Synthesis, and biological evaluation of 2-(4-aminophenyl)benzothiazole derivatives as photosensitizing agents

Article abstract of DOI:10.1016/j.bmc.2010.04.082

Photodynamic therapy (PDT) employing exogenous photosensitizers is currently being approved for treatment of basal cell carcinoma (BCC). 2-(4-Aminophenyl)benzothiazoles (6) consist of chromophoric structure and absorb light in the UVA (315-400 nm). These results encouraged us to design and synthesize a diversity of 2-phenylbenzothiazoles (6). Studies on the apoptotic mechanism involved in photosensitive effects induced by UVA-activated 6 in BCC cells are carried out in the present article. 6-UVA-treated cells displayed several features of apoptosis, including an increase in the sub-G1 population, a significantly increased annexin V binding, and activation of caspase-3. 6-UVA induced a decrease in mitochondrial membrane potential (Δψ _mt) and ATP via enhanced ROS generation and promoted phosphorylation of extracellular signal-regulated kinase (ERK) and p38 MAPK expression. These results suggest that 6-UVA elicits photosensitive effects in mitochondria processes which involve ERK and p38 activation, and ultimately lead to BCC cell apoptosis.

Full text of DOI:10.1016/j.bmc.2010.04.082

Bioorganic & Medicinal Chemistry 18 (2010) 6197–6207
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, and biological evaluation of 2-(4-aminophenyl)benzothiazole
derivatives as photosensitizing agents
Wan-Ping Hu a,c, Yin-Kai Chen a, Chao-Cheng Liao f, Hsin-Su Yu b,c, Yi-Min Tsai f, Shu-Mei Huang b,
Feng-Yuan Tsai d, Ho-Chuan Shen f, Long-Sen Chang e, Jeh-Jeng Wang f,
*
a Department of Biotechnology, College of Life Science, Kaohsiung Medical University, Kaohsiung, Taiwan
b Department of Dermatology, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
c Center of Excellence for Environmental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
d Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Miaoli, Taiwan
e Institute of Biomedical Science, National Sun Yat-Sen University, Kaohsiung, Taiwan
f Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung City 807, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Photodynamic therapy (PDT) employing exogenous photosensitizers is currently being approved for
treatment of basal cell carcinoma (BCC). 2-(4-Aminophenyl)benzothiazoles (6) consist of chromophoric
structure and absorb light in the UVA (315–400 nm). These results encouraged us to design and synthe-
size a diversity of 2-phenylbenzothiazoles (6). Studies on the apoptotic mechanism involved in photosen-
sitive effects induced by UVA-activated 6 in BCC cells are carried out in the present article. 6-UVA-treated
cells displayed several features of apoptosis, including an increase in the sub-G1 population, a signifi-
cantly increased annexin V binding, and activation of caspase-3. 6-UVA induced a decrease in mitochon-
Received 24 February 2010
Revised 23 April 2010
Accepted 24 April 2010
Available online 29 April 2010
Keywords:
Photodynamic therapy
2-(4-Aminophenyl)benzothiazoles
UVA
Mitochondria
MAPK
drial membrane potential (Dwmt) and ATP via enhanced ROS generation and promoted phosphorylation
of extracellular signal-regulated kinase (ERK) and p38 MAPK expression. These results suggest that 6-
UVA elicits photosensitive effects in mitochondria processes which involve ERK and p38 activation,
and ultimately lead to BCC cell apoptosis.
Apoptosis
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
tyrosine kinase inhibitors modeled on structural comparisons with
the flavone quercetin and the isoflavone genistein, which compete
Nonmelanoma skin cancer (NMSC) is an increasing problem in
the world.1 Basal cell carcinoma (BCC) is the most common type
of NMSC, and most frequently occurs in people with fair skin.2 Pho-
todynamic therapy (PDT), a noninvasive treatment and with excel-
lent cosmetic results, is used for the prevention and treatment of
BCC cells.3–5 It employs a combination of a photosensitizing agent
and light.6 Light activation of a photosensitizer accumulates in the
tumor, and in the presence of molecular oxygen, leads to reactive
oxygen species (ROS) generation, which ultimately kills the target
cells.7 Nevertheless, reported cure rates vary, and the transdermal
penetration levels for both the photosensitizer and its activating
light source are listed as limiting factors. These limitations have
prompted the research for new photosensitizers.
at the ATP-binding sites of tyrosine kinases.10,11 However, other
biological profiles may also be involved in this complicated biolog-
ical phenomenon and further investigation is needed to address
this issue. The synthesis and biological evaluation of 6 was carried
out in our laboratory. Due to these compounds having chromophoric
structure and light absorption in the UVA range (320–400 nm), this
in vitro study analyses the photosensitive effect of UVA-activated 6
in BCC cells. One can speculate that UVA will produce side effects
such as carcinogenesis and photoaging, but in our system, these do
not appear because the exposure time required for 6 activation
was very short and did not lead to chronic exposure to UVA.
Mitochondria are well known to participate actively in the pro-
duction of ROS which might be harmful if produced excessively,
2-Phenylbenzothiazoles (A, Fig. 1) represent a novel class of po-
tent and selective antitumor agents.8,9 For instance, 2-(4-amino-
phenyl)benzothiazoles (6, Scheme 1) were originally prepared as
synthetic intermediates within a programme to design potential
S
N
A
* Corresponding author. Tel.: +886 7 312 1101x2275; fax: +886 7 312 5339.
E-mail address: jjwang@kmu.edu.tw (J.-J. Wang).
Figure 1. Structures of 2-phenyl-benzothiazoles A.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.082

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W.-P. Hu et al. / Bioorg. Med. Chem. 18 (2010) 6197–6207
COX
R1
O
R1
1. method a
R2
2
+
N
R2
1. method b
H
NO2
NH2
NO2
3
2
1
R2
S
R1
S
N
3
R2
R1
NO2
N
H
NO2
5
4
6a: R1 = H, R2 = H;
6c: R1 = 6-Et, R2 = H;
6e: R1 = 6-OMe, R2 = H;
6g: R1 = H, R2 = CH3;
6i: R1 = 6-OCH3, R2 = CH3; 6j: R1 = 6-CF3, R2 = CH3
6b: R1 = 6-Me, R2 = H
6d: R1 = 7-OMe, R2 = H
6f: R1 = 6-CF3, R2 = H
6h: R1 = 6-Et, R2 = CH3
R2
3'
1
7
4
S
4
1'
R1
NH2
N
3
5'
6
Scheme 1. Total synthesis of 2-(4-aminophenyl)benzothiazoles 6. Reagents and conditions: (1) method a: X = Cl, pyridine, reflux; method b: X = OH, SOCl2, benzene, reflux.
(2) Lawesson’s reagent, chlorobenzene, 135 °C. (3) K3Fe(CN)6 aq, NaOH, 90 °C. (4) H2/Pd/C 10%, CH2Cl2, 25 °C.
and are critically involved in the regulation of cell death
pathways.12 Permeabilization of the mitochondrial outer mem-
brane and subsequent release of proapoptotic proteins from the
intermembrane space are viewed as decisive events in the initia-
tion and/or execution of apoptosis.13 In addition, recent evidence
has indicated that ROS play a pivotal role in UVA-induced cell dam-
age.14 Consequently, cell death induced by UVA-activated 6 might
be correlated with mitochondrial depolarization. The mitogen-
activated protein kinase (MAPK) family consists of extracellular
signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and
p38 MAPK. JNK and p38 MAPK pathways are known to be activated
by a variety of environmental stresses and chemicals,15 while the
ERK cascade is activated by mitogenic stimuli and is critical for
proliferation and survival.16 However, ERK signaling has been sug-
gested to be proapoptotic in cells undergoing apoptosis.17,18 In this
regard, we have initiated experiments aimed at characterizing the
above signaling molecules in the process of apoptosis after BCC
cells were treated with 6-UVA.
2.2. Cell viability
The effect of UVA-activated 6 on BCC cell viability was evalu-
ated by MTT assay. BCC cells were treated with different concen-
trations of 6f for 4 h followed by 1 J/cm2 UVA irradiation.
Twenty-four hours after exposure, cell viability was determined.
As shown in Figure 2A, the inhibitory effect is dependent on drug
concentration. At concentrations of 4 lM 6f, the cell survival was
below 50% after 1 J/cm2 UVA irradiation. To elucidate whether
1 J/cm2 UVA irradiation is the most effective dosage in this study,
cells were pretreated with 4 lM 6f before UVA treatment. Com-
pared with that of the untreated controls, the cell viability of BCC
cells was 71%, 65%, and 45% after irradiation with 0.25, 0.5, and
1 J/cm2 UVA treatment respectively. In addition, our data also
showed that 6-UVA exhibited a higher inhibitory activity more
than either UVA irradiation alone or treatment with exogenous 6
alone, and 6f alone does not have cytotoxicity against BCC cells
(Fig. 2B).
The aim of this study was to evaluate the newly synthesized 6
as photosensitizing agents and investigate the apoptotic mecha-
nisms induced by 6-UVA on BCC cells.
2.3. Cellular sub-G1 accumulation
To investigate the effects of compound 6 on cell cycle progres-
sion of BCC cells, the DNA content of cell nuclei was measured by
flow cytometric analysis. Agent action resulted in cells having a
hypodiploid DNA content (sub-G1 material) that is characteristic
of apoptosis and reflects fragmented DNA as shown in Figure 3.
2. Results and discussion
2.1. Syntheses
The preparation of 2-(4-aminophenyl)benzothiazoles 6 is shown
in Scheme 1.
BCC cells were treated with 4 l
M agents then 1 J/cm2 UVA irradi-
ation. Twenty-four hours after irradiation, the apoptotic effects in
2.2% (control), 6.5% (6a), 13.4% (6b), 29.2% (6c), 24.4% (6d), 6.5%
(6e), 35.4% (6f), 13.6% (6g), 13.8% (6h), 14.8% (6i) and 6.4% (6j) of
sub-G1 DNA peak were obtained. Because the compound 6f exhib-
ited the most sub-G1 accumulation on BCC cells, it was selected as
a model for further studies.
4-Nitro-N-phenyl benzamides 3 were obtained by the reaction
of anilines 1 with nitrobenzoyl chlorides 2 in pyridine under reflux
for 4 h (method A). Alternatively, anilines 1 were coupled to nitro-
benzoic acids 2 in the presence of thionyl chloride in benzene un-
der reflux condition to give benzamides 3 in high yields (method
B). The benzamides 3 were treated with Lawesson’s reagent in
chlorobenzene under reflux to form 4-nitro-N-phenylthiobenza-
mides 4 in good yields. Cyclization of compound 4 promoted by
potassium ferricyanide to produce 2-(4-nitrophenyl)benzothiaz-
oles 5,19 followed by catalytic reduction of nitro group of 5 with
palladium on charcoal in methanol generated the target com-
pounds 6 in excellent yields.
2.4. Apoptosis detection
Caspase-3 has been shown to be one of the most important cell
executioners for apoptosis.20,21 The expressions of caspase-3
activity were determined using colorimetric assay. Compared with

W.-P. Hu et al. / Bioorg. Med. Chem. 18 (2010) 6197–6207
6199
is an early event in the process of cell death. The decrease of fluo-
rescence intensity reflects the collapse of mt, which generally
defines an early but already irreversible stage of apoptosis. There-
fore, we examined whether the initial ROS generation after 6f-UVA
alters Dwmt. BCC cells were harvested after 0, 2, and 4 lM 6f-UVA
treatment, then analyzed by flow cytometry after DiOC6 dye label-
ing. The dye binds to the inner and outer membrane of mitochon-
dria and undergoes a red shift in fluorescence during membrane
depolarization. As demonstrated in Figure 5B, cells treated with
150
100
50
A
B
D
w
*
*
**
4
**
8
0
1
2
Concentration ( M)
dosages equal to or greater than 2
lM exhibited significant decline
125
100
75
50
25
0
Control
of mt in BCC cells. ATP is the central parameter of cellular ener-
D
w
UVA
6f
getics, metabolic regulation and cellular signaling; therefore,
determination of intracellular ATP is worthwhile in the character-
ization of cellular physiology. Compared with that of untreated
control, the intracellular ATP content of BCC cells decreased about
6f-UVA
30% and 45% after 2 and 4
(Fig. 5C).
lM of 6f-UVA treatment, respectively
2.6. Activation of MAPKs pathways
To determine the potential involvement of various protein ki-
nase pathways in 6f-UVA-induced apoptosis, MAPK activities were
evaluated by measuring phosphorylation of MAPK subfamilies.
Compared with the untreated control group, the phosphorylation
of ERK (p-ERK) and p38 (p-p38) of BCC cells increased after treat-
0
0.25
0.5
1
Laser dosage (J/cm2)
ment with 6f-UVA at a concentration of 4 lM. In contrast to ERK
Figure 2. Effect of 6-UVA cell viability. (A) Dose–response curves for compound 6f
tested against BCC cells. Cells were seeded in a 96-well plate at 2500 cells per well
and cultivated overnight until cell attachment. 6f-UVA at the indicated concentra-
tion was added into the culture media in triplicate and incubated for 4 h before
1 J/cm2 UVA irradiation. Twenty-four hours after irradiation, the MTT reagent was
added into each well. The absorbance is directly proportional to the number of
living cells. (B) To clarify whether 6-UVA has more cytotoxicity than either UVA
irradiation alone or treatment with 6 alone, cells were cultured with or without
and p38, treatment of BCC cells with 6f-UVA did not stimulate
the phosphorylation of JNK MAPK (Fig. 6A). Moreover, to elucidate
whether the activity of MAPK might be due to the decrease in cel-
lular ATP synthesis, cells were pretreated with oligomycin (a mito-
chondria-specific F0F1 ATP synthase inhibitor) or ATP before 4 lM
6f-UVA treatment. Our results showed that pretreatment with oli-
gomycin but not ATP increased the ERK activation (Fig. 6B).
4 lM 6f before different dosage of UVA irradiation. *p <0.05, **p <0.01 as compared
with the control.
3. Conclusions
Previous reports have shown that 2-(4-aminophenyl)benzo-
thiazoles have the potency of growth inhibition activity against
cancer cell lines. In this study, we provide evidence indicating that
most of the 6, under UVA light exposure, induced BCC cell apopto-
sis. This novel finding raised our interest and prompted us to
elucidate the signaling mechanism of BCC apoptosis induced by
6-UVA treatment. We found that when cells were pretreated with
6 for 4 h before 1 J/cm2 UVA irradiation, 6-UVA exhibited a cyto-
toxicity effect on BCCs, and this inhibitory activity is dependent
on drug concentration. We hypothesized that the antiproliferative
effect of 6-UVA may be associated with cellular apoptosis. Our re-
sults showed that 6-UVA induces a markedly increased accumula-
tion of sub-G1 phase and triggers apoptosis as revealed by the
increased annexin V-FITC cells and the caspase-3 activation.
Many photochemical experiments have indicated that the pri-
mary event in any photosensitization process is the absorption of
photon energy and the following generation of ROS. Our data indi-
cated that 6f-UVA promotes H2O2 (a representative ROS) generation.
In addition, application of catalase (H2O2 scavenger) abrogated the
enhanced ROS of BCC cells treated with 6f-UVA. Because mitochon-
the untreated control group, the caspase-3 activity of BCCs in-
creased after 6f-UVA treatment (p <0.01) (Fig. 4A). Moreover, treat-
ment of BCC cells with 0, 2, and 4 l
M 6f at 1 J/cm2 UVA irradiation
induced apoptotic cells (annexin V+/PIꢀ) at levels of 0.2%, 19.5%,
and 27.5%, respectively. In contrast, there was no obvious change
of necrotic cells (annexin V+/PI+) (Fig. 4B). In addition, it would
be interesting to know the apoptotic activity of 6f-UVA on normal
cells. Thus, human dermal fibroblast cells were used to clarify this
issue. Data from morphology observation and annexin V/PI binding
assays showed that no significant apoptotic effect was seen after
6f-UVA treatment as compared with the control group (Fig. 4C).
These results encouraged us to further study the apoptotic mecha-
nism involved in photosensitive effects induced by 6f-UVA in BCC
cells.
2.5. Mitochondrial dysfunction
Growing evidence suggests that mitochondrial dysfunction
plays a key role in oxidative stress,22 which induces production
of ROS, and can lead to the apoptotic mode of cell death.23 To
determine whether ROS was involved in 6f-UVA-induced mito-
chondria dependent apoptosis, we measured the production of
intracellular H2O2 using the DCFH-DA probe. Our result showed
that 6f-UVA significantly increases intracellular H2O2 levels. In
addition, catalase significantly abrogated the increased ROS pro-
dria are known to be a significant source of ROS and
mitochondrial homeostatic center, the assessment of
is worth investigating. Our experimental data showed that cells
treated with 6f-UVA exhibited a marked decrease in mt. A drop
in wmt promotes the opening of the permeability transition (PT),
D
wmt is the
D
wmt in cells
D
w
D
which leads to mitochondrial dysfunction. Moreover, mitochondrial
oxidative phosphorylation is the major ATP synthetic pathway in
eukaryotes. A study is in progress to investigate whether decreased
intracellular ATP is due to 6f-UVA treatment. Our study showed a
duction of BCC cells treated with 4
drial membrane potential ( mt) is an important parameter not
only for mitochondrial but also cellular status. A decline of
lM 6f-UVA (Fig. 5A). Mitochon-
D
w
D
wmt

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W.-P. Hu et al. / Bioorg. Med. Chem. 18 (2010) 6197–6207
Figure 3. Effect of compound tested on the cellular sub-G1 content. BCC cells were treated with 4 l
M agents for 4 h followed by 1 J/cm2 UVA. Twenty four hours after
irradiation, the PI reagent was used. Approximately 10,000 cells from each group were analyzed with the FACScan flow cytometer. Data represent the percentage of cell
counts and display sub-G1.
decrease of intracellular ATP content in response to 6f-UVA
treatment.
4. Experimental
4.1. Syntheses
Owing to studies suggesting that the activity of the MAPKs after
PDT are suspected to be of importance in the process of cell
death,24–26 this prompted us to investigate whether MAPK-signal-
ing pathways were involved in 6f-UVA-induced apoptosis. Our
study showed that 6f-UVA caused apoptosis in parallel with the
activation of ERK and p38 in BCC cells. Although PDT has been
shown to activate JNK with different photosensitizers,24,27 6f-
UVA did not affect the JNK-P expression, indicating that JNK MAPK
is not involved in 6f-UVA-induced apoptosis. The preceding re-
sults demonstrate that mitochondria and MAPK participate in
apoptosis of BCC cells. Nevertheless, the possible interrelation-
ships among these substances in 6f-UVA-induced apoptosis are
not clear. Thus, we investigated whether the ERK activation corre-
lated with alterations in ATP levels. Cells were pretreated with
oligomycin or ATP before 6f-UVA treatment. Our result showed
that increased ERK phosphorylation was caused by reduced cellu-
lar ATP synthesis.
Chemical reagents were obtained without further purification.
Solvents free distillated prior to use. Reactions were monitored by
thin layer chromatography, using Merck plates 60 F254. Flash chro-
matography was carried out on Merck Silica Gel 60 (40–63 lm)
using the indicated solvents. Melting points were determined using
Fargo MP-2D and are uncorrected. 1H NMR and 13C NMR spectra
were recorded on Varian UNITY plus-400 at 400 and 100 MHz,
respectively, using CDCl3 as a solvent. 1H NMR chemical shifts are
referenced to TMS or CDCl3 (7.26 ppm). 13C NMR was referenced
to CDCl3 (77.0 ppm). Mass spectra were recorded with Bruker APEX
II spectrometer. Elemental analyses were performed on Elementar
vario EL III analyzer, and the results were found to be 0.4% of the
theoretical values. Purity of tested compounds was >95%.
4.1.1. General procedure for the syntheses of benzamides
(3a–3f) (method a)
The data gathered in this study demonstrate that cells exposed
to 6f-UVA initiate mitochondrial changes including an increased
ROS generation, a decline in Dwmt, and a reduction of ATP. In addi-
To a stirred solution of aniline (225 mmol, 1.2 equiv) in pyridine
(450 mL) was added 4-nitrobenxzoyl chloride (186 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 the corresponding
compounds 3.
tion, 6f-UVA treatment also induced ERK and p38 MAPK phosphor-
ylation. We propose that 6f-UVA treatment induces cell apoptosis
through the mitochondrial pathway, which subsequently activates
p38 and ERK. In the present article, we describe the effect of UVA-
activated 6 on BCC cells in vitro. Our findings suggest that 6 have
photosensitive activity for BCC cells. Further studies are required
to elucidate the precise mechanism of the photosensitive effect
of 6 in vivo at the molecular biological level.
4.1.1.1. 4-Nitro-N-phenylbenzamide 3a. White solid; 91% yield;
mp 214–216 °C; 1H NMR (CDCl3, 400 MHz) d 9.42 (br s, NH), 8.38

W.-P. Hu et al. / Bioorg. Med. Chem. 18 (2010) 6197–6207
6201
250
200
150
100
50
**
A
**
0
0
2
4
Concentration ( M)
B
C
Fibroblasts
M
0
4
Annexin-FITC
Figure 4. Effect of 6f-UVA treatment on cell apoptosis. (A) The increased enzymatic activities of the caspase-3 in apoptotic were determined by colorimetric reaction. The
cleavage of peptide by the caspase releases the chromophore pNA (p-nitroaniline), which can be quantified spectrophotometrically at a wavelength of 405 nm. (B) Dot plots
for BCC cells treated with various concentrations of 6f-UVA and then stained with PI and an annexin V-FITC conjugate specifically detecting the exposure of PS residues at the
cell surface. (C) Morphological observation and annexin V/PI double stain were used for the detection of fibroblasts apoptosis. 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. **p <0.01 as compared with the control.
(dt, J = 2.4 and 3.6 Hz, 2H), 8.23 (dt, J = 2.4 and 3.6 Hz, 2H), 7.85–
7.82 (m, 2H), 7.40–7.35 (m, 2H), 7.17–7.13 (m, 1H); 13C NMR
(CDCl3, 100 MHz) d 165.3, 151.2, 142.6, 140.5, 130.5, 130.3,
100 MHz) d 163.8, 149.3, 140.7, 135.3, 134.4, 129.3, 128.7, 123.4,
120.8, 20.8; HRMS (ESI, m/z) for C14H12N2O3Na calcd 279.0746,
found 279.0744. Anal. Calcd for C14H12N2O3: C, 65.62; H, 4.72; N,
10.93. Found: C, 65.71; H, 5.00; N, 10.92.
125.8, 125.1, 121.8; HRMS (EI, m/z) for
C13H10N2O3 calcd
242.0691, found 242.0692. Anal. Calcd for C13H10N2O3: C, 64.46;
H, 4.16; N, 11.56. Found: C, 64.72; H, 4.25; N, 11.54.
4.1.1.3. N-(4-Ethylphenyl)-4-nitrobenzamide 3c. White solid;
96% yield; mp 186–188 °C; 1H NMR (CDCl3, 400 MHz) d 8.32 (dd,
J = 5.2 and 1.6 Hz, 2H), 8.03 (d, J = 8.4 Hz, 2H), 7.92 (br s, NH),
8.23 (d, J = 8.0 Hz, 2H), 7.22 (d, J = 8.4 Hz, 2H), 2.65 (q, J = 7.6 Hz,
2H), 1.24 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3, 100 MHz) d 163.6,
149.6, 141.5, 140.6, 134.8, 128.6, 128.2, 124.0, 120.6, 28.4, 15.6;
4.1.1.2. N-(4-Methylphenyl)-4-nitrobenzamide 3b. White solid;
93% yield; mp 201–203 °C; 1H NMR (CDCl3, 400 MHz) d 9.37 (br s,
NH), 8.26 (d, J = 8.8 Hz, 2H), 8.11 (d, J = 8.4 Hz, 2H), 7.58 (d,
J = 8.0 Hz, 2H), 7.16 (d, J = 8.0 Hz, 2H), 2.34 (s, 3H); 13C NMR (CDCl3,

6202
W.-P. Hu et al. / Bioorg. Med. Chem. 18 (2010) 6197–6207
Control
A
Catalase (4 M)
2 M
4
M
0
M
2
M
4 M
B
1.4 0.1%
34.1 1.7%
43.5 2.2%
C
150
100
50
**
*
0
0
2
4
Concentration ( M)
Figure 5. Effect of 6f-UVA on the mitochondrial function. (A) 6f-UVA induced ROS generation in BCC cells. Histogram of fluorescence vs cell count in BCC cells with various
concentrations of 6f-UVA and stained with DCFH-DA. As a control, ROS was measured in the presence of catalase, a H2O2.scavenger. (B) The
mt of BCC cells after exposure
to 6f-UVA. Cells were treated with 0, 2, and 4
M 6f for 4 h followed by 1 J/cm2 UVA irradiation, then stained with DiOC6 and analyzed immediately by flow cytometry as
described under Section 4. The number in M1 indicates the percentage of cells with reduced mt. Approximately 10,000 cells from each group were analyzed by flow
M level. Similar results were obtained in three independent experiments. *p <0.05, **p <0.01 as
D
w
l
D
w
cytometry. (C) Relative ATP levels were calculated as the percentage of the 0
compared with the control.
l
HRMS (ESI, m/z) for C15H15N2O3 calcd 271.1083, found 271.1084.
Anal. Calcd for C15H14N2O3:C, 66.66; H, 5.22; N, 10.36. Found: C,
66.48; H, 5.41; N, 10.15.
273.0873. Anal. Calcd for C14H12N2O4: C, 61.76; H, 4.44; N, 10.29.
Found: C, 61.88; H, 4.65; N, 10.14.
4.1.1.6. N-(4-Trifluoromethylphenyl)-4-nitrobenzamide 3f. White
solid; 95% yield; mp 194–196 °C; 1H NMR (DMSO, 400 MHz) d 10.87
(br s, NH), 8.37 (d, J = 8.4 Hz, 2H), 8.14 (d, J = 8.4 Hz, 2H), 7.97 (d,
J = 8.0 Hz, 2H), 7.70 (d, J = 8.4 Hz, 2H); 13C NMR (DMSO, 100 MHz) d
164.9, 149.6, 142.5, 140.3, 129.6, 126.3, 124.5 (q, J = 140 Hz, 1C),
123.9, 120.7, 114.1; HRMS (EI, m/z) for C14H9F3N2O3 calcd 310.0560,
found 310.0563. Anal. Calcd for C14H9F3N2O3: C, 54.20; H, 2.92; N,
9.03. Found: C, 54.50; H, 2.84; N, 9.25.
4.1.1.4. N-(3-Methoxyphenyl)-4-nitrobenzamide 3d. Yellow so-
lid; 80% yield; mp 158–160 °C; 1H NMR (CDCl3, 400 MHz) d 9.98
(br s, NH), 8.31–8.28 (m, 2H), 8.18 (d, J = 8.8 Hz, 2H), 7.94 (s,
1H), 7.32–7.23 (m, 2H), 6.72–6.69 (m, 1H), 3.82 (s, 3H); 13C NMR
(CDCl3, 100 MHz) d 163.8, 159.5, 149.0, 140.5, 139.2, 129.1,
128.7, 123.0, 112.8, 109.9, 106.3, 54.9; HRMS (ESI, m/z) for
C14H12N2O4Na calcd 295.0695, found 295.0694. Anal. Calcd for
14H12N2O4: C, 61.76; H, 4.44; N, 10.29. Found: C, 61.49; H, 4.59;
C
N, 10.33.
4.1.2. General procedure for the syntheses of benzamides
(3g–3j) (method b)
4.1.1.5. N-(4-Methoxyphenyl)-4-nitrobenzamide 3e. Yellow so-
lid; 80% yield; mp 196–197 °C; 1H NMR (CDCl3, 400 MHz) d 9.35
(br s, NH), 8.22 (d, J = 8.0 Hz, 2H), 8.07 (d, J = 8.0 Hz, 2H), 7.68 (d,
J = 8.4 Hz, 1H), 6.95 (d, J = 8.4 Hz, 2H), 3.75 (s, 3H); 13C NMR (CDCl3,
100 MHz) d 163.7, 156.6, 149.2, 140.7, 130.9, 128.6, 123.3, 122.5,
113.9, 55.3; HRMS (ESI, m/z) for C14H13N2O4 calcd 273.0875, found
To a stirred solution of 4-nitrobenzoic acid (82 mmol) in ben-
zene (165 mL) was treated with thionyl chloride (58 mL,
820 mmol, 10 equiv) under nitrogen at room temperature then
the mixture was reflux for 4 h. After removal of solvent, the ben-
zoyl chloride intermediate was added slowly to a solution of the
appropriately substituted aniline (123 mmol, 1.5 equiv) in pyridine

W.-P. Hu et al. / Bioorg. Med. Chem. 18 (2010) 6197–6207
6203
found 285.1241. Anal. Calcd for C16H16N2O3: C, 67.59; H, 5.67; N,
9.85. Found: C, 67.50; H, 5.45; N, 9.85.
6f ( M)
0
2
4
A
p-ERK
4.1.2.3. N-(4-Methoxyphenyl)-3-methyl-4-nitrobenzamide 3i.
White solid; 71% yield; mp 152–154 °C; 1H NMR (CDCl3,
400 MHz) d 7.98 (d, J = 8.4 Hz, 1H), 7.94 (br s, NH), 7.81 (s, 1H),
7.75 (d. J = 8.0 Hz, 1H), 7.52 (d, J = 8.8 Hz, 2H), 6.89 (d, J = 9.2 Hz,
2H), 3.81 (s, 3H), 2.62 (s, 3H); 13C NMR (CDCl3, 100 MHz) d 163.7,
157.0, 150.8, 138.8, 134.1, 131.7, 130.2, 125.3, 124.9, 122.3,
114.3, 114.2, 55.4, 20.2; HRMS (ESI, m/z) for C15H15N2O4 calcd
287.1032, found 287.1033. Anal. Calcd for C15H14N2O4: C, 62.93;
H, 4.93; N, 9.79. Found: C, 62.77; H, 5.17; N, 9.82.
ERK
p-JNK
JNK
4.1.2.4. 3-Methyl-4-nitro-N-(4-trifluoromethyl-phenyl)benzam-
ide 3j. White solid; 75% yield; mp 154–156 °C; 1H NMR (CDCl3,
400 MHz) d 9.38 (br s, NH), 8.01 (d, J = 8.8 Hz, 1H), 7.92 (s, 1H),
7.87–7.83 (m, 2H), 7.61 (d, J = 8.4 Hz, 2H), 2.64 (s, 3H); 13C NMR
(CDCl3, 100 MHz) d 164.8, 150.8, 141.0, 138.4, 133.8, 132.1, 126.1
(q, J = 38 Hz, CF3–C), 125.8, 124.7, 123.2 (q, J = 140 Hz, CF3),
p-p38
p38
120.3, 120.2 20.0; HRMS (EI, m/z) for
324.0722, found 324.0725. Anal. Calcd for
C
15H11N2O2F3 calcd
Actin
C
15H11F3N2O3: C,
55.56; H, 3.42; N, 8.64. Found: C, 55.50; H, 3.48; N, 8.64.
oligomycin
ATP
+
B
6f (uM)
4.1.3. General procedure for the syntheses of thiobenzamide
(4a–4j)
+
4
0
4
4
A
mixture of the substituted 4-nitro-N-phenylbenzamide
(41 mmol) and Lawesson’s reagent (8.52 g?21 mmol, 0.51 equiv)
in chlorobenzene (30 mL) was heated at reflux for 4–6 h, after
which it was concentrated, purified by column chromatography
(CH2Cl2/hexane = 2:3) to give the corresponding compounds 4.
p-ERK
4.1.3.1. 4-Nitro-N-phenylthiobenzamide 4a. Yellow solid; 60%
yield; mp 154–156 °C; 1H NMR (CDCl3, 400 MHz) d 9.08 (br s,
NH), 8.26 (d, J = 8.4 Hz, 2H), 7.95 (d, J = 8.4 Hz, 2H), 7.77 (d,
J = 7.6 Hz, 2H), 7.47 (t, J = 8.0 Hz, 2H), 7.34 (t, J = 8.0 Hz, 1H); 13C
NMR (CDCl3, 100 MHz) d 195.5, 148.9, 148.2, 138.5, 129.2, 127.7,
ERK
Figure 6. Immunoblot analysis showed the effect of 6f-UVA on the protein
expression of MAP kinases on BCC cells. (A) After exposure to different concentra-
tion of 6f-UVA, cell lysates were collected and immunoblotted with specific
antibodies as indicated. For the internal control, the same amounts of protein
127.5, 123.9, 123.5; HRMS (EI, m/z) for
C13H10N2O2S calcd
258.0463, found 258.0463. Anal. Calcd for C13H10N2O2S, C, 60.45;
H, 3.90; N, 10.85. Found: C, 60.44; H, 4.06; N, 10.75.
extract were also probed with antibody against actin. (B) As
a
control, the
M)
expression level of p-ERK was determined in the presence of oligomycin (25
l
and ATP (1 ꢁ 10ꢀ4 M).
4.1.3.2. N-(4-Methylphenyl)-4-nitrothiobenzamide 4b. Yellow
solid; 60% yield; mp 195–196 °C; 1H NMR (CDCl3, 400 MHz) d
9.15 (br s, NH), 8.21 (d, J = 8.4 Hz, 2H), 7.91 (d, J = 8.8 Hz, 2H),
7.60 (d, J = 8.4 Hz, 2H), 7.26–7.23 (m, 2H), 2.38 (s, 3H); 13C NMR
(CDCl3, 100 MHz) d 195.27, 148.8, 148.0, 137.6, 135.9, 129.7,
127.7, 123.8, 123.6, 21.2; HRMS (ESI, m/z) for C14H12N2O2SNa calcd
295.0517, found 295.0516. Anal. Calcd for C14H12N2O2S: C, 61.75;
H, .4.44; N, 10.29. Found: C, 61.81; H, 4.55; N, 10.29.
(165 mL). The mixture was refluxed under nitrogen at room tem-
perature for 4 h. After being cooled room temperature, the solution
was poured into ice/water. The resulting precipitate was filtered
and recrystallized from methylene chloride to give the correspond-
ing compounds 3.
4.1.2.1. N-(Phenyl)-3-methyl-4-nitrobenzamide 3g. White so-
lid; 82% yield; mp 149–151 °C; 1H NMR (CDCl3, 400 MHz) d 8.01
(d, J = 8.4 Hz, 1H), 7.98 (br s, NH), 7.84 (s, 1H), 7.78 (d, J = 8.4 Hz,
1H), 7.63 (d, J = 7.6 Hz, 2H), 7.40–7.36 (m, 2H), 7.21–7.17 (m,
1H), 2.64 (s, 3H); 13C NMR (CDCl3, 100 MHz) d 163.9, 150.9,
138.8, 137.27, 134.2, 131.8, 139.2, 125.4, 125.3, 125.0, 120.4,
20.3; HRMS (ESI, m/z) for C14H13N2O3 calcd 257.0926, found
257.0925. Anal. Calcd for C14H12N2O3: C, 65.62; H, 4.72; N, 10.93.
Found: C, 65.53; H, 4.60; N, 11.00.
4.1.3.3. N-(4-Ethylphenyl)-4-nitrothiobenzamide 4c. Yellow so-
lid; 65% yield; mp 142–144 °C; 1H NMR (CDCl3, 400 MHz) d 9.07
(br s, NH), 8.25 (d, J = 8.4 Hz, 2H), 7.94 (d, J = 8.8 Hz, 2H), 7.66 (d,
J = 8.4 Hz, 2H), 7.28 (d, J = 8.0 Hz, 2H), 2.69 (q, J = 8.0 Hz 2H), 1.27
(t, J = 7.6 Hz, 3H); 13C NMR (CDCl3, 100 MHz) d 195.2, 148.9,
148.2, 143.9, 136.1, 128.9, 128.6, 127.7, 123.9, 123.5, 28.5, 15.3;
HRMS (ESI, m/z) for
C15H15N2O2S calcd 287.0854, found
287.0856. Anal. Calcd for C15H14N2O2S: C, 62.92; H, .4.93; N, 9.78.
Found: C, 62.91; H, 4.95; N, 9.60.
4.1.2.2. N-(4-Ethylphenyl)-3-methyl-4-nitrobenzamide 3h. White
solid; 80% yield; mp 137–139 °C; 1H NMR (CDCl3, 400 MHz) d 8.03
(br s, NH), 7.97 (d, J = 8.4 Hz, 1H), 7.82 (s, 1H), 7.75 (d. J = 8.4 Hz,
1H), 7.52 (d, J = 8.4 Hz, 2H), 7.29 (d, J = 8.0 Hz, 2H), 2.67–2.61 (m,
5H), 1.24 (t, J = 8.4 Hz, 3H); 13C NMR (CDCl3, 100 MHz) d 163.8,
150.8, 141.4, 138.9, 134.9, 134.1, 131.8, 128.5, 125.4, 125.0, 120.6,
28.3, 20.3, 15.6; HRMS (ESI, m/s) for C16H17N2O3 calcd 285.1239,
4.1.3.4. N-(3-Methoxyphenyl)-4-nitrothiobenzamide 4d. Yellow
solid; 64% yield; mp 138–140 °C; 1H NMR (CDCl3, 400 MHz) d
10.61 (br s, NH), 8.23 (d, J = 8.8 Hz, 2H), 7.96 (d, J = 8.8 Hz, 2H),
7.60 (s, 1H), 7.36–7.30 (m, 2H), 6.86–6.83 (m, 1H), 3.83 (s, 3H);
13C NMR (CDCl3, 100 MHz) d 195.3, 159.8, 148.8, 148.2, 146.2,
129.6, 128.2, 123.3, 115.8, 112.7, 109.2, 55.4; HRMS (ESI, m/z) for
C14H12N2O3SNa calcd 311.0466, found 311.0464. Anal. Calcd for

6204
W.-P. Hu et al. / Bioorg. Med. Chem. 18 (2010) 6197–6207
C14H12N2O3S: C, 58.32; H, .4.20; N, 9.72. Found: C, 58.25; H, 4.36;
N, 9.61.
sium ferricyanide (13.16 g, 4 equiv) in water (50 mL) at 90 °C. The
reaction mixture was heated for a further 1 h and then cooled in
ice. The resulting precipitate was filtered and washed with water,
then subjected to flash chromatography (CH2Cl2/hexane = 1:4) to
give the corresponding compounds 5.
4.1.3.5. N-(4-Methoxyphenyl)-4-nitrothiobenzamide 4e. Yellow
solid; 62% yield; mp 174–175 °C; 1H NMR (CDCl3, 400 MHz) d 9.98
(br s, NH), 8.31–8.28 (m, 2H), 8.18 (d, J = 8.8 Hz, 2H), 7.94 (s, 1H),
7.32–7.23 (m, 2H), 6.72–6.69 (m, 1H), 3.82 (s, 3H); 13C NMR (CDCl3,
100 MHz) d 163.8, 159.5, 149.0, 140.5, 139.2, 129.1, 128.7, 123.0,
112.8, 109.9, 106.3, 54.85; HRMS (EI, m/z) for C14H12N2O3S calcd
288.0569, found 288.0571. Anal. Calcd for C14H12N2O3S: C, 58.32;
H, .4.20; N, 9.72. Found: C, 58.29; H, 4.42; N, 9.47.
4.1.4.1. 2-(4-Nitrophenyl)benzothiazole 5a. White solid; 68%
yield; mp 228–230 °C; 1H NMR (CDCl3, 400 MHz) d 8.35 (dt,
J = 9.2 and 2.0 Hz, 2H), 8.27 (dt, J = 9.2 and 2.0 Hz, 2H), 8.13 (d,
J = 8.0 Hz, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.56 (dt, J = 8.0 and 1.2 Hz,
1H), 7.47 (dt, J = 8.0 and 1.2 Hz, 1H); 13C NMR (CDCl3, 100 MHz) d
164.8, 154.1, 149.0, 139.1, 135.4, 128.2, 126.9, 126.2, 124.3,
123.9, 121.8; HRMS (EI, m/z) for C13H8N2O2S calcd 256.0306, found
256.0308. Anal. Calcd for C13H8N2O2S: C, 60.93; H, 3.15; N, 10.93.
Found: C, 60.89; H, 3.34; N, 10.75.
4.1.3.6. N-(4-Trifluoromethylphenyl)-4-nitrothiobenzamide 4f.
Yellow solid; 71% yield; mp 174–175 °C; 1H NMR (CDCl3,
400 MHz) d 11.49 (br s, NH), 8.25 (d, J = 8.4 Hz, 2H), 8.07 (d,
J = 8.8 Hz, 2H), 7.99 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 8.0 Hz, 2H); 13C
NMR (CDCl3, 100 MHz) d 196.1, 148.5, 148.0, 142.4, 128.4, 125.8,
125.8, 123.5, 123.1, 122.3 (q, J = 140 Hz, CF3); HRMS (ESI, m/z) for
C14H10N2O2F3S calcd 326.0337, found 326.0335. Anal. calcd for
4.1.4.2. 6-Methyl-2-(4-nitrophenyl)benzothiazole 5b. White so-
lid; 65% yield; mp 148–150 °C; 1H NMR (CDCl3, 400 MHz) d 8.31 (s,
1H), 8.23 (s, 2H), 7.99 (d, J = 6.4 Hz, 1H), 7.72 (s, 1H), 7.36 (s, 1H),
2.52 (s, 3H); 13C NMR (CDCl3, 100 MHz) d 163.7, 152.2, 148.8,
139.3, 136.6, 135.6, 128.5, 128.0, 124.2, 123.3, 121.4, 21.6; HRMS
(EI, m/z) for C14H12N2O2S calcd 295.0517, found 295.0516. Anal.
Calcd for C14H10N2O2S: C, 62.21; H, 3.73; N, 10.36. Found: C,
62.37; H, 3.91; N, 10.31.
C14H10N2O2F3S: C, 51.53; H, 2.78; N, 8.59. Found: C, 51.90; H,
2.86; N, 8.63.
4.1.3.7. N-(Phenyl)-3-methyl-4-nitrothiobenzamide 4g. Yellow
solid; 72% yield; mp 118–120 °C; 1H NMR (CDCl3, 400 MHz) d
9.09 (br s, NH), 7.99 (d, J = 8.4 Hz, 1H), 7.78–7.71 (m, 4H), 7.46 (t,
J = 8.0 Hz, 2H), 7.33 (t, J = 7.6 Hz, 1H), 2.64 (s, 3H); 13C NMR (CDCl3,
100 MHz) d 195.7, 146.6, 138.5, 134.2, 131.2, 129.2, 127.4, 125.1,
4.1.4.3. 6-Ethyl-2-(4-nitrophenyl)benzothiazole 5c. White so-
lid; 72% yield; mp 151–152 °C; 1H NMR (CDCl3, 400 MHz) d 8.34
(dd, J = .8 and 2.0 Hz, 2H), 8.25 (dd, J = 4.8 and 2.0 Hz, 2H), 8.02 (d,
J = 8.4 Hz, 1H), 7.76 (d, J = 0.8 Hz, 1H), 7.40 (dd, J = 8.4 and 1.6 Hz,
1H), 2.28 (q, J = 7.6 Hz, 2H), 1.33 (t, J = 7.6 Hz, 3H); 13C NMR (CDCl3,
100 MHz) d 163.8, 152.4, 148.8, 143.0, 139.3, 135.7, 128.0, 127.6,
124.3, 123.5, 120.3, 29.0, 15.7; HRMS (ESI, m/z) for C15H12N2O2S
calcd 285.0698, found 285.0695. Anal. Calcd for C15H12N2O2S: C,
63.36; H, 4.25; N, 9.85. Found: C, 63.40; H, 4.55; N, 9.53.
124.8, 123.5, 20.5; HRMS (ESI, m/z) for
C14H13N2O2S calcd
273.0698, found 273.0700. Anal. Calcd for C14H13N2O2S: C, 61.75;
H, 4.44; N, 10.29. Found: C, 62.01; H, 4.39; N, 10.26.
4.1.3.8. N-(4-Ethylphenyl)-3-methyl-4-nitrothiobenzamide 4h.
Yellow solid; 70% yield; mp 160–162 °C; 1H NMR (CDCl3,
400 MHz) d 9.03 (br s, NH), 7.99 (d, J = 8.4 Hz, 1H), 7.78–7.65 (m,
3H), 7.28 (d, J = 8.4 Hz, 2H), 2.72–2.64 (m, 5H), 1.26 (t, J = 7.6 Hz,
3H); 13C NMR (CDCl3, 100 MHz) d 195.4, 150.1, 146.6, 143.8,
136.1, 134.2, 131.3, 128.5, 125.0, 124.81 123.5, 28.5, 20.5, 15.4;
4.1.4.4. 7-Methoxy-2-(4-nitrophenyl)benzothiazole 5d. White
solid; 25% yield; mp 228–230 °C; 1H NMR (CDCl3, 400 MHz), d
8.33 (d, J = 8.8 Hz, 2H), 8.25 (d, J = 8.8 Hz, 2H), 7.75–7.73 (m, 1H),
7.49 (t, J = 8.4 Hz, 1H), 6.89 (d, J = 8.4 Hz, 1H), 4.03 (s, 3H); 13C
NMR (CDCl3, 100 MHz) d 165.4, 155.6, 154.3, 148.9, 139.2, 128.2,
127.8, 124.3, 116.4, 105.9, 56.0; HRMS (ESI, m/z) for C14H10N2O3S-
Na calcd 309.0310, found 309.0308. Anal. Calcd for C14H10N2O3S: C,
58.73; H, 3.52; N, 9.78. Found: C, 58.94; H, 3.70; N, 9.65.
HRMS (ESI, m/z) for
C16H17N2O2S calcd 301.1011, found
301.1012. Anal. Calcd for C16H17N2O2S: C, 63.98; H, 5.37; N, 9.33.
Found: C, 63.99; H, 5.39; N, 9.30.
4.1.3.9. N-(4-Methoxyphenyl)-3-methyl-4-nitrothiobenzamide
4i. Yellow solid; 61% yield; mp 160–162 °C; 1H NMR (CDCl3,
400 MHz) d 10.81 (br s, NH), 7.96 (d, J = 8.1 Hz, 1H), 7.83 (dd,
J = 1.6 and 0.8 Hz, 1H), 7.77 (dd, J = 8.4 and 2.0 Hz, 1H), 7.69 (dt,
J = 8.8 and 3.2 Hz, 2H), 6.94 (dt, J = 8.8 and 3.2 Hz, 2H), 3.83 (s,
3H), 2.63 (s, 3H); 13C NMR (CDCl3, 100 MHz) d 195.0, 157.9,
149.5, 146.2, 133.3, 132.2, 131.7, 125.4, 124.3, 113.7, 55.2, 20.2;
HRMS (ESI, m/z) for C15H15N2O3S calcd 303.0803, found 303.0804.
Anal. Calcd for C15H15N2O3S: C, 59.59; H, 4.67; N, 9.27. Found: C,
59.95; H, 4.90; N, 9.07.
4.1.4.5. 6-Methoxy-2-(4-nitrophenyl)benzothiazole 5e. Yellow
solid; 63% yield; mp 214–216 °C; 1H NMR (CDCl3, 400 MHz) d
8.33 (dt, J = 9.2 and 2.0 Hz, 2H), 8.20 (dt, J = 9.2 and 2.0 Hz, 2H),
8.00 (d, J = 9.2 Hz, 1H), 7.38 (d, J = 2.4 Hz, 1H), 7.15 (dd, J = 9.2
and 2.4 Hz, 1H), 3.92(s, 3H); 13C NMR (CDCl3, 100 MHz) d 162.2,
158.5, 148.7, 148.6, 139.3, 137.0, 127.8, 124.5, 124.2, 116.6,
104.0, 55.8; HRMS (ESI, m/z) for C14H11N2O3S calcd 287.0490,
found 287.0492. Anal. calcd for C14H10N2O3S: C, 58.73; H, 3.52;
N, 9.78. Found: C, 58.68; H, 3.47; N, 9.75.
4.1.3.10. 3-Methyl-4-nitro-N-(4-trifluoromethylphenyl)thioben-
zamide 4j. Yellow solid; 74% yield; mp 170–172 °C; 1H NMR
(CDCl3, 400 MHz) d 8.04–7.98 (m, 3H), 7.81–7.68 (m, 4H), 2.65 (s,
3H); 13C NMR (CDCl3, 100 MHz) d 208.5, 196.6, 149.8, 146.4,
142.2, 133.5, 131.5, 125.8, 125.3, 124.4, 123.5, 122.3 (q,
J = 140 Hz, CF3), 29.7, 20.0; HRMS (ESI, m/z) for C15H11N2O2SF3
calcd 340.0493, found 340.0494. Anal. Calcd for C15H11N2O2SF3:
C, 52.94; H, 3.26; N, 8.23. Found: C, 52.68; H, 3.47; N, 8.27.
4.1.4.6. 2-(4-Nitrophenyl)-6-trifluoromethyl-benzothiazole 5f.
Yellow solid; 61% yield; mp 149–151 °C; 1H NMR (CDCl3,
400 MHz) d 8.35 (dt, J = 9.2 and 2.0 Hz, 2H), 8.27–8.23 (m, 3H),
8.19 (d, J = 8.4 Hz, 1H), 7.77 (dd, J = 8.4 and 2.0 Hz, 1H); 13C NMR
(CDCl3, 100 MHz) d 167.8, 155.8, 149.4, 138.3, 135.4, 128.4, 128.2,
124.3, 124.2, 123.9, 123.8, 119.5; HRMS (ESI, m/z) for C14H7N2O2SF3
calcd 324.0175, found 324.0178. Anal. Calcd for C14H7N2O2SF3: C,
51.85; H, 2.18; N, 8.64. Found: C, 51.86; H, 2.22; N, 8.69.
4.1.4. General procedure for the syntheses of 2-(4-nitrophenyl)-
benzothiazole (5a–5j)
A solution of substituted 4-nitrothiobenzamides (50 mmol) in
three drops of 95% EtOH and 30% aqueous sodium hydroxide solu-
tion (10.6 mL, 8 equiv) was added dropwise to a solution of potas-
4.1.4.7. 2-(3-Methyl-4-nitrophenyl)benzothiazole 5g. White so-
lid; 71% yield; mp 163–165 °C; 1H NMR (CDCl3, 400 MHz) d 8.13–
8.08 (m, 3H), 8.02 (dd, J = 8.0 and 2.0 Hz, 1H), 7.95 (dd, J = 8.0 and

W.-P. Hu et al. / Bioorg. Med. Chem. 18 (2010) 6197–6207
6205
1.2 Hz, 1H), 7.55 (dt, J = 8.0 and 1.2 Hz, 1H), 7.45 (dt, J = 8.0 and
1.2 Hz, 1H), 2.71 (s, 3H); 13C NMR (CDCl3, 100 MHz) d 165.1,
153.9, 150.1, 137.4, 135.4, 134.7, 131.4, 126.8, 126.0, 125.8,
125.5, 123.7, 121.8, 20.6; HRMS (ESI, m/z) for C14H11N2O2S calcd
271.0541, found 271.0541. Anal. calcd for C14H11N2O2S: C, 62.21;
H, 3.73; N, 10.36. Found: C, 62.07; H, 3.88; N, 10.26.
C14H12N2S: C, 69.97; H, 5.03; N, 11.66. Found: C, 69.84; H, 5.01;
N, 11.70.
4.1.5.3. 2-(4-Aminophenyl)-6-ethylbenzothiazole 6c. Yellow so-
lid; 92% yield; mp 154–156 °C; 1H NMR (CDCl3, 400 MHz) d 7.90–
7.86 (m, 3H), 7.65 (t, J = 0.4 Hz, 1H), 7.27 (dd, J = 7.6 and 2.0 Hz,
1H), 6.81 (dt, J = 4.8 and 2.0 Hz, 2H), 3.98 (br s, NH2), 2.76 (q,
J = 7.6 Hz, 2H), 1.29 (t, J = 7.6 Hz, 3H); 13C NMR (CDCl3, 100 MHz)
d 167.6, 152.5, 149.0, 141.0, 134.7, 129.0, 126.5, 124.1, 122.1,
114.7, 28.87, 15.8; HRMS (ESI, m/z) for C15H14N2OSNa calcd
277.0775, found 277.0776. Anal. calcd for C15H14N2S: C, 70.83; H,
5.55; N, 11.01. Found: C, 70.62; H, 5.29; N, 10.90.
4.1.4.8. 6-Ethyl-2-(3-methyl-4-nitrophenyl)benzothiazole 5h.
Yellow solid; 75% yield; mp 118–120 °C; 1H NMR (CDCl3,
400 MHz)
d 8.10 (d, J = 8.8 Hz, 1H), 8.08 (s, 1H), 8.02 (d,
J = 8.4 Hz, 1H), 8.01 (dd, J = 8.4 and 2.0 Hz, 1H), 7.75 (d, J = 2.0 Hz,
1H), 7.38 (dd, J = 8.4 and 2.0 Hz, 1H), 2.83 (q, J = 7.6 Hz, 2H), 2.71
(s, 3H), 1.33 (t, J = 7.6 Hz, 3H); 13C NMR (CDCl3, 100 MHz) d
164.1, 152.4, 149.9, 142.8, 137.7, 135.6 134.6, 131.3, 127.4, 125.6,
125.5, 123.3, 120.3, 29.0, 20.6, 15.7; HRMS (ESI, m/z) for
4.1.5.4. 2-(4-Aminophenyl)-7-methoxybenzothiazole 6d. Yellow
solid; 95% yield; mp 142–144 °C; 1H NMR (CDCl3, 400 MHz) d 7.90
(d, J = 8.4 Hz, 2H), 7.62 (d, J = 8.0 Hz, 1H), 7.39 (t, J = 8.0 Hz, 1H),
6.78 (d, J = 8.0 Hz, 1H), 6.73 (d, J = 8.4 Hz, 2H), 3.99 (s, 5H); 13C
NMR (CDCl3, 100 MHz) d 169.1, 15.9, 154.2, 149.2, 129.1, 126.9,
124.0, 123.0, 115.3, 114.8, 104.7, 55.9; HRMS (ESI, m/z) for
C16H15N2O2S calcd 299.0854, found 299.0856. Anal. Calcd for
C16H15N2O2S: C, 64.41; H, 4.73; N, 9.39. Found: C, 64.20; H, 4.80;
N, 9.14.
4.1.4.9. 6-Methoxy-2-(3-methyl-4-nitrophenyl)benzothiazole 5i.
Yellow solid; 64% yield; mp 195–197 °C; 1H NMR (CDCl3,
400 MHz) d 8.07 (d, J = 8.8 Hz, 1H), 8.02 (d, J = 0.8 Hz, 1H), 7.98 (d,
J = 9.2 Hz, 1H), 7.95 (dd, J = 8.4 and 2.0 Hz, 1H), 7.36 (d, J = 2.4 Hz,
1H), 7.14 (dd, J = 9.2 and 2.4 Hz, 1H), 3.90 (s, 3H), 2.71 (s, 3H); 13C
NMR (CDCl3, 100 MHz) d 162.3, 158.4, 149.6, 148.6, 137.6, 136.8,
134.6, 130.9, 125.5, 125.3, 124.3, 116.4, 103.9, 55.8, 20.6; HRMS
(ESI, m/z) for C15H14N2O3S calcd 301.0647, found 301.0648. Anal.
calcd for C15H14N2O3S: C, 59.93; H, 4.03; N, 9.33. Found: C, 60.37
H, 4.13; N, 9.36.
C
C
14H13N2OS calcd 257.0749, found 257.0748. Anal. Calcd for
14H12N2OS: C, 65.60; H, 4.72; N, 10.93. Found: C, 65.66; H, 4.86;
N, 10.92.
4.1.5.5. 2-(4-Aminophenyl)-6-methoxybenzothiazole 6e. Yellow
solid; 96% yield; mp 174–176 °C; 1H NMR (CDCl3, 400 MHz) d 7.88
(s, 1H), 7.84 (dt, J = 4.8 and 2.8 Hz, 2H), 7.32 (d, J = 2.4 Hz, 1H),
7.04 (dd, J = 4.8 and 2.4 Hz, 1H), 7.73 (dt, J = 4.8 and 2.8 Hz, 2H),
3.98 (br s, NH2), 3.87 (s, 3H); 13C NMR (CDCl3, 100 MHz) d 166.1,
157.3, 148.8, 148.8, 135.9, 135.9, 128.8, 124.2, 123.0, 115.1, 114.8,
104.3, 55.8; HRMS (ESI, m/z) for C14H13N2OS calcd 257.0749, found
257.0748. Anal. Calcd for C14H12N2OS: C, 65.60; H, 4.72; N, 10.93.
Found: C, 65.35; H, 4.90; N, 10.67.
4.1.4.10. 2-(3-Methyl-4-nitrophenyl)-6-trifluoromethylbenzo-
thiazole 5j. Yellow solid; 30% yield; mp 99–101 °C; 1H NMR
(CDCl3, 400 MHz) d 8.21–8.15 (m, 2H), 8.08–8.06 (m, 2H), 8.00
(dd, J = 8.8 and 1.6 Hz, 1H), 7.76 (dd, J = 8.8 and 1.6 Hz, 1H), 2.69
(s, 3H); 13C NMR (CDCl3, 100 MHz) d 168.0, 155.7, 150.4, 136.6,
135.2 134.6, 131.60, 127.9 (q, J = 32.6 Hz, 1C), 125.9 (d,
J = 43.2 Hz, 1C), 125.49, 124.08, 123.8, 122.2, 119.4 (q, J = 4.5 Hz,
1C), 20.4; HRMS (ESI, m/z) for C15H10N2O2F3S calcd 339.0415, found
339.0414. Anal. Calcd for C15H9N2O2F3S: C, 53.25; H, 2.68; N, 8.28.
Found: C, 53.21; H, 2.73; N, 8.14.
4.1.5.6. 2-(4-Aminophenyl)-6-trifluoromethylbenzothiazole 6f.
Yellow solid; 91% yield; mp 181–183 °C; 1H NMR (CDCl3,
400 MHz) d 8.11 (s, 1H), 8.03 (d, J = 8.4 Hz, 1H), 7.90 (dt, J = 4.8
and 2.4 Hz, 2H), 7.67 (dd, J = 6.8 and 1.6 Hz, 1H), 6.73 (dt, J = 4.8
and 2.4 Hz, 2H), 4.08 (br s, NH2); 13C NMR (CDCl3, 100 MHz) d
169.9, 158.1, 149.9, 132.3, 129.4, 123.1, 123.0, 122.5, 119.0,
119.0, 114.7; HRMS (ESI, m/z) for C14H9F3N2S calcd 294.0439,
found 294.0438. Anal. Calcd for C14H9F3N2S: C, 57.14; H, 3.08; N,
9.52. Found: C, 57.20; H, 3.18; N, 9.49.
4.1.5. General procedure for the syntheses of 2-(4-aminophenyl)-
benzothiazole (6a–6j)
To a solution of 2-(4-nitrophenyl)benzothiazole (1 g, 3.9 mmol)
in CH2Cl2 (30 mL) was added 10%Pd/C (0.1 g) under hydrogen at
room temperature for 4 h. The resulting solution was concentrated
and subjected to flash chromatography (CH2Cl2) to give the corre-
sponding compounds 6.
4.1.5.7. 2-(4-Amino-3-methylphenyl)benzothiazole 6g. Yellow
solid; 94% yield; mp 147–149 °C; 1H NMR (CDCl3, 400 MHz) d
7.99 (d, J = 8.0 Hz, 1H), 7.85–7.83 (m, 2H), 7.75 (dd, J = 8.0 and
2.0 Hz, 1H), 7.44 (td, J = 8.0 and 1.2 Hz, 1H), 7.31 (td, J = 8.0 and
1.2 Hz, 1H), 6.71 (d, J = 8.0 Hz, 1H), 3.94 (br s, NH2), 2.23 (s, 3H);
13C NMR (CDCl3, 100 MHz) d 168.7, 154.2, 147.5, 134.5, 129.7,
126.9, 126.0, 124.3, 123.8, 122.4, 122.1, 121.3, 114.5, 17.1; HRMS
(ESI, m/z) for C14H12N2OSNa calcd 263.0619, found 263.0618. Anal.
Calcd for C14H12N2OS: C, 69.97; H, 5.03; N, 11.66. Found: C, 69.84;
H, 5.01; N, 11.70.
4.1.5.1. 2-(4-Aminophenyl)benzothiazole 6a. Yellow solid; 94%
yield; mp 130–132 °C; 1H NMR (CDCl3, 400 MHz) d 7.99 (m, 1H),
7.89 (m, 2H), 7.84 (m, 1H), 7.44 (m, 1H), 7.32 (m, 1H), 6.72 (dt,
J = 4.2 and 2.0 Hz, 2H), 4.00 (br s, NH2); 13C NMR (CDCl3,
100 MHz) d 168.5, 154.2, 149.2, 134.6, 129.1, 126.0, 124.4, 123.9,
122.5, 121.4, 114.8; HRMS (ESI, m/z) for
C13H10N2S calcd
4.1.5.8. 2-(4-Amino-3-methylphenyl)-6-ethylbenzothiazole 6h.
Yellow solid; 93% yield; mp 171–173 °C; 1H NMR (CDCl3,
400 MHz) d 7.90 (d, J = 8.4 Hz, 1H), 7.80 (d, J = 1.6 Hz, 1H), 7.32
(dd, J = 6.0 and 2.4 Hz, 1H), 7.65 (d, J = 0.8 Hz, 1H), 7.27 (dd,
J = 6.8 and 1.6 Hz, 1H), 6.70 (d, J = 8.4 Hz, 1H), 3.92 (br s, NH2),
2.76 (q, J = 8.0 Hz, 2H), 2.22 (s, 3H), 1.29 (t, J = 7.6 Hz, 3H); 13C
NMR (CDCl3, 100 MHz) d 167.8, 152.5, 147.4, 140.9, 140.9, 134.7,
129.6, 126.8, 126.5, 124.1, 122.1, 122.0, 120.0, 114.6, 28.9, 17.2,
15.8; HRMS (ESI, m/z) for C16H17N2OS calcd 269.1112, found
269.1110. Anal. Calcd for C16H16N2S: C, 71.61; H, 6.01; N, 10.44.
Found: C, 72.01; H, 6.36; N, 10.52.
226.0565, found 226.0567. Anal. Calcd for C13H10N2S: C, 69.00; H,
4.45; N, 12.38. Found: C, 69.01; H, 4.69; N, 12.29.
4.1.5.2. 2-(4-Aminophenyl)-6-methylbenzothiazole 6b. Yellow
solid; 95% yield; mp 181–183 °C; 1H NMR (CDCl3, 400 MHz) d
7.88–7.85 (m, 3H), 7.63 (s, 1H), 7.25 (dd, J = 6.8 and 1.6 Hz, 1H),
6.71 (dd, J = 4.4 and 2.4 Hz, 1H), 7.32 (m, 1H), 6.72 (dt, J = 4.2 and
2.0 Hz, 2H), 4.00 (br s, NH2), 2.46 (s, 3H); 13C NMR (CDCl3,
100 MHz) d 167.5, 152.3, 149.0, 134.7, 134.5, 129.5, 129.0, 127.6,
126.3, 124.1, 122.0, 121.2, 114.8, 21.5; HRMS (ESI, m/z) for
C14H13N2S calcd 241.0799, found 241.0798. Anal. Calcd for

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W.-P. Hu et al. / Bioorg. Med. Chem. 18 (2010) 6197–6207
4.1.5.9. 2-(4-Amino-3-methylphenyl)-6-methoxybenzothiazole
6i. Yellow solid; 95% yield; mp 151–153 °C; 1H NMR (CDCl3,
400 MHz) d 7.87 (d, J = 8.8 Hz, 1H), 7.77 (d, J = 2.0 Hz, 1H), 7.69
(dd, J = 6.0 and 2.4 Hz, 1H), 7.30 (d, J = 2.8 Hz, 1H), 7.03 (dd,
J = 6.0 and 2.4 Hz, 1H), 6.69 (d, J = 8.4 Hz, 1H), 3.86 (s, 5H), 2.22
(s, 3H); 13C NMR (CDCl3, 100 MHz) d 166.4, 157.2, 148.7, 147.2,
135.8, 129.4, 126.6, 124.0, 122.9, 122.2, 115.0, 114.6, 104.3, 55.8,
17.2; HRMS (ESI, m/z) for C15H15N2OS calcd 271.0905, found
271.0906. Anal. Calcd for C15H14N2OS: C, 66.64; H, 5.22; N, 10.36.
Found: C, 66.68; H, 5.38; N, 10.48.
4.5. Sub-G1 region analysis
BCC cells were treated with compounds at 4 l
M and 1 J/cm2
UVA. Twenty-four hours after irradiation, cells were harvested by
trypsinization and centrifugation. Cell pellets were resuspended
in 50% cold ethanol and fixed at ꢀ20 °C. After fixation, cells were
washed once with cold PBS and incubated in 0.5 mL of PBS contain-
ing 100
centrifugation at 400g for 5 min, and 250
50 g/mL propidium iodide (PI) was added to the pellet. Thirty
lg/mL RNase A for 20 min at 37 °C. Cells were harvested by
lL of PBS containing
l
minutes later, 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 Docu-
ment Interface software (WinMDI). Cells with DNA content less
than that in untreated cells in G0/G1 were considered apoptotic.
4.1.5.10. 2-(4-Amino-3-methylphenyl)-6-trifluoromethylbenzo-
thiazole 6j. Yellow solid; 91% yield; mp 152–154 °C; 1H NMR
(CDCl3, 400 MHz) d 8.12 (s, 1H), 8.04 (d, J = 8.8 Hz, 1H), 7.83 (d,
J = 1.2 Hz, 1H), 7.77 (dd, J = 6.0 and 2.4 Hz, 1H), 7.66 (dd, J = 6.8
and 1.6 Hz, 1H), 6.72 (d, J = 8 Hz, 1H), 4.20 (br s, NH2), 2.24 (s,
3H); 13C NMR (CDCl3, 100 MHz) d 171.1, 156.3, 148.3, 134.6,
130.0, 127.3, 126.4 (q, J = 65.5 Hz, 1C), 123.0 (q, J = 5.6 Hz, 1C),
122.4, 122.1, 118.9 (q, J = 5.6 Hz, 1C), 114.5, 17.2; HRMS (ESI, m/
z) for C15H12F3N2S calcd 308.0595, found 308.0597. Anal. Calcd
for C15H11F3N2S: C, 58.43; H, 3.60; N, 9.09. Found: C, 58.50; H,
3.60; N, 9.09.
4.6. Caspase-3 colorimetric assay
Twenty-four hours after irradiation, cells were collected by cen-
trifugation, washed once with PBS, and cell pellets were counted
and resuspended in 25
l
L/1 ꢁ 106 cells of cold lysis buffer and
homogenized. Homogenates were centrifuged at 12,000 rpm for
10 min at 4 °C, supernatants were used for measuring caspase-3
activity using an ELISA-based assay, according to the manufac-
turer’s instructions (R&D Systems, Minneapolis, MN). The results
were presented as mean SD.
4.2. Cell culture
Fibroblasts were obtained from adult foreskin specimens as
previously described28 and human basal cell carcinoma (BCC), pur-
chased from American Type Culture Collection (Manassas, VA), was
maintained in RPMI1640 medium supplemented with 10% FCS and
4.7. Annexin V and PI binding assay
To assess the simultaneous observation of early phase of apop-
totic and necrotic features, BCC cells were treated with various
100 U/mL penicillin G, and 100 lg/mL streptomycin sulfate (Gibco,
BRL). BCC cells were passaged at confluence after treatment with
5 mM EDTA (Gibco, BRL) and incubated at 37 °C in a humidified
atmosphere containing 5% CO2.
concentrations (0–4 lM) of 6f for 4 h before irradiation. Twenty-
four hours after irradiation, cells were harvested by trypsinization
and centrifugation and measured by cytometry by adding annexin
V-FITC to 106 cells per sample according to the manufacturer’s
specifications (Bender MedSystems, Vienna, Austria). Simulta-
neously, the cells were stained with PI. Flow cytometry data were
analyzed by the WinMDI software.
4.3. UVA irradiation
The method for UVA irradiation was described in our previous
study.29 For UVA irradiation, a specific UVA lamp emitting a peak
wavelength of 365 nm with intensity of 0.75 mW/cm2 (Vilber
Lourmat, Moune La Vallee, Cedex, France) was used. The cultured
4.8. Morphology observation
BCC or fibroblasts (5 ꢁ 105 cells/well) seeded on six well plate.
cells were pretreated with different agents at 4
lM or pretreated
Cells were treated with 4 l
M 6f for 4 h followed by 1 J/cm2 UVA
with various concentrations (0–4 M) of 6 for 4 h before UVA irra-
l
irradiation. Twenty four hours after exposure, take photos by using
microscope at 200ꢁ phase.
diation. The cultured cells were rinsed with phosphate-buffered
saline (PBS) and then irradiated with 1 J/cm2 UVA in PBS to avoid
the formation of medium-derived toxic photoproducts induced
by UV exposure. The doses of irradiation were measured using a
UVX radiometer (UVP, San Gabriel, CA, USA). Immediately after
phototreatment, PBS was removed and media were added to the
cells. All the following experiments were performed three times
in triplicate.
4.9. Determination of intracellular ROS level
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 rap-
idly oxidized to the highly fluorescent DCF by intracellular H2O2 or
nitric oxide. In addition, catalase (Sigma), an effective H2O2 scaven-
ger, was also used in this study. Cells were pretreated with catalase
4.4. Cell viability
Cell viability was assessed by the MTT assay, a mitochondrial
function assay based on the ability of viable cells to reduce the re-
dox indicator MTT to insoluble formazan crystals by mitochondrial
dehydrogenase. Briefly, cells were seeded in a 96-well plate at the
cell density of 2500 cells/well. After an overnight incubation, the
(800 U/mL) before 6f (0–4
lM) treatment. After indicated irradia-
tion, DCFH-DA (10 M) was immediately added into cultured cells
l
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.
cells were treated with compounds at 4
24 h. The medium was then discarded and replaced with 10
MTT dye. Plates were incubated at 37 °C for 2 h. The resulting for-
mazan crystals were solubilized in 100 L DMSO, and the optical
lM and incubated for
l
L of
4.10. Assessment of mitochondrial membrane potential (Dwmt)
l
density was read at 540 nm with a microplate reader (MRX-II, Dy-
nex technology, Chantilly, VA).
BCC cells were cultured in 35-mm dishes and allowed to reach
exponential growth for 24 h before treatment. Cells were pre-

W.-P. Hu et al. / Bioorg. Med. Chem. 18 (2010) 6197–6207
6207
treated with 0, 2, and 4
l
M 6f for 4 h before 1 J/cm2 UVA irradia-
scanning of X-ray films and analyzed by Eagle Eye Image System
(Stratagene, La Jolla, CA).
tion. The medium was removed and the adherent cells trypsinized.
The cells were pelleted by centrifugation at 400g for 5 min and
stained in a 100 nM/mL DiOC6 dye (Molecular Probes, Eugene,
OR) for 30 min at room temperature and washed with PBS twice
and resuspended in PBS. The samples were analyzed immediately
for fluorescence (FL-1 detector, filter 530/30 nm band pass) on a
FACScan flow cytometer (Elite ESP, Beckman Coulter, Brea, CA).
Histograms were analyzed using Windows Multiple Document
interface software (WinMDI).
4.13. Statistical analysis
The results were expressed as means SD and analyzed by
using the statistical analysis system (SPSS, SPSS Inc., Chicago, IL).
Differences among groups were analyzed by Student’s t-test. P val-
ues <0.05 were considered as significant for all statistical tests.
Acknowledgments
4.11. ATP content bioluminescence assay
We thank the National Science Council of the Republic of China,
KMU Center of Excellence for Environmental Medicine (KMU-EM-
97-2.1c), and Kaohsiung Medical University Research Foundation
(KMU-M099014) for financial support.
The amount of intracellular ATP was determined by biolumines-
cent assay based on the measurement of the light output of the
luciferin–luciferase reaction. After treatment with various concen-
trations of 6f-UVA, total cell extracts from cultured BCC cells were
obtained immediately by lysing solution. After centrifugation to re-
move cell debris, we collected supernatants for ATP measurement.
The total amount of intracellular ATP was determined according to
the protocol provided with the ATPLite assay kit (Perkin–Elmer,
Boston, MA).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.04.082.
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the blocking test, 25 lM oligomycin (Sigma, a mitochondria-spe-
cific F0F1 ATP synthase inhibitor) or 1 ꢁ 10ꢀ4 M ATP (Sigma) was
added to the media 30 min before treated with 6f-UVA, respec-
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amounts of specific proteins were quantified by densitometry