In vitro evaluation of triazenes: DNA cleavage, antibacterial activity and cytotoxicity against acute myeloid leukemia cells

Article abstract of DOI:10.1590/S0103-50532010001200009

The asymmetric diazoamines 1-(2-chlorophenyl)-3-(4-carboxyphenyl)triazene (1), 1-(2-fluorophenyl)-3-(4-carboxyphenyl)triazene (2) and 1-(2-fluorophenyl)- 3-(4-amidophenyl) triazene (3) were evaluated for their ability to cleave pUC18 and pBSKII plasmid DN

Full text of DOI:10.1590/S0103-50532010001200009

J. Braz. Chem. Soc., Vol. 21, No. 12, 2226-2237, 2010.
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In Vitro Evaluation of Triazenes: DNA Cleavage, Antibacterial Activity and
Cytotoxicity against Acute Myeloid Leukemia Cells
Vanessa O. Domingues,a Rosmari Hörner,*,a Luiz G. B. Reetz,a Fábio Kuhn,a
Virgínia M. Coser,b Jacqueline N. Rodrigues,b Rita Bauchspiess,b
Waldir V. Pereira,b Gustavo L. Paraginski,c Aline Locatelli,c
Juliana de O. Fank,c Vinícius F. Giglioc and Manfredo Hörner*,c
aUniversidade Federal de Santa Maria, Departamento de Análises Clínicas e Toxicológicas,
97105-970 Santa Maria-RS, Brazil
bHospital Universitário de Santa Maria, Departmento de Hematologia-Oncologia,
97105-970 Santa Maria-RS, Brazil
cUniversidade Federal de Santa Maria, Departamento de Química, CP 5031,
97105-970 Santa Maria-RS, Brazil
As diazoaminas assimétricas 1-(2-clorofenil)-3-(4-carboxifenil)triazeno (1), 1-(2-fluorofenil)-
3-(4-carboxifenil)triazeno (2) e 1-(2-fluorofenil)-3-(4-amidofenil)triazeno (3) foram avaliadas
quanto à capacidade de clivagem do DNA plasmidial pUC18 e pBSKII, atividade antibacteriana e
citotoxicidade in vitro frente a células de leucemia mielóide aguda e leucócitos normais utilizando
o bioensaio da redução do sal brometo de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazólio (MTT).
Os triazenos analisados demonstraram capacidade de clivagem dos dois tipos de DNA plasmidial:
triazeno 1 em pH 8,0 e 50 °C; triazeno 2 em pH 6,5 a 37 e 50 °C; triazeno 3 em pH 6,5 e 37 °C. Os
compostos apresentaram atividade citotóxica frente a células leucêmicas. O composto 1 mostrou
alta atividade frente a B. cereus (CIM = 32 mg mL-1). A associação molecular através de ligações
de hidrogênio no estado sólido do composto 3 com base na análise estrutural por difratometria de
raios X em monocristal, bem como os resultados das análises espectroscópicas nas regiões do IV
e UV-Vis dos compostos 1, 2 e 3 são discutidos no presente trabalho.
The asymmetric diazoamines 1-(2-chlorophenyl)-3-(4-carboxyphenyl)triazene (1),
1-(2-fluorophenyl)-3-(4-carboxyphenyl)triazene (2) and 1-(2-fluorophenyl)-3-(4-amidophenyl)
triazene (3) were evaluated for their ability to cleave pUC18 and pBSKII plasmid DNA, antibacterial
activity and in vitro cytotoxicity against acute myeloid leukemia cells and normal leukocytes using
the bioassay of reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).
The triazenes showed ability to cleave the two types of plasmid DNA: triazene 1 at pH 8.0 and
50 °C; triazene 2 at pH 6.5 and 37 and 50 °C; triazene 3 at pH 6.5 and 37 °C. The compounds
presented cytotoxic activity against myeloid leukemia cells. Compound 1 showed high activity
against B. cereus (MIC = 32 mg mL-1). The observation of intermolecular hydrogen bonding in
the solid state of compound 3, based on the structural analysis by X-ray crystallography, as well
as the results of IR and UV-Vis spectroscopic analyses of compounds 1, 2 and 3 are discussed in
the present work.
Keywords: triazenes, DNA cleavage, antibacterial activity, cytotoxicity
Introduction
in an acyclic arrangement.1 Diazoamino compounds
can be obtained by diverse synthetic ways, the classical
method including the coupling reaction of a diazonium
salt, [R-N≡N]+X-, with an amine (R'-NH2) in acidic
medium. This is followed by formation of the respective
diazoamino compound R-N=N-N(H)-R' (R = R' for
Triazenes or diazoamines constitute a class of
compounds containing three consecutive nitrogen atoms
*e-mail: rosmari.ufsm@gmail.com, hoerner.manfredo@gmail.com

Vol. 21, No. 12, 2010
Domingues et al.
2227
symmetric triazenes; R ≠ R' for asymmetric compounds)
by stepwise neutralization of the reaction system. They have
diverse applications as DNA alkylating agents in tumor
therapy, protecting groups in natural product synthesis and
combinatorial chemistry, as well as precursors incorporated
into polymer and oligomer synthesis.2-8
Hydrolysis of DNA or RNA catalyzed by relevant
enzymes is an important subject in biotechnology, medicine
and drug development.9,10 Synthetic nucleases which
rapidly cleave nucleic acids under mild conditions have
many important potential applications ranging from the
synthesis of custom-designed artificial restriction enzymes
to the development of new antitumor agents.11 To mimic
the biological function of enzymes, triazene compounds
that cleave DNA are interesting for the development of
artificial nucleases. In this context, N-(3-hydroxypropyl)
berenil represents one example of a triazene capable to
promote DNA cleavage.12
Triazenes also act as chemotherapeutic agents for
many tumors, such as brain, leukemia, melanoma,
lymphoma and sarcoma.13-17 Among all chemotherapeutic
agents tested against metastatic melanoma, DTIC
(5-(3,3-dimethyltriazene)imidazol-4-carboxamide) - an
FDA-approved pro-drug activated by N-demethylation in
liver microssomes (microsomal enzyme CYP450) - is the
most active single agent and thus considered a reference
drug.18,19
Temozolomide (TMZ), i.e., 8-carbamoyl-3-
methylimidazol[5,1-d]-1,2,3,5-tetrazin-4(3H)-one, is
another example of a methylating triazene like DTIC, which
acts under physiological conditions against primary and
metastatic tumors in the brain.20 Despite the need for more
clinical experiments for the use of triazene compounds
in acute leukemia and myelodysplasia treatments, some
studies have shown that both DTIC and TMZ have
antineoplastic activity in patients with leukemia.21-23
Recently the antimicrobial susceptibility profile of
fifteen triazenes was evaluated by our group based on the
reference broth microdilution method according to CLS
guidelines.24
biological importance. From this point of view, diverse
biological reaction mechanisms can be related to hydrogen
bonding due to its energy range situated between van der
Waals interactions and covalent bonds.26,27
The present study deals with the synthesis and
characterization of 1-(2-chlorophenyl)-3-(4-carboxyphenyl)
triazene(1), 1-(2-fluorophenyl)-3-(4-carboxyphenyl)triazene
(2) and 1-(2-fluorophenyl)-3-(4-amidophenyl)triazene (3)
(Figure 1). The study also demonstrates that the compounds
are able to cleave pUC18 and pBSKII plasmid DNA
through a possible hydrolytic mechanism. The antibacterial
activity and in vitro cytotoxicity of the three triazenes
against acute myeloid leukemia (AML) cell lines and
control cell (without AML) using the bioassay of reduction
of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) are also analyzed. The single crystal
structural analysis of 1-(2-fluorophenyl)-3-(4-amidophenyl)
triazene (3), including its self-assembly in the solid state
throughhydrogenbonding, andthevibrationalandelectronic
spectra of compounds 1, 2 and 3 are also presented.
Experimental
Materials
All reagents and solvents used in the study were reagent
grade and were used without further purification. For
biological assays, water was purified by passage through
a Millipore water purification system with a resistance
of 18 MW and sterilized by autoclave. Reaction solutions
for DNA cleavage studies, determination of minimum
inhibitory concentrations and cytotoxicity essays were
prepared according to standard sterile techniques. The DNA
bands were stained, visualized on a UV transilluminator at
254 nm, photographed using a digital photographic camera
and quantified using Scion Image Beta 4.03 software (Scion
Corp.).
1H and 13C NMR spectra were recorded on a Bruker
DPX 200 instrument. IR spectra were recorded on a Bruker
FT-IR Spectrometer TENSOR 27 instrument with samples
embedded in KBr pellets. UV-Vis spectra were measured
on a Shimadzu UV-1650-PC spectrophotometer.
The mechanism of cytotoxicity of triazenes involves
mainly methylation of the O6 position of guanine in DNA.25
Ontheotherhand, itiswellknownthatmolecularassociation
plays an important role in biological systems. Strategic
modifications of electronic and geometric characteristics
applied in molecular design determine the dimensionality
of molecular association by diverse types of intermolecular
secondary non-bonding interactions such as the large palette
of hydrogen bonding. Hydrogen bonding is an important
non-covalent interaction in structural investigations relative
to molecular recognition and reactivity of molecules with
Synthesis of 1-(2-chlorophenyl)-3-(4-carboxyphenyl)
triazene (1)
The synthesis of triazene 1 was formerly reported
in 1956.28 The preparation was made with a solution of
4-aminobenzoic acid (1.00 g, 7.30 mmol) in 10 mL HCl
6 mol L-1 previously cooled to -2 °C and added throughout
10 min, under continuous stirring, to a solution of sodium

2228
In Vitro Evaluation of Triazenes: DNA Cleavage, Antibacterial Activity and Cytotoxicity
J. Braz. Chem. Soc.
Figure 1. Molecular structure of 1-(2-chlorophenyl)-3-(4-carboxyphenyl)-triazene (1), 1-(2-fluorophenyl)-3-(4-carboxyphenyl)triazene (2),
1-(2-fluorophenyl)-3-(4-amidophenyl)triazene (3), 1,3-bis(4-carboxyphenyl)triazene (4), 1,3-bis(4-fluorophenyl)triazene (5) and 1,3-bis(4-acetamidophenyl)
triazene(6).
nitrite (0.60 g, 8.70 mmol) in water (10 mL). The reaction
mixture was kept at -2 °C and a solution of 2-chloroaniline
(0.93 g, 7.30 mmol) in 6 mol L-1 HCl (5 mL) was slowly
added under continuous stirring. Stirring was continued
for 20 min at a temperature between -2 and -5 °C. A cold
sodium acetate solution (10%) was then added in small
amounts until pH 7.0 was reached while a yellow solid
precipitated. The crude product, separated by filtration, was
washed with small amounts of cold water and purified by
column chromatography after its adsorption onto silica gel
(70/230 mesh) using 5% acetone in hexane as eluent. The
yellow product, in form of fine microcrystalline needles,
was dried under vacuum. Yield 53% (0.95 g, 3.84 mmol)
based on 4-aminobenzoic acid, mp 172 °C; IR nmax/cm-1,
KBr pellets: 3174 (N−H), 2986 (O−H), 1682 (C=O), 1597
(C=C), 1510 (d(N−H), deformation), 1430 (d(O−H),
deformation in plane), 1414 (N=N), 1312 (C-O), 1209
(N-N), 756 (d(C−H), deformation). 1H NMR (200 MHz,
dmso-d6/tms) d 12.33 (br, s, 1H, OH), 7.16-7.70 (m, 8H,
Ph); 13C NMR (200 MHz, dmso-d6) d 146.6 (C=O), 130.6,
129.4, 129.2, 129.0, 128.9, 128.5, 125.4, 121.4, 121.3,
120.1, 119.4, 119.2 (Ph); lmax/nm (MeOH) 203, 242, 361
(e/dm3 mol-1 cm-1 18.6 × 103).
sodium nitrite solution (0.60 g, 8.70 mmol) in water (10 mL)
was added under continuous stirring throughout 10 min.
The reaction mixture was kept at -2 °C and a solution of
2-fluoroaniline (0.81 g, 7.30 mmol) in 6 mol L-1 HCl (5 mL)
was slowly added under continuous stirring. Stirring was
continued for 20 min at a temperature between -2 and
-5 °C.A cold sodium acetate solution (10%) was then added
in small amounts until pH 7.0 was reached while a yellow
solid precipitated. The crude product, separated by filtration,
was washed with small amounts of cold water and purified
by column chromatography after its adsorption onto silica
gel (70/230 mesh) using diethyl ether as eluent. The yellow
product in form of fine microcrystalline needles was dried
under vacuum. Yield 64% (1.21 g, 4.70 mmol) based on
4-aminobenzoic acid, mp 175 °C. IR nmax/cm-1, KBr pellets:
3175 (N−H), 2985 (O−H), 1680 (C=O), 1607 (C=C), 1491
(d(N−H), deformation), 1431 (d(O−H), deformation in
plane), 1404(N=N), 1312(C-O), 1201(N-N), 756(d(C−H),
deformation). 1H NMR (200 MHz, dmso-d6/tms) d 13.06 (br,
s, 1H, OH), 7.21-7.96 (m, 8H, Ph); 13C NMR (200 MHz,
dmso-d6) d 166.9 (C=O), 131.1, 128.5, 128.4, 124.9, 124.8,
119.1, 116.8, 116.4, 114.0 (Ph); lmax/nm (MeOH) 202, 237,
361 (e/dm3 mol-1 cm-1 21.9 × 103).
Synthesis of 1-(2-fluorophenyl)-3-(4-carboxyphenyl)
Synthesis of 1-(2-fluorophenyl)-3-(4-amidophenyl)triazene
triazene (2)
(3)
To a solution of 4-aminobenzoic acid (1.00 g, 7.30 mmol)
in 10 mL 6 mol L-1 HCl previously cooled to -2 °C, a
To a solution of 2-fluoroaniline (0.89 g, 8.01 mmol)
in 10 mL of 6 mol L-1 HCl previously cooled to -2 °C, a

Vol. 21, No. 12, 2010
Domingues et al.
2229
sodium nitrite solution (0.58 g, 8.41 mmol) in water (5 mL)
was added under continuous stirring throughout 10 min.
The reaction mixture was kept at -2 °C and a solution of
4-aminobenzamide (1.09 g, 8.00 mmol) in acetone (10 mL)
was slowly added under continuous stirring. Stirring was
continued for 20 min at a temperature between -2 and
-5 °C.A cold concentrated sodium carbonate solution was
then added in small amounts until pH 7.0 was reached while
a yellow solid precipitated. The crude product, separated
by filtration, was washed with small amounts of cold water,
followed by small amounts of cold ethanol and purified by
column chromatography after its adsorption onto silica gel
(70/230 mesh) using hexane:ethyl acetate (3:7) as eluent.
The microcrystalline product was recrystallized from a
hexane:ethyl acetate mixture (1:3). Yellow vitreous bar-
shaped crystals of 3 suitable for X-ray diffraction analysis
were obtained by slow evaporation of the solvent mixture
within five days. Yield 86% (1.48 g, 5.7 mmol) based on
2-fluroaniline, mp 200 °C. IR nmax/cm-1, KBr pellets: 3493
(N−H, amide), 3404 (N−H, amide), 3167 (N−H, amine),
1665 (C=O), 1611 (C=C), 1489 (d(N−H), deformation),
1427 (d(O-H), deformation in plane), 1391 (N=N), 1312
(C-O), 1202 (N-N), 752 (d(C-H), deformation).1H NMR
(200 MHz, dmso-d6/tms) d 13.05 (br, s, 1H, OH), 7.22-
7.97 (m, 8H, Ph); 13C NMR (200 MHz, dmso-d6) d 167.9
(C=O), 144.4, 129.6, 129.1, 128.8, 125.4, 125.3, 119.6,
117.2, 116.9, 114.0 (Ph); lmax/nm (MeOH) 202, 233, 361
(e/dm3 mol-1 cm-1 26.7 × 103).
the cleavage ability in absence of atmospheric oxygen
according to the methodology described.29
Bacterial strains
In vitro antimicrobial activities of the triazene
compounds were evaluated against 30 test organisms,
including 14 Gram-positive and 16 Gram-negative bacterial
strains. The groups included nine bacteria of theAmerican
Type Culture Collection (ATCC): Pseudomonas aeruginosa
ATCC 27853, Escherichia coli ATCC 25922, Escherichia
coli ATCC 35218, Salmonella choleraesuis ATCC 10708,
Klebsiella pneumoniae ATCC 700603, Staphylococcus
aureus ATCC 25923, Staphylococcus epidermidis ATCC
12228, Enterococcus faecalis ATCC 29212 and Bacillus
cereus ATCC 10987. Twenty one clinical bacteria
from the Microbiology Laboratory of the Hospital
Universitário de Santa Maria, Brazil, were identified by a
Siemens MicroScan automated system: Shigella sonnei,
Enterobacter cloacae, Klebsiella oxytoca, Bacillus cereus,
Micrococcus sp., Streptococcus pyogenes, Acintobacter
baumannii, Ralstonia pickettii, Serratia marcescens,
Enterococcus sp., Listeria monocytogenes, Aeromonas
hidrophyla, Enterobacter aerogenes, Streptococcus
agalactiae, Salmonella tiphymurium, Corynebacterium sp.,
Salmonella sp., Staphylococcus epidermidis, Acinetobacter
lwoffii, Klebsiella pneumoniae, Staphylococcus hominis
subsp. hominis.
DNA cleavage studies
Determination of minimum inhibitory concentrations
(MICs)
DNA cleavage was measured by the conversion of
supercoiled pUC18 and pBSKII plasmid DNA (Stratagene)
to nicked circular and linear DNA forms. The screening
assay to achieve nucleic acids cleavage was performed
in a final volume of 20 mL where the following reagents
were present: 1 mg of plasmid DNA and different
concentrations of tested compounds (3.75, 1.875, 1.25,
0.75 and 0.375 mmol L-1) at pH 6.5 (200 mmol L-1 PIPES
buffer) and 8.0 (200 mmol L-1 Tris-HCl buffer) at 37 and
50 °C for 24 h; all assays were made twice. Samples were
submitted to agarose gel (0.8%) electrophoresis stained
with ethidium bromide. The resulting gels were digitalized
with a photodocumentation system.
In order to verify the probable mechanism of DNA
strand scission, the assays were repeated in the presence
of free radical scavengers (thiourea 40 mmol L-1, glycerol
0.1% and 1.0% and dimethyl sulfoxide 0.04 mol L-1) in the
same cleavage conditions (pUC18 and pBSKII plasmids
at pH 6.5 and 8.0, 37 and 50 °C for 24 h). Cleavage assays
were also performed in argon atmosphere in order to verify
Antimicrobial susceptibility testing was performed by
the broth microdilution method using cation-supplemented
Mueller Hinton broth (Difco, Detroit, MI), according
to Clinical and Laboratory Standards Institute (CLSI)
guidelines.30 Inoculumsuspensionswerepreparedbydiluting
the broth culture supernatant with sterile saline with density
of 0.5 (McFarland standard) using a spectrophotometer
(Bausch & Lomb Spectronic 20). All compounds were
tested at concentrations ranging from 0.125 to 128 mg mL-1
and dissolved in 20% of EtOH. The plates were incubated
without agitation at 35 2 °C for 24 h. The technique is
based on the growth of the test organisms (inoculums +
compounds) relative to a positive control (bacteria
without compound), with addition, after 24 h, of 10 mL
of 2% 2,3,5-triphenyltetrazolium chloride (TTC). TTC is
colorless in the oxidized form and red when reduced. Live
microorganismsreduceTTCbyenzymaticaction, originating
formazan kept inside granules in the cells, which become
red.31 MIC values were defined as the lowest concentration

2230
In Vitro Evaluation of Triazenes: DNA Cleavage, Antibacterial Activity and Cytotoxicity
J. Braz. Chem. Soc.
of the triazene compounds that inhibited visible growth of
microorganisms. All tests were made three times.
The IC50 value, defined as the drug concentration causing
a 50% reduction in cellular viability, was calculated for each
compound. The experimental data were transformed to
sigmoid dose-response curves using nonlinear regression
analysis (GraphPad Prizm 5), which enabled the calculation
of the corresponding IC50.
Cell lines
Leukemic cells were obtained from three acute
myeloid leukemia patients without treatment diagnosed
at the Hematology-Oncology Laboratory of the Hospital
Universitário de Santa Maria, Brazil, by light microscopic
examination of the bone marrow as well as flow cytometry
to diagnose the presence of leukemia, to differentiateAML
from other types of leukemia and to classify the subtype of
disease. Cell lines of control patient (without AML) was
obtained by collecting peripheral blood with EDTA. The
mononuclear cells from bone marrow (1 mL) and peripheral
blood (6 mL) were isolated by Ficoll-Paque PLUS (Sigma)
density separation (centrifuged at 4000 rpm for 20 min).
After isolation, the cells were washed three times in
RPMI-1640 (Sigma) and the erythrocytes were lysed with
hemolytic buffer. The cells were grown in RPMI with
20% fetal bovine serum (Sigma), penicillin 100 U mL-1,
streptomycin 100 mg mL-1, HEPES buffer and sodium
bicarbonate during 24 h at 37 °C, with 5% CO2.
Crystal structure determination
A single crystal on a glass fiber was used for the X-ray
data collection. Data were collected at -123 °C with a
Bruker APEX II CCD area-detector diffractometer and
graphite monochromatized Mo-Ka radiation using COSMO
program.32 No crystal decay was observed. Crystal system
and space group were unequivocally determined from the
data sets and reciprocal space images. Cell refinement,
data reduction and absorption correction were performed
using SAINT and SADABS programs, respectively.32 The
structure of 3 was solved by direct methods and refined
on F2 with anisotropic temperature parameters for all
non-hydrogen atoms.33,34 The H atoms in the structure of 3
were identified in Fourier difference maps and refined as
individual atoms with isotropic displacement parameters.
Table 1 shows the crystallographic data and details of data
collection and refinement, and Table 2 the selected bond
lengths and angles. Graphical representations were drawn
with the DIAMOND software.35
MTT assay
MTT was dissolved in phosphate buffered saline (PBS)
at 5 mg mL-1 and filtered to sterilize and remove a small
amount of insoluble residue. Trypan blue (1% in distilled
water) and cell suspensions were mixed in 1:6 quantities
(50 mL of cells + 10 mL trypan blue). The quantity of
cells was estimated by hemocytometry and (blue) stained
cells were scored as dead. About 3 × 106 cells mL-1 were
grown with compounds for 24 h at 37 °C and 5% CO2. The
triazene doses varied from 12.5, 50 and 100 mmol mL-1; the
compounds were dissolved in dimethyl sulfoxide, which did
not exceed 0.25% in the cell cultures. The DTIC solution
was prepared in water. The microculture plates included
90 mL cell suspension and 10 mL of the compound solution.
Control cultures received cells in absence of assayed
compounds. After 20 h the MTT solution was added to all
wells and plates were incubated at 37 °C for 4 h. At the
end of the incubation period, the formazan product was
dissolved in 100 mL of dimethyl sulfoxide. The optical
density (OD) of each well was measured using a Fisher
Bio-Tek BT2000 MicroKinetics reader (l = 570 nm). Each
assay was carried out three times. The cell survival fraction
was calculated by the following equation:
Results and Discussion
Spectroscopic characterization
Infrared spectra
The infrared spectra of 1, 2 and 3 were analyzed in the
spectral region 4000-400 cm-1. The main absorptions and
vibrational assignments were compared with literature
data.36-38 Amide derivatives are characterized by two
absorptions in sequence with medium intensities and
120-200 cm-1 spacing in the 3550-3150 cm-1 region. In
compound 3 these absorptions were assigned at 3493 and
3404cm-1.36,37 Theshifttolowerfrequencycanbeattributedto
electronicdelocalizationtowardstheterminalarylsubstituents
of the [-N=N-N(H)-] diazoamino group. The characteristic
N–H stretching at 3181 cm-1 for the [-N=N-N(H)-]
triazene group was observed as weak to medium signals at
3167-3175cm-1 in1, 2and3. Comparedwith1,3-bis(phenyl)
triazene the [-N=N-N(H)-] diazoamino group in 1, 2 and
3 was undoubtedly characterized by strong absorptions
at 1489-1510 cm-1, 1391-1414 cm-1 and 1201-1208 cm-1
corresponding to N-H in plane deformation, nas(N=N) and
ns(N-N), respectively.38
Cell survival = (OD of the treated well / mean OD of the
control wells) × 100%

Vol. 21, No. 12, 2010
Domingues et al.
2231
Compounds 1 and 2 could be analyzed as R-C(=O)OH
carboxylic derivatives, usually characterized in the
solid state by a strong, very broad O-H stretching near
3000 cm-1.36 In compounds 1 and 2 this broad band
was observed at 2986 and 2985 cm-1, respectively. In
compound 3 this absorption was absent. Carboxylic acids
demonstrating their efficient chromophoric behavior.
The electronic spectra of 1, 2 and 3 were compared with
those recorded in methanol for the compounds 1,3-bis(4-
carboxyphenyl)triazene (4), 1,3-bis(4-fluorophenyl)
triazene (5) and 1,3-bis(4-acetamidophenyl)triazene
(6) respectively (Figure 1). The electronic spectra of
compounds 4, 5 and 6 dissolved in solvents with different
polarities showed solvatochromism related to the n→p*
absorption due to the electron-withdrawing or donating
property of fragments attached to terminal aryl substituents
of the [-N=N-N(H)-] diazoamino group. Dissolved in
methanol, 4, 5 and 6 showed a lmax value of 348, 351 and
369 nm assigned to this n → p* absorption, respectively.38
These values agree with the maximum observed of 361 nm
for 1, 2 and 3. Further, the band observed at 242, 237 and
233 nm in the spectra of 1, 2 and 3 respectively is attributed
to the p → p* transition for aromatic rings, which were
observed for 4 and 5 at 221 and 231 nm, respectively.38 A
third intense band observed near 200 nm for 1, 2 and 3 was
attributed to the n → s* transition of the carbonyl group.40
…
and derivatives usually exist as (R-C=O H-O-C-R)2
hydrogen-bonded dimers. If the dimer unit is considered,
two carbonyl stretching frequencies, symmetric and
asymmetric, are observed. The former is Raman active,
and the asymmetric will be IR active only, assigned
for most carboxyl dimers at 1720-1680 cm-1.36 Such
observation agrees with the absorptions observed at
1665-1682 cm-1 assigned to nas(C=O) for 1, 2 and 3. Further,
the carboxyl dimer presents a characteristic infrared
band at 960-875 cm-1 which corresponds to out-of-plane
36
…
OH O hydrogen deformation. In the case of 1 and 2, this
characteristic absorption is observed near 850 cm-1 probably
due to electronic delocalization towards the terminal aryl
substituents of the [-N=N-N(H)-] diazoamino group. In
the case of 3 the crystal structure shows intermolecular
…
associations promoted by C-O H-N hydrogen bonding
X-ray crystallography
between the carbonyl group of the amido substituent and
Since the molecular association promoted by hydrogen
bonding plays an important role in biological systems,
especially for molecular recognition and reactivity of
molecules with biological importance, the crystal structure
analysis of one of the triazene derivatives was carried out to
help understand the hydrogen bond network established by
the terminal aryl group substituents of the [-N=N-N(H)-]
diazoamino moiety, as well as the influence of this non-
covalent interaction on the planarity of the molecule in the
solid state. Attempts to obtain single crystals suitable for
structural analysis of 1 and 2 are in progress.
Crystal data and experimental parameters of the
structural analysis of 3 are presented in Table 1. Selected
bond distances and angles are listed in Table 2. Figure 2
shows a projection of the molecular structure of
FC6H4N=N(H)C6H4C(=O)NH2 (3), while Figure 3
represents a section of the one-dimensional assembly of
the [-N=N-N(H)-] diazoamino group (Figure 3), which
…
can be associated to the out-of-plane OH N hydrogen
…
deformation in analogy with the out-of-plane OH
O
hydrogen deformation of R-C(=O)OH derivatives. This
explains the well-defined absorption observed at 843 cm-1
in the spectra of 3. Besides the characteristic out-of-plane
…
…
OH O hydrogen deformation, (R-C=O H-O-C-R)2
carboxyl dimers are characterized by O−H in plane
deformation and C-O stretching in the spectral region
of 1440-1395 cm-1 and 1315-1280 cm-1, respectively.36
Compounds 1 and 2 show the d(O−H) vibration at 1430 and
1431 cm-1, respectively, while in compound 3 the strong
absorption at 1427 cm-1 was assigned to a N−H deformation
…
in plane correspondent to the C-O H-N intermolecular
hydrogen bonding (Figure 3). All compounds showed the
C-O stretching at 1312 cm-1.
Strong bands observed for 1, 2 and 3 near 750 cm-1 were
attributed to the C-H out-of-plane deformation and the
very strong absorptions observed at 1597-1611 cm-1 were
assigned to the C=C axial deformation, both characteristic
of aromatic compounds.38
the molecules via secondary N-H O, N-H F, N-H N
…
…
…
…
and C-H O hydrogen bonds, in a projection slightly tilted
toward the [001] crystallographic direction.
Compound 3 crystallizes in the monoclinic crystal
system, the non-centrosymmetric polar space group Cc. To
test if a higher symmetry was eventually missed, an attempt
was made to solve the structure in the centrosymmetric C2/c
group, with negative results. Since the molecule of 3 is built
only by light atoms that do not cause anomalous scattering,
no significant intensity difference between the Friedel pairs
hkl and -h-k-l could be observed in the collected reflection
data. In other words, no conclusions could be drawn
UV-Vis spectra
The UV-Vis spectra of 1, 2 and 3 dissolved in methanol
were analyzed in the 190-440 nm spectral region. The
assignments made for the main absorptions are based
on literature data.39,40 Aryl and/or phenyl-substituted
triazenes show maximum absorptions at 190-440 nm

2232
In Vitro Evaluation of Triazenes: DNA Cleavage, Antibacterial Activity and Cytotoxicity
J. Braz. Chem. Soc.
Table 1. Crystal and structure refinement data for 3
Table 2. Selected bond lengths [Å] and angles [°] for 3
Empirical formula
Formula weight
T (K)
C13H11FN4O
258.26
Bond lengths
C1-O1
[Å]
Bond lengths
C2-O2
[Å]
1.248(2)
1.327(2)
1.485(2)
1.361(2)
0.81(3)
1.02(3)
1.01(3)
1.280(2)
1.327(2)
[°]
1.234(2)
1.336(3)
1.490(2)
1.349(2)
0.83(3)
150(2)
C1-N1
C2-N2
Radiation, l (Å)
Crystal system, space group
0.71073
C1-C24
C2-C44
C32-F2
monoclinic, Cc
C12-F1
Unit cell dimensions, a, b, c (Å) a = 27.630(2)
b = 5.0682(4)
N1-H3
N2-H5
N1-H4
N2-H6
1.06(3)
c = 18.7030(17)
N13-H1
N23-H2
N21-N22
N22-N23
Bond angles
0.88(3)
b = 112.570(8)°
N11-N12
1.271(2)
1.335(2)
[°]
Volume (Å3)
2418.5(3)
N12-N13
Z
8
Bond angles
O1-C1-N1
O1-C1-C24
N1-C1-C24
F1-C12-C13
F1-C12-C11
C25-C24-C1
C23-C24-C1
C1-N1-H3
C1-N1-H4
H3-N1-H4
N11-N12-N13
N12-N13-H1
C21-N13-H1
Calculated density (g cm-3)
Absorption coefficient (mm-1)
F(000)
1.419
121.29(17) O2-C2-N2
120.06(15) O2-C2-C44
118.60(14) N2-C2-C44
118.12(18) F2-C32-C33
118.44(17) F2-C32-C31
119.31(14) C45-C44-C2
121.82(15) C43-C44-C2
121.04(17)
119.87(17)
119.08(16)
118.48(16)
118.54(15)
118.40(16)
123.14(16)
120.0(19)
121.9(16)
118(2)
0.105
1072
Crystal size (mm)
Theta range (°)
0.43 × 0.39 × 0.22
1.60 to 30.19
Index ranges
-38 ≤ h ≤ 38, -7 ≤ k ≤ 7, -26 ≤ l ≤ 26
24746
Reflections collected
Independent reflections
Completeness to theta
Max. and min. transmission
Refinement method
Data / restraints / parameters
Goodness-of-fit on F2
Final R indices [I >2s (I)]
R indices (all data)
7113 [Rint = 0.0362]
99.5 %
117.8(17)
113.2(14)
129(2)
C2-N2-H5
C2-N2-H6
H5-N2-H6
0.9772 and 0.9561
Full-matrix least-squares on F2
7113 / 2 / 431
112.22(14) N21-N22-N23
111.02(14)
120.9(19)
118.2(19)
118(2)
122(2)
N22-N23-H2
C41-N23-H2
1.067
R1 = 0.0433, wR2 = 0.0994
R1 = 0.0595, wR2 = 0.1084
Largest diff. peak and hole (e.Å-3) 0.231* and 0.195*
*Highest peak: 0.231 (e.Å-3) at 0.1935, 0.6264, 0.0511 [0.77Å from H4].
*Deepest hole: -0.195 (e.Å-3) at 0.0306, 0.3744, 0.3387 [0.68 Å from
N12].
about the orientation of the molecules relative to the polar
axes in the unit cell of 3. Two symmetrically independent
molecules of 3 were observed in the asymmetric unit of the
unit cell (Figure 2). The observed intermolecular hydrogen
bonding involving the amide group made the independent
molecules deviate from planarity, becoming chiral. The
lack of planarity can be observed by the interplanar angles
O1C1N1/C21-C26 [22.5(3)°; whole molecule r.m.s.
deviation = 0.1610 Å] and O2C2N2/C41-C46 [2.7(3)°;
whole molecule r.m.s. deviation = 0.0310 Å].
The molecular structure corresponds to the expected
trans stereochemistry about the N11=N12 and N21=N22
double bonds. Diaryltriazenes typically present a
delocalization of the p electrons of the triazene group
towards the terminal aryl substituents. This property is
supported by the deviations observed from normal N-N
Figure 2. Molecular structure with atom-labeling scheme of the two
symmetrically independent FC6H4N=N(H)C6H4C(=O)NH2 molecules of
3 in the asymmetric unit of the space group Cc. Displacement ellipsoids
are drawn at the 50% level.

Vol. 21, No. 12, 2010
Domingues et al.
2233
and Caryl-N bond lengths. Accordingly, the N11=N12
[1.280(2) Å] and N21=N22 [1.271(2) Å] bond lengths
are longer than the characteristic value for a double
bond (1.236 Å), whereas N12-N13 [1.327(2) Å] and
N22-N23 [1.335(2)Å] bond distances are shorter than the
characteristicvalueforasinglebond(1.404Å).41 Ontheother
hand, C11-N11 [1.414(2) Å] and C21-N13 [1.393(2) Å],
aswellasC31-N21[1.416(2)Å]andC41-N23[1.398(2)Å]
bonds are shorter than the characteristic N-Caryl single
chains along the crystallographic direction [010]. Table 3
lists the geometric parameters of the intermolecular
hydrogen-bonding in 3. The molecules are associated
through hydrogen bonds including a bifurcated acceptor
…
geometry (D1-H1, D2-H2) A (D = donor, A = acceptor
…
atom), (N13-H1, C22-H22) O2', and a bifurcated
donor geometry D-H (A1, A2), N2-H6 (F1''', N11''');
symmetry codes: (') -1/2+x, -3/2-y, -1/2+z, (''') 1/2+x,
-3/2-y, 1/2+z. The angle F1''' H6 N11''' is 70.4(8)° and
the elevation of H6 from the plane defined by the atoms F1''',
N2, N11''' is 0.02(3)Å.Atom O2' deviates 0.054(2)Å from
the best plane defined by the atoms C22, C2', N13, while
…
…
…
…
2
bonds (secondary amines, R2NH, R = Csp ; 1.452Å).42 These
values agree with those observed in similar compounds:
1,3-bis(2-trifluoromethylphenyl)triazene [N=N 1.281(2)
and N-N 1.302(2) Å],40 1,3-bis(2,4-dibromophenyl)
triazene [N=N 1.267(7) and N-N 1.332(7) Å],44
and 1-(4-bromophenyl)-3-(4-nitrophenyl)triazene
[N=N 1.257(8) and N-N 1.326(10) Å].43 The bond angles
of the N=N-N triad [N11-N12-N13 112.22(14)°] and
[N21-N22-N33 111.02(14)°] are very close to the
angles found in 1,3-bis(2-trifluoromethylphenyl)triazene
[111.89(14)°] and 1-(4-bromophenyl)-3-(4-nitrophenyl)
triazene [110.9(7)°].43,45
…
…
the angle H1 O2' H22 is 63(1)°. The one-dimensional
arrangement of each symmetrically independent molecule
of 3 along the crystallographic direction [010] results from
…
the classical N1-H3 O1'' hydrogen bond, symmetry code:
('') x, -1+y, z. Due to the symmetry independence of each
molecule of 3, which originates the 1-D arrangement, both
chains running in opposite directions to each other along the
crystallographic b axis are also symmetrically independent,
as indicated in Figure 3.
The PLATON software was used to analyze the
intermolecular hydrogen bonding observed in the crystal
of 3, as shown in Figure 3.46
The crystal structure shows that the symmetrically
independent FC6H4N=N(H)C6H4C(=O)NH2 molecules
are individually related to each other by translation into
Table 3. Hydrogen-bonding geometric parameters (Å, °) for 3
D-H∙∙∙A
D-H
H∙∙∙A
D∙∙∙A
∠D-H∙∙∙A
162(3)
136(2)
123(2)
166(2)
139(2)
N13-H1∙∙∙O2'
C22-H22∙∙∙O2'
N2-H6∙∙∙F1'''
N2-H6∙∙∙N11'''
N1-H3∙∙∙O1''
1.01(3)
0.97(3)
1.06(3)
1.06(3)
0.81(3)
1.78(3) 2.758(2)
2.52(2) 3.295(2)
2.51(3) 3.223(2)
2.10(3) 3.142(2)
2.31(3) 2.963(2)
(D = donor atom, A = acceptor atom). Symmetry codes: (') -1/2+x, -3/2-y,
-1/2+z, ('') x, -1+y, z, (''') 1/2+x, -3/2-y, 1/2+z.
Biological activity
The ability of triazenes 1 and 2 to cleave supercoiled
pUC 18 and pBSKII plasmids in pH 8.0 (200 mmol L-1
Tris-HCl buffer) upon incubation at 50 °C for 24 h is
evidenced in Figure 4, while Figure S1 (see Supplementary
Information) shows that triazene 3 promotes DNA cleavage
in pH 6.5 (200 mmol L-1 PIPES buffer) after 24 h at 37
and 50 °C. Compounds 1 and 2 are able to cleave plasmid
DNA by transforming the supercoiled form (FI) into the
circular relaxed form II by single strand scission, while
compound 3 was not able to show cleavage activity in the
same conditions employed for 1 and 2. However, compound
3 has demonstrated more cleavage efficiency in pH 6.5 after
24 h at 37 and 50 °C, due to additional cleavage of form II
into III (linear form) as shown in Figure S1. The shape of the
linear form can be attributed to an increase in the number of
random cleavage events indicating low selectivity.47
Figure3. Sectionoftheone-dimensional(1-D)assemblingofthemolecules
inthecrystalstructureofFC6H4N=N(H)C6H4C(=O)NH2 (3). Intermolecular
hydrogenbondsincludingbifurcatedacceptorgeometry(D1-H1,D2-H2)∙∙∙A
(D = donor, A = acceptor atom), (N13-H1, C22-H22)∙∙∙O2’,
bifurcated donor geometry D-H∙∙∙(A1, A2), N2-H6∙∙∙(F1’’’, N11’’’), and
classical N1-H3∙∙∙O1’’ hydrogen bonding along the crystallographic
direction [010] are drawn with dashed lines. Symmetry transformations
used to generate equivalent atoms: (') -1/2+x, -3/2-y, -1/2+z,
('') x, -1+y, z, (''') 1/2+x, -3/2-y, 1/2+z.

2234
In Vitro Evaluation of Triazenes: DNA Cleavage, Antibacterial Activity and Cytotoxicity
J. Braz. Chem. Soc.
Figure 4. Total cleavage (%) of pUC18 and pBSKII plasmid DNA by triazenes 1 and 2. Lane 1: control plasmid DNA (untreated pUC18 and pBSKII);
lanes 2-6: addition of compounds in different concentrations: 3.75, 1.875, 1.25, 0.75 and 0.375 mmol L-1. Incubation for 24 h at 50 °C, pH 8.0.
In order to verify the plasmid DNA cleavage mechanism
for 1, 2 and 3, assays were carried out in the presence of
0.04 mol L-1 dmso or 0.1-1.0% glycerol as hydroxyl radical
scavengers. The experiments showed that these conditions
did not affect the cleavage activity of 1, 2 and 3, suggesting
that free hydroxyl radicals do not participate in the cleavage
reaction.48,49 To confirm that molecular oxygen did not
participate in the reaction, other assays were conducted
under anaerobic conditions, showing that the cleavage
ability of 1, 2 and 3 is not based on an oxidative mechanism.
In conclusion, the plasmid DNA cleavage activity of 1, 2
and 3 is probably based on a hydrolytic mechanism. It is
important to point out that all natural nucleases act through
a hydrolytic mechanism.47
against the tested organisms are comparable to those of the
similar compounds 1-(2-fluorophenyl)-3-(4-acetylphenyl)
triazene, 1-(4-ethoxyphenyl)-3-(4-carboxyphenyl)triazene,
1-(4-nitrophenyl)-3-(4-carboxyphenyl)triazene and
1-(4-acetylaminophenyl)-3-(4-carboxyphenyl)triazene.25
Other studies have shown that triazenes have better activity
against gram-positive than gram-negative bacteria,24 given
the fact that the gram-negative bacterial membrane is more
complex than the gram-positive one and presents a barrier
to the penetration of numerous antimicrobial molecules.
Also, the periplasmic space contains large concentrations
of enzymes which are able to decompose exogenous
molecules. However, the mode of action of the reported
compounds is still undetailed.
The MIC values of 1, 2 and 3 showed weak activity
against Gram-positive and Gram-negative bacteria. Two
susceptibility endpoints were recorded for each isolate.
The MIC is the lowest concentration of the compound in
which the microorganism does not demonstrate visible
growth. Minimum bactericidal concentration (MBC), in
its turn, is defined as the lowest concentration yielding
negative subculture or only one colony. The results
show that 1 is the most active triazene, inhibiting the
growth of some strains with MIC values of 128 mg mL-1
(Enterococcus faecalis ATCC 29212, Klebsiella oxytoca
and Aeromonas hidrophyla) and 32 mg mL-1 for Bacillus
cereus. Compound 2 revealed a MIC value of 64 mg mL-1
for Streptococcus agalactiae, while triazene 3 did not
show promising antimicrobial activity (MIC values higher
than 128 mg mL-1 against all bacteria assessed). The MBC
values for 1, 2 and 3 were higher than 128 mg mL-1 for all
organisms investigated. The observed MICs of 1, 2 and 3
Most anticancer agents act on DNA or its precursors,
inhibiting their synthesis or causing irreparable damage
to their molecules. Examples are alkylating agents,
cisplatin and antibiotics with antitumor activity, which
are able to act at various cell cycle phases and are called
non phase-specific. In this context, the bleomycins A2 and
B2 form an important group of antibiotics able to cleave
DNA through an oxidative mechanism.50 The cytotoxic
action of bleomycin transformed it into an anticancer
drug with significant activity against squamous cell
carcinoma of the uterine cervix, lymphomas and testicular
tumors. Other examples of antibiotics used in anticancer
therapy are dactinomycin, daunorubicin, doxorubicin,
epirubicin, mitoxantrone, idarubicin and doxorubicin (or
adriamycin).50,51 The default schema for the Hodgkin’s
disease treatment consists of the combination chemotherapy
of adriamycin, bleomycin, vinblastine and dacarbazine
(ABVD).52

Vol. 21, No. 12, 2010
Domingues et al.
2235
The ability of the triazenes 1, 2 and 3 to kill human
cancer cells was investigated using marrow bone cultures
of leukemia patients and peripheral blood of control patient
with a standard bioassay, MTT. Cells were exposed to 1,
2 and 3 during 24 h in order to evaluate the effects of the
investigated triazenes as described in the experimental
section. For comparison purposes, DTIC cytotoxicity
was evaluated under the same conditions. Based on the
concentration-response curve, the cell survival did not
depend on the triazene concentration for compounds 1, 2
and 3 (Figure 5).
Table 4 shows the results of the antiproliferative
effect of 1, 2 and 3 on acute myeloid leukemia cells
and control patient cells. IC50 values in the range of
1.83-129.30 mmol mL-1 were observed for the leukemia
cells. The compounds 1, 2 and 3 demonstrated cytotoxicity
very similar against AML3, while compound 1 appeared
to be more cytotoxic than 2 and 3 against AML1. These
results suggest that the replacement of fluorine by chlorine
in compound 1 increased the antileukemic activity of the
triazenes in all leukemic cells tested. Against AML3 cells
the compounds presented the best anti-leukemic activity and
against AML2 the worst. However, the three compounds
were more active against AML2 cells compared to DTIC.
The correct dose is one of the most important aspects to
achieve the best antitumor effect without increasing toxicity
too much, considering that the therapeutic window of most
antineoplastic agents is narrow. The fact that the IC50 of
the three compounds for AML3 is half the CP allows us
to suggest the possibility of administering a lower dosage,
reducing toxicity. Data also showed that the compounds
evaluated were highly toxic to normal leukocytes. The
results of the MTT-dye reduction assay indicated that 1, 2
and 3 had a significant cytotoxic/antiproliferative effect in
comparison with other triazenes.53,54
The three triazene compounds described in this work
belong to the alkylating agents group with similar chemical,
physical, antimicrobial, nuclease and antitumor properties.
DTIC and TMZ also belong to this group, showing an
activity mechanism mainly related to the methylation of
Table 4. Inhibition of cell growth by triazenes 1, 2 and 3 and DTIC
assessed after 24 h exposure (MTT-dye reduction assay)
Compound
Tumor cell
AML1(b)
AML2(c)
AML3(d)
CP(e)
IC50 (mmol mL-1)(ª)
2.33
1
116.6
1.99
3.17
2
AML1(b)
AML2(c)
AML3(d)
CP(e)
10.73
129.3
1.95
2.17
3
AML1(b)
AML2(c)
AML3(d)
CP(e)
9.67
47.86
1.83
4.44
DTIC
AML2(c)
132.4
Figure 5. Concentration dependence of the effect of the 1, 2 and 3
triazenes on cell proliferation of acute myeloid leukemia (AML1,AML2,
AML3) and normal leukocytes (CP). Cells were exposed and viability
determined after 24 h. Each data point represents the arithmetic mean
standard deviation.
(ª) IC50 (dose required to achieve 50% decrease in cell growth). (b)AML1 =
acute myeloid leukemia cells of patient number 1. (c)AML2 = acute
myeloid leukemia cells of patient 2. (d)AML3 = acute myeloid leukemia
cells of patient 3. (e) CP = control patient cells of peripheral blood.

2236
In Vitro Evaluation of Triazenes: DNA Cleavage, Antibacterial Activity and Cytotoxicity
J. Braz. Chem. Soc.
O6-guanine, mediated by the methyldiazonium ion, a highly
reactive derivative of these chemotherapeutics agents. The
antitumor effects of alkylating agents and cell resistance
to them depend on at least three DNA repair systems:
a)increaseinO6-methyl-guaninemethyl-tranferase(MGMT),
also called O6-alkylguanine-DNA-alkyltranferase; b) loss of
mismatch repair (the MMR system recognizes and repairs
base-base mismatches and small insertion-deletions loops,
IDLs); and c) base excision repair (BER) deficiency.
Acknowledgements
M. H. thanks CNPq (proc. 305254/2009-0), F. K. thanks
CNPq/PIBIC, A. L. thanks CNPq and V. F. G. thanks
CAPES for grants. The diffractometer was supported by
the Financiadora de Estudos e Projetos (FINEP, CT-INFRA
03/2001). The authors are grateful to PRPGP/UFSM/Pró-
Publicações Internacionais for the language review of the
manuscript.
Conclusions
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The 1-(2-chlorophenyl)-3-(4-carboxyphenyl)triazene
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and 1-(2-fluorophenyl)-3-(4-amidophenyl)triazene (3)
compounds assessed in the present study are capable to
cleaveplasmidDNAunderdefinedpH, temperatureandtime
conditions. Since the efficiency of plasmid DNA cleavage by
the investigated triazenes was not inhibited in the presence
of free hydroxyl scavengers and anaerobic atmosphere
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natural nucleases. The experimental results indicated that
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positive bacteria. All compounds tested had pronounced
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normal leukocytes. Further studies to establish whether
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Submitted: October 25, 2009
Published online: August 10, 2010

J. Braz. Chem. Soc., Vol. 21, No. 12, S1-S2, 2010.
Printed in Brazil - ©2010 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
In Vitro Evaluation of Triazenes: DNA Cleavage, Antibacterial Activity and
Cytotoxicity against Acute Myeloid Leukemia Cells
Vanessa O. Domingues,a Rosmari Hörner,*,a Luiz G. B. Reetz,a Fábio Kuhn,a
Virgínia M. Coser,b Jacqueline N. Rodrigues,b Rita Bauchspiess,b
Waldir V. Pereira,b Gustavo L. Paraginski,c Aline Locatelli,c
Juliana de O. Fank,c Vinícius F. Giglioc and Manfredo Hörner*,c
a Universidade Federal de Santa Maria, Departamento de Análises Clínicas e Toxicológicas,
97105-970 Santa Maria-RS, Brazil
bHospital Universitário de Santa Maria, Departmento de Hematologia-Oncologia,
97105-970 Santa Maria-RS, Brazil
c Universidade Federal de Santa Maria, Departamento de Química, C.P. 5031,
97105-970 Santa Maria-RS, Brazil
Table S1. Minimum inhibitory concentration values (mg mL-1) determined for the triazenes 1, 2 and 3
Bacterial strain
Compound 1
> 128
> 128
> 128
> 128
> 128
> 128
> 128
= 128
> 128
> 128
> 128
= 128
= 32
Compound 2
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
= 64
Compound 3
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
Pseudomonas aeruginosa ATCC 27853
Escherichia coli ATCC 25922
Escherichia coli ATCC 35218
Salmonella choleraesuis ATCC 10708
Klebsiella pneumoniae ATCC 700603
Staphylococcus aureus ATCC 25923
Staphylococcus epidermidis ATCC 12228
Enterococcus faecalis ATCC 29212
Bacillus cereus ATCC 10987
Shigella sonnei
Enterobacter cloacae
Klebsiella oxytoca
Bacillus cereus
Micrococcus sp.
> 128
> 128
> 128
> 128
> 128
> 128
> 128
= 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
Streptococcus pyogenes
Acintobacter baumannii
Raustonia pickettii
Serratia marcescens
Enterococcus sp.
Listeria monocytogenes
Aeromonas hidrophyla
Enterobacter aerogenes
Streptococcus agalactiae
Salmonella tiphymurium
Corynebacterium sp.
> 128
>128
>128
> 128
> 128
> 128
> 128
Salmonella sp.
Staphylococcus epidermidis
Acinetobacter lwoffii
Klebsiella pneumoniae
Staphylococcus hominis subsp. hominis
*e-mail: rosmari.ufsm@gmail.com, hoerner.manfredo@gmail.com

S2
In Vitro Evaluation of Triazenes: DNA Cleavage, Antibacterial Activity and Cytotoxicity
J. Braz. Chem. Soc.
Figure S1. Total cleavage (%) of pUC18 and pBSKII plasmid DNA by triazene 3. Lane 1: control plasmid DNA (untreated pUC18 and pBSKII); Lanes
2-6: addition of compounds in different concentrations: 3.75, 1.875, 1.25, 0.75 and 0.375 mmol L-1. Incubation for 24 h at 37 and 50 °C, pH 6.5.