Synthesis of 6-cinnamoyl-2H-benzo[b][1,4]oxazin-3(4H)-ones and their effects on A549 lung cancer cell growth

Article abstract of DOI:10.1016/j.ejmech.2014.03.087

A series of novel 6-cinnamoyl-2H-benzo[b][1,4]oxazin-3(4H)-one derivatives was synthesized. The structures of compounds were characterized by ¹H NMR, IR, and MS. Moreover, representative crystal structure was determined by X-ray diffraction ana

Full text of DOI:10.1016/j.ejmech.2014.03.087

European Journal of Medicinal Chemistry 79 (2014) 95e101
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of 6-cinnamoyl-2H-benzo[b][1,4]oxazin-3(4H)-ones and
their effects on A549 lung cancer cell growth
,
,
,
*
Xue-Wen Zhou a 1, Han-Lin Ma b 1, Xuan Zhang a, Shi-Yao Jing a, Jun-Ying Miao b
,
Bao-Xiang Zhao a
,
*
a Institute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, PR China
b Institute of Developmental Biology, School of Life Science, Shandong University, Jinan 250100, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 6-cinnamoyl-2H-benzo[b][1,4]oxazin-3(4H)-one derivatives was synthesized. The
structures of compounds were characterized by 1H NMR, IR, and MS. Moreover, representative crystal
structure was determined by X-ray diffraction analysis. The preliminary biological evaluation of all these
compounds showed that compounds 3ae3d would suppress the growth of A549 lung cells effectively by
inducing autophagy and cell cycle arrest.
Received 16 January 2014
Received in revised form
28 March 2014
Accepted 31 March 2014
Available online 1 April 2014
Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Benzoxazinone
Chalcone
A549 lung cells
Autophagy
1. Introduction
Compounds of benzoxazinone class, with the modifiable skel-
eton and relative accessibility, have attracted much attention of
With the increasingly serious environmental problems and the
dramatic increase of smoking rate, lung cancer is becoming primary
cause of cancer-related mortality not only in China but also around
the world. It has been believed that the primary modality for the
treatment of lung cancer is chemotherapy. Consequently, much
attention has recently been paid to the discovery and development
of new, more selective anticancer agents [1e4].
The common method of lung cancer chemotherapy issue
effective drugs for the treatment of patient [5]. Different mecha-
nisms of drug-induced death of cancer cells are not the same.
Therefore, the understanding of mechanisms of cancer cells death
can help us discover more convenient therapeutic strategies [6].
Several mechanisms of cell death including necrosis, apoptosis and
autophagy have been known [7]. However, we cannot expect the
mechanism by which a new small molecule inhibits cancer cell
growth. Therefore, it needs to design and synthesize new com-
pounds and screen effective lung cancer cell growth inhibitor.
chemists [8e11]. Benzoxazinone derivatives have a broad range of
biological activities, such as treatment of cardiovascular diseases
[12e14], antibacterial [15,16], antifungal [17e19], anticancer activ-
ity [20e22] and protein methyltransferases inhibitors [23].
Chalcones consist of two aryl rings linked by a,b-unsaturated
carbonyl system, which are members of flavonoids. Chalcone de-
rivatives have occupied an important position in drug discovery,
such as anti-inflammatory [24,25], antimicrobial activity [26e29],
anti-parasitic activity [30,31], acetylcholine esterase inhibition [32],
antidyslipidemic [33] and anticancer activity [34e38].
Based on the advantages of benzoxazinone and chalcone moi-
eties, we are interested in combining these active moieties to
design and synthesize new compounds and test their biological
activity. Herein we would like to report the synthesis, crystal
structure of 6-cinnamoyl-2H-benzo[b][1,4]oxazin-3(4H)-ones and
the findings of their biological activities against A549 lung cancer
cell growth and possible function mechanisms.
2. Chemistry
2.1. Synthesis
* Corresponding authors.
E-mail addresses: miaojy@sdu.edu.cn (J.-Y. Miao), bxzhao@sdu.edu.cn,
sduzhao@hotmail.com (B.-X. Zhao).
We designed and synthesized compounds 3ae3g as shown in
Scheme 1. Compound 2 was obtained by FriedeleCrafts acylation,
1
Equal contribution.
http://dx.doi.org/10.1016/j.ejmech.2014.03.087
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

96
X.-W. Zhou et al. / European Journal of Medicinal Chemistry 79 (2014) 95e101
Scheme 1. Synthesis of 6-cinnamoyl-2H-benzo[b][1,4]oxazin-3(4H)-ones.
and then, reacted with corresponding aldehyde to get the target
products.
2.2. Single-crystal structural characterization by X-ray
The spatial structure of compound 3a was determined by X-ray
diffraction analysis. The single crystal was grown from ethyl acetate
solution at room temperature. The molecular view of 3a is shown in
Fig. 1. The molecule of 3a consists three fragments, an aryl ring, a
benzoxazinone, and a,b-unsaturated carbonyl system.
Fig. 1. The X-ray crystal structure of compound 3a.
3. Pharmacology
Screening of synthesized substances was carried out using lung
cancer A549 cell line. Proliferation percentage was determined by
the SRB assay. Cells were incubated with compounds at 1, 5 and
mechanism [39]. Paradoxically, autophagy plays an important role
both in protective process and cell death [40]. Therefore, it is very
complex between autophagy and cancer treatment due to the pro-
survival and pro-death functions of autophagy. In the early stage of
cancer progression, enhanced autophagy has been regarded as the
inhibitor of tumor genesis through degradation of oncogenic pro-
teins. On the other hand, in the late stages of oncogenesis, auto-
phagy may assist tumor cells against metabolic stress and resist
death caused by chemotherapeutic [40e42].
10 mM for 48 h and the cell proliferation/viability determination
using the survival percentage obtained with the cell treated only
with the solvent (DMSO at 0.1%) as reference. The results are
expressed as the average of triplicate assays.
4. Results and discussion
4.1. Compounds 3ae3g inhibited proliferation of A549 lung cancer
cells
In order to determine the changes of autophagy in the growth
inhibitory effect of compounds 3ae3d, firstly, we performed acri-
dine orange (AO) staining. After treatment with compounds 3ae3d
In order to examine the anti-cancer activity of compounds 3ae
3g, we firstly observed the morphological changes of A549 cells
after treatment with the compounds for 24 h or 48 h with a phase
contrast microscope (Fig. 2). We found no obvious morphological
(5 mM) for 48 h (Fig. 4), compounds 3c, 3d could apparently increase
the acidic vesicles accumulation in A549 cells compared with
control, however, there was no clear facilitation effect in com-
pounds 3a, 3b treated groups. These results suggested that com-
pounds 3ae3d may have different effect on autophagy of
A549 cells.
To exhibit a far more explicit function of compounds 3ae3d on
autophagy of A549 cells, we detected the protein level of LC3-II
(Fig. 5), the up regulation of which means the promotion of auto-
phagy [43]. Western blotting assay showed that the expression of
changes in A549 cells treated with 5 mM dose of each compound for
48 h. However, the cell density greatly decreased in response to
compounds 3ae3d compared with control group. SRB assay
showed that compounds 3ae3d inhibited the growth of A549 cells
in dose dependent manner after treatment with the compounds for
48 h (Fig. 3). Among the four compounds, compound 3c (10 mM)
showed the most obvious effect and could inhibit the viability of
A549 cells to 24% compared with control group. Table 1 showed
compounds 3a, 3c and 3d have lower IC50 value.
LC3-II promoted after treatment with compounds 3c, 3d (5 mM) for
48 h, whereas compound 3b had no effect on LC3-II protein level.
Interestingly, in contrast with compounds 3c, 3d, compound 3a
could inhibit the expression of LC3-II protein. The study of struc-
tureeactivity relationship showed that compounds with multiple
methoxy groups and no substituent group in aryl moiety altered
the antitumor effect significantly. The underlying mechanism by
which compounds 3ae3d function on the autophagy of A549 cells
needed to be further investigation.
4.2. Compounds 3ae3d showed different effect on autophagy of
A549 cells
Autophagy is a basic homeostatic process working in almost
every mammalian cell types and plays a key role in cell survival

X.-W. Zhou et al. / European Journal of Medicinal Chemistry 79 (2014) 95e101
97
Fig. 2. Effects of the compounds 3ae3g on morphology of A549 cells. A549 cells were treated with 3ae3g (5 mM) or 0.1% DMSO (control) for 24 h or 48 h.
the compounds 3ae3d (5
mM) did not induce apoptosis in
A549 cells at 48 h significantly.
4.5. Compounds 3ae3d did not cause necrosis in A549 cells
To determine whether the proliferation inhibitory effects of
compounds 3ae3d were due to necrosis or not, which is undesired
in cancer therapy, we detected the LDH level after treatment with
compounds 3ae3d (10
an indicator of necrosis. Results showed that compounds 3ae3d
(10 M) did not cause necrosis in a certain period of time (48 h) in
mM) for 48 h, the up regulation of which is
m
A549 cells (Fig. 8).
Fig. 3. Effects of compounds 3ae3g on A549 cell viability. Cells were treated with
compounds 3ae3g, 5-FU and 0.1% DMSO (control) at the concentration of 1, 5 or 10
for 48 h. Cell viability was analyzed by SRB assay. Results were presented as
mM
5. Conclusion
mean ꢁ SE (n ¼ 3, *p < 0.05 vs. control; **p < 0.01 vs. control; ##p<0.01vs. normal).
In summary, we have synthesized a series of novel 6-cinnamoyl-
2H-benzo[b][1,4]oxazin-3(4H)-ones by the condensation of 6-
acetyl-2H-benzo[b][1,4]oxazin-3(4H)-one and corresponding aro-
matic aldehydes. Compounds 3ae3d can suppress the growth of
A549 lung cancer cells by inducing autophagy and cell cycle arrest.
4.3. Compounds 3ae3d induced G1-phase arrest
One main feature of cancer cells is multiply uncontrollably. In
tumors, there are much more cells that are in active cell division
compared with the normal tissues [44]. So, cell cycle arrest be-
comes a critical therapy target in oncotherapy [45,46]. Flow cyto-
6. Experimental
metric analysis indicated that compounds 3ae3d (5
mM) could
6.1. Reagents and apparatus
induce A549 cell cycle arrest in G1-phase after treatment for 48 h.
Compound 3c showed the best effect and could enhance the G1
population by 24.76% compared with control group. Meanwhile,
the portion of cells in G2-phase had a slight increase after treat-
All reagents were of analytical grade or chemically pure.
Analytical TLC was performed on silica gel 60 F254 plates (Merck
KGaA). Melting points were determined on an XD-4 digital micro-
melting point apparatus. HRMS spectra were recorded on a LTQ
Orbitrap Hybrid mass spectrograph. 1H NMR spectra were recorded
on a Bruker Avance 400 (400 MHz) spectrometer or Bruker Avance
300 (300 MHz) spectrometer, using DMSO as solvent and tetra-
methylsilane (TMS) as an internal standard.
ment with compounds 3ae3d (5 mM) for 48 h (Fig. 6).
4.4. Compounds 3ae3d did not induce apoptosis in A549 cells
Apoptosis, the process of programmed cell death (PCD), is an
important therapy target for cancer chemotherapy. Biochemical
events cause characteristic cell changes, including nuclear frag-
mentation, chromatin condensation and the fragmentation of
chromosomal DNA [47,48]. Hoechst 33258 staining was performed
to detect the effect of compounds 3ae3d on apoptosis of A549 cells
6.2. Preparation of 6-acetyl-2H-benzo[b][1,4]oxazin-3(4H)-ones (2)
Compound 1 (20 mmol), aluminum chloride (100 mmol) and
100 ml dry CH2Cl2 were added in round-bottom flask equipped
with calcium chloride guard tube under ice bath for 30 min, then
Ac2O (20 mmol) was added to the mixture. The reaction mixture
was refluxed for 5 h, monitored by TLC until completion. The
mixture was diluted with water and filtered. The filtrate was
washed with saturate NaHCO3 solution (100 ml ꢀ 2), and water
(100 ml ꢀ 1), dried over anhydrous Na2SO4, and filtered. The filtrate
was concentrated under reduced pressure. Recrystallization from
ethanol afforded the pure products 2.
(Fig. 7). After treatment with compounds 3ae3d (5 mM) for 48 h,
there were no obvious chromatin condensation and increase in the
formation of apoptotic bodies compared with control group. Thus,
Table 1
IC50 of the compounds 3ae3d and 5-FU for A549 cells at 48 h.
Compounds
IC50 M)
5-FU
0.98
3a
3b
3c
3d
(
m
5.46
16.31
3.29
6.49

98
X.-W. Zhou et al. / European Journal of Medicinal Chemistry 79 (2014) 95e101
Fig. 4. Effects of compounds 3ae3d (5
microscope (Nikon).
mM) on acidic vesicles accumulation in A549 cells after treatment for 48 h. Microscopic photographs (200ꢀ) were taken under a fluorescent
6.4.2. 6-(3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)-2H-benzo[b][1,4]
oxazin-3(4H)-one(3b)
Yield 45.2%, yellow solid, mp 267e268 ꢂC. IR (KBr),
n
/cmꢃ1
1H NMR (DMSO,
¼ 10.86 (s, 1H, NH), 7.91 (dd, J1 ¼ 2.1, J2 ¼ 8.4 Hz, 1H,
CH), 6.98e7.77 (m, 6H, ArH), 7.08 (d, J ¼ 8.4 Hz, 1H, CH), 6.11 (s, 2H,
CH2), 4.70 (s, 2H, CH2). 13C NMR (DMSO, 75 MHz):
:
3444 (NH), 1693 (C]O), 1651 (C]O) cmꢃ1
300 MHz):
.
d
d
¼ 187.14, 164.13,
149.48, 148.07, 147.24, 143.55, 132.20, 129.21, 127.46, 125.72, 124.80,
119.77, 115.83, 115.58, 108.50, 106.88, 101.62, 66.75. HRMS calcd for
C
18H14NO5 [MþH]þ: 324.0872, found: 324.0859.
6.4.3. 6-(3-(3,5-Dimethoxyphenyl)acryloyl)-2H-benzo[b][1,4]
oxazin-3(4H)-one (3c)
Yield 63.8%, light yellow solid, mp 240e241 ꢂC. IR (KBr), /cmꢃ1
n :
3443 (NH), 1697 (C]O), 1656 (C]O). 1H NMR (DMSO, 400 MHz):
¼ 10.88 (s, 1H, NH), 7.93 (dd, J1 ¼ 2.04, J2 ¼ 8.44 Hz, 1H, CH), 6.59e
7.52 (m, 6H, ArH), 7.10 (d, J ¼ 8.40 Hz, 1H, CH), 4.72 (s, 2H, CH2), 3.81
(s, 6H, CH3). 13C NMR (DMSO, 75 MHz):
d
d
¼ 187.51, 164.24, 160.85
(2C), 147.56, 143.78, 136.76, 132.08, 127.66, 125.14, 122.46, 116.01,
115.80, 106.78 (2C), 102.91, 66.90, 55.56 (2C). HRMS calcd for
C
19H18NO5 [MþH]þ: 340.1185, found: 340.1180.
6.4.4. 6-(3-(2,4-Dimethoxyphenyl)acryloyl)-2H-benzo[b][1,4]
oxazin-3(4H)-one (3d)
Yield 53.4%, yellow solid, mp 222e223 ꢂC. IR (KBr),
n :
/cmꢃ1
Fig. 5. Effects of compounds 3ae3d (5
mM) on the protein level of LC3-II. Cells were
3449 (NH), 1697 (C]O), 1642 (C]O). 1H NMR (DMSO, 300 MHz):
¼ 10.85 (s, 1H, NH), 7.79 (dd, J1 ¼ 1.8, J2 ¼ 8.4 Hz, 1H, CH), 6.61e
7.96 (m, 6H, ArH), 7.09 (d, J ¼ 8.4 Hz, 1H, CH), 4.70 (s, 2H, CH2), 3.91
(s, 3H, CH3), 3.85 (s, 3H, CH3). 13C NMR (DMSO, 75 MHz):
treated with compounds 3ae3d (5 M) for 48 h. The protein levels of LC3-II were
m
determined by Western blotting (A). The relative levels of LC3-II were normalized to
the level of GAPDH and represented as percent of control (B) (n ¼ 3, *P < 0.05 vs.
control).
d
d
¼ 187.28,
164.15, 163.00, 159.96, 147.04, 138.50, 132.44, 130.33, 127.38, 124.38,
118.86, 115.93, 115.57, 106.31, 98.34, 66.75, 55.81, 55.51. HRMS calcd
for C19H18NO5 [MþH]þ: 340.1185, found: 340.1172.
6.3. Preparation of 6-cinnamoyl-2H-benzo[b][1,4]oxazin-3(4H)-
ones (3ae3g)
6.4.5. 6-(3-(4-(Benzyloxy)phenyl)acryloyl)-2H-benzo[b][1,4]
Compound 2 (1.5 mmol), corresponding aldehyde (1.8 mmol),
sodium hydroxide (3 mmol) and 40 ml methanol were added into
round-bottom flask, then the reaction mixture was refluxed for
12 h. The reaction mixture was cooled to room temperature and
filtered, the residue was washed with water three times, then
washed with methanol two times and dried under infrared lamp to
get corresponding product 3.
oxazin-3(4H)-one (3e)
Yield 53.8%, yellow solid, mp 258e259 ꢂC. IR (KBr),
n :
/cmꢃ1
3445 (NH), 1704 (C]O), 1646 (C]O). 1H NMR (DMSO, 400 MHz):
¼ 10.86 (s, 1H, NH), 7.79 (dd, J1 ¼1.96, J2 ¼ 8.44 Hz, 1H, CH), 7.07e
7.84 (m, 6H, ArH), 7.09 (d, J ¼ 8.4 Hz, 1H, CH), 5.19 (s, 2H, CH2), 4.70
(s, 2H, CH2). 13C NMR (DMSO, 75 MHz):
d
d
¼ 187.19, 164.14, 160.36,
147.20, 143.44, 136.68, 132.25, 130.62 (2C), 128.43 (2C), 127.91,
127.73 (2C), 127.52, 127.49, 124.66, 119.41, 115.86, 115.62, 115.3 (2C),
69.38, 66.75. HRMS calcd for C24H20NO4 [MþH]þ: 386.1392, found:
386.1366.
6.4. Spectroscopy data of compounds 3ae3g
6.4.1. 6-Cinnamoyl-2H-benzo[b][1,4]oxazin-3(4H)-one (3a)
Yield 55.2%, yellow solid, mp 245e247 ꢂC. IR (KBr), /cmꢃ1: 3456
n
(NH), 1692 (C]O), 1651 (C]O). 1H NMR (DMSO, 400 MHz):
6.4.6. 6-(3-(4-Chlorophenyl)acryloyl)-2H-benzo[b][1,4]oxazin-
d
¼ 10.87 (s, 1H, NH), 7.91 (dd, J1 ¼ 2.04, J2 ¼ 8.36 Hz, 1H, CH), 7.46e
3(4H)-one (3f)
7.89 (m, 8H, ArH), 7.10 (d, J ¼ 8.36 Hz, 1H, CH), 4.72 (s, 2H, CH2). 13
C
Yield 63%, light yellow solid, mp 297e298 ꢂC. IR (KBr), /cmꢃ1
n :
NMR (DMSO, 100 MHz):
d
¼ 187.82, 164.63, 147.89, 144.04, 135.17,
3463 (NH), 1706 (C]O), 1651 (C]O). 1H NMR (DMSO, 400 MHz):
¼ 10.88 (s, 1H, NH), 7.92 (d, J ¼ 8.5 Hz, 1H, CH), 7.52e7.92 (m, 7H,
132.45, 131.03, 129.40 (2C), 129.26 (2C), 128.01, 125.42, 122.29,
116.43, 116.13, 67.25. HRMS calcd for C17H14NO3 [MþH]þ: 280.0974,
found: 280.0973.
d
ArH), 7.10 (d, J ¼ 8.40 Hz, 1H, CH), 4.72 (s, 2H, CH2). HRMS calcd for
C
17H13ClNO3 [MþH]þ: 314.0584, found: 314.0540.

X.-W. Zhou et al. / European Journal of Medicinal Chemistry 79 (2014) 95e101
99
Fig. 6. Effects of compounds 3ae3d (5 mM) on cell cycle distribution of A549 cells after treatment for 48 h. The values represented three independent experimental results.
6.4.7. 6-(3-(Naphthalen-2-yl)acryloyl)-2H-benzo[b][1,4]oxazin-
3(4H)-one (3g)
wavelength of 540 nm using a SpectraMAX190 microplate spec-
trophotometer (GMI Co, USA).
Yield 41.3%, white solid, mp 272e274 ꢂC (ref. 272e273 ꢂC
[14,49]). IR (KBr),
NMR (DMSO, 400 MHz)
ArH, CH), 7.12 (d, J ¼ 8.40 Hz, 1H, CH), 4.73 (s, 2H, CH2). HRMS calcd
n
/cmꢃ1: 3448 (NH), 1696 (C]O), 1649 (C]O). 1H
6.8. LDH assay
d
¼ 10.89 (s, 1H, NH), 7.60e8.55 (m, 11H,
Cell culture medium was gathered after 48 h treatment with
compounds 3ae3d (10 mM) or 0.1% DMSO (as control). LDH assay
for C21H16NO3 [MþH]þ: 330.1130, found: 330.1086.
was performed using a Lactate Dehydrogenase (LDH) kit (Nanjing
6.5. X-ray crystallography
Jiancheng Co, China), according to the manufacturer’s instructions.
Suitable single crystals of 3a for X-ray structural analysis were
obtained by slow evaporation of a solution of the solid in ethyl
acetate at room temperature for 5 days. The crystals 3a were
6.9. Acridine orange (AO) staining
After treatment with compounds 3ae3d (5
A549 cells were stained with acridine orange (5 g/ml) for 1 min at
room temperature. Then cells were washed with PBS twice and
observed under an Olympus (Japan) BH-2 fluorescence microscope.
mM) for 48 h,
mounted on a Bruker Smart Apex II CCD equipped with a graphite
m
ꢀ
monochromated MoK
a
radiation (
l
¼ 0.71073 A) by using 4 and
u
scan modes and the data were collected at 298(2) K. The structure
of the crystal was solved by direct methods and refined by full-
matrix least-squares techniques implemented in the SHELXTL-97
crystallographic software.
6.10. Western blot analysis
As described previously, cells were washed twice with ice cold
PBS and were then lysed in protein lysis buffer (Shanghai beyotime
Co., China). The protein concentration of the cells was determined
by Bradford method. Following electrophoresis, the resolved pro-
tein was electrophoretically transferred to a Polyvinylidene Fluo-
ride (PVDF) membrane (Millipore, MA, USA). The membrane was
blocked with 5% non-fat milk in PBST (PBS containing 0.05% Tween
20) for 1 h at room temperature. Subsequently the membrane was
probed with Rabbit anti-human LC3b antibody and anti-GAPDH
mouse monoclonal antibody overnight at 4 ꢂC, and was then
washed 3 times with PBST, each time for 5 min. The membrane was
subsequently incubated with HRP-conjugated goat anti-rabbit IgG
or polyclonal rabbit anti-mouse immunoglobulins/HRP for 1 h at
room temperature and then washed 3 times with PBST. Then the
membrane was incubated with HRP substrate for 5 min and the
fluorescence signals were detected with X-ray films. The relative
6.6. Cell culture
Human lung cancer cell line A549 were cultured in RPMI-1640
medium at 37 ꢂC in 5% CO2, supplemented with 10% (v/v) bovine
calf serum and 80 U/ml penicillin/streptomycin. The cells were
seeded onto 24-well plates or other appropriate dishes at a number
of 20,000 cells/ml.
6.7. Cell viability assay (SRB)
As previously reported, A549 cells were seeded onto 96-well
plates, then treated with 0.1% DMSO (as control) or the com-
pounds 3ae3g at 1, 5 and 10 mM for 48 h, respectively. Cell viability
was determined by sulforhodamine B (SRB) assay, according to the
method of Skehan. Light Absorption was measured at the

100
X.-W. Zhou et al. / European Journal of Medicinal Chemistry 79 (2014) 95e101
Fig. 7. Effects of compounds 3ae3d (5
m
M) on A549 cell apoptosis after treatment for 48 h. Microscopic photographs (200ꢀ) were taken under a fluorescent microscope (Nikon).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.03.087.
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with 10
m
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Acknowledgments
This study was supported by National Natural Science Founda-
tion of China (20972088 and 90813022).

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