Accepted Manuscript In vitro and in silico studies of antioxidant activity of 2-thiazolylhydrazone derivatives Vinícius Gonçalves Maltarollo, Marina Ferrara de Resende, Thales Kronenberger, Cleudiomar Inácio Lino, Maria Clara Pinheiro Duarte Sampaio, Maira Galdino da Rocha Pitta, Moacyr Jesus Barreto de Melo Rêgo, Renata Adriana Labanca, Renata Barbosa de Oliveira PII:
To appear in:
Journal of Molecular Graphics and Modelling
Received Date: 8 June 2018 Revised Date:
17 September 2018
Accepted Date: 8 October 2018
Please cite this article as: Viní.Gonç. Maltarollo, M.F. de Resende, T. Kronenberger, Cleudiomar.Iná. Lino, M.C. Pinheiro Duarte Sampaio, M.G. da Rocha Pitta, M.J.B. de Melo Rêgo, R.A. Labanca, R.B. de Oliveira, In vitro and in silico studies of antioxidant activity of 2-thiazolylhydrazone derivatives, Journal of Molecular Graphics and Modelling (2018), doi: https://doi.org/10.1016/j.jmgm.2018.10.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ACCEPTED MANUSCRIPT 1
IN VITRO AND IN SILICO STUDIES OF ANTIOXIDANT ACTIVITY OF 2-
Vinícius Gonçalves Maltarolloa#, Marina Ferrara de Resendeb#, Thales Kronenbergerc,
Cleudiomar Inácio Linoa, Maria Clara Pinheiro Duarte Sampaiod, Maira Galdino da Rocha
Pittad, Moacyr Jesus Barreto de Melo Rêgod, Renata Adriana Labancab, Renata Barbosa de
Department of Pharmaceutical Products, Pharmacy Faculty, Federal University of
Minas Gerais, 6627 Antônio Carlos AVE, 31270-901, Belo Horizonte, Minas Gerais,
Antônio Carlos AVE, 31270-901, Belo Horizonte, Minas Gerais, Brazil.
Strasse 14, Tübingen, DE 72076.
Therapeutical Aproaches, Federal University of Pernambuco, Prof. Moraes Rêgo AVE,
50670-901, Recife, Pernambuco, Brazil.
Department of Internal Medicine VIII, University Hospital Tübingen, Otfried-Müller-
Laboratory of Immunomodulation
# Vinícius G. Maltarollo and Marina F. de Resende contributed equally to this work.
R. B. Oliveira
Av. Antônio Carlos 6627,
31270-901, Belo Horizonte, Minas Gerais, Brazil
E-mail: [email protected]
28 29 30 31
Departament of Biochemistry,
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Department of Foods, Pharmacy Faculty, Federal University of Minas Gerais, 6627
ACCEPTED MANUSCRIPT 32 33
The antioxidant potential of a series of thiazolylhydrazone derivatives was investigated
using three different methods namely DPPH, ABTS and FRAP assays. In general, the
tested compounds showed higher or comparable activity to that of curcumin, used as
positive control. Chemometric analyses demonstrated that the presence of hydrazone
moiety is required for the activity of this class of compounds. From these results,
compound 4 was identified as the most promising molecule and was then selected for
further studies. The antiproliferative effect of compound 4 was evaluated, being active
in three (T47D, MDA-MB-231 and SKMEL) of the six cancer cell lines tested, with
IC50 values ranging from 15.9 to 31.3 µM. Compound 4 exhibited no detectable
cytotoxic effect on peripheral blood mononuclear cells (PBMC) when tested at a
concentration of 100 µM, demonstrating good selectivity. From these results, it is
possible to infer that there is a correlation between antioxidant capacity and anticancer
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52 53 54 55 56 57 58 59 60 61 62 63 64 65
Keywords: antioxidant activity; 2-thiazolylhydrazone, chemometric; cytotoxic activity
ACCEPTED MANUSCRIPT 66 67 68 69
Thiazolylhydrazone derivatives are a class of compounds with a diverse spectrum of
biological activity. Representative examples of this class of compounds were reported
to exhibit anticancer, antibacterial, antifungal and antioxidant activities.1-6 According to
Mei-Hsiu Shih and co-workers, the scavenging activity of 2-thiazolylhydrazones is
related to the presence of N-H group in the hydrazone moiety, which has the ability to
donate a hydrogen atom and to reduce the DPPH radical (Figure 1).6
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Figure 1 - Schematic representation of the reaction between DPPH radical and 2-
There are several examples of chemometric techniques explaining antioxidant effects,
especially for relevant chemical classes such as the flavonoids.7,8 Choudhary et al. used
2D and 3D quantitative structure-activity relationship (QSAR) models to guide the
synthesis of a series of chalcone derivatives, which were then evaluated on their
antioxidant activity. It was suggested that the introduction of electron-releasing groups
and bulky heteroatoms at specific positions of the benzylideneacetophenone nucleus
increased the activity of the molecules.9 Another example resides in QSAR models
employing a series of antioxidant curcumin derivatives, which reported that the higher
ACCEPTED MANUSCRIPT activity could be attributed to their increased chemical stability. The chemical stability
was suggested by the lower hardness, higher softness and higher highest occupied
molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energies
gap for the molecules.10 Specifically, it was reported that hydrazine thiazole derivatives
exhibit potent DPPH radical-scavenging activity, on levels comparable with that of
vitamin E.11 This series was employed to generate descriptor-based quantitative
structure-activity relationship and classification models, which showed that fragments
bearing an aromatic carbon attached to three heteroatoms can contribute to explain the
high antioxidant activity.12
Based on the scavenging ability of this class of compounds, we evaluated the
antioxidant activity of a series of 2-thiazolylhydrazones derivatives, previously
synthesized in our laboratory.3 In addition, chemometric analyses were carried out to
establish a relationship between the molecular and electronic properties with the
antioxidant activity of these compounds. Since a correlation between antioxidant and
anticancer activities has been reported,13-15 the compound with the most promising
antioxidant activity was tested for cytotoxicity activity against different tumor cell lines.
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109 110 111
2. Materials and Methods
2.1 Chemometric analyses
The 2-thiazolylhydrazones derivatives 1-9 employed in multivariate analyses had their
3D structures determined by conformational analyses, using OMEGA 188.8.131.52
software16,17 with MMFF94 force-field18. Compounds 8 and 9 were simulated by using
R configuration for convention due the experimentally obtained compounds were
racemic mixtures. Then, the lowest energy conformers were employed in quantum
mechanics calculations, using the software Jaguar (v9.8 release 11)19 implemented in
the Maestro (2017v4, Schrodinger, NY, USA) package. All molecules underwent
geometry optimization calculations which were carried out with Density Functional
Theory (DFT) method with B3LYP functional and 6-311g (d,p) basis.20-22 The absence
of imaginary frequencies was used as a criterion to ensure that the optimized structures
represented the minimum in the potential energy surface. Subsequently, electronic
ACCEPTED MANUSCRIPT properties as frontier molecular orbital energies, hardness, softness, electronegativity,
dipole moment, atomic charges derived from electrostatic potential and related
properties, as well as the molecular surface area were calculated. Further,
physicochemical and molecular properties as n-octanol/water partition coefficient
(logP), molecular weight, number of hydrogen-bond acceptors and donors, topological
polar surface area, number of rotatable bonds, and hybridization ratio were calculated
with PaDEL descriptor software.23
Hierarchical Cluster Analysis (HCA) studies were carried out using Euclidean distance
as similarity measurement and average linking method and using values of experimental
antioxidant activities. In order to select the variables that better represent antioxidant
activity, the Pearson correlation coefficients between each independent variable and
DPPH, ABTS and FRAP values were calculated. Variables having low correlation with
experimental activities (R < 0.7), as well as high correlated variables, were not further
pursued in the study. This decision aimed to reduce overfitting due to the inclusion of
two properties related to the same physical phenomenon.24 The selected properties were
autoscaled before PCA. Both HCA and Principal Component Analysis (PCA) were
carried out with Chemoface 1.6 software.25
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The 2-thiazolylhydrazones derivatives 1-7 were obtained by reaction between an
aldehyde or ketone with thiosemicarbazide, followed by cyclization in the presence of
substituted or unsubstituted 2-bromoacetophenone, as previously described by us.3 The
starting ketones used in the synthesis of 8 and 9 were synthesized from cyclohexanone
by base-catalyzed aldol reaction by adaptation of the method reported in the literature.26
Using the procedure described in literature,3 8 was obtained as an orange solid in 99 %
yield. Mp: 182.1-182.9 °C; IR (cm-1): 3376, 3037, 2936, 2857, 1619, 1536, 1348, 1479,
1445, 737; 1H NMR (200 MHz, DMSO-d6), δ/ppm: 8.3 (1H, s); 8.2 (1H, d); 7.9-7.8
(3H, m); 7.6-7.6(2H, m); 7.4 (2H, d); 7.3 (1H, s); 5.2 (1H, d); 2.6(2H, m); 1.7-1.2 (8H,
ACCEPTED MANUSCRIPT 13
C NMR (50 MHz, DMSO-d6), δ/ppm: 169,6, 156.8, 149.3, 147.4, 143.2, 132.8,
132.5, 132.3, 128.4, 128.1, 126.5, 121.9, 121.4, 102.7, 74.2, 50.0, 30.0, 26.8, 24.7, 23,7;
HRMS (m/z) 457.1096 [M+H]+, calcd457.1101 C22H22ClN4O3S+.
Using the procedure described in literature,3 9 was obtained as a brown solid in 88%
yield. Mp: 215.3-215.8 °C; IR (cm-1): 3393, 3102, 2939, 2854, 1577, 1567, 1473, 730;
(1H, s), 5.1(1H, s), 5.0 (1H, m), 2.7 (1H, m), 2.2-1.3 (8H, m);
DMSO-d6), δ/ppm: 170.2, 154.7 153.4, 149.0, 133.7, 131.7, 128.6, 127.1, 121.8, 104.1,
70.3, 50.1, 27.3, 26.7, 25.9, 22.8; HRMS (m/z) 413.1197 [M+H]+, calcd413.1203
H NMR (200 MHz, DMSO-d6), δ/ppm: 8.4 (2H, m); 7.8 (2H, d), 7.4-7.3(4H, m), 7.0
C NMR (50 MHz,
2.3 In vitro antioxidant activity
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DPPH radical scavenging assay
The ability of synthetic compounds to scavenge 2,2-diphenyl-1-picrylhyadrazyl
(DPPH•) radical was measured using the method described by Lue et al.27 with
modifications by Bhullar et al.,28 in triplicate. The absorbance was measured at 515 nm
on a spectrophotometer with automatic microplate reader (Molecular Devices, Versa
Max Program, Sunnyvale, California, USA) and the percentage of DPPH that has
reacted was calculated using the following formula:
% antioxidant activity = ((Acontrol- Asample)/Acontrol)*100
being A = absorbance at 515 nm.
Curcumin and the Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid)
were used as positive control.
2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assay
The ABTS•+ scavenging capacity was evaluated according to the method described by
Re et al.29 with modifications,30 in triplicate. The absorbance was measured at 734 nm
in a spectrophotometer with automatic microplate reader (Molecular Devices,
VersaMaxProgram, Sunnyvale, California, USA). Curcumin and the Trolox were used
ACCEPTED MANUSCRIPT 187
as positive control. The amount of radical scavenged by the samples was calculated
using the following formula, and the result was expressed in percentage of antioxidant
% antioxidant activity = ((Acontrol- Asample)/Acontrol)*100 being A = absorbance at 734 nm.
Ferric reducing antioxidant power (FRAP) assay
To assess the antioxidant capacity based on ferric reduction, the method described by
Benzie and Strain31 was used with modifications,28,32 in triplicate. After reading the
absorbance at 593 nm in a spectrophotometer with automatic microplate reader
(Molecular Devices, VersaMaxProgram, Sunnyvale, California, USA), the antioxidant
potentials of the analyzed compounds were calculated based on Trolox standards at
concentrations of 50, 100, 150, 200, 250, 300 and 350 µM. The results were expressed
as Trolox equivalentes, or TE, in µM Trolox/ 100 µM sample. Curcumin was also
included in the assay as positive control.
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2.4 Cytotoxic activity
Cells were seeded at a density of 1 x 104 cells/well in 96-well plates with flat bottom in
the final volume of 100 µL. After 24 hours, cells were treated with RI83 at different
concentrations (1 to 100 µM) for 72 hours at 37º C with 5% CO2. Then, 20 µL per well
of (3-(4,5-dimethylthiazol-2-)-2,5-diphenyltetrazolium bromide (MTT) were added, for
three hours later, followed by 130 µL of 20% SDS. The absorbance was measured 24
hours later using ELx808 Microplate Reader (Biotek, USA) at a wavelength of 570 nm.
Non-treated cells were used as negative control, DMSO (0.1%) as vehicle control and
Doxorubicin as positive control. Experiments were performed three independent times
2.5 Selectivity assay
Peripheral blood mononuclear cells (PBMC) were obtained from whole blood of
healthy volunteers (N = 3). All donors signed a consent form and the study was
approved by Human Research Ethics Committee from Federal University of
Pernambuco (CEP/CCS/UFPE-11006). Cells were isolated using a density-gradient
method with Ficoll-Paque PLUS (GE Healthcare). At the end of the process, the cell
ACCEPTED MANUSCRIPT pellet was resuspended in RMPI-1640 medium supplemented with 10% fetal bovine
serum, 10 mM HEPES and 200 U/mL streptomycin and penicillin. Briefly, cells were
seeded in 96-well plates (5x105 cells per well) and treated with RI83 (1-100 µM) for 48
hours. After incubation, steps were performed as previously described in cytotoxic
activity topic. Selectivity index (SI) was calculated according to Islam et al (2016).33
Compounds with high selectivity have SI value > 3.33
3. Results and Discussion
Antioxidant activity of the compounds 1-9 (Figure 2) was assessed in vitro by three
different methods: 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay,
2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS•+) radical scavenging assay
and ferric reducing antioxidant power (FRAP) assay. Curcumin and the Trolox, a water-
soluble analog of vitamin E, were used as positive control. In these assays, it is
observed that some 2-thiazolylhydrazones showed better or similar results than that of
positive controls. The assays results are shown in Table 1.
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Figure 2 – Chemical structure of the 2-thiazolylhydrazones derivatives 1-9
Table 1 – Comparison of the results obtained for the in vitro antioxidant assays: DPPH radical scavenging assay; ABTS radical cation scavenging assay and ferric reducing antioxidant power (FRAP) assay FRAP (µM TE*/ 100
40.1 ± 1.4f
30.2 ± 2.0a
102.5 ± 0.8a
47.4 ± 1.5e
19.5 ± 0,9b
62.1 ± 2.3d 68.7 ± 2.1d
10.7 ± 0.7
61.6 ± 1.8b
11.4 ± 1.5c
53.9 ± 1.9d
10.6 ± 1.7c
37.2 ± 2.4f
32.6 ± 1.9a
57.3 ± 0.4c
16.1 ± 2.6g
67.7 ± 1.3a
38.9 ± 0.9f
39.3 ± 0.4f
38.3 ± 0.7
75.7 ± 0.8c
82.4 ± 0.9b
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62.9 ± 0.5d
22.7 ± 4.7b
28.9 ± 1.4g
9.8 ± 2.6c
53.8 ± 0.3f
18.3 ± 3.1b
64.1 ± 2.4d
9.1 ± 1.1c
58.6 ± 1.7e
18.7 ± 1.3b
*TE = Trolox equivalents. Means followed by the same lower case letter in columns do
not differ according to the Tukey’s test at 5% significance.
DPPH and ABTS assay methods measures the DPPH and ABTS radical consumption
rate by radical scavenger compounds. In DPPH assay, four compounds (2, 3, 5, and 7)
showed superior antioxidant activity than both curcumin and Trolox, and other four (1,
4, 8 and 9) showed similar antioxidant activity to that of curcumin. Regarding IC50
values in DPPH radical scavenging assay, seven compounds showed lower values than
that of curcumin (IC50 = 167.7 µM), being thus more active: 7 (IC50 = 97.1 µM), 3 (IC50
= 110.6 µM), 9 (IC50 = 118.8 µM), 1 (IC50 = 126.0 µM), 2 (IC50 = 126.6 µM), 8 (IC50 =
139.2 µM) and 5 (IC50 = 162.1 µM).
In ABTS assay, only the compound 4 showed comparable activity to that of curcumin
and compounds 4, 5, 6 and 8 exhibited higher or comparable scavenging potency than
ACCEPTED MANUSCRIPT FRAP assay is based on the ability of tested compounds to reduce ferric-
tripyridyltriazine (FeIII-TPTZ) to ferrous-tripyridyltriazine (FeII-TPTZ), a blue-colored
product. The results were expressed as Trolox equivalents (TE), in µM Trolox/ 100 µM
sample. Unfortunately, in this assay, no compound showed greater activity than that of
curcumin. However, among the compounds tested, the 2-thiazolylhydrazone 4 displayed
the best result.
After evaluation of antioxidant activity of the compounds, chemometric analyses were
performed to correlate such activity with a set of chemical and electronic descriptors
calculated from the molecular structure of the compounds. Initially, Hierarchical Cluster
Analysis (HCA) were applied using DPPH, ABTS and FRAP antioxidant activity
values to cluster compounds according to its antioxidant capacity. This step was
performed due to the divergence of experimental results of the antioxidant activity.
Compounds were separated into two major clusters (Figure 3A), the first cluster
containing compound 6, which is the least active (according to DPPH and FRAP
assays), selected as the dendrogram root, and compound 4 with the most antioxidant
activity, according ABTS and FRAP assays. The second cluster grouped all the other
molecules, which have a similar antioxidant profile.
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Figure 3. Dendrogram of HCA from antioxidant activities analysis (A); loadings plot
from PCA analysis showing the importance of each property for each principal
component (B) and; scores plot of the compound set from PCA using selected
properties highlighting two clusters from the hierarchical cluster analysis (C).
Subsequently, we searched for suitable calculated chemical or experimental properties,
which could explain the difference in antioxidant ability, by employing a correlation
matrix. Among the 39 calculated properties, only four (Table 2) were selected for
further studies, based on correlation with biological activities: (1) HOMO energy; (2)
ACCEPTED MANUSCRIPT 280
ESP balance: distribution of atomic charges along the molecular surface; (3) atomic
ESP charge in the sulphur atom and; (4) atomic ESP charge on the C1 atom.
Table 2 - Calculated properties for dataset compounds. C1 (a.u.)
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Cpd HOMO (a.u.) ESP balance S (a.u.)
From loadings plot of PCA (Figure 3B), is possible to note that atomic charges on S
and C1 atoms has the highest contributions to the first principal component, and ESP
balance and HOMO energy has the highest contribution to the second PC. Is also
possible to note that compounds 4 and 6 are separated from other along PC1 and all
compounds are separated from compound 6 along PC2 (Figure 3C). Altogether, it is
suggested that ESP balance and HOMO energy could explain the lack of activity of
compound 6 and atomic charges on S and C1 atoms could explain the differences of the
antioxidant profile of compound set. From raw calculated data, HOMO energies seem
to be almost invariable (ranging from -0.22 to -0.19 atomic unities). However, when
considering that all variables were auto-scaled before the chemometrics studies and its
conversion for a usual unity as kcal/mol (-138.05 to -119.23 kcal/mol), it is noteworthy
the large variation on electron donor ability of studied compounds.
The correlation between antioxidant activity and atomic charge properties34,35 and the
molecular orbital energies36 was already proposed in the literature by chemometrics
studies. The relevance of atomic charges of S and C1 atoms to antioxidant activity could
be related to the stabilization of the formed radical as well as the orientation of a
preferably oxidation site near thiazolylhydrazone moiety. The HOMO energy is an
important electronic parameter for describing the antioxidant ability of a molecule
ACCEPTED MANUSCRIPT because it is related to electron transfer reactions, in practical terms a compound with
low values of HOMO energy has a weak electron donating ability, as well as a
compound with high HOMO energy would is good electron-donor molecule.37-39 Then,
a plot of HOMO for all compounds was analyzed in terms of the atomic contributions
and we observed that this molecular orbital is mainly located at thiazolylhydrazone
moiety as illustrated by three representative compounds of dataset (Figure 4). All these
findings corroborate the proposed antioxidant mechanism would involve the hydrazone
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Figure 4. General structure of dataset compounds (A) and plot of HOMO of
representative compounds of dataset (B, C and D).
The results found in the present study are in agreement with those described by Mitra et
al.12 These authors generated several QSAR models with different methods which its
findings corroborate our results. From their work, the thiazolylhydrazone moiety as well
as aromatic rings are described to be structurally important to antioxidant activity and;
the hydrazine region has a molecular interaction field contour generate by a 3D-QSAR
model (calculations derived from atomic partial charges) indicating its electrostatic
importance for biological activity.12
Based on the results obtained, compound 4 was considered the most promising hit for
further studies. The cytotoxic activity of 4 was then evaluated on six cancer cell lines
ACCEPTED MANUSCRIPT 327
(T47D = human breast tumor; MDA-MB-231 = human breast adenocarcinoma; UACC
= human melanoma; A375 = human melanoma; SKMEL = human melanoma; PANC =
human pancreatic cancer cells). Peripheral blood mononuclear cells (PBMC) were used
as models for healthy cells to evaluate the selectivity of compound 4. The results
obtained are shown in Table 3.
Table 3 - Cytotoxicity of compound 4 on different cell lines. IC50 (µM) 25.3+0.3 15.9+2.4 >100 >100 31.3+4.0 >100 >100
SI* > 3.9 > 6.3 > 3.2 -
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Cell lines T47D MDA-MB-231 UACC A375 SKMEL PANC PBMC
Compound 4 exhibited significant antiproliferative effects against both breast cancer
cell lines, T47D and MDA-MB-231, and one of the melanoma cell lines (SKMEL)
tested, with IC50 values ranging from 15.9 to 31.3 µM. In addition, it should be noted
that 4 had no cytotoxic effect on non-malignant cells (PBMC), indicating good
selectivity. In these experiments, doxorubicin, a chemotherapeutic drug, was used as
positive control and showed IC50 values from 5 to 10 µM against all tumor cell lines
tested. It is worth mentioning that doxorubicin exhibit significant toxicity on PBMC
with IC50 value of 15.2 + 3.5 µM, displaying lower selectivity than 4.
Although still controversial, some studies indicate the effectiveness of the use of
antioxidants for the treatment of cancer. For example, antioxidant agents can lead to the
death of cancer cells from ROS-induced survival signaling pathways.40 ROS are potent
activators of nuclear factor κB (NF-κB), which plays a significant role in malignancy
and cancer progression. Therefore, there is a correlation between ROS, NF-κB
activation and tumor progression and compounds displaying antioxidant activity against
free radicals may exert antitumor activity by modulating these pathways.15
From these results, it is possible to infer that there is a correlation between antioxidant
capacity and anticancer effects.
ACCEPTED MANUSCRIPT 355
In the current work, a promising antioxidant compound was identified from a series of
2-thiazolylhydrazone derivatives. Compound 4 has been shown to have antioxidant and
antitumor potential, being active on three cancer cell lines tested, with an acceptable
selectivity index. The results obtained herein revealed compound 4 as a hit for the
development of new drugs, whose mechanism of action is ascribed to their antioxidant
The authors would like to thank OpenEye Scientific Software for OMEGA academic
license. This work was supported by the Fundação de Amparo à Pesquisa de Minas
Gerais (FAPEMIG), Conselho Nacional de Desenvolvimento Científico e Tecnológico
(CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
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Antioxidant potential of a series of thiazolylhydrazone derivatives was evaluated. Chemometric analyses showed that the hydrazone moiety is required for the activity. The results indicated the compound 4 as being the most promising one. The antiproliferative effect of compound 4 was also evaluated.