Biscoumarin derivatives: Synthesis, crystal structure, theoretical studies and induced apoptosis activity on bladder urothelial cancer cell

Article abstract of DOI:10.1016/j.molstruc.2014.12.024

In this study, five new biscoumarin derivatives (1-5) were synthesized and compound 4 inhibited the proliferation of the bladder urothelial cells (J82 cell line) obviously after 48 h treatment at different concentration (1, 5 and 10 μmol/L), and J82 cells

Full text of DOI:10.1016/j.molstruc.2014.12.024

Accepted Manuscript
Biscoumarin derivatives: Synthesis, crystal structure, theoretical studies and in-
duced apoptosis activity on bladder urothelial cancer cell
Jia-jia Xin, Jing Li, Zi-dan Zhang, Xing-bin Hu, Ming-kai Li
PII:
S0022-2860(14)01227-7
DOI:
http://dx.doi.org/10.1016/j.molstruc.2014.12.024
MOLSTR 21174
Reference:
To appear in:
Journal of Molecular Structure
Received Date:
Revised Date:
Accepted Date:
31 August 2014
5 December 2014
5 December 2014
Please cite this article as: J-j. Xin, J. Li, Z-d. Zhang, X-b. Hu, M-k. Li, Biscoumarin derivatives: Synthesis, crystal
structure, theoretical studies and induced apoptosis activity on bladder urothelial cancer cell, Journal of Molecular
Structure (2014), doi: http://dx.doi.org/10.1016/j.molstruc.2014.12.024
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Biscoumarin derivatives: synthesis, crystal structure,
theoretical studies and induced apoptosis activity on bladder
urothelial cancer cell
1
§
2§
4
1*
3*
Jia-jia Xin , Jing Li , Zi-dan Zhang , Xing-bin Hu , Ming-kai Li
1
2
3
4
. Department of Blood Transfusion, Xijing Hospital, the Fourth Military Medical University, Xi’an, China
. School of Chemistry and Chemical Engineering, Xi’an University, Xi’an, China
. Department of Pharmacology, School of Pharmacy, the Fourth Military Medical University, Xi’an, China
. Department of Physics, School of Science, Tianjin University, Tianjin, China
§
These authors contributed equally to this work.
*
Corresponding author: mingkai@fmmu.edu.cn (Ming-kai Li) and hxbsxk@fmmu.edu.cn (Xing-bin Hu)
Abstract
In this study, five new biscoumarin derivatives (1~5) were synthesized and compound 4 inhibited the
proliferation of the bladder urothelial cells (J82 cell line) obviously after 48 hours treatment at different
concentration (1, 5 and 10 µmol/L), and J82 cells were predominantly induced to apoptotic cell death after
compound 4 treatment. Morphologic changes of bladder urothelial cancer cells were also observed under
transmission electron microscopy (TEM) after compound 4 treatment. In addition, compound 4 had much less
toxicity to human umbilical vein endothelial cells. To explore the possible anti-cancer mechanism of compound 4,
two classical intramolecular O—H···O hydrogen bonds (HBs) in their structures and the corresponding HB
energies were performed with the density functional theory (DFT) [B3LYP/6-31G*] method. Anti-bladder cancer
activity of compound 4 is possible due to the intramolecular weakest HB energies.
Key Words Biscoumarin, Crystal, Bladder urothelial cancer, Apoptosis, Chemotherapy
1
. Introduction
Bladder urothelial cancer is diagnosed at an increasing rate in the world, which is the second most common
[1-3]
urologic malignancy, while the clinical outcomes remain highly controversial
. Treatment for the bladder
urothelial cancer takes different approaches depending on the conditions, and chemotherapy is one of the
important treatments, which may be used before surgery, after surgery, or instead of surgery for those cases in
[
4-6]
which surgery is considered unsuitable
. However, chemotherapeutic agents against bladder urothelial cancer
are still limited, more novel anti-cancer agents with great selectivity and specificity need to be developed for
bladder urothelial cancer treatment.
Biscoumarins have received considerable attention in the past few years for their versatile biological and
[
7-9]
medical properties, such as antioxidant, anti-inflammatory, antibacterial, and anticancer activities
. It was
reported that biscoumarin derivatives can strongly inhibit tubulin aggregation and played an efficient role against
cancer, so the cancer cells were able to prevent progression through the cell cycle. Other mechanism of
[
10-12]
anti-cancer activity was due to the antiangiogenesis and promotion of apoptosis
. These results indicated that
coumarin derivatives might represent interesting novel drug candidates.
In
this
work,
we
synthesized
five
new
biscoumarin derivatives, namely,

3
3
3
3
,3′-benzylidene-bis-(4-hydroxycoumarin)
,3′-(3-nitrobenzylidene)-bis-(4-hydroxycoumarin)
,3′-(2-chloro-5-nitrobenzylidene)-bis-(4-hydroxycoumarin)
(1),
3,3′-(2-nitrobenzylidene)-bis-(4-hydroxycoumarin)
(2),
(3),
and
(4)
,3′-(3-nitro-4-hydroxybenzylidene)-bis-(4-hydroxycoumarin) (5) (Fig. 1), and then evaluated their anti-cancer
activities on two human bladder urothelial cell lines. In addition, the cell cycle analysis and apoptosis change
induced by compound treatment were also observed.
OH HO
OH HO
OH HO
O
O
O
O
O
O
O
O
O
O
O
O
NO2
NO2
2
3
1
OH HO
OH HO
O
O
O
O
O
O
O
O
Cl
NO2
NO2
OH
4
5
Fig. 1 Chemical structures of compounds 1~5
2
. Experimental
2
.1 Apparatus and materials
-
1
1
IR spectra (400-4000 cm ) were obtained using a Brucker Equinox-55 spectrophotometer. H NMR spectra
were obtained using a Varian Inova-400 spectrometer (at 400 MHz). Mass spectra were obtained using a
micrOTOF-Q II mass spectrometer. The melting points were taken on a XT-4 micro melting apparatus, and the
thermometer was uncorrected.
The bladder urothelial carcinoma cell line J82 and human umbilical vein endothelial cells were purchased
from ATCC (Manassas, VA, USA). Cells were initially transferred into uncoated plastic tissue plates and were
grown in Eagle’s minimal essential medium (EMEM) with Earle’s balanced salt solution (BSS) and 2 mM
L-glutamine, modified to contain 1.0 mM sodium pyruvate, 0.1 mM nonessential amino acids, 1.5 g/L sodium
bicarbonate, and 10% FBS. Cells were incubated at 37 °C in 95% air/5% CO
2
. Medium was refreshed 3 times per
week, and the cancer cells were harvested when they formed a confluent monolayer on the tissue plate.
2
.2 Synthesis and characterization of compounds 1~5
[13]
Compounds 1~5 were synthesized according to a reported procedure
. A mixture of benzaldehyde
(
(
2-nitrobenzaldehyde, 3-nitrobenzaldehyde, 2-chloro-5-nitrobenzaldehyde and 3-nitro-4-hydroxybenzaldehyde)
10 mmol) and 4-hydroxycoumarin (20 mmol) was dissolved in 100 mL of EtOH. A few drops of piperidine were
added, and the mixture was stirred for 3 h at room temperature. After reaction completion as determined by TLC,

water was added until precipitation occurred. After filtering the precipitates, they were sequentially washed with
ice-cooled water and ethanol and then dried in a vacuum.
-
1
3
,3′-Benzylidene-bis-(4-hydroxycoumarin) (1): m.p. 208-209 °C. IR (KBr pellet cm ): 3446 (OH), 1652
-
1
1
(
C=O), 1615 (C-O), 1572 (C=C) cm . H NMR (CDCl
3
, δ, ppm): 11.528 (s, 1H, OH), 11.299 (s, 1H, OH),
+
7
.994-8.080 (q, 2H), 7.606-7.649 (m, 2H), 7.215-7.421 (m, 9H), 6.104 (s, 1H, CH). HRMS (ESI ): m/z: calcd for
+
C
25
H
16
O
6
: 435.0839 [M+Na ]; found: 435.0899.
-
1
3
,3′-(2-Nitrobenzylidene)-bis-(4-hydroxycoumarin) (2): m.p. 218-219 °C. IR (KBr pellet cm ): 3446
-
1 1
(
OH), 1652 (C=O), 1615 (C-O), 1520 (C=C) cm . H NMR (CDCl
3
, δ, ppm): 11.550 (s, 1H, OH), 11.219 (s, 1H,
OH), 7.978-8.073 (m, 2H), 7.612-7.658 (m, 3H), 7.540-7.582 (m, 1H), 7.386-7.464 (m, 6H), 6.628 (s, 1H, CH).
+
+
HRMS (ESI ): m/z: calcd for C25
8
H15NO : 480.0690 [M+Na ]; found: 480.0611.
-
1
3
,3′-(3-Nitrobenzylidene)-bis-(4-hydroxycoumarin) (NBH): m.p. 220-221 °C. IR (KBr pellet cm ): 3446
-
1 1
(
3
OH), 1652 (C=O), 1615 (C-O), 1555 (C=C) cm . H NMR (CDCl , δ, ppm): 11.579 (s, 1H, OH), 11.384 (s, 1H,
OH), 8.137-8.162 (m, 1H), 8.070-8.104 (t, 2H), 7.990-8.008 (d, 1H), 7.651-7.690 (t, 2H), 7.568-7.591 (m, 1H),
+
7
.496-7.536 (t, 1H), 7.385-7.453 (m, 4H), 6.129 (s, 1H, CH). HRMS (ESI ): m/z: calcd for C25
H
15NO
8
: 480.0690
+
[
M+Na ]; found: 480.0689.
-
1
3
,3′-(2-Chloro-5-nitrobenzylidene)-bis-(4-hydroxycoumarin) (CBH): m.p. 150-151 °C. IR (KBr pellet cm ):
-
1 1
3
1
4
489 (OH), 1684 (C=O), 1623 (C-O), 1520 (C=C) cm . H NMR (CDCl
3
, δ, ppm): 11.752 (s, 1H, OH), 11.006 (s,
H, OH), 8.359-8.365 (t, 1H), 8.064-8.144 (m, 3H), 7.640-7.683 (m, 2H), 7.533-7.555 (d, 1H), 7.402-7.438 (t,
+
+
H), 6.192 (s, 1H, CH). HRMS (ESI ): m/z: calcd for C25
8
H14ClNO : 514.0300 [M+Na ]; found: 514.0321.
3
,3′-(3-Nitro-4-hydroxybenzylidene)-bis-(4-hydroxycoumarin) (NHH): m.p. 238-239 °C. IR (KBr pellet
-
1
-1 1
cm ): 3251 (OH), 1655 (C=O), 1582 (C-O), 1536 (C=C) cm . H NMR (CDCl
3
, δ, ppm): 11.593 (s, 1H, OH),
1
1
4
1.396 (s, 1H, OH), 10.545-10.558 (d, 1H), 7.944-8.108 (m, 3H), 7.686 (s, 2H), 7.402-7.466 (m, 4H), 7.287 (s,
+
+
H), 7.142-7.176 (q, 1H), 6.046 (s, 1H, CH). HRMS (ESI ): m/z: calcd for C25
H
9
15NO : 496.0639 [M+Na ]; found:
96.0632.
2
.3 Crystal structure determination
For X-ray diffraction experiments, single crystals of compound 4 were both grown from methanol. The X-ray
diffraction data were collected on a Bruker SMART APEX II CCD diffractometer equipped with a graphite
monochromated Mo Kα radiation (λ = 0.71073 Å) by using the ω-2θ scan technique at room temperature. The
structure was solved by direct methods using SHELXS-97 and refined using the full-matrix least squares method
2
[14]
on F with anisotropic thermal parameters for all non-hydrogen atoms by using SHELXL-97 . Hydrogen atoms
were generated geometrically. The crystal data and details concerning data collection and structure refinement are
given in Table 1. Molecular illustrations were prepared using the XP package. Parameters in CIF format are
available as Electronic Supplementary Publication from Cambridge Crystallographic Data Centre.
Table 1 Crystal data, data collection and structure refinement of compound 4
Formula
Mr
25 8
C H14ClNO
491.0408
293(2)
Temperature / K
Crystal system
Space group
a / Å
Triclinic
Pī
9.7912(9)
11.2742(8)
12.0934(13)
64.872(9)
79.063(9)
84.199(7)
b / Å
c / Å
α / °
β / °
γ / °

3
V / Å
1186.36(19)
4
Z
-
3
D
calc / g·cm
1.506
-
1
μ(Mo Kα) / mm
θ range / °
0.221
2.60 to 25.00
7797
Reflections collected
No. unique data[R(int)] 4179
No. data with I ≥ 2σ(I) 3229[0.0210]
R
1
0.0657
0.2044
1009072
2
ωR (all data)
CCDC
2
.4 Quantum chemical calculations
[15]
All calculations were carried out using the Gaussian 09 package
. Density functional theory (DFT),
Becke’s three-parameter hybrid function (B3LYP), and LYP correlation function were used to fully optimize all
the geometries on the energy surface without constraints. Frequency calculations using the same basis sets have
been performed to confirm that the stationary points are minima (zero imaginary frequencies) on the potenial
energy surface. To obtain precise results that are in conjunction with experimental results, three basis sets, namely
6
-31G*, 6-31+G**, and 6-311G*, were tested. Frequency calculations at the B3LYP (with basis sets 6-31G*)
level of theory were carried out to confirm stationary points as minima and to obtain the zero-point energies and
the thermal correlation data at 1 atm and 298 K.
2
.5 Anti-cancer and cytotoxicactivity assay
The anti-cancer activity of compounds to the bladder urothelial carcinoma cells (J82 cell line), and the in
vitro cytotoxicity of compound to the human umbilical vein endothelial cells (HUVECs) was determined by
[16]
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) staining as described previously
.
3
Briefly, cells (5×10 cells/well) were seeded in a 96-well plate with 100 μL EMEM media with 20% fetal bovine
serum (FBS) in every well for 12 hours, and then treated with or without compounds at various concentrations for
2
4 or 48 hours. After treatment, MTT solution (final concentration, 0.5%) was added and cells were incubated for
another 4 hours at 37°C. 150 μL DMSO was added to each well after removal of the supernatant and the
absorbance at 490 nm was measured with a microplate reader.
2
.6 Detection of apoptosis by flow cytometry
The apoptotic ratios of cells were determined with the Annexin V-FITC apoptosis detection kit (Sigma, St.
[17]
Louis, MO, USA) . Briefly, after 48 hours compound 4 treatment, the cells were collected and washed twice
with cold PBS buffer, resuspended in 100 mL of binding buffer, incubated with 5 mL of Annexin V conjugated to
FITC and 10 mL PI for 15 min at room temperature, and analyzed by flow cytometry. Cells treated with DMSO
were used as the negative control.
2
.7 Transmission electron microscopy (TEM) observation
Bladder urothelial cancer cells were seeded and grown at 5×10 /ml in three flasks. Cells after treated with
7
compound 1 (1, 5 and 10 µmol/L) were harvested and washed with 1xPBS twice, and then added to 2.5%
glutaraldehyde fixative for microtome sectioning using ultramicrotome (LKB-V; JEOL Co; Japan). TEM was
performed with a Transmission Electron Microscope (JEM-2000EX; JEOL Co; Japan).
3
Results
3
.1 Molecular structure
The crystal structure of compound 4 is given in Fig. 2. The selected bond lengths, bond angles and partial
values of torsional angles are listed in Table 2. From the diagram of the asymmetric unit including the atomic

numbering scheme of compound 4, we can see an ethanol solvent molecule incorporated in the asymmetric unit.
The title compound is a 4-hydroxycoumarin dimer, consisting of two monomeric building blocks of
4
-hydroxycoumarin and a 2-chloro-5-nitrophenyl ring on the central methylene linker. Two 4-hydroxycoumarin
residues arranged in a position that permits the formation of two classical intramolecular hydrogen bonds between
a hydroxyl group of one coumarin fragment and a lacton carbonyl group of another coumarin fragment help
3 4 1 6
stabilize the whole structure [d(O -O ) = 2.575 Å and d(O -O ) = 3.429 Å].
Fig. 2 Molecular structure of 3,3′-(2-chloro-5-nitrobenzylidene)-bis-(4-hydroxycoumarin) (4).
3
.2 Quantum chemical calculations
3
.2.1 Geometric parameters of compounds 1~5
The fully optimized molecular structures of compounds 1~5 with atomic numbering calculated at B3LYP
level of theory are shown in Fig. 3. For compound 4, selected calculated geometric parameters under three
different basis sets (6-31G*, 6-31+G**, and 6-311G*) and experimental geometric parameters are presented in
Table 2.
R=
H3
NO2
O3
O4
1
R=
R
H
O N
2
C3
C1
C19
O
Cl
R=
R=
4
NO2
OH
2
3
O
C12
NO2
R=
O1
O6
H6
5
Fig. 3 Schematic presentation of compounds 1~5
As seen in Table 2, the calculated geometric parameters of compound 4 agree with available experimental
data. Under three different basis sets, the maximum deviation of the selected bond lengths and bond angles are
very close; the average discrepancy of the selected bond lengths between theoretical and experimental data is less
than ±0.02Å, and the average discrepancy of the selected bond angles is less than ±2°. B3LYP/6-31G* exhibited
sufficient agreement with experimental data and lower computational cost, so further theoretical study was
performed at this level.

Table 2 Experimental and calculated parameters of the selected bond lengths and bond angles of
3
,3′-(2-chloro-5-nitrobenzylidene)-bis-(4-hydroxycoumarin) (4).
Name definition
X-ray
1.221
1.205
1.370
1.374
1.358
1.370
1.525
6-31G* 6-31+G** 6-311G*
R(C19＝O
4
)
1.232
1.233
1.370
1.368
1.372
1.367
1.539
1.235
1.236
1.367
1.370
1.370
1.369
1.540
1.224
R(C
R(C
R(C
1
1
9
＝O
1
)
1.226
-O
-O
2
2
)
)
5
5
1.369
1.367
R(C19-O
R(C18-O
)
)
1.372
1.365
R(C10-C20
)
1.538
A(C
1
3
-C
2
2
-C10
)
)
115.33 114.05 114.05
125.55 126.24 126.41
120.46 119.91 119.77
119.94 120.73 121.01
113.33 112.18 111.97
115.24 114.66 115.18
113.45 116.95 116.38
113.80
126.43
119.35
121.19
112.02
115.02
116.41
61.22
A(C
-C
-C10
A(C10-C11-C19
A(C10-C11-C12
)
)
A(C
2
2
-C10-C11
-C10-C20
)
)
A(C
A(C11-C10-C20
)
D(C
D(C
D(C
3
2
1
-C
2
-C10-C11
-C10-C11-C19
-C10-C20
)
56.57
98.57
62.11
62.34
)
102.87 102.33
103.45
101.33
46.12
-C
2
)
104.79 102.23 102.67
40.32 45.22 45.33
D(C19-C11-C10-C20
)
3
.2.2 Estimation of the single and total HB energies in compounds 1~5
To obtain single and total HB energies of the five compounds, structure optimization was performed to
elucidate stable PES structures.
We took compound 4 for example to estimate single and total HB energies. The global minimum structure is
stabilized by two HBs (4ab); however, two higher energy structures is stabilized by one HB (4a or 4b)
respectively.
The relative energies of different structures were calculated to estimate the strengths of the HBs formed. The
O
6
—H
6
···O
1
HB energy was estimated from the energy difference between 4ab and 4a,
coor
4ab
coor
E(O ⎯H …Ο ) = E − E4a , calculated to be -42.905921 kJ/mol (Table 3). 4a is a global minimum
6
6
1
structure with one HB (O
3 3 4 . 3 3 4
—H ···O ) The O —H ···O HB energy was estimated from the energy difference
coor
4ab
coor
between 4ab and 4b, E(O ⎯H …Ο ) = E − E4b , calculated to be -50.231066 kJ/mol (Table 3). 4b was
3
3
4
obtained from the global minimum structure, but H
3
was rotated around the C
. The HB energies obtained predicted that O —H ···O
which is consistent with the fact that the distance of O —O is greatly shorter than that of O
3
—O
3
bond until O
3
—H
3
···O
4
HB
[
18, 19]
rupture occurred
3
3
4
HB is stronger than O
6
—H
6
···O ,
1
3
4
6
—O
1
. The total HB
coor
coor
coor
energy in compound 4 was estimated to be -93.136987 kJ/mol by the equation 2E4ab −(E4a + E4b ) (Table 3).
For compounds 1~3 and compound 5, their total HB energies are -118.4546835, -118.215763, -115.5666335
-
1
and -120.3450435 kJ·mol , respectively (Table 3).

Table 3 Total electronic energies (in hartree) and HB energies (in kJ/mol) of hydrogen bonded conformers of
compounds 1~5 calculated at B3LYP/6-31G* level of theory.
a
System Total electronic energies E(O
6
—H
6
···O
1
)
E(O
3
—H
3
···O
4
)
E(total HB)
-1413.328042
-1413.308121
-1413.302846
-1617.817382
-1617.799287
-1617.790451
-1617.827653
-1617.807773
-1617.803516
-2077.424463
-2077.408121
-2077.405331
-118.4546835
1
ab
-52.3025855
-47.5084225
-52.19494
1
a
-66.152098
1
b
-118.215763
-115.5666335
-93.136987
2ab
2
a
-70.7073405
-63.3716935
-50.231066
2
b
3ab
3
a
3
b
4ab
-42.905921
4
a
4
b
5
ab
-
1567.108786
1567.088785
-120.3450435
5
a
-
-52.5126255
5
b
-1567.08295
-67.832418
a
ZP corrected.
3
.3 Effect of compounds on the viability of bladder urothelial cancer cells
After treatment with compounds 1~5 (0.1, 1, 5 and 10 µmol/L) for 48 h, cell viability was decreased in
bladder urothelial cell line J82 at 1, 5 and 10 µmol/L significantly(Table 4), and compound 4 showed the most
potent anti-cancer activities on the J82 cells. A probity analysis of the dose–response functions showed the IC50
value of compound 4 was 7.36±2.47µmol/L.
Table 4 Effect of compounds 1~5 on the J82 cell viability
Value relative to control (%)
Compounds
(µmol/L)
0.1
1
5
10

1
2
3
4
5
99.36±8.57
97.94±5.83
97.42±6.65
90.36±8.05
98.56±6.16
91.21±8.84
86.38±7.56
88.51±6.74
80.12±7.74*
92.58±5.56
88.36±6.95
80.86±6.51
79.57±5.88
36.46±5.33*
83.16±5.42
79.38±8.42
72.68±7.85
70.13±4.69
33.31±6.06**
71.38±7.26
3
.4 Induction of apoptosis by compound 4 in J82 cell line
To determine whether the treatment of cells using compound 4 may lead to apoptotic cell death, Annexin
V-FITC/PI staining flow cytometric analysis was performed. After compound 4 treatment for 48 hours, cell
apoptosis was observed in J82 cell line. Apoptosis was seen in only 2.34± 0.38% of vehicle treated control cells
(
Fig. 4), after treatment with 1 µmol/L compound 4, the apoptotic cells ration was 11.17± 0.44%, and when the
cells were treated with 5 and 10 µmol/L compound 4, it was increased dramatically to 63.17±6.38 and 66.35±8.17,
respectively.
Fig. 4 Compound 4 induce apoptosis in J82 cell line. A: Control J82 cells without treatment of compound 4; B:
Cells were treated with 1 µmol/L compound 4 for 48 hours; C: Cells were treated with 5 µmol/L compound 4 for
4
8 hours; D: Cells were treated with 10 µmol/L compound 4 for 48 hours. Induction of apoptosis was determined
using Annexin V FITC/propidium iodide staining. Data was from at least three independent experiments.
3
.5 Induction of apoptosis by compound 4 in J82 cell
Under the observation of transmission electron microscope, bladder urothelial cancer J82 cells were round and
regular, with abundant organelles and normal double-membrane nuclei (Fig. 5A). After exposing to compound 4
for 24 hours, early stage apoptosis could be observed in 1 µmol/L (Fig. 5B) compound 4 treatment, and more
apoptosis change could be observed in 5 µmol/L (Fig. 5C) and 10 µmol/L (Fig. 5D) compound 4 treatment group.
It showed that nuclear membrane was domed outward with a sharp angle, and the nuclei chromatin was
concentrated and clustered on the inner border of cells. The endoplasmic reticulum became dilated in the inner
segment.

Fig. 5 Transmission electron microscopy showed J82 cell morphologic change of compound 4 treatment. A:
Control; B: Treated with 1µmol/L compound 4; C: Treated with 5µmol/L compound 4; D: Treated with 10
µmol/L compound 4.
3
.6 Cytotoxic activity of compound 4 on HUVECs
To further explore the safety of compound 4, we investigated its cytotoxicity to the HUVECs in vitro. As
shown in Fig. 6, after treated with the compound 4 for 24 hours or 48 hours, there was no significant difference on
cell viability between the control group and compound 4 treated group under concentration of 100 µmol/L (P>
0
.05). These results implied that compound 4 had much less toxicity to mammalian normal endothelial cells, and
had a relatively wide safety range for potential anticancer applications.
Fig. 6 Effect of compound 4 on the viability of HUVECs cells as determined by MTT assay. Data
are represented as mean percentage of viable cells in bars ± SD of at least three replicates
in six independent tests.
4
. Discussion
Bladder urothelial cancer patients are suffering from limited treatment options due to late diagnosis and poor
[20, 21]
drug tolerance
. Therefore, it is significant for us to seek novel chemotherapy agent which can effectively
prevent the disease and even eradicate the progression and metastasis of bladder urothelial cancer.
In this study, we have demonstrated that new biscoumarin derivatives suppressed cell proliferation in J82
bladder urothelial cancer cells. In addition, it was discovered firstly that the compound 4 had bladder urothelial
cancer cell cytotoxicity by promoting the apoptosis death. In agreement with our results, it was reported that

coumarin derivative could induce the apoptosis and inhibit proliferation in the other cancers such as lung cancer
[22, 23]
and colon cancer
.
X-ray structural analysis also showed that all the compounds were stabilized by two asymmetrical
intramolecular O—H···O HBs, which were considered as important factors in assisting the molecule to attain the
[22, 23]
correct configuration for biological activity
. Our study also firstly demonstrated that the energy of hydrogen
bonds in compounds 1~5 were in agreement with their anti-proliferation activity in vitro. The total HB
stabilization energies in compounds 1~5 were estimated to be -118.4546835, -118.215763, -115.5666335,
-93.136987, -120.3450435 kJ/mol, respectively. These values suggest that the most potent anti-cancer activity in
compound 4 was consistent with its weakest HB strengths.
5
. Conclusion
Taken together, our study demonstrated the in vitro therapeutic effect of biscoumarin derivatives on human
bladder urothelial cancer J82 cell line, suggesting that biscoumarin derivative is a potential therapeutic drug for
bladder urothelial cancer. Furthermore, biscoumarin derivatives can inhibit bladder urothelial cancer cells growth
through anti-proliferation and induce apoptosis. In compounds 1~5, compound 4 had the most potent anti-cancer
2
efficiency, possibly because two strong electron-withdrawing groups (NO and Cl) on the phenyl ring further
weakens the HB strengths. However, it will be beneficial and necessary for further study to reveal the underlying
mechanism and study the effect in vivo.
Acknowledgments
This work was supported by grants from the project of science and technology of Shaanxi Province
(
2012K13-02-26). The authors thank the High Performance Computing Center of Tianjin University and Prof.
Xuehao He for the services provided.
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Highlights
1
2
3
4
． Five new biscoumarin derivatives (1~5) were synthesized.
． Their anti-cancer activities on two human bladder urothelial cell lines were evaluated.
． The cell cycle analysis and apoptosis change of the compounds were also observed.
． The HB energies of compounds 1~5 were calculated.