (m, 5H, Qu-6,7-H, Ar–CH=, F–Ar–CH=, F–Ar-
2-H), 7.57–7.62 (m, 3H, F–Ar-6-H, Ar-3,5-H), 7.33 (d,
J = 7.6 Hz, 2H, F–Ar-3,5-H), 6.99–7.11 (m, 4H, F–Ar–
C=CH, Ar–C=CH, Ar-2,6-H), 2.77 (s, 1H, CH3); 13C NMR
(125 MHz, CDCl3) d: 189.3 (C=O), 167.1 (C-4), 161.3 (C-
26), 154.3 (C-2), 153.0 (C-12), 151.2 (C-9), 143.6 (C-18),
142.3 (C-22), 136.6 (C-7), 134.8 (C-8), 133.3 (C-15), 132.6
(C-24, 28), 129.9 (C-23), 129.6 (C-14, 16), 129.4 (C-6),
127.4 (C-19), 125.7 (C-21), 122.7 (C-13, 17), 121.3 (C-5),
116.2 (C-10), 110.6 (C-25, 27), 17.8 (CH3); Anal. Calcd for
C26H19FN2O2: C, 76.08; H, 4.67; N, 6.83; Found: C, 76.40;
H, 4.43; N, 7.12.
(1E,4E)-1-(2,4-dichlorophenyl)-5-(4-((8-methylquinazolin-
4-yl)oxy)phenyl)penta-1,4-dien-3-one (5k)
Yellow powder; mp 203–205 °C; yield: 62.5 %; ESI–MS:
461 [M?H]?; IR (KBr, cm-1) m: 1653, 1618, 1576,1506,
1477, 1400,1356, 1221, 934; H NMR (500 MHz, CDCl3)
1
d: 8.82 (s, 1H, Qu-2-H), 8.22 (d, J = 8.0 Hz, 1H, Cl–Ar–
CH=), 8.04 (d, J = 16.0 Hz, 1H, Ar–CH=), 7.73–7.79 (m,
4H, Cl–Ar-3-H, Qu-5,6,7-H), 7.65 (d, J = 8.6 Hz, 1H, Cl–
Ar-6-H), 7.57 (d, J = 8.6, Hz, 1H, Cl–Ar-5-H), 7.46 (d,
J = 2.4 Hz, 1H, Ar–C=CH), 7.28–7.33 (m, 3H, Cl–Ar–
C=CH, Ar-3,5-H), 7.07 (d, J = 16.0 Hz, 1H, Ar-2-H), 7.02
(d, J = 16.0 Hz, 1H, Ar-6-H), 2.77 (s, 3H, CH3); 13C NMR
(125 MHz, CDCl3) d: 188.5 (C=O), 167.0 (C-4), 154.4 (C-
2), 153.1 (C-12), 151.0 (C-9, 22), 142.9 (C-18), 137.9 (C-
24), 136.6 (C-7), 136.5 (C-8), 134.5 (C-15), 132.6 (C-23),
131.7 (C-28), 130.2 (C-14, 16), 130.0 (C-25), 128.5 (C-6),
128.4 (C-27), 127.7 (C-26), 127.4 (C-21), 125.1 (C-19),
122.7 (C-13, 17), 121.2 (C-5), 116.3 (C-10), 17.7 (CH3);
Anal. Calcd for C26H18Cl2N2O2: C, 67.69; H, 3.93; N,
6.07; Found: 67.92; H, 3.56; N, 5.79.
(1E,4E)-1-(4-chlorophenyl)-5-(4-((8-methylquinazolin-4-
yl)oxy)phenyl)penta-1,4-dien-3-one (5i)
Yellow powder; mp 188–190 °C; yield: 51.5 %; ESI–MS:
427 [M?H]?; IR (KBr, cm-1) m: 1653, 1620, 1581, 1481,
1
1359, 1223, 984; H NMR (500 MHz, CDCl3) d: 8.82 (s,
1H, Qu-2-H), 8.21–8.23 (d, J = 10.0 Hz, 1H, Cl–Ar–
CH=), 7.68–7.79 (m, 4H, Qu-5,6,7-H, Ar–CH=), 7.54–7.58
(m, 4H, Cl–Ar-2,3,5,6-H), 7.34–7.57 (m, 4H, Ar-3, 5-H,
Cl–Ar–C=CH, Ar–C=CH), 7.04–7.07 (d, J = 10.0 Hz, 2H,
Ar-2,6-H), 2.77 (s, 3H, CH3); 13C NMR (125 MHz,
CDCl3) d: 188.6 (C=O), 167.0 (C-4), 154.3 (C-2), 153.1
(C-12), 151.0 (C-9), 142.6 (C-18), 142.1 (C-22), 136.5 (C-
7), 134.5 (C-8), 133.3 (C-26), 132.6 (C-23), 129.9 (C-15),
129.6 (C-14, 16), 129.4 (C-24, 28), 127.4 (C-25, 27), 125.9
(C-6), 123.4 (C-21), 122.7 (C-19), 121.2 (C-13, 17), 120.4
(C-5), 116.3 (C-10), 17.7 (CH3); Anal. Calcd for
C26H19ClN2O2: C, 73.15; H, 4.49; N, 6.56; Found: C,
72.99; H, 4.81; N, 6.77.
(1E,4E)-1-(2,5-dimethoxyphenyl)-5-(4-((8-
methylquinazolin-4-yl)oxy)phenyl)penta-1,4-dien-
3-one (5l)
Yellow powder; mp 171–173 °C; yield: 53.4 %; ESI–MS:
453 [M?H]?; IR (KBr, cm-1) m: 1658, 1616, 1576,1506,
1
1478, 1356, 1220, 983; H NMR (500 MHz, CDCl3,) d:
8.83 (s, 1H, Qu-2-H), 8.23 (d, J = 8.0 Hz, 1H, CH3O-Ar–
CH=), 8.05 (d, J = 8.5 Hz, 1H, Ar–CH=), 7.74–7.78 (m,
4H, Ar-3-H, Qu-5,6,7-H), 7.57 (d, J = 7.5 Hz, 1H, Ar-5-
H), 7.30–7.34 (m, 2H, CH3O-Ar–CH=, Ar–CH=),
7.06–7.15 (m, 3H, Ar-2,6-H, CH3O-Ar-6-H), 6.87–6.93
(m, 2H, CH3O-Ar-3,4-H), 3.86 (s, 6H, 2CH3O), 2.77 (s,
3H, CH3); 13C NMR (125 MHz, CDCl3) d: 189.4 (C=O),
167.1 (C-4), 155.2 (C-2), 154.1 (C-12), 153.6 (C-27), 153.2
(C-22), 150.3 (C-24), 148.4 (C-9), 141.9 (C-18), 138.6 (C-7),
136.5 (C-8), 129.9 (C-15), 127.4 (C-14, 16), 126.7 (C-6),
(1E,4E)-1-(2-methoxyphenyl)-5-(4-((8-methylquinazolin-4-
yl)oxy)phenyl)penta-1,4-dien-3-one (5j)
Yellow powder; mp 156–158 °C; yield: 52.6 %; ESI–MS:
423 [M?H]?; IR (KBr, cm-1) m: 1647, 1616, 1582, 1481,
1400,1356, 1219, 989; 1H NMR (500 MHz, CDCl3) d: 8.81
(s, 1H, Qu-2-H), 8.21 (d, J = 7.0 Hz, 1H, CH3O-Ar–CH=),
8.07 (d, J = 16.6 Hz, 1H, Ar–CH=), 7.74–7.77 (m, 4H,
123

Med Chem Res (2014) 23:2584–2595
2593
125.4 (C-19), 124.4 (C-21), 122.6 (C-13, 17), 121.2 (C-5),
117.6 (C-10, 23), 113.2 (C-25), 112.6 (C-26, 28), 56.2
(OCH3), 55.9 (OCH3), 17.7 (CH3); Anal. Calcd for
C28H24N2O4: C, 74.32; H, 5.35; N, 6.19; Found: C, 74.20;
H, 5.43; N, 6.08.
Biology, China Academy of Science. PC3 is prostate
cancer cell line; Bcap-37 is breast cancer cell line; MGC-
803 is gastric cancer cell line, and NIH3T3 is mouse
embryo fibroblast cell line. The entire cancer cell lines
were maintained in the RPMI 1640 medium, whereas the
NIH3T3 cell line was maintained in DMEM medium. They
were supplemented with 10 % heat-inactivated fetal bovine
serum (FBS). All cell lines were maintained at 37 °C in a
humidified 5 % carbon dioxide and 95 % air incubator.
(1E,4E)-1-(2-chloro-5-nitrophenyl)-5-(4-((8-
methylquinazolin-4-yl)oxy)phenyl)penta-1,4-dien-
3-one (5m)
Yellow powder; mp 214–216 °C; yield: 62.5 %; ESI–MS:
472 [M?H]?; IR (KBr, cm-1) m: 1653, 1626, 1579, 1479,
1402,1348, 1221, 1165, 1111, 979, 766; 1H NMR
(500 MHz, CDCl3) d: 8.83 (s, 1H, Qu-2-H), 8.60 (s, 1H, Cl–
Ar-6-H), 8.08–8.23 (m, 4H, Cl–Ar-4-H, Qu-5,6,7-H),
7.76–7.85 (m, 4H, Cl–Ar-3-H, Ar–CH=, Cl–Ar–CH=, Cl–
Ar–C=CH), 7.57–7.65 (m, 2H, Ar-3,5-H), 7.36 (d,
J = 8.5 Hz, 1H, Ar–C=CH), 7.23 (d, J = 16.5 Hz, 1H, Ar-
2-H), 7.08 (d, J = 16.5 Hz, 1H, Ar-6-H), 2.77 (s, 3H, CH3);
13C NMR (125 MHz, CDCl3) d: 187.8 (C=O), 167.0 (C-4),
154.6 (C-2), 153.1 (C-12), 151.0 (C-22), 146.8 (C-9), 144.2
(C-27), 143.7 (C-18), 141.8 (C-24), 136.6 (C-7), 136.6 (C-
8), 134.5 (C-23), 131.4 (C-15), 130.2 (C-14, 16), 130.1 (C-
6), 129.8 (C-25), 127.4 (C-26), 125.5 (C-21), 125.2 (C-19),
122.8 (C-28), 122.6 (C-13, 17), 121.2 (C-5), 116.3 (C-10),
17.8 (CH3); Anal. Calcd for C26H18ClN3O4: C, 66.18; H,
3.84; N, 8.90; Found: C, 66.42; H, 3.78; N, 8.65.
MTT assays
All tested compounds were dissolved in DMSO (1-100 lM
solution) and subsequently diluted in the culture medium
before treatment of the cultured cells. Tested cells were
maintained in 96-well plates at a density 2 9 103 cells/
well/100 lL of the proper culture medium and treated with
the compounds at 1 to 100 lM for 72 h. In parallel, the
cells treated with 0.1 % DMSO served as control. A MTT
assay (Roche Molecular Biochemicals, 1465-007) was
performed 30 h later according to the instructions provided
by Roche. This assay was based on the cellular cleavage of
MTT into formazane which is soluble in cell culture
medium. Any absorbance caused by formazan was mea-
sured at 595 nm with a microplate reader (BIO-RAD,
model 680), which is directly proportional to the number of
living cells in culture. The experiment was performed in
triplicate.
(1E,4E)-1-(5-chloro-1,3-dimethyl-1H-pyrazol-4-yl)-5-
(4-((8-methylquinazolin-4-yl)oxy) phenyl)penta-1,4-dien-
3-one (5n)
AO/EB staining
Six coverslips were first soaked in 75 % alcohol for 6 h and
then put into 6 well plates and dried by alcohol lamp; MGC-
803 cell suspension (4 mL) was digested and mixed steadily
in a centrifuge tube (15 mL). Then 800 lL cell suspensions
to per coverslips, 6 well plates were put into incubator. Add
1,200 lL RPMI 1640 culture medium containing 10 %FBS
to each well after the cells, cultivate cells continuatively.
Add test compounds to 6 well plates when the cells grow to
80 %. The cells were observed after add drug. The cells can
be stain when cells morphology changed, add 20 lL stains
to glass slide, coverslips in 6 wells plate were covered the
surface of stains. The cells were observed and photographed
by fluorescence microscope.
Yellow powder; mp 202–204 °C; yield: 62.3 %; ESI–MS:
445 [M?H]?; IR (KBr, cm-1) m: 1651, 1616, 1582, 1479,
1359, 1223, 982; H NMR (500 MHz, CDCl3) d: 8.82 (s,
1
1H, Qu-2-H), 8.21 (d, J = 10.0 Hz, 1H, pyrazole-CH=),
7.72–7.78 (m, 3H, Qu-5,6,7-H), 7.55–7.63 (m, 3H, Ph-3-H,
Ar–CH=, pyrazole-C=CH), 7.31–7.33 (m, 2H, Ar–
C=CH,Ar-5-H), 6.96–7.04 (m, 2H, Ar-2,6-H), 2.39 (s, 3H,
Qu-CH3), 2.77 (s, 3H, pyrazole-CH3), 3.82 (s, 3H, N–
CH3); 13C NMR (125 MHz, CDCl3) d: 188.7 (C=O), 167.1
(C-4), 154.2 (C-2), 153.1 (C-12), 150.9 (C-9, 22), 148.9 (C-
27), 141.9 (C-18), 136.5 (C-7), 134.5 (C-8), 132.8 (C-24),
129.9 (C-15), 129.2 (C-21), 127.4 (C-14, 16), 126.0 (C-6),
123.5 (C-19), 122.6 (C-13, 17), 121.2 (C-5), 116.3 (C-10),
112.5 (C-23), 36.3 (CH3), 17.7 (CH3), 14.2 (CH3); Anal.
Calcd for C25H21ClN4O2: C, 67.49; H, 4.76; N, 12.59;
Found: C, 67.24; H, 4.98; N, 12.42.
Hoechst 33258 staining
The procedures of inoculating well plates and adding drugs
are same with AO/EB staining. 0.5 mL fixative solution
was added into each well; cells were fixed for 10 min. The
wells were washed twice (3 min/once) with 1 9 PBS after
drain fixative solution. 0.5 mL Hoechst 33258 staining
solution was added into each well; the cells were stained for
Cell lines and culture
PC3, Bcap-37, MGC-803, and NIH3T3 cell lines were
obtained from the Institute of Biochemistry and Cell
123

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5 min on shaking table. Then the wells were washed twice
(3 min/once) with 19 PBS after staining. Later, coverslips
having cells were covered the glass slide; the cells were
detected by the violet ray of fluorescence microscope.
DNA ladder
Detect apoptosis by DNA Ladder. The exponential phase
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which diameter was 10 cm. The compounds were added
into the cells for inducing apoptosis when the cells grow to
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Flow cytometry analysis
Prepared MGC-803 cells (1 9 106/mL) were washed twice
with cold PBS and then resuspended gently in 500 lL
binding buffer. Then the cells were stained in 5 lL
Annexin V-FITC and shaked well. 5 lL PI was added to
these cells and incubated for 20 min in a dark place, ana-
lyzed by FACS.
Statistical analysis
All statistical analysis were performed using SPSS 10.0, and
the data were analyzed using one-way ANOVA. The mean
separations were performed using the least significant dif-
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Acknowledgments The authors wish to thank the Guizhou Province
Production Technology & Innovation Capacity Building for Fine
Fruits (No. 20104009) and the Science and Technology Major Spe-
cialized Projects for ‘‘Significant New Drugs Creation’’ of the 12th
5-year Plan (2012ZX09101231-004) for the financial support.
¨
Senft C, Polacin M, Priester M, Seifert V, Kogel D, Weissenberger J
(2010) The nontoxic natural compound curcumin exerts anti-
proliferative, anti-migratory, and anti-invasive properties against
malignant gliomas. BMC Cancer 10:491
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Takahashi Y, Watanabe M, Takano H, Ishioka C, Noda T,
Iwabuchi Y (2009) Newly synthesized curcumin analog has
improved potential to prevent colorectal carcinogenesis in vivo.
Cancer Sci 100:956–960
Conflict of interest None.
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