Synthesis and biological evaluation of thiobenzanilides as anticancer agents

Article abstract of DOI:10.1016/j.bmc.2008.03.003

A series of novel thiobenzanilides is described. These compounds have been previously found to show strong biological activity such as antimycotic and antifungal actions. This is the first demonstration on the mechanism of the anticancer effect of thiobenzanilide agents (4a-c) on human melanoma A375 cells. The cytotoxic studies of compounds 4a-c on human melanoma A375 cells indicate thiobenzanilides induced higher cytotoxicity than nitrobenzanilides (3a-c). In addition, DNA flow cytometric analysis shows that 4a-c displays a significant G2/M phase arrest, which progresses to early apoptosis as detected by flow cytometry after double-staining with annexin V and propidium iodide (PI). Because cellular apoptosis is often preceded by the disruption of mitochondrial function, the assessment of mitochondrial function in 4a-c-treated cells is worthy of investigation. Our data revealed that treatment of A375 cells with 4a-c resulted in the loss of mitochondrial membrane potential (ΔΨ_mt), a reduction of ATP synthesis, increased reactive oxygen species (ROS) generation, and activation of caspase-3. Thus, we suggest that 4a-c agents are potent inducers of cell apoptosis in A375 cells.

Full text of DOI:10.1016/j.bmc.2008.03.003

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5295–5302
Synthesis and biological evaluation of thiobenzanilides
as anticancer agents
Wan-Ping Hu,a Hsin-Su Yu,b Yan-Ren Chen,c Yi-Min Tsai,c Yin-Kai Chen,a
Chao-Cheng Liao,c Long-Sen Changd,e and Jeh-Jeng Wangc,d,
*
aFaculty of Biotechnology, College of Life Science, Kaohsiung Medical University, Kaohsiung, Taiwan
bDepartment of Dermatology, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
cFaculty of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung, Taiwan
dNational Sun Yat-Sen University – Kaohsiung Medical University Joint Research Center, Kaohsiung, Taiwan
eInstitute of Biomedical Science, National Sun Yat-Sen University, Kaohsiung, Taiwan
Received 8 January 2008; revised 29 February 2008; accepted 1 March 2008
Available online 6 March 2008
Abstract—A series of novel thiobenzanilides is described. These compounds have been previously found to show strong biological
activity such as antimycotic and antifungal actions. This is the first demonstration on the mechanism of the anticancer effect of thio-
benzanilide agents (4a–c) on human melanoma A375 cells. The cytotoxic studies of compounds 4a–c on human melanoma A375
cells indicate thiobenzanilides induced higher cytotoxicity than nitrobenzanilides (3a–c). In addition, DNA flow cytometric analysis
shows that 4a–c displays a significant G2/M phase arrest, which progresses to early apoptosis as detected by flow cytometry after
double-staining with annexin V and propidium iodide (PI). Because cellular apoptosis is often preceded by the disruption of mito-
chondrial function, the assessment of mitochondrial function in 4a–c-treated cells is worthy of investigation. Our data revealed that
treatment of A375 cells with 4a–c resulted in the loss of mitochondrial membrane potential (DWmt), a reduction of ATP synthesis,
increased reactive oxygen species (ROS) generation, and activation of caspase-3. Thus, we suggest that 4a–c agents are potent induc-
ers of cell apoptosis in A375 cells.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
melanoma cell line A375 was selected as a model, it is a
highly metastasizable cell line resistant to radio- and
chemo-therapy.2 Meikrantz and Schlegel reported the
control of cell death is linked to the cell cycle.3 Cells with
a defective cell cycle are more vulnerable to some anti-
cancer agents according to numerous preclinical studies.
Recently, perturbation of mitochondrial function has
been shown to be a key event in the apoptotic cascade.4
Anticancer drugs may damage the mitochondria by
increasing the permeability of the outer mitochondrial
membrane, which is associated with the collapse of
mitochondrial membrane potential (DWmt). Disruption
in DWmt can be measured using rhodamine 123, a
cationic lipophilic fluorochrome5; the extent of fluores-
cent dye uptake reflects the redox potential across the
mitochondrial membrane.6 This is because a decline in
DWmt can disturb the intracellular ATP synthesis, cause
generation of reactive oxygen species (ROS), alter the
mitochondrial redox ratio, and lead to caspase-3 activa-
tion, which is one of the major promoters of apoptotic
effect.
Much attention has recently been paid to the discovery
and development of new, more selective anticancer
agents. Thiobenzanilides are found to show strong bio-
logical activity as antimycotic and antifungal agents.1
However, other biological profiles such as antitumor
action may also be involved in this complicated biolog-
ical phenomenon and further investigation is needed to
address this issue. The synthesis of thiobenzanilides
(4a–c) with 4-nitrobenzyl moiety and the differently
modified N-aryl fragment was carried out in our
laboratory.
In the present study, we report the synthesis and biolog-
ical evaluation of benzanilides (3a–c, 4a–c). The human
Keywords: Thiobenzanilides; Human melanoma; Mitochondria;
Apoptosis.
*
Corresponding author. Tel.: +886 7 312 1101x2275; fax: +886 7 312
5339; e-mail: jjwang@kmu.edu.tw
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.03.003

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The aim of this study was to investigate whether 4a–c
agents induced antiproliferation, leading to cell growth
cycle perturbation, an aberrant mitochondrial function
and subsequent apoptotic cell death.
at any cell cycle phases by 4a–c-induced apoptosis in
A375 cells, cells were treated with 10 lM agents for
24 h. As shown in Figure 1, the majority of control cells
exposed to DMSO of the cell cycle were in the G1 phase
69.8 2.5%; S phase 11.4 1.0%, and G2/M phase
18.8 1.6%. After treatment with compounds 4a–c for
24 h, cells progressed to the G2/M phase, and the major-
ity of the cell population was arrested at the G2/M
phase. Consistent with this cell cycle, 58.4 0.6%,
55.4 0.9%, and 59.9 2.5% of the cells were found
at the G1 phase, and 12.7 1.1%, 17.1 0.6%, and
11.3 0.6% in S phase, with 29.0 0.4%, 27.6 1.5%,
and 28.9 1.9% in G2/M phase, respectively. Our data
indicate that 4a–c had cytotoxic effects on human mela-
noma A375 cells, and the cytotoxic effects may be
through apoptosis induction.
2. Results and discussion
2.1. Syntheses
The thiobenzanilides (4) were synthesized in two steps
(Scheme 1). Anilines (1) and 4-nitrobenzoyl chloride
(2) were reacted in the presence of pyridine under reflux
conditions to form benzanilides (3). In the second step,
the benzanilides were converted to thiobenzanilides (4)
with Lawesson’s reagents in refluxing chlorobenzene.
2.2. In vitro cytotoxic effects
2.4. Mitochondrial membrane potential (DWmt) disruption
We evaluated the cytotoxicity of the benzanilides (3a–c,
4a–c) in various human cell lines using an MTS cell pro-
liferation assay. The activity of mitochondrial dehydro-
genase enzymes, detectable by catalyzing MTS reagent,
correlated with cell viability.7 As shown in Table 1, thio-
benzanilides (4a–c) were potently cytotoxic in sensitive
human melanoma A375, K562 leukemia, and 293T kid-
ney cell lines compared to that of nitrobenzanilides (3a–
c). Because the agents 4a–c exhibited a higher inhibitory
activity on A375 cells compared to other cell lines at a
concentration of 10 lM, the A375 cells were selected
as a model for further studies.
Previous studies have suggested that a decline of DWmt
may be an early event in the process of cell death. There-
fore, we investigated whether DWmt disruption was in-
volved in agent-induced apoptosis. A375 cells were
treated with 4a–c at a concentration of 10 lM for
24 h, and then analyzed by flow cytometry after rhoda-
mine 123-dye labeling. The dye binds to the inner and
outer membrane of mitochondria and undergoes a red
shift in fluorescence during membrane depolarization.8
Compared with the untreated control group, com-
pounds 4a–c exhibited a marked reduction in cellular
uptake of the fluorochrome (Fig. 2). The decrease of
fluorescence intensity reflects the collapse of DWmt,
which generally defines an early but already irreversible
stage of apoptosis.9 These results reveal that exposure of
melanoma A375 cells to 4a–c inducing the drop of DWmt
may be a possible cause of the apoptotic process.
2.3. Cell cycle effects
To verify whether cell damage might be attributable to
the cell cycle program or might have become arrested
R1
O
R1
ClOC
pyridine
reflux, 4hr
+
N
H
NH2
NO2
S
1
2
NO2
3a: R1 = Br
3b: R1 = CF3
3c: R1 = NO2
R1
Lawession's
reagent
chlorobenzene,
reflux, 4hr
N
H
NO2
4a: R1 = Br
4b: R1 = CF3
4c: R1 = NO2
Scheme 1. Synthesis of thiobenzanilide analogs 4a–c.
Table 1. Compounds against human-derived cancer cell lines in vitro
Survival (% control)
Compound (10 lM)
3a
3b
3c
4a
4b
4c
A375 (melanoma)
K562 (leukemia)
293T (kidney)
81.1 4.8
89.1 6.6
86.9 1.6
87.2 1.3
81.3 6.8
87.7 0.8
70.5 2.9
71.3 4.8
85.2 3.5
43.0 4.1
42.9 1.6
48.4 0.7
36.2 4.9
41.4 2.3
41.2 1.4
39.1 4.9
47.9 1.6
44.0 2.4
Cells were cultured with agents at a concentration of 10 lM for 24 h before growth and viability were assessed using the MTS cell proliferation assay.

W.-P. Hu et al. / Bioorg. Med. Chem. 16 (2008) 5295–5302
5297
2.5. Intracellular ATP content
ATP is the central parameter of cellular energetics, met-
abolic regulation, and cellular signaling; therefore,
determination of intracellular ATP is worthwhile in
the characterization of cellular physiology. Intracellular
ATP was measured by continuously monitoring ATP
production by firefly luciferase luminescence. Following
exposure of cells to 10 lM 4a–c, the intracellular ATP
content of A375 cells was 66.1 3.8% (4a),
63.1 0.3% (4b), and 83.6 1.5% (4c). A representative
histogram is shown in Figure 3.
2.6. ROS generation
To determine whether ROS is involved in 4a–c-induced
apoptosis, we measured the production of intracellular
H2O2 using DCFH-DA probe. Our result showed that
4a–c agents significantly increase intracellular H2O2 lev-
els. In addition, it was also observed that catalase signif-
icantly abrogated the increased ROS production of
A375 cells treated with 10 lM 4a (Fig. 4A and B).
Figure 1. Cell cycle distribution of A375 cells after treating with
compounds 4a–c. The percentage of the cells in each phase was
calculated by using the WinMDI software for the flow cytometry.
2.7. Apoptosis detection
Fluorescein isothiocyanate (FITC)-conjugated annexin
V has been utilized to detect the externalization of phos-
phatidylserine that occurs at an early stage of apoptosis.
Propidium iodide (PI) is used as a marker of necrosis
due to cell membrane destruction.10 To elucidate
whether compound-induced cell death involved apopto-
sis or necrosis, we performed a biparametric cytofluori-
metric analysis using annexin V and PI double-staining
as shown in Figure 5. Treatment of A375 cells with
10 lM agents for 24 h induced apoptosis effects (annexin
V+/PIꢀ) in 1.7% (Control), 7.5% (4a), 33.0% (4b), and
16.3% (4c) of annexin V-FITC cells. Our results showed
that 4a–c agents induce more apoptotic cells than that of
the untreated control; in contrast, there was no obvious
change of necrotic cells (annexin V+/PI+).
2.8. Caspase-3 activation
Figure 2. Effect of 4a–c on the mitochondrial membrane potential
(DWmt). Cells were cultured with different agents at a concentration of
10 lM for 24 h, then stained with rhodamine 123 and analyzed
immediately by flow cytometry as described under Section 4. The
Caspase-3 has been shown to be one of the most impor-
tant cell executioners for apoptosis.11,12 The expressions
of caspase-3 activity were determined using Western
blotting analysis. Compared with the untreated control
group, the caspase-3 activity of A375 cells increased
after 10 lM 4a–c treatment (Fig. 6).
number in M1 indicates the percentage of cells with reduced DWmt
.
120
100
80
3. Conclusions
**
**
**
Previous reports have shown that thiobenzanilides are a
promising group of compounds of a wide range of activ-
ities including antifungal and antimycotic activities. In
this study, we provide evidence indicating that thioben-
zanilides (4a–c) induce A375 melanoma cell apoptosis.
This novel finding raised our interest and prompted us
to elucidate the signaling mechanism of A375 cell apop-
tosis induced by 4a–c treatment. First, we used an MTS
cell proliferation to evaluate the cytotoxicity of tested
compounds in three human cell lines, A375, K562, and
60
40
20
0
Control
4a
4b
4c
Figure 3. Effect of compound tested on the intracellular ATP levels.
A375 cells were treated with 10 lM agents for 24 h. Relative ATP
levels were calculated as the percentage of the 0 lM level. **p < 0.01 as
compared to the control.

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W.-P. Hu et al. / Bioorg. Med. Chem. 16 (2008) 5295–5302
Figure 4. Effect of compounds 4a–c on reactive oxygen species (ROS) generation in A375 cells. (A) Cells were untreated or incubated with 10 lM
agents for 4 h, and the ROS production (dichlorofluorescein, DCF fluorescence) was measured. (B) As a control, ROS was measured in the presence
Å
of catalase, a H2O2 scavenger. Approximately 10,000 cells from each group were analyzed by flow cytometry. Similar results were obtained in three
independent experiments.
293T. Our results indicated that 4a–c agents are more
effective as an antiproliferative agent than 3a–c. We
hypothesized that the antiproliferative effect of 4a–c
may be associated with cellular apoptosis. Because
hypodiploid DNA content (sub-G1 material) is charac-
teristic of apoptosis and reflects fragmented DNA, the
sub-G1 population in A375 cells after compound treat-
ment was measured. Our results showed that com-
pounds 4a–c induces more apoptotic cells than 3a–c
(data not shown). Additionally, our results also showed
that 4a–c agents induce an arrested G2/M phase and
trigger apoptosis as revealed by the externalization of
annexin V-targeted PS residues at the periphery of the
cells.
Owing to studies suggesting that a decline of DWmt may
be an early event in the process of cell death, this
prompted us to investigate whether DWmt disruption
was involved in 4a–c-induced apoptosis. In this study,
compounds 4a–c exhibited a marked reduction in DWmt.
Mitochondrial oxidative phosphorylation is the major
ATP synthetic pathway in eukaryotes. A study is in pro-
gress to investigate whether decreased intracellular ATP
is due to 4a–c treatment. Our study showed a decrease of
intracellular ATP content in response to 4a–c treatment.
While mitochondria-dependent depletion of ATP would
by itself damage cells due to the failure to provide en-
ergy for membrane pumps, a greater hazard is the gen-
eration of ROS. Accumulation of ROS has been

W.-P. Hu et al. / Bioorg. Med. Chem. 16 (2008) 5295–5302
5299
Figure 5. Thiobenzanilide induces externalization of PS. Dot plots for A375 cells treated with agents at a concentration of 10 lM for 24 h and then
stained with PI and an annexin V-FITC conjugate specifically detecting the exposure of PS residues at the cell surface. Approximately 10,000 cells
from each group were analyzed by flow cytometry. Data shown are of a representative experiment repeated three times with similar results.
Based on our results, compound 4b is the most effective
one in preliminary anticancer activity. Further investiga-
tion is needed to address this issue. Our results, never-
theless, confirm for the first time that thiobenzanilides
induce apoptosis in A375 cells and that thiobenzani-
lide-induced apoptosis may involve mitochondrial dys-
function. We expect our studies can provide an
important mechanistic insight into the action of
thiobenzanilides.
Figure 6. Western blot analysis showed the effect of 4a–c treatment on
activation of caspase-3. After exposure to 10 lM 4a–c for 24 h, cell
lysates were collected and Western blotted with specific antibodies as
indicated. For the internal control, the same amounts of protein
extract were also probed with antibody against actin. Similar results
were observed in three separate experiments.
4. Experimental
4.1. Syntheses
shown to be an effective mechanism to activate pro-
grammed cell death.13 We found that hydrogen peroxide
(H2O2, a representative ROS) was involved in apoptotic
effect induced by 4a–c treatment. In addition, applica-
1H NMR and 13C NMR spectra were recorded at 400
and 100 MHz, respectively. Chemical shifts are reported
relative to TMS (0.0 ppm) and are designated as s (sin-
glet), d (doublet), t (triplet). Coupling constants (J val-
ues) are reported in Hertz. Mass spectra and high
resolution mass spectra (HRMS) were measured using
the electron-impact (EI, 70 eV) or electron-spray-impact
(ESI) technique. Flash chromatography was carried out
on Silica Gel 60 (E. Merck, 230–400 mesh).
Å
tion of catalase, that H2O2 scavenger, abrogated the en-
hanced ROS of A375 cells treated with 4a. A recent
report has shown that H2O2-induced cell death is pre-
dominantly a caspase-mediated apoptosis.14 Caspases
that are involved in the execution of apoptosis exist in
living cells as inactive zymogens that become activated
through intracellular caspase cascades, especially the
caspase-3, which is a straight promoter of apoptosis.15,16
We observed that an increase of caspase-3 activity in 4a–
c-induced A375 cells.
4.1.1. General procedure for synthesis of nitrobenzamides
(3). To a stirred solution of aniline 1 (60 mmol) in pyri-
dine (200 mL) was added 4-nitrobenzoyl chloride

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W.-P. Hu et al. / Bioorg. Med. Chem. 16 (2008) 5295–5302
(66 mmol) under nitrogen at room temperature, then the
mixture was refluxed for 4 h. After being cooled to room
temperature, the solution was poured into ice/water. The
resulting precipitate was filtered and recrystallized from
methylene chloride to give products 3.
100 MHz) d: 196.1 (s), 148.6 (s), 148.0 (s), 142.4 (s),
128.4 (d), 128.1 (q, 32.2 Hz), 125.8 (q, 3.2 Hz), 123.7
(q, 270 Hz), 123.5 (d), 123.2 (d). HRMS (EI, m/z) for
C14H9F3N2O2S: calcd 326.0337; found: 326.0335.
4.1.2.3. N-(4-Nitrophenyl)-4-nitrothiobenzamide (4c).
Light orange solid. Yield 3.7 g (61%). Mp: 175–178 ꢁC.
1H NMR (DMSO-d6, 400 MHz) d: 12.51 (s, 1H, NH),
8.33 (d, J = 8.8 Hz, 2H), 8.31 (d, J = 8.8 Hz, 2H), 8.22
(d, J = 8.8 Hz, 2H), 8.01 (d, J = 8.8 Hz, 2H). 13C
NMR (DMSO-d6, 100 MHz) d: 196.8, 148.6, 147.9,
145.3, 144.6, 128.9, 124.5, 123.9, 123.5. HRMS (EI, m/
z) for C13H9N2O4S: calcd 303.0314; found: 303.0312.
4.1.1.1. N-(4-Bromo-phenyl)-4-nitrobenzamide (3a).
Yellow solid. Yield 17.9 g (93%). Mp 247–249 ꢁC. H
1
NMR (DMSO-d6, 400 MHz) d 10.68 (s, 1 H, NH),
8.37 (d, J = 8.8 Hz, 2H), 8.17 (d, J = 8.4 Hz, 2 H), 7.76
(d, J = 8.4 Hz, 2 H), 7.57 (d, J = 8.8 Hz, 2 H). 13C
(DMSO-d6,100 MHz) d: 164.1, 149.2, 140.3, 138.1,
131.6, 129.3, 123.6, 122.4, 116.0. Anal. Calcd for
C13H9BrN2O3: C, 48.62; H, 2.82; N, 8.72. Found: C,
48.58; H, 2.85; N, 8.69.
4.2. Cell culture
4.1.1.2. N-(4-Trifluoromethyl-phenyl)-4-nitrobenza-
mide (3b). Light yellow solid. Yield 17.7 g (95%). Mp
209–212 ꢁC. H NMR (CDCl3 + DMSO-d6, 400 MHz)
d: 10.30 (s, 1H, NH), 8.32 (d, J = 8.8 Hz, 2H), 8.20 (d,
J = 8.8 Hz, 2H), 7.96 (d, J = 8.8 Hz, 2H), 7.61 (d,
J = 8.8 Hz, 2H). 13C NMR (DMSO-d6, 100 MHz)
d:164.2 (s), 149.3 (s), 141.4 (s), 140.1 (s), 128.9 (d),
125.7 (q, 31 Hz), 125.6 (q, 3.8 Hz), 123.8 (q, 270 Hz),
123.1 (d), 120.2 (d), HRMS (EI, m/z) for C14H9F3N2O3:
calcd 310.0560; found: 310.0563.
Three human cell lines, A375 (melanoma), K562 (leuke-
mia), and 293T (kidney), purchased from American
Type Culture Collection (Manassas, VA), were main-
tained in DMEM (A375, 293T) or RPMI1640 (K562)
medium supplemented with 10% FCS and 100 U/mL
penicillin G and 100 lg/mL streptomycin sulfate (Gibco,
BRL) at 37 ꢁC in a humidified atmosphere containing
5% CO2.
1
4.3. MTS cell proliferation assay
4.1.1.3. N-(4-Nitrophenyl)-4-nitrobenzamide (3c).
Light yellow solid. Yield 15.5 g (90%). Mp 267–
269 ꢁC. 1H NMR (DMSO-d6, 400 MHz) d: 11.07 (s,
1H, NH), 8.37 (d, J = 8.8 Hz, 2H), 8.27 (d, J = 8.8 Hz,
2H), 8.19 (d, J = 8.4 Hz, 2H), 8.04 (d, J = 8.8 Hz, 2H).
13C NMR (DMSO-d6, 100 MHz) d: 164.9, 149.6,
145.0, 143.0, 140.0, 129.6, 124.9, 123.7, 120.3. HRMS
(EI, m/z) for C13H9N3O5: calcd 287.0542; found:
287.0542.
A commercially available kit (CellTiter96 Aqueous pro-
liferation assay kit, Promega, Madison, WI) was used to
detect the proliferation according to the manufacturer’s
instruction. Cells were seeded in a 96-well plate at the
cell density of 2500 cells/well. After an overnight incuba-
tion the 4a–c agents at indicated concentrations was
added to the culture media and incubated for 24 h.
The MTS reagent contains tetrazoliumsalt, [3-(4,5-
dimethylthiazol-2-y1)-5-(3-carboxymethoxyphenyl)-2(4-
sulfophenyl)-2H-tetrazolium], premixed with the elec-
tron coupling reagent (phenazine ethosulfate) were
added into each well at 20 lL. The plate was then incu-
bated for 1–2 h at 37 ꢁC. The optical density value was
detected by a microplate reader (MRX-II, Dynex tech-
nology, Chantilly, VA), whose detecting and reference
wavelengths were set at 490 and 690 nm, respectively.
4.1.2. General procedure for synthesis of thiobenzamides
(4). To a stirred solution of nitrobenzamides (3)
(20 mmol) in chlorobenzene (20 mL) was added Lawes-
son’s reagent (10.2 mmol) under nitrogen at room tem-
perature, then the mixture was refluxed for 4 h. After
being cooled to room temperature, hexane (60 mL)
was added to the above solution. The resulting precipi-
tate was filtered and recrystallized from acetone to give
products 4.
4.4. Cell cycle analysis
A375 cells were treated with compounds at 10 lM for
24 h. Cells were harvested by trypsinization and centri-
fugation. After being washed with PBS, the cells were
fixed with ice-cold 70% ethanol for 30 min, washed with
PBS, and then treated with 1 mg/mL of RNase A solu-
tion (containing 0.112 mg/mL of trisodium citrate) at
37 ꢁC for 30 min. Cells were harvested by centrifugation
at 400g for 5 min and further stained with 250 lL of
DNA staining solution (10 mg of propidium iodide
(PI), 0.1 mg of trisodium citrate, and 0.03 mL of Triton
X-100 were dissolved in 100 mL H2O) at room temper-
ature for 30 min in the dark. After loading 750 lL of
PBS, the DNA contents of 10,000 events were measured
by FACSscan flow cytometer (Elite ESP, Beckman
Coulter, Brea, CA). Histograms were analyzed using
Windows Multiple Document interface software
(WinMDI).
4.1.2.1.
N-(4-Bromo-phenyl)-4-nitrothiobenzamide
(4a). Orange solid. Yield 4.1g (61%). Mp 174–177 ꢁC.
1H NMR (DMSO-d6, 400 MHz) d: 12.18 (s, 1H, NH),
8.28 (d, J = 8.4 Hz, 2H), 7.96 (d, J = 8.4 Hz, 2H), 7.78
(d, J = 8.8 Hz, 2H), 7.57 (d, J = 8.8 Hz, 2H). 13C
NMR (DMSO-d6, 100 MHz) d: 195.9, 148.7, 148.0,
139.1, 132.0, 129.0, 126.4, 123.8, 119.3. HRMS (ESI,
m/z) for C13H10BrN2O2S: calcd 336.9646; found:
336.9674.
4.1.2.2. N-(4-Trifluoromethyl-phenyl)-4-nitrothioben-
zamide (4b). Bright yellow solid. Yield 4.8 g (73%). Mp
182–185 ꢁC. H NMR (CDCl3 + DMSO-d6, 400 MHz)
d: 11.50 (s, 1H, NH), 8.25 (d, J = 8.4 Hz, 2H), 8.08 (d,
J = 8.0 Hz, 2H), 8.00 (d, J = 8.4 Hz, 2H), 7.68 (d,
J = 8.0 Hz, 2H). 13C NMR (CDCl3 + DMSO-d6,
1

W.-P. Hu et al. / Bioorg. Med. Chem. 16 (2008) 5295–5302
5301
4.5. Assessment of mitochondrial membrane potential
(DWmt)
PBS, 1% NP-40, 0.5% sodium deoxycholate, and 0.1%
SDS) containing 100 lg/mL PMSF, 2 lg/mL aprotinin,
2 lg/mL leupeptin, and, 100 lg/mL NaF. After centrifu-
gation at 14,000g for 30 min, protein in the supernatants
was quantified by the Bradford method (Bio-Rad).
Forty micrograms of protein per lane was applied in
10% SDS–polyacrylamide gel. After electrophoresis,
protein was transferred from the gel to the polyvinyli-
dene difluoride (PVDF) membrane (Millipore, Bedford,
MA). The membranes were blocked at room tempera-
ture for 1 h in PBS + 0.1% Tween 20 (PBS-T) containing
5% skim milk. After briefly rinsing with PBS-T, the
membrane was incubated with primary antibody at
room temperature for 2 h or at 4 ꢁC overnight. Rabbit
polyclonal antibodies against caspase-3 (p20, active
form) was purchased from Santa Cruz Biotechnology.
Mouse monoclonal antibody against actin was pur-
chased from Chemicon Int. Inc. (Temecula, CA). The
membrane was incubated with the corresponding horse-
radish peroxidase-labeled secondary antibody (Santa
Cruz Biotechnology) at room temperature for 1 h.
Membranes were washed with PBS-T four times for
15 min, and the protein blots were visualized with Wes-
tern Lightning Chemiluminescence Reagent Plus (Per-
kin-Elmer Life Sciences, Boston, MA). The relative
amounts of specific proteins were quantified by densi-
tometry scanning of X-ray films and analyzed by Eagle
Eye Image System (Stratagene, La Jolla, CA).
A375 cells were cultured in 6-well plates and allowed to
reach exponential growth for 24 h before treatment. The
cells were harvested 24 h after treatment with compounds
at a concentration of 10 lM. The medium was removed
and the adherent cells trypsinized. The cells were pelleted
by centrifugation at 400g for 5 min and further resus-
pended in 1 mL of rhodamine 123 (10 lg/mL) for
30 min at room temperature and washed with PBS twice
and resuspended in PBS. The samples were analyzed for
fluorescence (FL-1 detector, filter 530/30 nm band pass)
by using the WinMDI software for the flow cytometry.
4.6. ATP content bioluminescence assay
The amount of intracellular ATP was determined by
bioluminescent assay based on the measurement of the
light output of the luciferin–luciferase reaction. After
treatment with 4a–c agents at a concentration of
10 lM for 24 h, total cell extracts from cultured A375
cells were obtained immediately by lysing methods.
After centrifugation to remove cell debris, we collected
supernatants for ATP measurement. The total amount
of intracellular ATP was determined according to the
protocol provided with the ATP lite assay kit (Perkin-
Elmer, Boston, MA).
4.7. Determination of intracellular ROS level
4.10. Statistical analysis
To evaluate intracellular reactive oxygen species (ROS)
levels, 20,70-dichlorofluorescein diacetate (DCFH-DA,
Molecular Probes) fluorescent dye was used to clarify
this issue. The nonpolar DCFH-DA is converted to
the polar derivative DCFH by esterases when it is taken
up by the cell. DCFH is nonfluorescent but is rapidly
oxidized to the highly fluorescent DCF by intracellular
H2O2 or nitric oxide. In addition, catalase (Sigma), an
effective H2O2 scavenger, was also used in this study.
Cells were pretreated with catalase (800 U/mL) before
0 and 10 lM agents treatment. Four hours later,
DCFH-DA (10 lM) was immediately added into cul-
tured cells for 30 min at 37 ꢁC. The fluorescence of the
samples was measured with a flow cytometer. The
20,70-dichlorofluorescein (DCF) data were recorded
using FL-1 photomultiplier.
All results were expressed as means values standard
deviation (SD) and analyzed by using the statistical
analysis system (SPSS, SPSS Inc., Chicago, IL). Differ-
ences among groups were analyzed by Student’s t-test.
P values <0.05 were considered as significant for all sta-
tistical tests.
Acknowledgments
We would like to thank the National Science Council of
the Republic of China, Kaohsiung Medical University
Research Foundation (Q095008), and National Sun
Yat-Sen University-Kaohsiung Medical University Joint
Research Center for financial support.
4.8. Annexin V and PI binding assay
Supplementary data
To assess the simultaneous observation of early phase of
apoptotic and necrotic features, A375 cells were treated
with 4a–c agents at a concentration of 10 lM for 24 h,
then cells were measured by cytometry by adding annexin
V-FITC to 106 cells per sample according to the manufac-
turer’s specifications (Bender MedSystems, Vienna, Aus-
tria). Simultaneously, the cells were stained with PI. Flow
cytometry data were analyzed by the WinMDI software.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.
2008.03.003.
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