Synthesis, DNA interaction and genotoxic evaluation of a photoactive benzothiadiazole with potential application in photovoltaic paint

Article abstract of DOI:10.21577/0103-5053.20170241

A new fluorophore containing 2,1,3-benzothiadiazole unit with alkylated tetrazole terminal groups as electron donors connected through a π-conjugated system of the type (D-π-A-π-D) was synthesized using the Sonogashira coupling reaction with good yields.

Full text of DOI:10.21577/0103-5053.20170241

http://dx.doi.org/10.21577/0103-5053.20170241
J. Braz. Chem. Soc., Vol. 29, No. 7, 1388-1399, 2018
Printed in Brazil - ©2018 Sociedade Brasileira de Química
Article
Synthesis, DNA Interaction and Genotoxic Evaluation of a Photoactive
Benzothiadiazole with Potential Application in Photovoltaic Paint
Adriani P. Damiani,a Paula Rohr,a José L. Westrup,b Rodrigo C. Duarte,c
Luiza M. Longaretti,a Bruna M. da Rocha,a Giulia Strapazzon,a Lara C. Venturini,a
Emanuel Souza,a Claus T. Pich,d Tiago E. A. Frizon,b Vanessa M. de Andrade,a
Fabiano S. Rodembusch*,c and Alexandre G. Dal-Bó*,b
aLaboratório de Biologia Celular e Molecular, Programa de Pós-Graduação em Ciências da Saúde,
Unidade Acadêmica de Ciências da Saúde, Universidade do Extremo Sul Catarinense,
Av. Universitária, 1105, 88806-000 Criciúma-SC, Brazil
bPrograma de Pós-Graduação em Ciência e Engenharia de Materiais, Universidade do Extremo Sul Catarinense,
Av. Universitária, 1105, 88806-000 Criciúma-SC, Brazil
cGrupo de Pesquisa em Fotoquímica Orgânica Aplicada, Instituto de Química,
Universidade Federal do Rio Grande do Sul,
Avenida Bento Gonçalves, 9500, 91501-970 Porto Alegre-RS, Brazil
dCentro de Biologia Molecular Estrutural (CEBIME), Universidade Federal de Santa Catarina (UFSC),
Campus Trindade, 88040-900 Florianópolis-SC, Brazil
A new fluorophore containing 2,1,3-benzothiadiazole unit with alkylated tetrazole terminal
groups as electron donors connected through a π-conjugated system of the type (D-π-A-π-D) was
synthesized using the Sonogashira coupling reaction with good yields. The genotoxicity of the
two intermediate compounds and the final product was evaluated in vitro in consideration of the
potential application of the new compound in the chemical production of photovoltaic paints. The
new fluorophore did not show significant biological effects in the cell viability test and the comet
assay when compared with the intermediate compounds that were obtained synthetically despite
the ability to interact with deoxyribonucleic acid (DNA) as demonstrated in circular dichroism
assays. Thus, the compound does not show toxic or genotoxic activity, and therefore, it can be
applied as a pigment in photovoltaic paint.
Keywords: cell viability, genotoxicity, mutagenicity, photovoltaic paint, circular dichroism
Introduction
acceptor 2,1,3-benzothiadiazole as the central unit bound to
electron donors22-29 are of great interest for the development
of photovoltaic devices because of their potential use in the
production of flexible polymeric films30 that can be processed
on a large scale. In addition, these π-conjugated compounds
exhibit good intramolecular charge transfer, thereby
reducing the band gap,3,18,31 which is a requirement for the
application of these derivatives as pigments in photovoltaic
paints.32 However, there is a lack of information regarding
their interaction with deoxyribonucleic acid (DNA) and
the in vitro toxicity and genotoxicity associated with the
composition and chemical structures of intermediates and
final compounds containing the 2,1,3-benzothiadiazole core.
These have biological applications in fungicidal formulations
as chemical activators of plant resistance. Since paints are
composed of a mixture of several components, they can
Interest in the application of organic compounds in
optoelectronic devices is currently increasing.1 These
compounds have been extensively investigated since they
could lead to smaller devices that are more flexible and of
lower cost. Examples of these applications include organic
photovoltaic cells (OPVs),1-8 organic light-emitting diodes
(OLEDs),9-12 organic field effect transistors (OFETs),3
organic thin film transistors (OTFTs)13-18 and organic solar
cells.19,20 In this sense, different polymers, small molecules,
oligomers and hybrid compounds have been investigated
for optoelectronic applications.21
In this context, compounds that contain the electron
*e-mail: adalbo@unesc.net; fabiano.rodembusch@ufrgs.br

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Damiani et al.
1389
generate reactive oxygen species (ROS) which can damage
biomolecules and increase the health risks to exposed
operators.33-37 The interaction with and damage to DNA
can alter cellular metabolism and genomic stability.35 The
evaluation of DNA interactions can be performed by several
methods, and circular dichroism (CD) is a well-established
method for this purpose.36 CD can detect alterations in the
optical activity of chiral molecules by their interaction
with circularly polarized electromagnetic rays. The B-form
conformation of calf thymus-DNA (CT-DNA) produces
two CD bands in the UV region, a positive band at 278 nm
due to base stacking and a negative band at 246 nm due to
polynucleotide helicity. The genetic damage that is caused by
exposure to compounds with different chemical, physical and
biological characteristics can be evaluated using genotoxicity
biomarkers, which play an important role in the prediction
of health risks,37,38 as is used in the comet assay.39 This
test allows the direct measurement of single- and double
stranded DNA breaks, alkali-labile sites, crosslinking of
DNA and incomplete excision repair sites.40 In addition,
the cell viability test is also an important tool for studying
genetic damage and for detecting non-viable cells that have
damaged membranes that affect cellular adhesion or lead
to cell death.41
ISR-2200 Integrating SphereAttachment in a spectral range of
250-600 nm. The baseline was obtained using BaSO4 (Wako
Pure Chemical Industries, Ltd.). Steady state fluorescence
spectra was performed on a Shimadzu spectrofluorometer
model RF-5301PC with a solid state holder. The experiments
were performed at room temperature.
5-(4-Bromophenyl)tetrazole (1)
Yellow-colored crystals; mp 259-260 °C;43,44 FTIR
(Fourier transform infrared spectroscopy) (KBr) nmax / cm-1
3444, 3086, 3061, 3000, 1900, 1602; ESI anal. calcd. for
C7H5BrN4: m/z 226.9, found: 246.9 [M + Na]+.
5-(4-Bromophenyl)-2-dodecanetetrazole (2)
White solid; mp 54-55 °C; FTIR (KBr) nmax / cm-1 2954,
1
2918, 2848, 1602; H NMR (400 MHz, CDCl3) d 8.02
(d, 2H, J 8.60 Hz), 7.62 (d, 2H, J 8.21 Hz), 4.62 (t, 2H,
J 7.03 Hz), 2.10-1.95 (m, 2H), 1.45-1.30 (m, 2H), 1.30-1.15
13
(m, 16H), 0.87 (t, 3H, J 6.64 Hz); C NMR (400 MHz,
CDCl3) d 156.8, 124.8, 120.9, 119.2, 117.2, 46.0, 24.6,
22.3, 22.0, 21.5, 19.0, 15.3, 6.8.
5-[4-(2-Methyl-3-butyne-2-ol-phenyl)]-2-dodecanetetrazole
(3)
Here, we report the DNA interaction and in vitro
toxicity and genotoxicity of a photoactive compound and
respective intermediates that contain alkylated tetrazole
rings as electron donor groups bound by a π-conjugated
system to a 2,1,3-benzothiadiazole group as an electron
donor unit. These compounds have potential applications
in photovoltaic paints, devices and electronic systems.
mp 64-65 °C; FTIR (KBr) nmax / cm-1 3313, 3055, 2958,
1
2920, 2850; H NMR (400 MHz, CDCl3) d 8.09 (d, 2H,
J 8.20 Hz), 7.52 (d, 2H, J 8.20 Hz), 4.64 (t, 2H, J 7.03 and
7.32 Hz), 2.55 (s, 1H), 2.06 (q, 2H, J 7.03 Hz), 1.65 (s,
6H), 1.45-1.32 (m, 2H), 1.32-1.22 (m, 16H), 0.89 (t, 3H,
J 6.59 Hz); 13C NMR (400 MHz, CDCl3) d 164.9, 164.4,
132.1, 127.1, 126.6, 124.6, 95.5, 81.6, 65.5, 53.3, 31.9,
31.4, 29.5, 29.3, 28.8, 26.3, 22.6, 14.1.
Experimental
1-Dodecanetetrazole-4-ethinylbenzene (4)
Materials and methods
mp 56-57 °C; FTIR (KBr) nmax / cm-1 3286, 2954, 2916,
2846; 1H NMR (400 MHz, CDCl3) d 8.10 (d, 2H, J 8.22 Hz),
7.59 (d, 2H, J 8.22 Hz), 4.63 (t, 2H, J 7.04 Hz), 3.18 (s,
1H), 2.04 (q, 2H, J 7.04 Hz), 1.40-1.30 (m, 2H), 1.30-1.17
All reagents and solvents were purchased from commercial
suppliers and were used without further purification. The
solvents were dried and distilled before use based on
procedures available in the literature.42 The melting point was
measured using aThermolyne apparatus and was uncorrected.
The vibrational spectra of the intermediates and the final
compound in KBr pellets were obtained using a Shimadzu
IR Prestige 21 with a resolution of 4 cm-1 between 400 and
13
(m, 16H), 0.86 (t, 3H, J 6.46 Hz); C NMR (400 MHz,
CDCl3) d 164.3, 132.5, 127.7, 126.6, 123.9, 83.1, 78.8, 53.2,
31.8, 29.5, 29.3, 28.8, 26.3, 22.6, 14.1; ESI anal. calcd. for
C21H30N4: m/z 338.2, found: 361.3 [M + Na]+.
2,1,3-Benzothiadiazole (5)
1
4000 cm-1. The H and 13C nuclear magnetic resonance
mp 43-44 °C;45 FTIR (KBr) nmax / cm-1 3088, 3053,
(NMR) spectra were obtained in CDCl3 at 400 and 100 MHz,
respectively. The chemical shifts (d) are reported in parts per
million (ppm) relative to tetramethylsilane(TMS) (0.00 ppm),
and the coupling constants (J) are reported in hertz (Hz). The
photostability was evaluated by UV-Vis absorption spectra
in film on a Shimadzu UV-2450 spectrophotometer using an
1613; 1H NMR (400 MHz, CDCl3) d 7.97 (dd, 2H, J 3.13
13
and 6.64 Hz), 7.56 (dd, 2H, J 3.13, 7.03 Hz); C NMR
(400 MHz, CDCl3) d 154.7, 129.2, 121.5.
4,7-Dibromo-2,1,3-benzothiadiazole (6)
mp 187-188 °C;45 FTIR (KBr) nmax / cm-1 3078, 3045,

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1634; 1H NMR (400 MHz, CDCl3) d 7.71 (s, 2H); 13C NMR
(400 MHz, CDCl3) d 152.9, 132.3, 113.8.
prepared from a stock solution containing 20 mg of each
compound to be evaluated when diluted in 1 mL of DMSO.
Three different amounts of stock solution were used: 100,
50 and 25 µL, containing 2, 1 and 0.5 mg, respectively, of
each compound.
4,7-bis(1-Dodecanetetrazole-4-ethynylbenzene)-
2,1,3-benzothiadiazole (7)
o
Yellow solid; yield 95%; mp 216-218 C; FTIR
(KBr) nmax / cm-1 3037, 2953, 2920, 2848, 2204; 1H NMR
(400 MHz, CDCl3) d 8.21 (d, 4H, J 8.20 Hz), 7.85 (s, 2H),
7.82 (d, 4H, J 8.20 Hz), 4.68 (t, 4H, J 7.03 Hz), 2.25-2.00
(m, 4H), 1.55-1.35 (m, 4H), 1.35-1.15 (m, 32H), 0.86 (t, 6H,
J 6.44 Hz); 13C NMR (400 MHz, CDCl3) d 164.3, 154.3,
132.6, 132.4, 127.9, 126.7, 124.2, 117.1, 97.0, 86.8, 53.3,
46.0, 31.8, 29.5, 29.4, 28.8, 22.6, 14.1, 8.6.
In vitro tests
In this experiment 4 mL of peripheral blood was
collected from 5 healthy volunteer subjects using
disposable sterile materials to minimize the risk of infection
and disease transmission. Blood samples were collected
in Falcon tubes (1 mL per tube) containing heparin and
were divided into four groups according to the exposure
concentration to be evaluated. The blood was stored in a
CO2 incubator at 37 ºC and samples were collected after
2, 6, 12 and 24 h of exposure to the compounds for the
cell viability test and comet assay. All procedures were
approved by the Ethics Committee in Research of the
Universidade do Extremo Sul Catarinense under report
No. 365 397.
Photostability
The photostability of compound 7 was studied in
film, which were prepared by spin coating under vacuum
using a Glove Box MBRAUN, model MB 200B with
glass plates as substrates. The compound 7 was dissolved
in chloroform and deposited onto glass substrate via
the spin-coating method at 100 rpm for 20 s. The films
were analyzed in UV radiation chamber, equipped with a
cooling device, six Philips bulbs (model Actinic BL) were
used, which emitted UVA radiation at a wavelength of
330 nm, with a radiation intensity of 2.01 mW cm-2. The
radiation measurements were recorded in the chambers
with a digital UV measurement device manufactured by
Instrutherm (model MRUR-202). The chamber with six
bulbs reproduced artificially an 824% incidence of UV-A.
Cell viability (trypan blue exclusion test)
In this experiment 5 µL of cell suspension and 5 µL
of trypan blue solution were mixed in a microtube. After
mixing, the suspension was transferred to a Neubauer
micro-camera and enclosed with a cover slip. A total of
100 cells were evaluated using an optical microscope at
a magnification of 400× to determine the percentage of
viable cells.47
Circular dichroism
Comet assay
The experiments were performed as described in
the literature46 with small modifications using a JASCO
510 instrument. One sample of CT-DNA in 10 mM
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(HEPES) buffer was titrated with the complex using
concentrations ranging from 19.80 to 181.82 mM (molar
ratios of 0.25 to 2.50) and 0 (negative control). The
screenings were recorded from 240 to 400 nm at 37 ºC
immediately after the addition of the complexes. The
results were plotted and graphics presenting the regions
of major interest were produced. Spectra containing only
the complexes and DNA with increasing concentrations
of solvent (dimethyl sulfoxide, DMSO) were collected as
control reactions.
The comet assays were performed under alkaline
conditions as previously described by Tice et al.48
Peripheral blood samples were collected in heparinised
tubes and were subsequently maintained on ice. Aliquots
(5 µL) of the blood cells were embedded in low melting
point agarose (0.75%, m/v; 95 µL). This mixture was
then placed on a microscope slide that was pre-coated
with normal melting point agarose (1.5%, m/v) and
furnished with a cover slip (two slides per sample). The
slides were briefly placed on ice to enable the agarose to
solidify and the cover slips to be removed carefully. The
base slide was then immersed in freshly prepared lysis
solution (2.5 M NaCl, 100 mM ethylenediaminetetraacetic
acid-EDTA, 10 mM Tris, pH 10.0-10.5). In the next step,
the slides were immersed for 20 min in freshly prepared
alkaline buffer (300 mM NaOH, 1 mM EDTA, pH > 13)
for electrophoresis analysis (15 min per 300 mA, 25 V,
Biological assays
For the biological assays, different concentrations were

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Damiani et al.
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0.7 V cm-1). These steps were performed under minimal
indirect light. Following electrophoresis the slides were
neutralized with 400 mMTris (pH 7.5) and stained with silver
nitrate. To calculate the damage index (DI), cells were visually
separated into five classes according to tail size (0 = no tails to
4 = maximum-length tails). Accordingly, a single DNA
damage score was obtained for each sample and subsequently
for each group studied. The DI for the group ranged from 0
(completely undamaged = 100 cells × 0) to 400 (maximum
damage = 100 cells × 4). The damage frequency (DF in
percentage) was calculated for each sample based on the
number of cells with tails compared to the number without
tails. Visual scoring in the comet assay is considered a
reliable method of evaluation and usually results in high
correlation values with computer-based image analysis
methods.49 All slides were coded and analyzed blind.
obtained for the damage index and damage frequency in the
comet assay and the percentage of viable cells assessed in
the cell viability tests was conducted by one-way analysis
of variance (one-way ANOVA) and two-way analysis of
variance (two-way ANOVA). The post hoc Bonferroni
test was applied to results which exhibited significant
differences.A p value of < 0.05 was considered statistically
significant. The statistical package used was Prism 5.0.50
Results and Discussion
Synthesis and spectroscopic characterization
The benzothiadiazole compound 7 was obtained using
a previously described51 synthetic method by reacting the
4,7-dibromo-2,1,3-benzothiadiazole (6) with the terminal
alkyne 4 in a Sonogashira coupling reaction (Scheme 1).
Figure 1 shows the infrared absorption spectra for the
precursors (3-4) and the final compound (7). Significant
structural changes are observed that corroborate the
proposed chemical structure of the studied compounds.
Statistical analysis
The normality of the variables was assessed using the
Kolmogorov-Smirnov test. Statistical analysis of the values
Scheme 1. Synthetic route used to produce benzothiadiazole (7).51

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Synthesis, DNA Interaction and Genotoxic Evaluation of a Photoactive Benzothiadiazole
J. Braz. Chem. Soc.
A vibrational mode that is due to aliphatic stretching bands
of the C12H25 moieties is present for all compounds and
is indicative of alkyl chains bound to the tetrazole ring.
For compound 3, the stretching band of the OH group is
located at 3310 cm-1, indicating a coupling between the
aryl bromide 3 and 2-methyl-3-butyne-2-ol. An alkyne
deprotection reaction was evidenced by the appearance of
a C−H stretching band of the terminal alkyne located at
3286 cm-1. In addition, a weak band located at 2204 cm-1
due to C≡C stretching of the internal alkyne can be observed
for compound 7.
1
In the H NMR spectrum for compound 7 (Figure 2)
there are eight signals, where two sets of equivalent
hydrogens of the aromatic system (named a and b)
at 8.21 ppm (d, 4H, J 9.00 Hz) and 7.82 ppm (d, 4H,
J 9.00 Hz), respectively, correspond to a system of type
A4X4. The singlet located at 7.85 ppm related to 2H, named
(f) refers to the equivalent hydrogens of the benzothiadiazole
central unit. In the region of the aliphatic hydrogens, three
sets of signals are shown. The first one, a triplet located at
4.68 ppm (4H, J 7.03 Hz) named (c), corresponds to the
methylene group directly bonded to the tetrazole ring. The
second set of signals, a multiplet integrating 40H can be
observed between 2.25 and 1.15 ppm (named d) is due to
the rest of the methylene chain; and the last one, a triplet
Figure 1. FTIR spectra (500-4000 cm-1) for compounds 3, 4 and 7.
located at 0.86 ppm (6H, J 6.44 Hz), named (e) corresponds
to the terminal methyl group.
In relation to the thermal stability of the compound 7,
it is worth mentioning that as reported in the literature,51
Figure 2. 1H NMR spectrum of compound 7 (CDCl3, 400 MHz). The aromatic and part of the aliphatic regions are shown in zoom.

Vol. 29, No. 7, 2018
Damiani et al.
1393
Figure 3. Photostability of compound 7 by (a) diffuse reflectance UV-Vis absorption spectra and (b) steady-state fluorescence emission.
this tetrazole presents in nitrogen atmospheres several
decomposition temperatures with an initial decomposition
temperature between 235-295 °C with a mass loss of 30%
and a maximum rate of decomposition at 279 °C. In an
oxidizing media, a similar thermal behavior was observed,
with an initial decomposition temperature between
254-294 °C with a mass loss of 12% and a maximum
rate of decomposition at 280 °C. These results show that
the investigated compound presents significant thermal
stability. In addition, its photostability was also investigated
by UV-Vis and fluorescence emission spectroscopies
(Figure 3). It can be observed a small decrease on the
absorption intensity between 350-475 nm. Moreover, it can
also be observed a decrease on the fluorescence emission
intensity after UV exposure. These preliminary results can
probably be related to photobleaching process observed for
7 after UV irradiation.
is present. In addition, no modification was observed when
the incubations were performed in the solvent DMSO in
the absence of the complex. In addition, the high molar
ratios of complex and CT-DNA necessary to observe the
CD assay
The results from the CD studies presented in Figure 4,
which analyze the ability of the complex to interact with
CT-DNA suggest that the complex is a weak groove
binder. The complex exhibited a significant decrease
in the DNA reference band at 275 nm that is related to
base stacking without expressing changes in the 245 nm
DNA band that is associated with right-handed helicity.
A redshift is also observed for the 275 nm band. These
observations are usually associated with a groove binding
interaction mode46,52,53 or, in some cases, to an external
interaction as can be observed in high salt concentrations
associated to electrostatic binding.54 For this compound,
the interaction with a DNA molecule is likely based
on electrostatic interactions with the sugar-phosphate
backbone, and allowed by the quadrupolar character that
Figure 4. (a) Circular dichroism spectra of compound 7 and CT-DNA
interactions showing a reduction of the DNA reference band at 275 nm
that is related to base stacking and groove binding interactions. The
redshift observed can be associated with this mode of interaction or to
external binding when the 245 nm band does not produce any modification;
(b) circular dichroism spectra of DMSO (solvent used in (a) and CT-DNA
interactions showing no alteration in either the 275 or 245 nm bands).

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Synthesis, DNA Interaction and Genotoxic Evaluation of a Photoactive Benzothiadiazole
J. Braz. Chem. Soc.
effects of the first (1 – 1 and higher) when compared to
other copper(II) complexes,46,53 characterize this complex
as a weak binder.
from compound 4 only at concentration 3. After 12 h of
exposure, a significantly lower number of viable cells
were observed for compound 2 at the lowest concentration
compared to the negative control and for compound 5
compared to the control group, as well as for compounds 4
and 7 at concentrations 2 and 3. Compound 6 produced
similar results, but only at the highest concentration. In
addition, it was observed that the highest concentration of
compound 5 led to lower viability compared to the control
and compound 7, while compound 6 at the intermediate
concentration differed from the control and compound 4.
After 24 h of exposure it was observed that compounds 2
and 5 differed from the negative control at the three
concentrations evaluated, while compound 1 differed only
at the intermediate concentration, compound 3 at the two
lowest concentrations and compound 6 only at the highest
concentration. It was also noted that compound 5 led to a
lower cell viability compared to compounds 4 and 7 at the
three concentrations, and compound 6 only at the highest
concentration.
There are three main mechanisms that induce DNA
damage, as follows: (i) environmental agents (e.g., UV);
(ii) products of normal metabolism, which influence
the precision of the replication of genetic material and
(iii) chemical, physical and biological agents, which bind
to DNA and tend to lead to breakage of the molecule,55
the latter being the focus of this study. Currently, in vitro
genotoxicity tests are based on established methods
for the characterization of the hazards associated with
chemical products.47,49,56,57 The genotoxicity evaluation of
Cell viability and comet assay
The viability test was performed in vitro using human
peripheral blood to evaluate the cytotoxicity of the
compounds that are included in the chemical composition
of photovoltaic paints. The relevant data are summarized
in Table 1. After 2 h of exposure, it was observed that
compounds 2, 5 and 6 exhibited a significant difference
relative to the negative control at the three concentrations
evaluated, with the exception of compounds 2 and 5 at the
highest and lowest concentrations, respectively, which did
not produce a significant difference. Compound 5 resulted
in lower cell viability compared to compounds 4 and 7 at the
two highest concentrations, and compared to compound 4,
only at the lowest concentration. In the case of compound 6
compared with compound 7, there were fewer viable cells
at the highest concentration.
After 6 h of exposure, compounds 1 to 3 produced
a lower viability at the intermediate concentration
compared to the negative control, while compounds 2, 5
and 6 exhibited the same trend at the highest and lowest
concentrations, with the exception of compound 2, which
differed from the negative control only at the highest
concentration tested (Table 1). Compound 5 also exhibited
a significantly lower viability compared to compounds 4
and 7 at the two highest concentrations, and differed
Table 1. Effect on cell viability observed following the in vitro exposure of human peripheral blood to the intermediate and final compounds, with potential
application in photovoltaic paints, obtained via a synthetic route
Compound
Negative
control
time / h
2
Concentration
1
2
3
4
5
6
7
1
2
3
1
2
3
1
2
3
1
2
3
85.6 1.1
93.0 1.2
96.8 1.3
84.8 1.5
89.4 1.1a
96.2 0.8
84.8 1.1
86.2 1.8
96.0 1.9
79.6 1.1
86.0 1.0a
95.8 0.8
73.4 2.9
91.4 1.3a
96.4 0.6a
68.8 1.3a
89.2 1.5a
95.4 0.6
69.2 0.8
87.2 1.5
90.2 1.1a
67.4 1.5a
85.4 1.5a
92.8 1.3a
73.2 1.5
93.2 1.9
97.6 1.1
73.8 1.3
90.2 1.3a
95.6 1.1
68.0 0.8
86.4 0.6
91.8 1.5
71.0 1.6
85.2 0.8a
92.8 1.5a
94.2 1.9 39.4 0.9a,c,d 40.6 0.6a,d
95.4 1.5 49.6 1.1a,c,d 90.0 0.7a
94.4 1.1
94.2 0.8
97.2 0.8
93.4 0.9
93.8 0.8
96.4 1.1
93.0 0.7
94.2 1.5
97.2 0.8
100
0
97.8 0.8
93.2 1.3
95.6 1.1
97.0 1.0
92.2 0.8
88.4 0.9a,c
37 1.2a,c,d
51.4 0.9c,d
89.0 0.7a,c
38.2 1.1a,d
97.0 0.7
40.0 0.7a
90.0 0.7
95.0 0.7a
37 0.7a,c,d
6
99.6 0.55
99.2 0.84
12
24
95.0 1.0 49.4 0.9a,c,d 70.0 0.7a,c
97.4 1.5 88.4 0.9a,c,d 94.0 0.7
93.4 1.1 30.2 0.8a,c,d 30.0 0.7a,c,d 92.0 1.6
96.0 0.7 39.0 1.4a,c,d 90.0 0.7
97.2 1.3 88.2 1.6a,c,d 94.0 0.7
98
0
94.2 1.5
97.2 0.8
Cell viability observed following the in vitro exposure of 3 different concentrations of each compound in human peripheral blood; concentration 1:
equivalent to 2 mg of each compound; concentration 2: equivalent to 1 mg of each compound; concentration 3: equivalent to 0.5 mg of each compound;
values expressed as average standard deviation; ap < 0.05 difference compared to the negative control with the same exposure time, one-way ANOVA,
Kruskal Wallis; bp < 0.05 difference compared to compound 3 with the same exposure time and concentration, one-way ANOVA, Kruskal Wallis; cp < 0.05
difference compared to compound 4 with the same exposure time and concentration, one-way ANOVA, Kruskal Wallis; dp < 0.05 difference compared to
compound 7 with the same exposure time and concentration, one-way ANOVA, Kruskal Wallis.

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Damiani et al.
1395
the compounds was performed using the comet assay, the
results for which are the parameters of damage index and
damage frequency, which are shown in Tables 2 and 3,
respectively. The comet assay, also known as single-cell gel
electrophoresis, is very useful and widely employed in the
evaluation of DNA damage and repair in individual cells.58
The primary goal of this test is to evaluate the damage that
is caused by oxidative stress, through the interaction of
DNA with reactive oxygen species.59
Compound 1 exhibited a significant difference
compared to the negative control after 2 h of exposure
at concentrations 2 and 3; after 24 h, only the highest
concentration produced a difference in both parameters
evaluatedinthecometassay. Furthermore, atconcentration2,
after 2 and 12 h, there was also a difference compared to
the negative control; however, only for the damage index.
Compound 2, on the other hand, differed significantly
from the control only at the highest concentration after
2 h for both the DI and DF (Tables 2 and 3) while after 6 h
of exposure, only the DI exhibited a difference from the
control at the highest concentration (Table 2).
In the case of compound 5, the amount of damage was
significantly greater after all exposure times at the three
concentrations when compared with the negative control
and compounds 4 and 7, according to both DI and DF.
However, with the exception that at the three concentrations
after 2 h of exposure, no difference was observed compared
to compound 4, both damage parameters at the lowest
concentration differed only from the negative control and
compound 3 (Tables 2 and 3). Additionally, after 6 h of
exposure at the intermediate concentration, the DF and DI
demonstrated no difference compared to compound 4, but
the DI differed from compound 3.
In comparison with concentrations 2 and 3 after
12 h, the DI exhibited no difference from compound 4
(Tables 2 and 3), and the DF values after 12 h at the same
concentrations also produced no significant differences. In
addition, at concentration 3 after 24 h, no difference was
detected in relation to compound 4 was detected (Table 3).
For compound 6, both the DI and the DF produced
significant differences at all concentrations after the
four exposure times compared to the negative control
(Tables 2 and 3). However, after 24 h of exposure at the
lowest concentration, no differences were found for the
DF compared to the control. At concentration 1 after 6 h
of exposure, the DI and DF were significantly higher
than at concentration 3, and at the same concentration
after 24 h, this behavior was observed only for the DI
(Tables 2 and 3). At concentrations 2 and 3 after 12 h
and at concentration 2 after 24 h there was a significant
difference in the DI and DF compared to compound 7,
while only the DF at concentration 1 after 24 h differed
from compound 7 (Tables 2 and 3). Compound 7 did not
produce significantly greater damage in the cell viability
test and comet assay compared to the other compounds
that are included in the chemical composition of the paint,
demonstrating that it does not have toxic or genotoxic
activity (Tables 1, 2 and 3).
On the other hand, only compounds 1 and 4 promote
reactions with biological substrates that can damage
Table 2. Effect of in vitro exposure of human peripheral blood to intermediate and final compounds, with potential application in photovoltaic paints,
obtained synthetically, on the damage index (DI) obtained in the comet assay
Compound
4
Negative
control
time / h
2
Concentration
1
2
3
5
6
7
1
2
3
1
2
3
1
2
3
1
2
3
51.2 14.4 86.6 15.7a 45.8 12.5
68.8 12.7a
61.8 41.1
54.2 26.0
37.4 23.5
32.2 9.7
321.4 26.5a,d 184.4 25.5a 42.6 19.4
252.6 57.2a,d 137.0 13.3a 24.8 11.1
160.4 34.7a,b 111.8 7.1a
335.0 8.4a,c,d 143.8 30.2a,c 46.2 3.8
6.8 3.7
10.2 2.4
12.2 1.1
12.0 2.8
30.6 9.6
25.4 6.0
36.6 15.5
20.6 8.2
34.2 10.8
77.4 13.4 86.8 16.2a 56.4 15.8
64.0 14.5a 39.2 10.7
56.0 11.0a
33.4 8.9
22.2 3.3
6
33.6 18.3
23.4 4.4
59.4 5.4
38.2 4.3
31.8 12.2
42.0 5.6
33.6 5.7
24.8 6.5
36.6 12.9 283.6 14.5a,b,d 108.0 18.0a 28.6 9.9
23.8 14.3 183.2 15.8a,c,d 91.6 11.5a
21.4 8.6
48.4 51.4 337.5 24.7a,c,d 158.8 12.8a 42.2 17.9
94.4 20.2 110.6 50.1
72.2 19.0a 46.4 22.5
12
24
31.8 19.2
32.8 24.4
282.4 10.5a,d 109.0 12.9a,d 19.8 2.9
101.5 7.8a,d 85.8 13.7a,d 16.4 4.8
51.6 11.9
80.6 6.5a
58.2 11.9
56.2 14.9
41.4 13.5
62.4 13.7
57.0 8.4
28.2 8.4
36.2 14.1 323.6 25.0a,c,d 190.8 24.3a,c 21.8 1.5
23.8 8.7
283.0 9.2a,c,d 102.3 17.8a,d 13.4 7.6
146.2 28.0a,d 64.0 11.2a 13.0 10.0
24.8 16.8
Cell viability observed following the in vitro exposure of 3 different concentrations of each compound in human peripheral blood; concentration 1:
equivalent to 2 mg of each compound; concentration 2: equivalent to 1 mg of each compound; concentration 3: equivalent to 0.5 mg of each compound;
values expressed as average standard deviation; ap < 0.05 difference compared to the negative control with the same exposure time, one-way ANOVA,
Kruskal Wallis; bp < 0.05 difference compared to compound 3 with the same exposure time and concentration, one-way ANOVA, Kruskal Wallis; cp < 0.05
difference compared to compound 4 with the same exposure time and concentration, one-way ANOVA, Kruskal Wallis; dp < 0.05 difference compared to
compound 7 with the same exposure time and concentration, one-way ANOVA, Kruskal Wallis.

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Synthesis, DNA Interaction and Genotoxic Evaluation of a Photoactive Benzothiadiazole
J. Braz. Chem. Soc.
Table 3. Effect of in vitro exposure of human peripheral blood to intermediate and final compounds, with potential application in photovoltaic paints,
obtained synthetically, on the damage frequency (DF) obtained in the comet assay
Compound
4
Negative
control
time / h
2
Concentration
1
2
3
5
6
7
1
2
3
1
2
3
1
2
3
1
2
3
17.0 4.9
19.2 5.9
13.4 3.4
25.2 3.7
25.4 5.5a
21.2 4.6a
30.2 4.6
24.4 5.5
18.8 5.6
25.0 1.6a
25.6 15.1
17.8 4.7
26.8 5.0a
13.2 4.8
12.0 2.1
26.6 4.8
14.4 4.6
14.6 2.6
31.8 13.1
16.2 6.1
13.2 3.2
22.4 5.0
19.0 3.5
11.6 3.8
16.4 3.7
12.4 4.2
7.2 3.3
20.2 4.3
12.4 4.0
8.6 1.5
20.4 2.1
13.0 1.2
10.6 3.4
17.2 3.5
11.0 2.2
10.6 3.9
19.4 9.9
17.6 6.1
13.8 4.6
87.2 5.8a,d
76.8 9.4a,d
50.0 6.4a
40.0 2.3a
13.2 4.6
9.2 2.4
10.6 2.6
15.6 1.7
10.2 3.0
9.2 3.0
14.4 5.2
7.8 1.9
7.0 1.4
10.4 2.3
6.2 1.8
8.0 5.2
4.8 2.49
6.2 1.30
6.6 0.55
6.6 0.89
46.6 9.6a,b 34.8 3.3a,b
12.0 2.7 88.2 1.7a,c,d 40.6 7.9a,c
6
13.4 3.5
9.8 3.6
77.0 7.9a,d
31.0 5.2a
50.2 4.7a,b,c,d 27.0 3.3a
14.4 12.1 88.5 4.0a,c,d 43.4 3.0a
11.2 6.7 75.4 1.8a,c,d 31.0 3.4a,d
12
24
10.6 5.6
12.4 3.0 84.8 4.7a,c,d 53.2 4.0a,d
29.8 2.1a,d 25.2 3.5a,d
9.6 3.5
9.8 3.6
74.2 2.3a,c,d 30.5 6.2a,d
41.6 6.4a,c,d 20.2 5.0
Cell viability observed following the in vitro exposure of 3 different concentrations of each compound in human peripheral blood; concentration 1:
equivalent to 2 mg of each compound; concentration 2: equivalent to 1 mg of each compound; concentration 3: equivalent to 0.5 mg of each compound;
values expressed as average standard deviation; ap < 0.05 difference compared to the negative control with the same exposure time, one-way ANOVA,
Kruskal Wallis; bp < 0.05 difference compared to compound 3 with the same exposure time and concentration, one-way ANOVA, Kruskal Wallis; cp < 0.05
difference compared to compound 4 with the same exposure time and concentration, one-way ANOVA, Kruskal Wallis; dp < 0.05 difference compared to
compound 7 with the same exposure time and concentration, one-way ANOVA, Kruskal Wallis.
biomolecules and consequently affect human health.
The most serious effects are from damage to DNA and
ribonucleic acid (RNA). If the DNA chain is broken, it can
be reconnected at another position, thus altering the order
of its bases. This is one of the basic processes involved
in mutation, and the accumulation of damaged bases can
initiate oncogenesis. The results of this study highlight the
importance of considering a compound’s concentration
and exposure time when evaluating their genotoxicity,
particularly those that are obtained early in the synthetic
route. An increase in the compound’s concentration is
directly proportional to the increase in the damage, and
inversely proportional to cell viability; that is, the higher the
concentration of the sample is, the lower the cell viability
and the greater the DNA damage will be.
There are several factors that could lead to variations
in the damage indicated by the present results in both
cell viability and the comet assay. It was observed that
compounds 5 and 6 led to greater damage to the genetic
material than did the final compound. Compounds 1, 2 and
6 contain in their chemical structures an electronegative
moiety (bromine) despite the benzothiadiazole group
in the final compound 7. The presence of a bromine
substituent in these compounds increases the possibility
that a tautomeric change will occur. If 5-bromouracil
(5-BU) is incorporated in place of thymine and a
tautomeric change to the enol form occurs, the 5-BU
will pair with guanine which, after a round of replication,
will result in a transition of A=T to G=C. In addition, the
presence of 5-BU in DNA increases the sensitivity of the
molecule to UV light, which is itself mutagenic. Bromine
has been shown to be carcinogenic in rats, hamsters and
mice.48,60-63 Additionally, its mutagenic effects in several
in vitro64-66 and in vivo67,68 test systems have been verified.
The mechanisms underlying the genotoxicity of bromine
are only partially understood. Thus, it is important to
evaluate the genotoxic action of compounds that are used
in the production of photovoltaic paint.
Figure 5 shows the data for the cell viability and the
DNA damage index and damage frequency obtained in vitro
for blood cells that are exposed to the final (D-π-A-π-D)
photoactive compound synthesized according to Scheme 1.
The cell viability was not significantly different for the
exposure times tested at any of the concentrations of the
photoactive benzothiadiazole dye.
With regard to the damage index, at concentration 1 after
2, 6 and 24 h of exposure, the DI resulted in a significant
difference compared to the negative control and compared
to the values obtained for concentrations 2 and 3 after 2, 6
and 12 h of exposure.Additionally, for exposure times of 2, 6
and 12 h, the DI differed statistically from the value obtained
at 24 h at the highest concentration (concentration 1). For
2 h of exposure, the DI values obtained at concentrations
2 and 3 differed from the negative control, while after 6 h,

Vol. 29, No. 7, 2018
Damiani et al.
1397
only the DI obtained at concentration 2 differed from the
negative control. The DI for an exposure time of 6 h exhibited
a statistically significant difference compared to that obtained
after 24 h at concentration 2.
In the case of the DF parameter that was evaluated in the
comet assay, the highest concentration after the first three
exposure times demonstrated significantly greater damage
compared to the negative control, from which the results for
concentration 2 after 2 and 6 h of exposure also differed, and
for those for concentration 3 after 6 and 12 h of exposure.
After 24 h of exposure, no significant differences were
observed for any of concentrations evaluated compared
to the DI or the DF, showing that within this period, this
compound did not produce genotoxicity at any of the
concentrations tested.
Figure 6 reports the results for compounds 1 and 5,
which begin the series in the synthetic route, together with
compound 7, which is the final product, i.e., the photoactive
(D-π-A-π-D) benzothiadiazole derivative with potential
application in photovoltaic paints. This figure shows
that from the cell viability and the parameters evaluated
in the comet assay, as the compounds pass through the
synthetic route they lose toxicity, with the final compound 7
exhibiting no sign of toxicity.
The results of this study show that blood samples in
contact with compound 7 remain viable, exhibiting no
significant difference compared to the negative control,
and the DI and DF were of minimal significance, even
at the highest concentration of the compound. It is worth
mentioning that at the lowest concentration, no cytotoxic
effect was observed.
Figure 5. Cell viability (top) and the DNA damage index (middle) and
damage frequency (bottom) obtained in vitro for blood cells exposed to
compound 7 at three concentrations (conc. 1-3: 2, 1 and 0.5 mg of 7,
respectively) and for four exposure times obtained using the comet assay.
Data are expressed as average standard deviation. ap < 0.05 difference
compared to the negative control, with the same exposure time, two-
b
way ANOVA 2, post hoc Bonferroni; p < 0.05 difference compared to
concentration 2, with the same exposure time and concentration, two-way
c
ANOVA 2, post hoc Bonferroni; p < 0.05 difference compared to
concentration 3, with the same exposure time and concentration, two-way
ANOVA 2, post hoc Bonferroni; dp < 0.05 difference compared to 24 h of
exposure at the same concentration, two-wayANOVA, post hoc Bonferroni.
Figure 6. Comparison of the parameters obtained for the final compound 7
and respective precursors 1 and 5.

1398
Synthesis, DNA Interaction and Genotoxic Evaluation of a Photoactive Benzothiadiazole
J. Braz. Chem. Soc.
In addition, there was almost no genotoxic damage to the
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à Pesquisa do Estado do Rio Grande do Sul (FAPERGS),
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