A novel nitro-substituted benzothiadiazole as fluorescent probe for tumor cells under hypoxic condition

Article abstract of DOI:10.1016/j.bmc.2013.10.019

Most of solid tumor cells are hypoxic and hard to trace and measure. A new compound, 4,7-bis(4-dodecylthiophen-2-yl)-5,6-dinitrobenzo[c][1,2,5]thiadiazole (BTTD-NO₂), was synthesized for labeling the hypoxic cells specially in this paper. BTTD-NO₂ showed no cytotoxicity to MG63 cells by MTT method. When MG63 cells were cultured with BTTD-NO₂ under hypoxic condition for 24 h, strong red fluorescence distribution in cytoplasm was observed. Flow cytometry results showed that 65% of MG63 cells were labeled with strong red fluorescence in hypoxic condition while only 2.4% in oxic condition. Furthermore, Real time RT-PCR proved that BTTD-NO₂ could stimulate high gene expression of the nitroreductase in the cells which could improve the conversion rate of BTTD-NO₂ to BTTD-NH₂ in turn. It proved that the fluorescence of BTTD-NO₂ was quenched by its two nitro groups, however, strong red fluorescence could emit in the cytoplasm after the reduction of its nitro groups to amino groups in the tumor cells under hypoxic condition. These results suggested that BTTD-NO₂ had the potential as a superior fluorescent probe for tumor detection.

Full text of DOI:10.1016/j.bmc.2013.10.019

Bioorganic & Medicinal Chemistry 21 (2013) 7735–7741
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A novel nitro-substituted benzothiadiazole as fluorescent probe for
tumor cells under hypoxic condition
Qian Jiang , Zhanyuan Zhang , Jiao Lu , Yan Huang , Zhiyun Lu , Yanfei Tan , Qing Jiang b,
a
a
b
a
a
b,
⇑
⇑
a
College of Chemistry, Sichuan University, Chengdu 610064, China
National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 July 2013
Revised 14 October 2013
Accepted 16 October 2013
Available online 1 November 2013
Most of solid tumor cells are hypoxic and hard to trace and measure. A new compound, 4,7-bis(4-dode-
cylthiophen-2-yl)-5,6-dinitrobenzo[c][1,2,5]thiadiazole (BTTD-NO ), was synthesized for labeling the
hypoxic cells specially in this paper. BTTD-NO showed no cytotoxicity to MG63 cells by MTT method.
When MG63 cells were cultured with BTTD-NO under hypoxic condition for 24 h, strong red fluores-
cence distribution in cytoplasm was observed. Flow cytometry results showed that 65% of MG63 cells
were labeled with strong red fluorescence in hypoxic condition while only 2.4% in oxic condition. Fur-
2
2
2
Keywords:
Benzothiadiazole
Hypoxic
Fluorescent probe
Nitroreductase
thermore, Real time RT-PCR proved that BTTD-NO
ductase in the cells which could improve the conversion rate of BTTD-NO
that the fluorescence of BTTD-NO was quenched by its two nitro groups, however, strong red fluores-
cence could emit in the cytoplasm after the reduction of its nitro groups to amino groups in the tumor
2
could stimulate high gene expression of the nitrore-
2
to BTTD-NH in turn. It proved
2
2
2
cells under hypoxic condition. These results suggested that BTTD-NO had the potential as a superior
fluorescent probe for tumor detection.
Ó 2013 Elsevier Ltd. All rights reserved.
1
. Introduction
mor. Hypoxic cells in some human solid tumors are thought to lim-
it the effectiveness of radiotherapy and chemotherapy, and in some
cases may contribute to failure to control the disease, as most of
8
Cancer is one of the diseases which are the most pressing public
health now. The great challenges for future personalized oncology
are to explore improved methodology for the early detection of
localized and disseminated tumor cells in patients, which is very
critical to the success of cancer therapy and improvement of pa-
tients’ survival rates. Compared to other technologies of tumor
detection, such as radioisotope labeling, magnetic resonance imag-
ing (MRI), electron spin resonance (ESR) spectroscopy, and electro-
chemical detection, fluorescence imaging has many advantages, for
example, it enables highly sensitive, less-invasive and safe detec-
tion using readily available instruments. Another advantage of
fluorescence imaging is that the fluorescence signal of a molecule
can be drastically modulated, so that probes relying on activation,
not just accumulation, can be utilized.1
solid tumor cells are hypoxic. Therefore, both before and during
therapy, tracing and measurement of the hypoxic cell fraction in
tumor would be considerable important in clinical treatment. Yin
9
et al. had designed a series of aliphatic N-oxide of naphthalimides
and fluorescence image analysis showed that one of compounds
had 17 times intensity of fluorescence in V79 cells under hypoxic
condition compared with oxic environment. Many other research-
ing works indicated that fluorescent detection for tumor cells could
be effective by distinguishing those tumor cells under hypoxic con-
1
0–12
dition.
In the past few years, we had developed a few families of ben-
zothiadiazole fluorophores which were optically stable and had
13,14
easily tunable properties.
Due to the electron-withdrawing
Many works had carried out on the fluorescence imaging by
using luminescence probes for tumor cells in vitro.2 Intravital
microscopic imaging approaches, based on the use of fluorescence
imaging, had been shown to allow the study of cell morphology
and cell–cell interaction with high (single-cell) resolution in living
properties and high fluorescence quantum yields both in solution
and in the solid state, benzo[c][1,2,5]thiadiazole (BTD) had been
used for developing optoelectronic materials, liquid crystal dis-
plays (LCDs), organic semiconductors and light technology.15 Pre-
–6
vious researches showed that benzothiadiazole could selective
7
16,17
organisms. However, distinguishing of tumor cells compared with
label the mitochondria of the cells,
hence, we attempted to in-
normal cells should be considered. With fast growth in the tumor
of cancer, hypoxic condition would aroused in the center of the tu-
duce the stem of benzothiadiazole and synthesize a new derivative
as a fluorescent probe for tumor cells.
After the new benzothiadiazole derivative was synthesized, we
hypothesized that this compound could be transmitted into cells
and emitted red fluorescence in the cells after bio-reduction under
⇑
Corresponding authors. Fax: +86 28 85415123 (Y.T.), +86 28 85410246 (Q.J.).
E-mail addresses: tanyf@scu.edu.cn (Y. Tan), jiangq@scu.edu.cn (Q. Jiang).
0
968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.10.019

7
736
Q. Jiang et al. / Bioorg. Med. Chem. 21 (2013) 7735–7741
S
S
N
N
N
N
HNO3/H2SO4
1:1
Br
Br
Br
O2N
Br
NO2
C1
S
N
N
Pd(PPh3)4/K2CO3
toluene
S
S
C12H25
C12H25
O2N
NO2
C12H25
C12H25
BTTD-NO2
1
2
n-BuLi/ B(OCH3)3
O
B
S
S
O
HO
HO
C2
Figure 1. Synthesis of target compound, BTTD-NO
2
.
0
0
0
0
.15
.10
.05
.00
1
1
20
00
BTTD-NO2
after
before
8
0
0
0
0
6
4
2
0
400
450
500
550
600
540 560 580 600 620 640 660 680 700
Wavelength (nm)
Wavelength (nm)
À5
Figure 2. Absorption spectra of the target compound BTTD-NO
methanol).
2
(10 mol/L
Figure 4. Fluorescence intensity of the target compound BTTD-NO
after chemical reduction (10 mol/L methanol), excited at 480 nm.
2
before and
À6
hypoxic condition. To testify the hypothesis, the osteosarcoma cell
line, MG63, was employed to the following biological experiments.
2.1.3. Fluorescence spectra of BTTD-NO
To study the fluorescent features, the BTTD-NO
firstly according to chemical reduction which was illustrated in
Figure 3. The results of fluorescence intensity of the BTTD-NO be-
fore and after reduction were shown in Figure 4, no fluorescence
was detectable before chemical reduction of BTTD-NO , whereas
BTTD-NH (chemical reduction from BTTD-NO ) had broad and
2
2
was reduced
2
2
2
. Results and discussion
2
.1. Synthesis and spectra analysis
2
2
2
bright red luminescence with the peak at 600 nm after it was ex-
cited at 480 nm. Previous reports had proved that emission wave-
length at 600 nm could greatly reduce the background
.1.1. Synthesis
The new benzothiadiazole derivative, 4,7-bis(4-dodecylthio-
phen-2-yl)-5,6-dinitrobenzo[c][1,2,5]thiadiazole (BTTD-NO ), was
2
synthesized from 4,7-dibromobenzo[c][1,2,5]thiadiazole and 3-
1
8
fluorescence and improve the resolution. The fluorescent fea-
tures of BTTD-NO
2
had reached our initial expectation.
dodecylthiophene as shown in Figure 1. Their structures were con-
1
13
firmed by H NMR, C NMR.
2
.1.4. Biological reduction of BTTD-NO
2
To further testify whether BTTD-NO
reductase, liver microsomes provided as nitroreductase (P450
reductase) in this experiment, the possible reaction principle was
shown in Figure 5. After different concentration of BTTD-NO
was treated by liver microsomes for 4 h, the fluorescence spectra
2
could be reduced by nitro-
2
.1.2. Absorption spectra of BTTD-NO
The absorption spectra of BTTD-NO
2
2
were shown in Figure 2. In
methanol solution, its maximal absorption band lie at 432 nm,
arising from
⁄
p–p
transitions (log
e
= 4.06).
2
S
S
N
N
N
N
S
S
S
S
Fe, 80 °C, 10 h
CH3COOH
C12H25
C12H25
C12H25
C12H25
H2N
NH2
O2N
NO2
BTTD-NO2
BTTD-NH2
Figure 3. Chemical reduction of target compound BTTD-NO2.

Q. Jiang et al. / Bioorg. Med. Chem. 21 (2013) 7735–7741
7737
S
S
N
N
N
N
S
S
S
S
nitroreductase
hypoxia
C12H25
C12H25
C12H25
C12H25
H2N
NH2
O2N
NO2
BTTD-NH 2
BTTD-NO 2
Non-fluorescent
Fluorescent
2
Figure 5. Proposed mechanism for fluorescent enhancement of BTTD-NO .
6
5
4
3
2
1
0
were detected and the results were shown in Figure 6(A). Sharply
red luminescence with the peak at 613 nm was observed after bio-
logical reduction of BTTD-NO and the fluorescence intensity in-
2
creased gradually with raised concentration according to analysis
by fluorescence spectrophotometer. In addition, to identify
A
2.0 × 10- mol
1
0
6
-
6
.0 × 10 mol
.5 × 10- mol
6
whether the results of the biological reduction of BTTD-NO
coincided with chemical reduction of BTTD-NO or not, fluores-
cence spectra of BTTD-NO by chemical reduction were compared
2
were
0 mol
2
2
with that of by biological reduction, the results were shown in Fig-
ure 6(B). They had the same peak at 613 nm and the shape of the
spectra was almost unchanged, it indicated that the nitro group
could be reduced to the amino group whatever the reduction
method we adopted. However, the fluorescent intensity in the bio-
logical reduction group was considerable lower than that of chem-
5
80
600
620
640
660
680
Wavelength (nm)
2
ical reduction group, which indicated BTTD-NO was not reduced
to BTTD-NH totally according to biological reduction.
2
1
1
2
0
8
6
4
2
0
B
Chemical reduction
Biological reduction
2
2
.2. Tracing tumor cells with BTTD-NO in vitro
2
.2.1. Cytotoxicity of BTTD-NO
After the MG63 cells were co-cultured with BTTD-NO
and 48 h, the MTT results showed that each concentration of
BTTD-NO did not inhibit the growth of MG63 cells (Fig. 7), no sig-
2
2
for 24
2
nificant differences (t-test, P < 0.05) were found in cellular prolifer-
ation between different concentration groups. The results
indicated that the BTTD-NO compound had good biocompatibility
2
as bio-imaging agents.
5
80
600
620
640
660
680
Wavelength (nm)
2
.2.2. Fluorescent labeling of hypoxic cell by BTTD-NO
To assess the potential applications as fluorescent probe of the
2
Figure 6. Fluorescence spectra excited at 500 nm. (A) The BTTD-NO
concentration after reduction by nitroreductase for 4 h, the fluorescence intensity
increased gradually with raised concentration of BTTD-NO (B) fluorescence
2
with different
compound BTTD-NO , fluorescent imaging inside the MG63 cells
2
treated by BTTD-NO under hypoxic or oxic condition was exam-
2
2
;
spectra of BTTD-NO
2
according to chemical reduction or biological reduction
À6
(
ultimate concentration: 2 Â 10 mol/L). They had the same peak at 613 nm and
ined at 4 and 24 h, respectively. After exchanging medium, laser
scanning confocal microscope (LSCM) observation was performed
and the results were shown in Figure 8. Strong fluorescence in
the MG63 cells was observed at 24 h under hypoxic condition com-
pared with oxic condition. It proved that the fluorescence could be
significant enhanced in the hypoxic cells according to LSCM
observation.
the similar spectra shape.
2
1
1
0
0
0
.0
.6
.2
.8
.4
.0
2
4hours
8hours
4
2
.2.3. Quantitative analysis of fluorescent cells by flow
cytometry
In order to further determine the proportion of fluorescent cells
labeled by BTTD-NO , cytometric analyses were performed by flow
2
cytometry and the results were shown in Figure 9. The proportion
of fluorescent labeling cells was reached 65% after cultured with
BTTD-NO
cells were recognized as fluorescent cells in control group (without
BTTD-NO ).
Yin and Zhu, et al.
2
for 24 h under hypoxic condition while only 2.4% of
2
0
0.5
1
2
9,11
had reported that the nitro group could
-6
Concentration of BTTD-NO (10 mol/L)
quench the fluorescence of the aromatic ring system, but when
the nitro group was bio-reduced to amino group by nitroreductase
in hypoxic cells, their naphthalimide and quinoxaline compounds
2
2
Figure 7. BTTD-NO had no cytotoxicity to MG63 cells according to MTT method.

7
738
Q. Jiang et al. / Bioorg. Med. Chem. 21 (2013) 7735–7741
2
Figure 8. Fluorescence microphotograph of MG63 cells incubated with BTTD-NO for 24 h under oxic condition (A–C) and hypoxic condition (D–I). Left pictures:
transmission channel; middle pictures: fluorescence channel; right pictures: composition. Strong fluorescence was observed in cytoplasm when these cells were cultured
under hypoxic condition (E and H) compared with that of under oxic condition (B).
2
Figure 9. Flow cytometry results. Only 2.4% of cells were recognized as fluorescent cells after cultured with BTTD-NO for 24 h under oxic condition (A), while the proportion
of fluorescent cells was reached 65% under hypoxic condition (B).

Q. Jiang et al. / Bioorg. Med. Chem. 21 (2013) 7735–7741
7739
5
4
3
2
1
0
stimulate the high gene expression of the nitroreductase in MG63
cells, which improved conversion rate of BTTD-NO to BTTD-NH
in turn. In conclusion, BTTD-NO had the potential as a superior
fluorescent probe for tumor detection.
without BTTD-NO
2
2
2
*
with BTTD-NO
2
2
4
. Experimental section
*
4.1. Synthesis of target compound (BTTD-NO
2
)
All the chemicals commercially available were used directly
without further purification unless otherwise stated. All the sol-
vents were of analytical grade and freshly distilled prior to use, ex-
cept spectrographically pure DMSO used for biological evaluation.
0
h
4h
24h
1
13
H NMR and C NMR spectra were recorded on a Bruker AVANCE-
4
3
00 spectrometer in CDCl using TMS as internal standard.
Figure 10. Gene expression of nitroreductase in MG63 cells cultured with or
⁄
The detailed synthetic routes were shown in Figure 1.
without BTTD-NO
2
.
the mRNA expression of nitroreductase increased significantly
(p < 0.05).
2
3
24
after 4 and 24 h by adding BTTD-NO
2
Pd(PPh
thiophene
3
)
4
,
4,7-dibromobenzo[c][1,2,5]thiadiazole,
3-dodecyl-
2
5
were synthesized according to the literature
procedures.
could reach 11 and 17 times differential fluorescence between oxic
and hypoxic cells. In this paper, based on the reduction of BTTD-
4
.1.1. Synthesis of 4,7-dibromo-5,6-
dinitrobenzo[c][1,2,5]thiadiazole (C1)
A mixture of concentrated H SO (140 mL) and fuming HNO
140 mL) was cooled to 0 °C. After stirring for 10 min at 0 °C, 4,7-
NO
2 2
to BTTD-NH , the proportion of fluorescent cells significantly
enhanced under hypoxic condition compared with oxic condition.
2
4
3
(
2
.2.4. Gene expression of nitroreductase in MG63 cells
To analyze the effect of BTTD-NO on the gene expression of the
dibromobenzo[c][1,2,5]thiadiazole (5.0 g, 17 mmol) was added in
small portions. The reaction mixture was stirred for 6 h at 0 °C,
then poured out into ice/water. The precipitate was filtrated,
washed with water and purified by column chromatography on sil-
ica gel to afford beige solid (1.5 g, 40%). Melting point: 201–202 °C.
2
nitroreductase in MG63 cells, quantitative RT-PCR was carried out
after 4 and 24 h. As shown in Figure 10. Real time RT-PCR analysis
revealed that, after the MG63 cells treated with BTTD-NO for 4
2
and 24 h, the gene expression level of reductase reached 2.5 times
and 4 times, respectively, compared with control group (without
4
1
.1.2. Synthesis of 2-(4-dodecylthiophen-2-yl)-5,5-dimethyl-
,3,2-dioxaborinane (C2)
BTTD-NO
2
).
These results indicated that BTTD-NO
2
could transfer into
3
-Dodecylthiophene (5.04 g, 20 mmol) was dissolved in 40 mL
MG63 cells and increase gene expression of nitroreductase, high
expression of nitroreductase could accelerate reduction of BTTD-
NO to BTTD-NH in turn, as a result, the MG63 cells showed sig-
2 2
nificant fluorescent intensity which were confirmed by CLSM
observation and Flow cytometry.
of anhydrous THF under inert atmosphere, then the solution was
cooled down to À78 °C, and 8.8 mL (22 mmol) of n-BuLi (2.5 M in
n-hexane) was added dropwise. The mixture was allowed to be
stirred at À78 °C for an additional hour, trimethyl borate
(
3.36 mL, 30 mmol) was added dropwise, followed by 1 h stirring
Previous reports had showed that thiophene fluorophores
were capable of staining the cytoplasm19 and some benzothiadi-
azole derivatives could selectively stain the mitochondria of the
at À78 °C, and 24 h stirring at room temperature. After that, the
mixture was cooled to 0 °C and 2 mol/L HCl (50 mL) was added un-
der stirring for 30 min, then extracted by ether. The organic layer
1
6,17
20–22
cells
or label the nucleus of the cells.
However, those
was washed with brine for three times, finally anhydrous MgSO
4
kinds of compounds could only be as fluorescent probes for all
kinds of cells and could not distinguish tumor cells specially. In
this paper, BTTD-NO constituted of both thiophene group and
2
the stem of benzothiadiazole, was also observed distribution in
the cytoplasm of MG63 cells. Furthermore, with nitro group on
(
4
40 mmol, 4.8 g) and 2,2-dimethyl-1,3-propanediol (40 mmol,
.72 g) were added. The reactant was stirred overnight. After evap-
orating off the solvent, the residue was purified by column chro-
matography on silica gel to afford white solid (4.8 g, 66%). 1
NMR (400 MHz, CDCl ): d (ppm) 7.46 (s, 1H), 7.19 (s, 1H), 3.78 (s,
H), 2.61 (t, J = 7.6 Hz, 2H), 1.60 (m, 2H), 1.30 (m, 18H), 0.99–
H
3
the stem, the BTTD-NO
cells with strong red fluorescence. The possible mechanism was
that BTTD-NO could be reduced by nitroreductase, and stimu-
lated the high gene expression of the nitroreductase in MG63
cells, which improved conversion rate of BTTD-NO to BTTD-
NH in turn.
Totally, the new benzothiadiazole derivative, BTTD-NO
2
could selectively trace the hypoxic tumor
4
0
1
1
3
3
.87 (m, 9H). CNMR (100 MHz, CDCl ): d (ppm) 144.6, 136.9,
2
26.5, 72.4, 32.0, 31.9, 30.1, 29.7, 29.6, 29.5, 29.4, 22.7, 21.9, 14.1.
2
4
.1.3. Synthesis of 4,7-bis(4-dodecylthiophen-2-yl)-5,6-
2
dinitrobenzo[c][1,2,5]thiadiazole (BTTD-NO
To a 100 mL two-neck flask C1 (0.38 g, 1.0 mmol), C2 (1.09 g,
.0 mmol), catalytic amount of Pd(PPh (0.5 mol %), aqueous
potassium carbonate (2 mol/L, 20 mL) and deoxygenated toluene
20 mL) were added under the protection of argon. The mixture
2
)
2
, had
the considerably potential as fluorescent probe in cancer cells
due to high specificity in hypoxic condition, negligible background
fluorescence signal, ease of handling, and solution stability.
3
3 4
)
(
was stirred for 48 h at 90 °C. After cooling to room temperature,
the reaction mixture extracted with toluene. The organic layer
was washed with water before being dried over anhydrous magne-
sium sulfate. After evaporating off the solvent, the residue was
3
. Conclusions
2
The new benzothiadiazole compound, BTTD-NO , had been suc-
cessfully synthesized and showed no cytotoxicity to MG63 cells. It
could significantly label the MG63 cells by emitting strong red
fluorescence under hypoxic condition. The possible mechanism
purified by column chromatography on silica gel to afford light yel-
1
low solid (0.47 g, 65%). Melting point: 68–69 °C.
(400 MHz, CDCl
1.68–1.61 (m, 2H), 1.33–1.26 (m, 18H), 0.89 (t, J = 6.4 Hz, 3H).
H NMR
3
): d (ppm) 7.32 (s, 2H), 2.68 (t, J = 7.6 Hz, 2H),
13
was that BTTD-NO
2
could be reduced by nitroreductase, and could
C

7
740
Q. Jiang et al. / Bioorg. Med. Chem. 21 (2013) 7735–7741
3
NMR (100 MHz, CDCl ): d (ppm) 152.3, 144.5, 141.8, 132.3, 129.3,
solve the formazen, and then the absorbance was measured at
1
2
26.5, 121.5, 32.2, 32.1, 30.5, 30.4, 29.8, 29.8, 29.7, 29.6, 29.5,
570 nm with a microplate reader (BIO-RAD, model 550).
À1
9.4. IR (KBr), cm : 2920, 2850, 1539, 1468, 1353, 827. ESI-MS:
+
+
m/z 765.2946 (M+K ); calcd for M
w
+K : 765.2944.
4.3.2. Fluorescence labeling of MG63 cells by using BTTD-NO
2
MG63 cells were cultured in DMEM medium with fetal bovine
4
4
4
.2. Spectroscopic feature of BTTD-NO
2
serum (10%, v/v) at 37 °C in 5% CO
2
. The cells (5 Â 10 cells) were
seeded in culture flasks (25 cm , Corning co.). After cultured in a
CO incubator for 24 h, the culture medium was exchanged to
2
2
.2.1. Absorption spectrophotometry
the DMEM medium with BTTD-NO
2
(ultimate concentration:
À6
The extinction co-efficient (
e
) of BTTD-NO
2
was calculated
2 Â 10 mol/L). The cells were then placed in a hypoxic chamber
according to the Lambert–Beer law. Absorption spectra of the sam-
2 2
flushed with 5% CO and 95% N at 37 °C for hypoxic condition.
ple were measured by a Perkin Elmer Lambda 950 UV/VIS Spec-
For oxic condition, the flasks with cells directly placed in incubator
at 37 °C. After treatment for 24 h, the cells were observed by Laser
Scanning Confocal Microscopy.
3
trometer in CH OH.
4
.2.2. Fluorescence spectrophotometry before and after
chemical reduction
4
.3.3. Quantitative analysis of fluorescent cells by flow
cytometry
In order to determine the proportion of fluorescent cells labeled
by BTTD-NO , cytometric analyses were performed according to
Reduction of BTTD-NO
2
by iron dust in acetic acid and heating
. The structure was
confirmed by H NMR and FTIR. 1H NMR (400 MHz, CDCl
): d
ppm) 7.18 (s, 2H), 7.12 (s, 2H), 4.39 (br, 4H), 2.71 (t, J = 8.0 Hz,
for 10 h at 80 °C, resulted in diamine BTTD-NH
2
1
3
2
(
4
flow cytometry. The MG63 cells (2 Â 10 cells) were seeded in cul-
4
6
H), 1.72–1.65 (m, 4H), 1.43–1.26 (m, 36H), 0.90 (t, J = 8.0 Hz,
H). IR (KBr), cm : 3330, 2922, 2851, 1445. The detailed synthetic
2
ture flasks (25 cm , Corning co.), treated with BTTD-NO
2
(ultimate
À1
À6
concentration: 2 Â 10 mol/L) for 24 h under oxic or hypoxic con-
dition. Then the cell pellet was washed with phosphate buffered
saline and centrifuged again. The pellet was resuspended in 1 mL
PBS buffer and measured in a flow cytometer (Becton Dickinson).
Data processing was carried out using the FACSDiva Version 6.1.1
Software (BD Biosciences).
routes were shown in Figure 3. Fluorescence spectra of BTTD-NO
before and after chemical reduction (10 mol/L methanol) were
measured with excitation at 480 nm in CH OH. Fluorescence spec-
3
tra were analyzed by a HITACHI F-7000 fluorescence spectropho-
tometer at 298 K.
2
À6
4
.2.3. Bioreductive activation of BTTD-NO
The liver microsomes were isolated from 10 rats (average
2
by nitroreductase
4
.3.4. Gene expression of nitroreductase in MG63
The BTTD-NO could be as substrate for the nitroreductase in
the cells, to determine whether the BTTD-NO could promote the
2
6–28
2
weight: 150 g) according to the method reported previously,
the liver microsomes provided as nitroreductase (P450 reductase)
in this experiment. After homogenization of the liver samples in
2
higher expression of the nitroreductase in cells. The gene expres-
sion of nitroreductase in MG63 cells was analyzed by RNA extrac-
tion followed by quantitative real-time polymerase chain reaction
2
0 mL of 50 mM phosphate (with 0.25 M sucrose, pH 7.4), the
microsomes were isolated by centrifugation at 10,000g to
00,000g, and resuspended in 5.0 mL of 0.1 M phosphate buffer
pH 7.4). The samples were prepared with microsomes (10 mg/
(
QRT-PCR) with SYBR green.
1
(
À6
After incubated for 4 and 24 h with BTTD-NO
2
(2 Â 10 mol/L),
À6
À6
À3
MG63 cells were collected for RNA extraction by Trizol (Invitrogen,
USA) according to the instructions of the manufacturer. Briefly,
cells were rinsed twice with PBS and lysed directly by adding
mL), BTTD-NO
2
(concentration: 0, 0.5 Â 10
,
1.0 Â 10
,
-
À6
À3
2
.0 Â 10 mol/L), NADPH (2 Â 10 mol/L) and MgCl
2
(3 Â 10
mol/L) in 0.1 M phosphate buffer pH 7.4. All these samples were
incubated at 37 °C for 4 h. After incubation these samples were
centrifuged at 10,000g and the fluorescence spectra of the superna-
tants were measured by the F-7000 FL Spectrophotometer (Hit-
achi). Furthermore, the fluorescence spectra of chemical reduced
1
8
mL sample of Trizol reagent, and followed by passing with an
# needle for several times. Then RNA samples was subsequently
isolated and were dissolved in 30 lL of RNase- and DNase-free
water, and the RNA concentration were measured using a Nano-
Drop (Thermo Scientific NanoDrop 2000, DE, USA).
Total RNA (1
tary DNA (cDNA) using an iScrip cDNA Synthesis Kit (Bio-Rad,
CA) in 20 reaction by following the protocol of the
manufacturer.
BTTD-NO
NO
2
was compared with that of biological reduced BTTD-
at concentration of 2.0 Â 10 mol/L.
À6
lg) was reverse transcribed into complemen-
2
a
lL
4
.3. Tracing tumor cells with BTTD-NO
2
in vitro
The PCR reactions were performed using SsoFast™ EvaGreenÒ
Supermix (Bio-Rad, CA), in a CFX96 real-time thermo cycler (Bio-
Rad, CA), and Triplicate PCR reactions were carried out. Relative
mRNA abundance was analyzed according to Bio-Rad CFX manager
software (version: 1.6.541) and reported as fold induction. GAPDH
abundance was used for normalization. The primer sequences of
the GAPDH were: (forward) GGAAG GTGAA GGTCG GAGTC and (re-
verse) TTAGGGTAGTGGTAGAAGGT; the primer sequences of nitro-
ductase were: (forward) GTCCTGAAACTGGGAACTAACA and
After the BTTD-NO
2
was synthesized, it was initially dissolved
À4
at 1 Â 10 mol/L in dimethyl sulfoxide (DMSO), followed by filtra-
tion with 0.22 lm filter membrane to eliminate contamination.
Small volumes were added to cell suspensions to give the appropri-
ate concentration in the following biological experiment (ultimate
concentration of DMSO <2%).
4
.3.1. Cytotoxicity of BTTD-NO
For cell cytotoxicity assay, MG63 cells were seeded in 96-well
2
(
reverse) TCCTCTTCTTCATCGGTGGTAA.
4
plates at a concentration of 1 Â 10 cells/well for 24 h, then differ-
À6
-6
-
ent concentration of BTTD-NO
2
(0, 0.5 Â 10 , 1 Â 10 and 2 Â 10
4.4. Statistical analysis
6
mol/L in ultimate concentration) were added in the well and cul-
tured for another 24 and 48 h. Cytotoxicity was determined by
MTT colorimetric method with 5 parallel wells at each time point.
After cells were incubated with 5 mg/mL of MTT for 4 h at 37 °C,
All data were expressed as means ± standard deviations (SD).
An unpaired Student’s t-test was adopted to test the significance
of the observed differences between the study groups. A value of
p < 0.05 was considered statistically significant.
supernatant was discarded, 200 lL/well of DMSO was added to dis-

Q. Jiang et al. / Bioorg. Med. Chem. 21 (2013) 7735–7741
7741
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Synth. Met. 2013, 175, 21.
The authors acknowledge the financial support for this work by
the National Natural Science Foundation of China (NSFC) (Grants
Nos. 20872103, 50902098, 50803040, 21072139, 21190031) and
National Key Technology R&D Program (2012BAI42G01).
1
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5. Neto, B. A.; Lapis, A. A.; da Silva, E. N.; Dupont, J. Eur. J. Org. Chem. 2013, 2013,
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Gatto, C. C.; de Oliveira, H. C. B.; Fasciotti, M.; Eberlin, M. N.; da Silva, E. N. J.
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Supplementary data
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7. Neto, B. A. D.; Carvalho, P. H. P. R.; Santos, D. C. B. D.; Gatto, C. C.; Ramos, L. M.;
Vasconcelos, N. M. d.; Correa, J. R.; Costa, M. B.; de Oliveira, H. C. B.; Silva, R. G.
RSC Adv. 2012, 2, 1524.
Supplementary data associated with this article can be found, in
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