Synthesis and evaluation of novel isoxazolyl chalcones as potential anticancer agents

Article abstract of DOI:10.1016/j.bioorg.2014.03.004

A series of novel isoxazolyl chalcones were synthesized and evaluated for their activities in vitro against four types of human non-small cell lung cancer cells, including H1792, H157, A549 and Calu-1 cells. The preliminary biological screening showed tha

Full text of DOI:10.1016/j.bioorg.2014.03.004

Bioorganic Chemistry 54 (2014) 38–43
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and evaluation of novel isoxazolyl chalcones as potential
anticancer agents
Maosheng Wan a,1, Linyan Xu , Li Hua , Ailing Li , Shuqing Li , Wenjing Lu , Yue Pang ,
b,1
c
c
c
a
a
a,
⇑
b,
⇑
d,
⇑
Chengbo Cao , Xiangguo Liu , Peifu Jiao
a
School of Chemistry and Chemical Engineering, Shandong University, Jinan, Shandong 250100, China
School of Life Sciences, Shandong University, Jinan, Shandong 250100, China
Shandong Drug and Food Vocational College, Weihai, Shandong 264210, China
Department of Chemistry, Qilu Normal University, Jinan, Shandong 250013, China
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 December 2013
Available online 15 March 2014
A series of novel isoxazolyl chalcones were synthesized and evaluated for their activities in vitro against
four types of human non-small cell lung cancer cells, including H1792, H157, A549 and Calu-1 cells. The
preliminary biological screening showed that compounds 5d and 5f–i exhibited significant cytotoxicity,
particularly, compounds 5f and 5h were identified as the most potent anticancer agents with IC50 values
Keywords:
Isoxazolyl chalcones
Synthesis
Human non-small cell lung cancer cells
Apoptosis
Death receptor 5
1.35–2.07 lM and 7.27–11.07 lM against H175, A549 and Calu-1 cell lines, respectively. Compounds 5f–i
could induce apoptosis in A549 cells by death receptor 5 (DR5) mediated extrinsic pathways. The preli-
minary structure–activity relationship study showed that compounds bearing electron withdrawing
groups (EWG) at the 2-position of the phenyl ring in Ar group were more effective than those with
EWG at 4-position. These results further demonstrated that the scaffolds designed in this work might
lead to the discovery of novel anti-lung cancer agents.
Ó 2014 Elsevier Inc. All rights reserved.
1
. Introduction
Lung cancer is a deadly disease in the world today and causes
amino groups of nucleic acids, a number of clinically useful anti-
cancer drugs have genotoxic effects. However, chalcones have
not been found to show such undesired side effects [27]. In order
to improving chalcone compound bioavailability, different substit-
uents chalcones on aryl or heteroaryl rings have been synthesized
[28].
In the design of new drugs, the combination of different phar-
macological monomers may lead to the finding of novel candidates
with interesting biological activity [29,30]. In this study, we aimed
to design and synthesize novel compounds with both isoxazole
and chalcone entities in one molecule and investigate their cyto-
toxicity to human non-small cell lung cancer cells.
more deaths than any other type of cancer [1]. Despite great ad-
vances in developing anticancer drugs in the past decades, the dis-
covery of novel anti-lung cancer agents with new mechanism of
action, high selectivity and excellent therapeutic efficacy is still a
major work to chemists.
Isoxazole derivatives as a special class of compounds exhibited
many interesting biological activities, for example, antituberculosis
activity [2], antioxidant activity [3], antidepressant [4], antifungal
activity [5], herbicidal activity [6], insecticidal activity [7,8],
DNA-intercalating agent [9] and anticancer activities [10–13].
Chalcones, which bear a three-carbon
a, b-unsaturated carbonyl
system between two aromatic rings, also showed a broad spectrum
of biological activities including anti-inflammatory [14], antima-
larial [15], anti-invasive [16], antibacterial [17], and anticancer
2
. Results and discussion
2.1. Chemistry
[
[
18–20]. In addition, Chalcones are capable of inducing apoptosis
21,22]. As a result, these compounds are regarded as promising
The synthesis of isoxazolyl chalcones 5a–i has been achieved by
anticancer agents (Fig. 1) [23–26]. Owing to interaction with the
the reaction between 1-(5-methyl-3-(4-nitrophenyl)isoxazol-4-
yl)ethanone 4 and aromatic aldehydes in the presence of ethanol
and sodium hydroxide at room temperature for 3–6 h as shown
in Scheme 1. The intermediate 4 was prepared by 1,3-dipolar
cycloaddition reaction at room temperature in dichloromethane.
⇑
Corresponding authors. Fax: +86 531 88392606.
E-mail address: cbcao@sdu.edu.cn (C. Cao).
Equal contribution.
1
http://dx.doi.org/10.1016/j.bioorg.2014.03.004
045-2068/Ó 2014 Elsevier Inc. All rights reserved.
0

M. Wan et al. / Bioorganic Chemistry 54 (2014) 38–43
39
Fig. 1. Examples of anticancer agents based on chalcones, already reported in literature.
Scheme 1. Synthesis of compounds 5a–i. (i) NH
2
OH.HCl, Na
2
CO
3
, EtOH, reflux, 6 h, 93%; (ii) DMF, NCS, 0 °C, then rt, 12 h; (iii) CH
2
Cl
2 3
, Et N, 30 min; acetylacetone, rt, 12 h,
8
0%; (iv) aromatic aldehydes, EtOH, NaOH (aq), rt, 3–6 h, 49–92%.
The crucial intermediate 3 was prepared from 4-nitrobenzalde-
hyde oxime 2 in the presence of N-chlorosuccinimide (NCS).
The structures of compounds 5a–i were made on the basis of
their spectral data and elemental analysis. The IR spectrum of com-
2
four aromatic proton signals in p-NO phenyl moiety appeared at
the range of d = 8.32 and d = 8.07 ppm as doublet peaks
(J = 8.44 Hz. Four aromatic proton signals in o-Cl phenyl moiety ap-
peared at the range of d = 7.20–7.84 ppm as multiplet peaks
(J = 8.44 Hz). Two proton signals in double bond appeared at
d = 7.87 and d = 7.09 ppm as doublet peaks (J = 16.00 Hz). Three
protons of methyl moiety appeared at the range of d = 2.75 ppm
À1
pound 5f showed the absorption band at 1666 cm assigned to
C@O stretching mode. The presence of N@C had been identified
À1
by the characteristic vibration at 1603 cm . The presence of NO
2
À1
13
and CACl had been identified at 1523, 1352 and 753 cm , respec-
tively. The H NMR spectra (400 MHz) of compound 5f indicated
as single peak. The C NMR spectrum of compound 5f showed
1
3
characteristic signals at 13.13 (CH ), 116.43, 123.34, 124.53,

4
0
M. Wan et al. / Bioorganic Chemistry 54 (2014) 38–43
1
(
27.96, 128.18, 131.57, 134.20 (CACl), 139.07, 148.37, 160.18
C@N), 174.30 and 184.74 (C@O) accounting for the presence of
different kinds of carbon atoms in the molecule.
properties (IC50) for compounds 5a–i are listed in Table 1. IC50 val-
ues were determined from log plots of percent of control vs
concentration.
2
.3. Compounds 5d and 5f–i induce apoptosis in A549 cells by cell
2
.2. Effects of compounds 5a–i on the Viability of Human Non-small
cycle analysis
Cell Lung Cancer Cells
According to the result of SRB assay, we found that 5d, 5f, 5g, 5h
and 5i were the effective compounds against cancer cells, so we
investigated the five compounds thoroughly. By flow cytometric
analysis of cell cycle, we found that 5f, 5g, 5h and 5i increased
The synthesized compounds were evaluated for their in vitro
anticancer activity using Sulforhodamine B (SRB) assays. The
growth-inhibitory experiments were undertaken in four human
non-small cell lung cancer cell lines, H1792, H157, A549 and
Calu-1 cells. The obtained data showed most compounds had
inhibitory effects on the growth of four cells in a dosage dependent
manner. Compounds 5a, 5b, 5c and 5e could mild inhibit the cell
the percentage of sub-G
/G phase, while 5d increased the percentage of sub-G
weakly in A549 cells. These results suggested that 5f, 5g, 5h and
0
phase and decreased the percentage of
G
0
1
0
phase
5
5
i induced apoptosis in A549 cells. In addition, we found that 5f,
g and 5h induced apoptosis in a dose dependent manner (Fig. 3).
growth at 40 lM after 48 h of the treatment. At 80 lM concentra-
tion, all compounds showed significantly inhibitory effects (Fig. 2).
Exposure of H1792, H157, A549 and Calu-1 cells to compounds 5d,
2
.4. Compounds 5f–i induce apoptosis in A549 cells
5
f, 5g and 5h at 20
ing from 100% to 79.9–51.5%, 58.1–20.5%, 60.9–20.9% and
0.5–9.0%. Exposure of H1792, H157, A549 and Calu-1 cells to
compounds 5d, 5f, 5g and 5h at 40 M for 48 h, the cell viabilities
reduced more significantly from 100% to 45.2–11.9%, 27.9–7.4%,
2.5–14.1% and 11.7–1.4%, respectively. When the concentration
of compounds 5f, 5g and 5h increased to 80 M, the cell viabilities
of H1792, H157, A549 and Calu-1 cells reduced to 28.4–7.9%, 5.9–
lM for 48 h resulted in cell viabilities decreas-
Then we determined whether 5f–i could induce apoptosis in
2
A549 cells by western blot. A549 cells were treated with concen-
trations (0, 20 and 40 M) of 5f–i respectively for 48 h, then the
l
l
cells were harvested and prepared for western blot analysis. We
found that 5f–i could induce the up-regulation of DR5 in a dose-
dependent manner (Fig. 4), suggesting that 5f–i activated the
extrinsic pathways of apoptosis. Next, we found that 5f–i induced
the cleaved bands of caspase 8, caspase 9, caspase 3 and PARP, indi-
cating that 5f–i induced apoptosis.
3
l
5
.2%, 10.3–3.9% and 5.3–0.5%.
In addition, we observed that the structure of the Ar group in
compounds 5a–i played an important role in their anticancer activ-
ity. When Ar was 4-nitrophenyl, 4-chlorophenyl and 4-bromophe-
nyl, the growth inhibitory property of these compounds was
inferior to that of compounds with 2-nitrophenyl, 2-chlorophenyl
and 2-bromophenyl, respectively. When Ar was 2-thienyl, phenyl
and 4-methylphenyl, the toxicity of these compounds was poor.
These results indicated that compounds bearing EWG at the 2-po-
sition of the phenyl ring were more effective. Growth inhibitory
3. Conclusion
In summary, we designed and synthesized a series of isoxazolyl
chalcones 5a–i. Most of the compounds showed significantly cyto-
toxic effect on human non-small cell lung cancer cells. Preliminary
structure–activity relationship study showed that compounds 5d,
Fig. 2. Cytotoxic effect of nine synthesized isoxazolyl chalcones on human non-small cell lung cancer cells. Cancer cells were treated with various concentrations (0, 5, 10, 20,
0, 80 M) of compounds 5a–i for 48 h. A viability assay was carried out. Experiments were performed in triplicate; data are expressed as means of the triplicate
determinations of a representative experiment in% cell viability of untreated cells (100%).
4
l

M. Wan et al. / Bioorganic Chemistry 54 (2014) 38–43
41
Table 1
as lead compounds to develop novel potent anti-lung cancer
agents.
Growth inhibitory properties IC50 (lM) for compounds 5a–i at 48 h.
Compound
Lung cancer cell lines
H1792
H157
90.22
A549
Calu-1
4. Experimental section
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
g
h
i
NDb
70.14
Inactive
50.67
19.32
72.33
1.48
12.87
11.07
17.71
83.57
Inactive
62.26
a
a
b
a
a
Inactive
80.55
28.20
Inactive
19.30
63.38
19.63
ND
Melting points were determined in open glass capillaries in
anelectrical melting point apparatus which was uncorrected. The
infrared (IR) spectra were recorded on Perkin–Elmer IR Spectro-
photometer using the KBr pellet technique. H NMR spectra was
6
recorded in DMSO-d on a Bruker NMR spectrometer at 400 MHz
60.38
17.13
68.05
1.35
12.14
b
ND
1
2.07
20.40
8.98
b
ND
7.27
12.77
using tetramethylsilane (TMS) as internal standard. Chemical shifts
are expressed in d, ppm. C NMR spectra were recorded in a Bru-
b
b
ND
ND
13
IC50: concentration of compound that inhibits cell proliferation by 50%.
6
ker Avance 400 (100 MHz, DMSO-d as solvent). The reactions were
a
Inactive: IC50 > 100
lM.
monitored by thin layer chromatography (TLC) on silica gel plates
using a mixture of petroleum ether and ethyl acetate. Chemicals
were obtained from commercial supplies and used without purifi-
cation. DR5 antibody was purchased from ProSci (Poway, CA). Cas-
pase 3 antibody was purchased from Imgenex (San Diego, CA).
Caspase 8, caspase 9 and PARP antibodies were purchased from
Cell Signaling Technology (Danvers, MA).
b
ND: not determined.
4
4
.1. Synthesis
.1.1. Synthesis of 4-nitrobenzaldehyde oxime (2)
4
-Nitrobenzaldehyde (0.2 mol, 30.2 g) and hydroxylamine
hydrochloride (0.2 mol, 13.9 g) were added to an ethanolic solution
250 mL) of anhydrous sodium carbonate (0.1 mol, 10.6 g) and the
(
reaction mixture was heated and refluxed for 6 h, and the reaction
was completed (TLC monitoring known). The ethanol was evapo-
rated, water was added and the mixture was extracted with dichlo-
romethane. The organic layer was dried over sodium sulfate and
after evaporation of the solvent the 4-nitrobenzaldehyde oxime
was obtained as a yellow solid (30.9 g, 93%), M.p. 122–124 °C
and it was used without further purification.
Fig. 3. A549 cell was treated with concentrations (0, 20 and 40 lM) of 5d and 5f–i
respectively for 48 h, then the cells were harvested and stained with propidium
iodide for detection of cell cycle by flow cytometry.
5
f and 5h bearing EWG at the 2-position of the phenyl ring in Ar
4.1.2. Synthesis of 1-(5-methyl-3-(4-nitrophenyl)isoxazol-4-
yl)ethanone (4)
1-Chloropyrrolidine-2,5-dione (NCS) (0.15 mol, 20 g) was added
in the solution of nitrobenzaldehyde oxime (0.15 mol, 25 g) and
100 mL N,N-dimethyl formamide (DMF) in ice bath. After the addi-
tion the reaction mixture was stirred for 12 h at room temperature
group were more effective than compounds 5e, 5g and 5i with
EWG at 4-position, respecitively. In A549 cells, compounds 5f–i
could induce apoptosis by death receptor 5 (DR5) mediated
extrinsic pathways. These results suggested isoxazolyl chalcones
bearing EWG at the 2-position of the phenyl ring could be used
Fig. 4. A549 cell was treated with concentrations (0, 20 and 40 lM) of 5f–i respectively for 48 h, then the cells were harvested and prepared for western blot analysis. b-Actin
expression were used as loading controls. CFs, cleaved forms.

4
2
M. Wan et al. / Bioorganic Chemistry 54 (2014) 38–43
and the reaction was completed (TLC monitoring known). After
that saturated NaCl solution (150 mL) and ethyl acetate (150 mL)
was added and stirred. The organic phase was separated and
washed with water (150 mL). The organic phase was dried over so-
dium sulfate and concentrated under reduced pressure. Dichloro-
methane (150 mL) and triethylamine (0.24 mol, 24 g) were added
to the residues and stirred at room temperature for 30 min. Then
acetyl acetone (0.2 mol, 20 g) was added and stirred for 12 h.
Water was added, the organic phase was separated, dried with so-
J = 16.00 Hz), 7.68–7.90 (m, 4H, PhH), 7.88 (d, 1H, @CH,
J = 16.00 Hz), 8.06 (d, 2H, PhH, J = 8.40 Hz), 8.35 (d, 2H, PhH,
J = 8.40 Hz); C NMR: (100 MHz, DMSO-d ): 13.04 (CH ), 116.24,
6 3
122.86, 123.69, 124.79, 128.61, 129.56, 130.31, 131.59, 134.48,
139.57, 148.45 (CAN), 160.16 (C@N), 174.18, 185.11 (C@O); Anal.
1
3
Calcd for C19
13 2 6
H N O : C, 60.16; H, 3.45; N, 11.08. Found: C,
60.52; H, 3.63; N, 10.93.
4
.1.3.5.
1-(5-Methyl-3-(4-nitrophenyl)isoxazol-4-yl)-3-(4-nitro-
: 3443, 1677, 1608, 1528, 1350; 1H NMR
, d): 2.77 (s, 3H, CH ), 7.08 (d, 1H, COCH@,
dium sulfate and concentrated under reduced pressure to give a
phenyl)prop-2-en-1-one (5e). Yield: 79%; M.p. 181–182 °C; Gray
À1
yellow powder (29.5 g, 80%), M.p. 134–136 °C. IR (KBr, cm
3
)
m:
À1
solid; IR (KBr, cm
400 MHz, DMSO-d
) m
443, 1677, 1609, 1529, 1361; 1H NMR (400 MHz, DMSO-d
.34 (s, 3H, CH ), 2.79 (s, 3H, COCH ), 7.84 (d, 2H, PhH,
6
, d):
(
6
3
2
3
3
J = 16.00 Hz), 8.06 (d, 2H, PhH, J = 8.44 Hz), 7.72 (d, 2H, PhH,
J = 8.44 Hz), 7.90 (d, 1H, @CH, J = 16.00 Hz), 8.07 (d, 2H, PhH,
13
J = 8.60 Hz), 8.33 (d, 2H, PhH, J = 8.60 Hz); C NMR: (100 MHz,
DMSO-d ): 13.63 (CH ), 30.46 (CH ), 116.72, 123.33, 130.72,
35.16, 148.28, 160.50 (C@N), 174.71, 191.82 (C@O).
13
6
3
3
J = 8.40 Hz), 8.36 (d, 2H, PhH, J = 8.40 Hz); C NMR: (100 MHz,
1
DMSO-d
6
): 13.58 (CH
30.25, 134.83, 140.34, 148.24 (CAN), 151.56, 160.51 (C@N),
74.26, 185.32 (C@O); Anal. Calcd for C19 : C, 60.16; H,
.45; N, 11.08. Found: C, 60.74; H, 3.42; N, 11.21.
3
), 116.69, 122.82, 123.32, 126.87, 129.19,
1
1
3
4.1.3. General procedure for the synthesis of compounds 5a–i
13 2 6
H N O
5
-Methyl-3-(4-nitrophenyl)-4-acetyl-iso-oxazole (8 mmol, 2 g)
and aromatic aldehydes (8 mmol) were added to the solution of
ethanol (150 mL) and 10% aq NaOH (3 mL). The reaction mixture
was stirred for 3–6 h at room temperature and the reaction was
completed (TLC monitoring known). The precipitate was filtered
and washed with water to obtain the compounds 5a–i. The pure
products were crystallizeed in ethanol.
4
.1.3.6. 3-(2-Chlorophenyl)-1-(5-methyl-3-(4-nitrophenyl)isoxazol-4-
yl)prop-2-en-1-one (5f). Yield: 66%; M.p. 162–164 °C; White solid;
IR (KBr, cm
À1
: 3443, 1666, 1603, 1523, 1352, 753; 1H NMR
) m
(
6 3
400 MHz, DMSO-d , d): 2.75 (s, 3H, CH ), 7.09 (d, 1H, COCH@,
J = 16.00 Hz), 7.20–7.84 (m, 4H, PhH), 7.87 (d, 1H, @CH,
J = 16.00 Hz), 8.07 (d, 2H, PhH, J = 8.44 Hz), 8.32 (d, 2H, PhH,
J = 8.44 Hz); C NMR: (100 MHz, DMSO-d ): 13.13 (CH ), 116.43,
6 3
4
.1.3.1. 1-(5-Methyl-3-(4-nitrophenyl)isoxazol-4-yl)-3-(thiophen-2-
13
yl)prop-2-en-1-one (5a). Yield: 65%; M.p. 163–164 °C; Yellow so-
lid; IR (KBr, cm
1
1
H
8
23.34, 124.53, 127.96, 128.18, 131.57, 134.20 (CACl), 139.07,
48.37, 160.18 (C@N), 174.30, 184.74 (C@O); Anal. Calcd for C19-
13ClN : C, 61.88; H, 3.55; N, 7.60. Found: C, 61.34; H, 3.81; N,
.08.
À1
) m
: 3442, 1659, 1591, 1524, 1353; 1H NMR
(
400 MHz, DMSO-d
J = 15.60 Hz), 7.14–7.81 (m, 3H, Thiophene ring), 7.74 (d, 1H,
CH, J = 15.60 Hz), 7.86 (d, 2H, PhH, J = 8.40 Hz), 8.34 (d, 2H,
6 3
, d): 2.69 (s, 3H, CH ), 6.58 (d, 1H, COCH@,
2 4
O
@
1
3
PhH, J = 8.40 Hz); C NMR: (100 MHz, DMSO-d
1
1
for C17H N O
12 2 4
3
6
): 12.93 (CH
16.35, 123.64, 126.81, 128.82, 130.50, 131.08, 134.72, 137.63,
39.00, 148.36, 159.99 (C@N), 173.71, 184.43 (C@O); Anal. Calcd
S: C, 59.99; H, 3.55; N, 8.23. Found: C, 59.93; H,
3
),
4.1.3.7. 3-(4-Chlorophenyl)-1-(5-methyl-3-(4-nitrophenyl)isoxazol-4-
yl)prop-2-en-1-one (5g). Yield: 87%; M.p. 217–220 °C; White solid;
À1
) m
: 3426, 1658, 1600, 1529, 1352, 784; 1H NMR
IR (KBr, cm
(
6 3
400 MHz, DMSO-d , d): 2.71 (s, 3H, CH ), 7.06 (d, 1H, COCH@,
.72; N, 8.15.
J = 16.00 Hz), 7.48 (d, 2H, PhH, J = 8.44 Hz), 7.61 (d, 2H, PhH,
J = 8.44 Hz), 7.67 (d, 1H, @CH, J = 16.00 Hz), 7.86 (d, 2H, PhH,
4
.1.3.2. 1-(5-Methyl-3-(4-nitrophenyl)isoxazol-4-yl)-3-phenylprop-2-
1
3
J = 8.40 Hz), 8.33(d, 2H, PhH, J = 8.40 Hz); C NMR: (100 MHz,
DMSO-d ): 13.02 (CH ), 116.40, 123.67, 124.64, 126.31, 128.60,
29.61, 130.44, 134.59, 135.51 (CACl), 149.40, 160.18 (C@N),
74.50, 185.40 (C@O); Anal. Calcd for C19 13ClN : C, 61.88; H,
en-1-one (5b). Yield: 81%; M.p. 180–183 °C; White solid; IR (KBr,
cm
DMSO-d
7
2
À1
: 3422, 1657, 1592, 1524, 1350; 1H NMR (400 MHz,
6
3
) m
1
1
6
, d): 2.70 (s, 3H, CH
.28–7.63 (m, 5H, PhH), 7.61 (d, 1H, @CH, J = 16.00 Hz), 7.84 (d,
H, PhH, J = 8.40 Hz), 8.46 (d, 2H, PhH, J = 8.40 Hz); 13C NMR:
): 12.99 (CH ), 116.45, 123.51, 125.67,
3
), 7.03 (d, 1H, COCH@, J = 16.00 Hz),
H
2 4
O
3.55; N, 7.60. Found: C, 61.29; H, 3.67; N, 8.34.
(
100 MHz, DMSO-d
6
3
4.1.3.8. 3-(2-Bromophenyl)-1-(5-methyl-3-(4-nitrophenyl)isoxazol-4-
1
1
C
28.75, 130.50, 131.18, 130.61, 130.61, 133.97, 134.66, 145.14,
48.32, 160.15 (C@N), 173.58, 185.46 (C@O); Anal. Calcd for
yl)prop-2-en-1-one (5h). Yield: 56%; M.p. 135–137 °C; White solid;
À1
) m
: 3416, 1666, 1602, 1523, 1353, 760; 1H NMR
IR (KBr, cm
19
14 2 4
H N O : C, 68.26; H, 4.22; N, 8.38. Found: C, 68.09; H, 4.74;
(
6 3
400 MHz, DMSO-d , d): 2.49 (s, 3H, CH ), 6.70 (d, 1H, COCH@,
N, 8.52.
J = 16.00 Hz), 6.89–7.87 (m, 4H, PhH), 7.69 (d, 1H, @CH,
J = 16.00 Hz), 8.06 (d, 2H, PhH, J = 8.44 Hz), 8.32 (d, 2H, PhH,
4
.1.3.3. 1-(5-Methyl-3-(4-nitrophenyl)isoxazol-4-yl)-3-p-tolylprop-2-
1
3
J = 8.44 Hz); C NMR: (100 MHz, DMSO-d
123.29 (C-Br), 124.59, 127.99, 128.27, 132.74, 134.53, 142.79,
48.09, 160.18 (C@N), 174.25, 184.73 (C@O); Anal. Calcd for C19-
13BrN : C, 55.23; H, 3.17; N, 6.78. Found: C, 55.31; H, 3.40; N,
.72.
6 3
): 13.41 (CH ), 116.20,
en-1-one (5c). Yield: 49%; M.p. 203–206 °C; White solid; IR (KBr,
cm
DMSO-d
COCH@, J = 16.00 Hz), 7.21 (d, 2H, PhH, J = 7.60 Hz), 7.50 (d, 2H,
PhH, J = 7.60 Hz), 7.63 (d, 1H, @CH, J = 16.00 Hz), 7.85 (d, 2H,
À1
: 3422, 1657, 1592, 1524, 1350; 1H NMR (400 MHz,
, d): 2.32 (s, 3H, CH ), 2.69 (s, 3H, CH ), 6.98 (d, 1H,
) m
1
H
6
6
3
3
2 4
O
13
PhH, J = 8.40 Hz), 8.32 (d, 2H, PhH, J = 8.40 Hz);
100 MHz, DMSO-d ): 12.95 (CH ), 21.02 (CH ), 116.48, 123.52,
24.68, 128.68, 129.67, 131.25, 134.69, 141.24, 145.27, 148.31,
60.11 (C@N), 173.43, 185.42 (C@O); Anal. Calcd for C20
C NMR:
(
1
1
6
3
3
4.1.3.9. 3-(4-Bromophenyl)-1-(5-methyl-3-(4-nitrophenyl)isoxazol-4-
yl)prop-2-en-1-one (5i). Yield: 92%; M.p. 198–200 °C; White solid;
À1
: 3443, 1660, 1600, 1529, 1353, 779; 1H NMR
H
16
N
2
O
4
:
IR (KBr, cm
)
m
C, 68.96; H, 4.63; N, 8.04. Found: C, 68.34; H, 4.96; N, 7.89.
6 3
(400 MHz, DMSO-d , d): 2.71 (s, 3H, CH ), 7.08 (d, 1H, COCH@,
J = 16.00 Hz), 7.60 (d, 2H, PhH, J = 8.40 Hz), 7.62 (d, 2H, PhH,
J = 8.40 Hz), 7.67 (d, 1H, @CH, J = 16.00 Hz), 7.87 (d, 2H, PhH,
4
.1.3.4. 1-(5-methyl-3-(4-nitrophenyl)isoxazol-4-yl)-3-(2-nitro-
1
3
phenyl)prop-2-en-1-one (5d). Yield: 77%; M.p. 188–190 °C; Gray
solid; IR (KBr, cm
J = 8.40 Hz), 8.33(d, 2H, PhH, J = 8.40 Hz); C NMR: (100 MHz,
DMSO-d ): 13.63 (CH ), 116.40, 123.67 (CABr), 124.63, 126.34,
128.44, 130.45, 132.27, 134.74, 143.75, 148.32, 160.16 (C@N),
À1
: 3442, 1660, 1605, 1521, 1350; 1H NMR
)
m
6
3
(
6 3
400 MHz, DMSO-d , d): 2.76 (s, 3H, CH ), 7.02 (d, 1H, COCH@,

M. Wan et al. / Bioorganic Chemistry 54 (2014) 38–43
43
1
3
73.66, 185.39 (C@O); Anal. Calcd for C19
.17; N, 6.78. Found: C, 55.23; H, 3.21; N, 7014.
H
13BrN
2
O
4
: C, 55.23; H,
conjugated secondary antibody at room temperature for 1 h; final-
ly, the antibody binding was detected by the ECL reagents follow-
ing the manufacturer’s protocol.
4.2. Cell culture
Acknowledgments
Human non-small cell lung cancer (NSCLC) cell lines H1792,
A549, H157 and Calu-1 were purchased from the American Type
Culture Collection and maintained in monolayer culture in RPMI-
We thank Wei Hua and Tao Wang for technical assistance. This
work was supported by the Shandong Province Natural Science
Foundation, China (ZR2013BQ010).
1
640 medium with 5% fetal bovine serum in a 5% CO
2
, humidified
incubator at 37 °C.
4.3. Cell survival assay
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