Synthesis and antioxidant activity of 1,3,4-oxadiazoles and their diacylhydrazine precursors derived from phenolic acids

Article abstract of DOI:10.1039/c6ra28787e

Eight 1,3,4-oxadiazole derivatives containing phenolic acid moieties (7a-h) and eight of their diacylhydrazine precursors (6a-h) were synthesized, characterized using spectroscopic methods and examined by scavenging of stable DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals. The most potent phenolic 1,3,4-oxadiazoles showed better DPPH scavenging activity in comparison with their corresponding diacylhydrazine precursors as a result of participation of both aromatic rings and a 1,3,4-oxadiazole moiety in resonance stabilization of the formed phenoxyl radical. Four diacylhydrazines (6d, 6e, 6g, and 6h) and four 1,3,4-oxadiazoles (7d, 7e, 7g and 7h) with the best DPPH scavenging activity, were chosen for further evaluation of their antioxidant potential through various assays. The investigated compounds exerted pronounced ABTS radical scavenging capacity, moderate to good H₂O₂ scavenging properties and strong ferric ion reducing capacity. Further in vitro evaluation of the antioxidant properties of the most active compounds demonstrated their protective effects in normal lung fibroblasts MRC-5 against hydrogen peroxide induced oxidative stress. Diacylhydrazine 6h increased two times the activity of glutathione peroxidase in treated cells in comparison with a control sample and did not affect the superoxide dismutase activity.

Full text of DOI:10.1039/c6ra28787e

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PAPER
Synthesis and antioxidant activity of 1,3,4-
oxadiazoles and their diacylhydrazine precursors
Cite this: RSC Adv., 2017, 7, 8550
derived from phenolic acids†
Nevena Mihailovic,a Violeta Markovic,a Ivana Z. Matic,b Nemanja S. Stanisavljevic,c
´
´
´
´
´
c
d
´
a
*
Zˇivko S. Jovanovic, Snezana Trifunovic and Ljubinka Joksovic
´
ˇ
Eight 1,3,4-oxadiazole derivatives containing phenolic acid moieties (7a–h) and eight of their diacylhydrazine
precursors (6a–h) were synthesized, characterized using spectroscopic methods and examined by scavenging
of stable DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals. The most potent phenolic 1,3,4-oxadiazoles showed
better DPPH scavenging activity in comparison with their corresponding diacylhydrazine precursors as a result
of participation of both aromatic rings and a 1,3,4-oxadiazole moiety in resonance stabilization of the formed
phenoxyl radical. Four diacylhydrazines (6d, 6e, 6g, and 6h) and four 1,3,4-oxadiazoles (7d, 7e, 7g and 7h) with
the best DPPH scavenging activity, were chosen for further evaluation of their antioxidant potential through
various assays. The investigated compounds exerted pronounced ABTS radical scavenging capacity,
moderate to good H2O2 scavenging properties and strong ferric ion reducing capacity. Further in vitro
evaluation of the antioxidant properties of the most active compounds demonstrated their protective
effects in normal lung fibroblasts MRC-5 against hydrogen peroxide induced oxidative stress.
Diacylhydrazine 6h increased two times the activity of glutathione peroxidase in treated cells in comparison
with a control sample and did not affect the superoxide dismutase activity.
Received 28th December 2016
Accepted 20th January 2017
DOI: 10.1039/c6ra28787e
rsc.li/rsc-advances
properties.6–8 The combination of the known phenolic antioxi-
dants with heterocyclic pharmacophores in the same unit can
1. Introduction
be an interesting approach to discover novel more potent
radical scavengers as a result of their synergistic effects.
Furthermore, one of such heterocycles, substituted 1,3,4-oxa-
diazole scaffold has been reported to exhibit a broad spectrum
of biological activity.9–12 Literature searches reveal that 2,5-
disubstituted 1,3,4-oxadiazole derivatives commonly obtained
from diacylhydrazines show antioxidant potential. This class of
compounds includes sulfonamidomethane linked 1,3,4-oxa-
diazoles,13 substituted bis(1,3,4-oxadiazoles),14 1,3,4-oxadia-
zoles possessing benzoxazole,15 1,3,4-oxadiazole tagged thieno
Reactive oxygen species (ROS) in the form of free radicals
(superoxide, singlet oxygen, hydroxyl radical) and neutral
molecules (hydrogen peroxide) induce damage of biological
macromolecules under oxidative stress and contribute to the
pathogenesis of many health problems such as cancer,
inammation, atherosclerosis, cardiovascular and neurode-
generative diseases.1–3 The antioxidants prevent the oxidation of
biological substrates, lowering oxidative stress, DNA mutations,
malignant changes and thus, block the loss of cell function.4
Owing to the possibility of facile hydrogen atom transfer and
resonance stabilization of the resulting phenoxyl radical,
phenolic acids act as chain-breaking antioxidants able to scav-
enge free radical species.5 Natural and synthetic phenolic acids
oen contain both hydroxyl and methoxy substituents and the
positioning of OH groups is crucial for their antioxidant
[2,3-d]pyrimidines,16
zoles,17
and 1,3,4-oxadiazoles
2-benzoylamino-5-hetaryl-1,3,4-oxadia-
containing 3-uoro-4-
methoxyphenyl moiety.18 To the best of our knowledge, there
is no available data regarding antioxidant activity of hydroxy-
substituted dibenzoylhydrazines in the literature. In this
study, a series of 1,3,4-oxadiazoles and their diacylhydrazine
precursors derived from phenolic acids was synthesized and
screened for radical scavenging properties using several assays.
a
´
Faculty of Science, Department of Chemistry, University of Kragujevac, R. Domanovica
12, 34000 Kragujevac, Serbia. E-mail: ljubinka@kg.ac.rs
bInstitute of Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade, Serbia
cInstitute of Molecular Genetics and Genetic Engineering, University of Belgrade,
Vojvode Stepe 444a, 11000 Belgrade, Serbia
2. Results and discussion
2.1. Chemistry
dFaculty of Chemistry, University of Belgrade, Studentski trg 12–16, 11000 Belgrade,
The synthesis of 1,3,4-oxadiazoles from 1,2-diacylhydrazines
requires various cyclodehydration agents such as SOCl2,19
Serbia
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c6ra28787e
POCl3,20,21
Burgess
reagent,22
and
2-chloro-1,3-
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Scheme 1 Reagents and conditions: (a) H2SO4, MeOH, 5 h, reflux; (b) N2H4 ꢀ H2O, EtOH, 12 h, reflux; (c) SOCl2, DMF, CH2Cl2, 2 h, r.t.; (d) THF,
6 h, r.t.; (e) SOCl2, 6 h, reflux.
dimethylimidazolinium chloride.23 A series of 2,5-disubstituted chloride (4a–h) were stirred at room temperature in dry THF,
1,3,4-oxadiazole antioxidants incorporating phenol moiety was giving the corresponding diacylhydrazines19 (6a–h) with satis-
prepared by reacting aryl hydrazides with substituted 4- factory purity without a need for further purication. It was
hydroxybenzoic acids in the presence of phosphorus oxy- possible to combine here the condensation of acid chloride
chloride.20 Nieddu et al. performed a synthesis of cytotoxic ArCOCl with acylhydrazine Ar0CONHNH2 or acid chloride
unsymmetrical 1,3,4-oxadiazoles also using POCl3 as cyclo- Ar0COCl with acylhydrazine ArCONHNH2. The selection of
dehydration agent.21 For cyclization of our 1,2-diacylhydrazines precursors for this type of reaction only depends on higher purity
containing more phenolic hydroxyl groups, thionyl chloride was and better yields of diacylhydrazines 6a–h. Finally, diacylhy-
the best choice, and for this purpose, we modied literature drazines 6a–h were cyclised to 1,3,4-oxadiazole derivatives 7a–h
procedure for the synthesis 1,3,4-oxadiazoles containing ada- by treatment with SOCl2 in the reux conditions.19
mantane ring19 replacing solvent and excluding base during the
preparation of 1,2-diacylhydrazines as well as excluding toluene was conrmed by means of H and 13C NMR spectroscopy (see
in the next cyclization step. ESI, Fig. S1–S32†). All aromatic protons for both classes of
The exact structure of synthesized compounds (6a–h and 7a–h)
1
The synthetic pathway for the targeted 1,3,4-oxadiazole compounds displayed signals in the characteristic region ranging
derivatives 7a–h is presented in Scheme 1. In the rst step, from 6.3 to 8.3 ppm. Signals present at the lower eld in 1H NMR
commercially available aromatic acids 1a–h were converted into spectra of compounds 6a–h correspond to OH and NH protons.
corresponding methyl esters 2a–h in the presence of H2SO4.24 The Their acidic nature was proven by the addition of two drops of D2O
obtained esters were then reacted with excess of hydrazine to NMR sample, which resulted in the disappearance of the cor-
hydrate yielding acylhydrazines (5a–h).25 In order to dissolve responding signals as a consequence of fast deuterium exchange.
1
polar impurities aer the evaporation of the solvent, small In H NMR spectra of 7a–h, due to the cyclization to 1,3,4-oxa-
amount of water was added to the crude residue and the formed diazole derivatives, signals attributed to NH protons disappeared
suspension was ltrated and dried. On the other side, commer- and remained only D2O exchangeable signals assigned to phenolic
cially available aromatic acids 3a–h were transformed into acid OH protons at the highest chemical shi values.
chlorides 4a–h in the reaction with thionyl chloride in the pres-
ence of catalytic amounts of N,N-dimethylformamide, according
to slightly modied procedure.26 The next step was performed
immediately due to the low stability of the formed acid chlorides.
The obtained acylhydrazine (5a–h) and corresponding acid
2.2. DPPH scavenging activity of diacylhydrazines and their
corresponding 1,3,4-oxadiazoles
The results of DPPH radical scavenging activity of the synthe-
sized diacylhydrazines and corresponding 1,3,4-oxadiazoles,
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Table 1 DPPH scavenging activity of diacylhydrazines and their cor-
1). In these compounds the formation of radicals is possible
through heterolytic OH and NH cleavage, as it was previously
demonstrated by DFT calculations for triazole derivatives.6 The
radical species formed from derivatives 6a–h aer the cleavage
of NH bond have no ability for resonance stabilization through
the corresponding aromatic ring. Therefore, their role in the
antioxidant activity of parent compounds is not signicant. On
the other hand, in the case of heterolytic OH-cleavage, the
unpaired electron of the formed radical can be delocalized
across the benzene ring (Scheme 2A), causing an easy formation
of phenoxyl radical with lower reactivity and higher stability.
The phenoxyl radical formed from the corresponding 1,3,4-
oxadiazole derivative (7a–h) can be highly stabilized through
resonance since the unpaired electron may be delocalized
across both aromatic rings and 1,3,4-oxadiazole part of the
molecule (Scheme 2B). All of the compounds which show
excellent antioxidant activity (7d, 7e, 7g and 7h) contain in their
structure two neighboring hydroxyl groups having the possi-
bility to form an intramolecular hydrogen bond (Scheme 2B).
The most active compound 7e possesses two hydroxyl groups in
m- and p-positions of the aromatic ring. Substitution of the m-
hydroxyl group with methoxy one (7c) leads to the loss of the
activity due to the less effective radical stabilization by intra-
molecular hydrogen bonding. Comparing the activity of
compound 7e with the activities of 7g and 7h it can be
concluded that the presence of additional hydroxyl groups
induces a slight decrease of activity. The presence of one or two
OH groups at the second benzene ring in 7g and 7h does not
contribute to resonance stabilization of radical due to the
negative inductive effect of the hydroxyl group. Compound 7f
exerts no antioxidant activity, as it was expected for the
responding 1,3,4-oxadiazolesa
Diacylhydrazines
Compound
1,3,4-Oxadiazoles
Compound
IC50 (mM) ꢁ SD
IC50 (mM) ꢁ SD
6a
6b
6c
6d
6e
6f
>1000
>1000
>1000
16.06 ꢁ 0.40
20.97 ꢁ 0.47
>1000
7a
7b
7c
7d
7e
7f
>1000
>1000
>1000
14.63 ꢁ 0.34
13.59 ꢁ 0.30
>1000
6g
6h
17.28 ꢁ 0.41
7g
7h
15.81 ꢁ 0.38
22.17 ꢁ 0.53
14.93 ꢁ 0.36
Referent antioxidants
Ascorbic acid
NDGA
38.78 ꢁ 0.60
20.83 ꢁ 0.23
a
Results are mean values ꢁ SD from three measurements.
expressed as concentration of tested compound that reduce
50% (IC50) of the DPPH radicals are presented in Table 1. From
all tested compounds, four diacylhydrazines and four 1,3,4-
oxadiazoles showed good antioxidant activity, with IC50 values
in the range of 13.59 to 22.17 mM, while IC50 values of ascorbic
acid and nordihydroguaiaretic acid (NDGA) were 38.78 and
20.83 mM, respectively.
Compared to the corresponding 1,3,4-oxadiazoles (7a–h),
their diacylhydrazine precursors derived from phenolic acids
(6a–h) have shown up to 50% weaker antioxidant activity (Table
Scheme 2 Resonance stabilization of radicals formed from compound 6e (A), and 7e (B), after heterolytic cleavage of OH bond, and loss of one
electron.
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Table 2 Antioxidant activities of selected diacylhydrazines and 1,3,4-oxadiazoles
FC reducing capacity
assay (gGAE per g)
Ferric ion reducinga capacity
H2O2 scavenging
assay IC50 (mM)
Compounds
assay (A700 nm
)
ABTS assay IC50 (mM)
6d
6e
6g
6h
7d
7e
7g
7h
1.141 ꢁ 0.013
1.256 ꢁ 0.007
1.107 ꢁ 0.003
1.470 ꢁ 0.019
0.918 ꢁ 0.008
1.153 ꢁ 0.016
1.450 ꢁ 0.006
1.209 ꢁ 0.006
—
0.296 ꢁ 0.009
0.478 ꢁ 0.011
0.461 ꢁ 0.008
0.478 ꢁ 0.009
0.270 ꢁ 0.005
0.367 ꢁ 0.015
0.313 ꢁ 0.005
0.426 ꢁ 0.004
0.173 ꢁ 0.007
120.1 ꢁ 5.7
104.4 ꢁ 11.6
133.3 ꢁ 35.7
102.6 ꢁ 7.5
118.4 ꢁ 31.2
79.3 ꢁ 2.6
37.41 ꢁ 0.89
62.24 ꢁ 3.97
43.92 ꢁ 0.38
57.00 ꢁ 1.43
100.33 ꢁ 5.89
107.58 ꢁ 9.02
84.89 ꢁ 0.70
75.17 ꢁ 5.23
22.40 ꢁ 0.60
137.7 ꢁ 30.4
120.6 ꢁ 18.5
242.6 ꢁ 7.3
Ascorbic acid
a
Concentrations of tested compounds and ascorbic acid are set at 500 mM.
molecule which contains two m-hydroxyl groups in its structure suitable for the estimation of antioxidant capacity.27 It could be
and it does not allow extended resonance stabilization.
particularly useful for comparison of reducing capacities of
natural products and newly synthesized compounds since the
results are oen expressed in mg or g of gallic acid or catechin
equivalents per gram of tested sample which allows direct
comparison with available literature data. In the present study
majority of investigated compounds exhibited a stronger
reduction of F–C complex than gallic acid which was used as
standard with the exception of compound 7d which was
approximately 10% less efficient in the reduction of F–C reagent
than the gallic acid. Sample 6h possessed the strongest
reducing capacity among tested compounds (Table 2).
Considering the results of ferric ion reducing capacity assay
(Table 2) it can be observed that all examined compounds
showed higher capacities for Fe3+ reduction than ascorbic acid
which was used as standard. In concordance with the previous
assay compound 7d exhibited the lowest reduction capacity
towards Fe3+ ion and two samples with the strongest reduction
capacity were 6e and 6h.
From the mechanistic standpoint discrepancies in two
previous reducing capacity tests and ABTS assay could be
explained either by different redox potential of counterparts
reacting with the tested antioxidant compounds or by different
reaction times needed for reaching the steady-state time point.
The difference in steric accessibility of antioxidant compounds to
ABTS radical cation, F–C complex and Fe3+ ion could be also the
possible reason for observed discrepancies between the assays.27
2.3. ABTS, H2O2 scavenging activity and reducing capacity
The compounds which showed the best DPPH radical scavenger
activities 6d, 6e, 6g, 6h, 7d, 7e, 7g and 7h have been chosen for
further examination of their in vitro antioxidant properties
through ABTS radical scavenging, peroxide scavenging assays
and reducing capacity. The obtained results are summarized in
Table 2.
Radical scavenging capacity of synthesized compounds was
tested using ABTS radical cation scavenging assay, which is
another widely used method for the estimation of in vitro
antioxidant ability. The obtained results (Table 2) showed that
all tested compounds possessed more pronounced scavenging
capacities than ascorbic acid (IC50 ¼ 242.6 mM). Similarly to
previous DPPH assay compound 7e (IC50 ¼ 79.3 mM) showed to
be the most potent scavenger of ABTS radical cation which was
followed by samples 6e and 6h. Meanwhile 6d and 7h exhibited
the weakest scavenging capacities (IC50 ¼ 120.1 mM and IC50
¼
120.6 mM respectively).
Hydrogen peroxide is a biologically relevant, oxidizing
species which is formed in numerous animal tissues through
several oxidative processes and can diffuse across biological
membranes, causing oxidative damage to DNA, proteins, lipids
and other biomolecules. According to our results presented in
Table 2 all tested compounds showed weaker H2O2 scavenging
properties than the ascorbic acid (IC50 ¼ 22.4 mM). Among
tested samples 6d showed the most prominent scavenging
capacity with IC50 value approximately two times higher than
Table 3 Concentrations of diacylhydrazines and 1,3,4-oxadiazoles
which induced 50% decrease in MRC-5 cell survival after 72 h
the ascorbic acid (IC50 ¼ 37.41 mM). The obtained results are treatment
comparable to data collected by Kotaiah et al.16 who examined
H2O2 scavenging capacities of 1,3,4-oxidazole tagged thieno[2,3-
Compound
IC50 ꢁ SD [mM]
d]pyrimidine derivatives using the same assay. On contrary to
our results all compounds examined in the mentioned study
6d
6e
6g
6h
7d
7e
7g
7h
118.35 ꢁ 9.41
194.27 ꢁ 2.69
86.33 ꢁ 2.41
91.83 ꢁ 8.57
26.88 ꢁ 1.66
37.49 ꢁ 0.20
34.88 ꢁ 1.93
25.59 ꢁ 1.76
9.24 ꢁ 0.18
exhibited H2O2 scavenging capacities near to the value obtained
for the ascorbic acid (IC50 ¼ 16.35).
Folin–Ciocalteu reducing capacity assay (F–C) has been
widespread assay for measurement of total phenolics in natural
products, although the basic mechanism is an oxidation–
reduction process, thus it can be considered as a method
Cisplatin
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Fig. 1 Effects of 24 h pretreatment of MRC-5 cells with diacylhydrazines and 1,3,4-oxadiazoles on ROS production induced by hydrogen
peroxide. (A) Fluorescence histograms; (B) fluorescence intensity of generated dichlorofluorescein in MRC-5 cells. Applied non-toxic
concentrations of compounds were: 50 mM for 6d, 6e, 6g and 6h, and 20 mM for 7d, 7e, 7g and 7h.
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Table 4 Activities of antioxidant enzymes in control MRC-5 cells and cells exposed for 24 h to non-toxic concentrations of investigated
diacylhydrazines and 1,3,4-oxadiazoles
Glutathione reductase
Glutathione peroxidase
activity (U mgꢂ1 protein)
Superoxide dismutase
activity (mU mgꢂ1 protein)
activity (U mgꢂ1 protein)
Control
6d
6e
6g
6h
7d
7e
7g
7h
10.09 ꢁ 0.44
11.18 ꢁ 0.76
7.58 ꢁ 1.02
5.67 ꢁ 0.33
3.48 ꢁ 0.07
3.59 ꢁ 0.01
2.19 ꢁ 0.06
0.86 ꢁ 0.17
1.18 ꢁ 0.11
1.17 ꢁ 0.16
0.86 ꢁ 0.11
0.87 ꢁ 0.09
0.05 ꢁ 0.02
2.26 ꢁ 0.21
0.66 ꢁ 0.08
0.67 ꢁ 0.12
0.37 ꢁ 0.07
0.37 ꢁ 0.10
2.70 ꢁ 0.24
2.66 ꢁ 0.21
2.53 ꢁ 0.14
2.57 ꢁ 0.23
2.66 ꢁ 0.31
2.64 ꢁ 0.16
2.76 ꢁ 0.15
2.73 ꢁ 0.22
2.79 ꢁ 0.30
progression of malignant tumors.28 Due to their prominent
antioxidant effects, and especially the ability to reduce intracel-
lular ROS levels, the examined 1,3,4-oxadiazoles and their diac-
ylhydrazine precursors might serve as an useful cancer
chemopreventive agents.
2.4. Cytotoxic activity of diacylhydrazines and 1,3,4-
oxadiazoles on MRC-5 cells
All synthesized compounds which exerted the most intensive
antioxidant effects as shown by DPPH assay, were chosen for
further evaluation of their protective properties in normal cells.
For that reason, we rst examined the cytotoxic activity of
selected diacylhydrazines 6d, 6e, 6g, 6h, and 1,3,4-oxadiazoles
7d, 7e, 7g and 7h against normal human lung broblasts MRC-5
2.6. Effects of diacylhydrazines and 1,3,4-oxadiazoles on the
activity of antioxidant enzymes
(results are presented in Table 3). Among tested compounds, Several enzymatic defense mechanisms attempt to minimize
1,3,4-oxadiazoles 7h and 7d exerted the highest intensity of the the production and the action of harmful oxidants, such as
cytotoxic activity on MRC-5 cells. The compounds 7g and 7e also superoxide dismutase (SOD), glutathione reductase (GR) and
showed pronounced cytotoxicity with IC50 values approximately glutathione peroxidase (GPX). SOD dismutates the free radical
30% higher than 7h and 7d. However, the examined diacylhy- superoxide by converting it to hydrogen peroxide, which in turn
drazines exhibited signicantly lower cytotoxic effects in is decomposed by catalase at high concentration, and by GPX at
comparison with 1,3,4-oxadiazoles. The cytotoxicity of low concentration. Most hydrogen peroxide in the cell is
compounds 6g and 6h was approximately three times lower generated through the dismutation of superoxide by SOD
than the most active compounds 7d and 7h. The compound 6d action, even though it can be produced by other superoxide-
showed very low cytotoxic activity, while the activity of 6e was generating enzymes.29,30 In our experiments we also investi-
quite poor.
gated the effects of 1,3,4-oxadiazoles and their diacylhydrazine
precursors derived from phenolic acids on the activities of
antioxidant enzymes, GR, GPX and SOD in MRC-5 cells (Table
4). It is evident that applied compounds drastically reduced the
activity of enzymes involved in glutathione metabolism – GR
and GPX. The only exception is diacylhydrazine 6h which
increased two times the GPX activity in exposed cells in
comparison with the control cell sample. However, no signi-
cant changes were noticed on SOD activity. The decrease in GPX
activity could suggest inactivation by ROS.31 Superoxide anions
have been shown to inhibit GPX.32 However, it is more likely
that such decrease in the activity of GR and GPX could be
attributed to other mechanisms, not to the production of ROS,
having in mind that there were no changes in SOD activity
during applied treatment. It is well known that some 1,3,4-
oxadiazoles, but only small group of them, could inhibit the
enzymes of glutathione metabolism, dominantly GR.33 That is
very important for using of 1,3,4-oxadiazoles and their deriva-
tives as anthelmintic agents.
2.5. Effects of pretreatment of MRC-5 cells with
diacylhydrazines and 1,3,4-oxadiazoles on ROS generation
induced by H2O2
To fully evaluate the antioxidant potential of synthesized diac-
ylhydrazines and 1,3,4-oxadiazoles, their possible cytoprotective
effects against the generation of ROS induced by hydrogen
peroxide were examined in normal human lung broblasts MRC-
5. As it could be seen in Fig. 1, pretreatment for 24 h of MRC-5
cells with non-toxic concentrations of all tested compounds (50
mM for diacylhydrazines and 20 mM for 1,3,4-oxadiazoles, as
determined for 24 h treatment) reduced the levels of ROS in MRC-
5 cells treated for 30 min with H2O2 compared with cells that were
grown in the nutrient medium during 24 h and then exposed to
H2O2. The percentages labeled in each histogram represent the
subpopulation of cells with increased uorescence levels, i.e. with
increased levels of ROS. The effectiveness of diacylhydrazines and
1,3,4-oxadiazoles in attenuating the oxidative stress in normal
broblasts in addition to the results of the antioxidant assays
suggest the signicant antioxidant potential of the investigated
3. Conclusion
compounds. Higher intracellular level of ROS is implicated in the Four diacylhydrazines (6d, 6e, 6g, and 6h) and four 1,3,4-oxa-
early steps of malignant transformation, as well as in the further diazoles (7d, 7e, 7g and 7h) showing the best DPPH scavenging
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activity, with IC50 values in the range of 13.59 to 22.17 mM were a small amount of water was added (2 mL) and the suspension
selected for further evaluation of their antioxidant potential was ltrated giving 5a–h.
through various assays. The excellent ABTS radical scavenging
4.2.2. Preparation of 7a–h. To the mixture of a corre-
potential displayed compounds 6h, 6e and 7e. Considering the sponding aromatic acid 3a–h (1.00 mmol) in dry dichloro-
structures of the compounds which showed the strongest methane (4.0 mL), SOCl2 (0.3 mL, 4.00 mmol) was slowly added,
antioxidant activity (6d, 6e, 6g, 6h, 7d, 7e, 7g and 7h), it can be followed by two drops of dimethylformamide. The resulting
noticed that they all contain two neighboring hydroxyl groups mixture was then stirred for 2 h at room temperature. Aer-
having the possibility to form an intramolecular hydrogen wards, the solvent was evaporated under reduced pressure, and
bond. The most potent compound 7e possesses two hydroxyl the excess of SOCl2 was removed by azeotropic distillation with
groups in m- and p-positions of the aromatic ring, while the toluene. To the formed acid chloride 4a–h (1.00 mmol), a cor-
presence of additional hydroxyl groups induces a slight responding acid hydrazide 5a–h (1.30 mmol) and dry tetrahy-
decrease of activity (7g and 7h). The compounds 6d, 6e, 6g and drofuran (10.0 mL) were added, and resulting mixture was
6h possessed moderate to good H2O2 scavenging properties; the stirred at room temperature for 6 h. Then, the solvent was
most potent was compound 6d. The diacylhydrazines 6e and 6h evaporated under reduced pressure, small amount of water was
showed the strongest ferric ion reducing capacities. In addition, added (2 mL) and the formed precipitate (6a–h) was isolated by
the majority of compounds exhibited a stronger reduction of ltration and dried over CaCl2. The mixture of 6a–h (1.00 mmol)
F–C complex than gallic acid; among them, compound 6h had in SOCl2 (6.00 mL) was then reuxed for 6 h. Aer cooling, the
the strongest capacity. The tested compounds applied at non- ice was added to the solution and formed suspension was stir-
toxic concentrations showed the ability to reduce intracellular red at room temperature for 30 min and le standing overnight
levels of oxidative stress induced by hydrogen peroxide in at 4 ꢃC and compound 7a–h was ltered off and dried over
normal human lung broblasts MRC-5. Non-toxic concentra- CaCl2. The nal compounds 7a–h needed further purication
tions of the examined compounds reduced the activities of by recrystallization from hot aqueous solution of 45% EtOH.
glutathione reductase and glutathione peroxidase in treated
4.2.2.1. N0-Benzoyl-2-hydroxybenzohydrazide ꢀ 0.5H2O (6a).
1
ꢃ
MRC-5 cells. The exception was diacylhydrazine 6h which White powder; yield: 0.24 g (89%); mp: >250 C; H NMR (200
increased two times the activity of glutathione peroxidase in MHz, DMSO-d6): 6.92–7.00, (m, 2H, Ar-H); 7.43–7.62, (m, 4H, Ar-
treated cells in comparison with control sample. However, the H); 7.93, (dd, 3H, J ¼ 7.9 and 1.2 Hz, Ar-H); 10.69, (s, 2H, NH);
compounds did not affect the superoxide dismutase activity. 11.95, (s, 1H, NH); 13C NMR (50 MHz, DMSO-d6): 114.7, 117.5,
Results of our study point out the signicant antioxidant 119.1, 127.6 (2C), 128.4, 128.6 (2C), 132.0, 132.8, 134.2, 159.4,
potential of selected 1,3,4-oxadiazole derivatives and their 165.7, 167.8; IR (KBr, cmꢂ1): 3304; 3058; 1657; 1635; 1548; 1480;
diacylhydrazine precursors, especially of compounds 6h and 7e. 1283; 759; anal. calcd for C14H12N2O3 ꢀ 0.5H2O (265.27 g
molꢂ1): C, 63.39; H, 4.94; N, 10.56; found: C, 63.53; H, 4.92; N,
10.60.
4.2.2.2. N0-Benzoyl-3-hydroxybenzohydrazide ꢀ 0.5H2O (6b).
4. Experimental
4.1. Physical measurements and methods
1
Beige powder; yield: 0.20 g (74%); mp: >250 C; for H and 13C
ꢃ
NMR see ref. 34 or our spectral data placed in ESI.†
4.2.2.3. N0-Benzoyl-4-hydroxy-3-methoxybenzohydrazide
ꢀ
Melting points were determined on a Mel-Temp capillary
melting points apparatus, model 1001 and are uncorrected.
Elemental (C, H, N, S) analysis of the samples was carried out in
the Center for Instrumental Analysis, Faculty of Chemistry,
Belgrade. UV spectra were recorded using an Agilent Technol-
ogies, Cary 300 Series UV-Vis Spectrophotometer. IR spectra
were obtained on a Perkin Elmer Spectrum One FT-IR spec-
0.5H2O (6c). Beige powder; yield: 0.18 g (60%); mp: 219–220 ꢃC;
1H NMR (200 MHz, DMSO-d6): 3.83, (s, 3H, CH3); 6.86, (d, 1H, J
¼ 8.2 Hz, Ar-H); 7.42–7.60, (m, 5H, Ar-H); 7.92, (dd, 2H, J ¼ 7.4
and 1.6 Hz, Ar-H); 9.73, (s, 1H, OH); 10.28, (s, 1H, NH); 10.42, (s,
1H, NH); 13C NMR (50 MHz, DMSO-d6): 55.8, 111.6, 115.1, 121.3,
123.6, 127.5 (2C), 128.5 (2C), 131.8, 132.9, 147.3, 150.2, 165.6,
166.0; IR (KBr, cmꢂ1): 3218; 3009; 1633; 1593; 1513; 1287; 690;
anal. calcd for C15H14N2O4 ꢀ 0.5H2O (295.29 g molꢂ1): C, 61.01;
H, 5.12; N, 9.49; found: C, 60.94; H, 5.14; N, 9.47.
1
trometer with a KBr disc. H and 13C-NMR spectra were taken
on a Varian Gemini 200 MHz spectrometer.
4.2.2.4. N0-Benzoyl-2,3-dihydroxybenzohydrazide
(6d). White powder; yield: 0.23 g (82%); mp: 226–227 ꢃC (Dec.);
ꢀ
0.5H2O
4.2. Procedure for the preparation of 7a–h
4.2.1. Preparation of 5a–h. The mixture of a corresponding 1H NMR (200 MHz, DMSO-d6): 6.76, (t, 1H, J ¼ 7.8 Hz, Ar-H);
aromatic acid 1a–h (16.00 mmol) and H2SO4 (0.7 mL) in 6.99, (dd, 1H, J ¼ 7.8 and 1.2 Hz, Ar-H); 7.39, (dd, 1H, J ¼ 8.0
methanol (10 mL) was reuxed for 5 h. Aer evaporation of and 1.2 Hz, Ar-H); 7.49–7.66, (m, 1H, Ar-H); 7.93, (dd, 2H, J ¼ 7.4
methanol, water is added and crude ester was extracted with and 1.6 Hz, Ar-H); 9.38, (s, 1H, OH); 10.64, (s, 1H, NH); 10.72, (s,
EtOAc (3 ꢀ 15 mL). The organic layer was washed with NaHCO3 1H, NH); 11.96, (s, 1H, OH); 13C NMR (50 MHz, DMSO-d6):
(15 mL) and H2O (15 mL), dried with Na2SO4, ltered and the 114.3, 117.7, 118.6, 119.5, 127.6 (2C), 128.6 (2C), 132.0, 132.5,
solvent was evaporated in vacuo giving the corresponding ester 146.3, 149.2, 165.8, 168.9; IR (KBr, cmꢂ1): 3434; 3224; 3057;
2a–h. The ester of the corresponding aromatic acid 2a–h (8.00 1636; 1602; 1579; 1535; 1310; 1265; 737; anal. calcd for
mmol) and hydrazine hydrate (1.2 mL, 24.00 mmol) in EtOH (30
mL) were reuxed for 12 h. Then, the solvent was evaporated, 9.96; found: C, 59.73; H, 4.67; N, 9.93.
C
14H12N2O4 ꢀ 0.5H2O (281.27 g molꢂ1): C, 59.78; H, 4.66; N,
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4.2.2.5. N0-Benzoyl-3,4-dihydroxybenzohydrazide
RSC Advances
150.6, 163.5, 164.4; IR (KBr, cmꢂ1): 3432; 3204; 1600; 1502; 1432;
1288; 726; anal. calcd for C15H12N2O3 ꢀ 2.5H2O (313.31 g molꢂ1):
C, 57.50; H, 5.47; N, 8.94; found: C, 57.53; H, 5.46; N, 8.96.
4.2.2.12. 3-(5-Phenyl-1,3,4-oxadiazol-2-yl)benzene-1,2-diol (7d).
Beige crystals; yield: 0.15 g (58%); mp: 172–173 ꢃC; for 1H and 13C
NMR see ref. 36 or our spectral data placed in ESI.†
ꢀ
1.5H2O
(6e). Beige powder; yield: 0.17 g (57%); mp: 166–167 ꢃC; 1H NMR
(200 MHz, DMSO-d6): 6.80, (d, 1H, J ¼ 8.2 Hz, Ar-H); 7.29, (d, 1H,
J ¼ 8.2 and 2.0 Hz, Ar-H); 7.34, (d, 1H, J ¼ 2.0 Hz, Ar-H); 7.46–
7.59, (m, 3H, Ar-H); 7.91, (dd, 2H, J ¼ 7.2 and 1.6 Hz, Ar-H); 9.25,
(s, 1H, OH); 9.59, (s, 1H, OH); 10.16, (s, 1H, NH); 10.36, (s, 1H,
NH); 13C NMR (50 MHz, DMSO-d6): 115.1, 115.5, 119.5, 123.9,
127.5 (2C), 128.5 (2C), 131.8, 132.9; 145.1, 149.0, 165.9, 166.0; IR
(KBr, cmꢂ1): 3215; 3055; 1618; 1600; 1576; 1516; 1489; 1296; 688;
anal. calcd for C14H12N2O4 ꢀ 1.5H2O (299.28 g molꢂ1): C, 56.38;
H, 4.73; N, 9.32; found: C, 56.40; H, 4.74; N, 9.34.
4.2.2.13. 4-(5-Phenyl-1,3,4-oxadiazol-2-yl)benzene-1,2-diol
ꢀ
2H2O (7e). Beige powder; yield: 0.16 g (56%); mp: 220–221 ꢃC; 1H
NMR (200 MHz, DMSO-d6): 6.93, (d, 1H, J ¼ 8.0 Hz, Ar-H); 7.45,
(dd, 1H, J ¼ 8.0 and 2.0 Hz, Ar-H); 7.50, (d, 1H, J ¼ 2.0 Hz, Ar-H);
7.61–7.64, (m, 3H, Ar-H); 8.05–8.10, (m, 2H, Ar-H); 9.58, (s, 1H,
OH); 9.86, (s, 1H, OH); 13C NMR (50 MHz, DMSO-d6): 113.8,
114.4, 116.4, 119.1, 123.7, 126.6 (2C), 129.5 (2C), 131.9; 146.0,
149.5, 163.4, 164.5; IR (KBr, cmꢂ1): 3421; 3253; 1608; 1506; 1448;
1296; 698; anal. calcd for C14H10N2O3 ꢀ 2H2O (290.28 g molꢂ1):
C, 57.92; H, 4.86; N, 9.65; found: C, 57.90; H, 4.87; N, 9.63.
4.2.2.14. 5-(5-Phenyl-1,3,4-oxadiazol-2-yl)benzene-1,3-diol (7f).
4.2.2.6. N0-Benzoyl-3,5-dihydroxybenzohydrazide (6f). Light
1
ꢃ
beige powder; yield: 0.23 g (84%); mp: >250 C; H NMR (200
MHz, DMSO-d6): 6.41, (t, 1H, J ¼ 2.2 Hz, Ar-H); 6.75, (d, 2H, J ¼
2.2 Hz, Ar-H); 7.47–7.60, (m, 3H, Ar-H); 7.91, (dd, 2H, J ¼ 8.0 and
1.6 Hz, Ar-H); 9.56, (s, 2H, OH); 10.26, (s, 1H, NH); 10.41, (s, 1H,
NH); 13C NMR (50 MHz, DMSO-d6): 105.8 (3C), 127.5 (2C), 128.5
(2C), 131.8, 132.8, 134.9; 158.5 (2C); 165.8; 166.2; IR (KBr, cmꢂ1):
3340, 3198; 1676; 1649; 1599; 1541; 1501; 1160; 706; anal. calcd
for C14H12N2O4 (272.26 g molꢂ1): C, 61.76; H, 4.44; N, 10.29;
found: C, 61.73; H, 4.45; N, 10.31.
1
ꢃ
Light yellow powder; yield: 0.19 g (71%); mp: >250 C; H NMR
(200 MHz, DMSO-d6): 6.46, (t, 1H, J ¼ 2.2 Hz, Ar-H); 6.98, (d, 2H, J
¼ 2.2 Hz, Ar-H); 7.62–7.66, (m, 3H, Ar-H); 8.06–8.11, (m, 2H, Ar-
H); 9.84, (s, 2H, OH); 13C NMR (50 MHz, DMSO-d6): 104.8 (2C),
106.2, 123.5, 124.7, 126.7 (2C), 129.5 (2C); 132.1; 159.3 (2C); 163.9;
164.3; IR (KBr, cmꢂ1): 3493, 3386; 3102; 1624; 1608; 1567; 1555;
1448; 1160; 853; anal. calcd for C14H10N2O3 ꢀ H2O (272.26 g
molꢂ1): C, 61.76; H, 4.44; N, 10.29; found: C, 61.75; H, 4.43; N,
10.27.
4.2.2.7. 3,4-Dihydroxy-N0-(2-hydroxybenzoyl)benzohydrazide ꢀ
1.5H2O (6g). Beige powder; yield: 0.28 g (88%); mp: >250 ꢃC; 1H
NMR (200 MHz, DMSO-d6): 6.81, (d, 1H, J ¼ 8.2 Hz, Ar-H); 6.91–
6.99, (m, 2H, Ar-H); 7.29, (dd, 1H, J ¼ 8.2 and 2.0 Hz, Ar-H); 7.34,
(d, 1H, J ¼ 2.0 Hz, Ar-H); 7.46, (td, 1H, J ¼ 8.0 and 1.6 Hz, Ar-H);
7.92, (dd, 1H, J ¼ 8.0 and 1.6 Hz, Ar-H); 9.27, (s, 1H, OH); 9.63, (s,
1H, OH); 10.32, (s, 1H, NH); 10.58, (s, 1H, NH); 12.03, (s, 1H, OH);
13C NMR (50 MHz, DMSO-d6): 114.7, 115.2, 115.4, 117.5, 119.1,
119.6, 123.5, 128.3, 134.2, 145.1, 149.2, 159.5, 165.6, 168.0; IR
(KBr, cmꢂ1): 3288, 3055; 2956; 1638; 1605; 1526; 1489; 1302; 1219;
754; anal. calcd for C14H12N2O5 ꢀ 1.5H2O (315.28 g molꢂ1): C,
53.34; H, 4.80; N, 8.88; found: C, 53.35; H, 4.79; N, 8.90.
4.2.2.8. N0-(3,5-Dihydroxybenzoyl)-3,4-dihydroxybenzohydrazide
ꢀ 0.5H2O (6h). Beige powder; yield: 0.19 g (62%); mp: >250 ꢃC; 1H
NMR (200 MHz, DMSO-d6): 6.39, (t, 1H, J ¼ 2.2 Hz, Ar-H); 6.73, (d,
1H, J ¼ 2.2 Hz, Ar-H); 6.79, (d, 1H, J ¼ 8.2 Hz, Ar-H); 7.27, (dd, 1H, J
¼ 8.2 and 2.0 Hz, Ar-H); 7.33, (d, 1H, J ¼ 2.0 Hz, Ar-H); 9.24, (s, 1H,
OH); 9.54, (s, 2H, OH); 9.58, (s, 1H, OH); 10.06, (s, 1H, NH); 10.11,
(s, 1H, NH); 13C NMR (50 MHz, DMSO-d6): 105.7, 105.8 (2C), 115.1,
115.5, 119.5, 124.0, 135.1, 145.1, 149.0, 158.4 (2C), 165.7, 166.3; IR
(KBr, cmꢂ1): 3385; 3279; 1598; 1508; 1299; 1168; 859; anal. calcd
for C14H12N2O6 ꢀ 0.5H2O (313.27 g molꢂ1): C, 53.68; H, 4.18; N,
8.94; found: C, 53.66; H, 4.19; N, 8.92.
4.2.2.15. 4-(5-(2-Hydroxyphenyl)-1,3,4-oxadiazol-2-yl)benzene-
1,2-diol ꢀ 0.5H2O (7g). Light brown powder; yield: 0.22 g (78%);
1
ꢃ
mp: >250 C; H NMR (200 MHz, DMSO-d6): 6.94, (d, 1H, J ¼
8.2 Hz, Ar-H); 7.00–7.12, (m, 2H, Ar-H); 7.42, (dd, 1H, J ¼ 8.2 and
1.6 Hz, Ar-H); 7.47–7.51, (m, 2H, Ar-H); 7.87, (dd, 1H, J ¼ 8.0 and
1.0 Hz, Ar-H); 9.60, (s, 1H, OH); 9.86, (s, 1H, OH); 10.28, (s, 1H,
OH); 13C NMR (50 MHz, DMSO-d6): 109.7, 113.8, 114.2, 116.4,
117.2, 119.1, 120.0, 128.4, 133.3, 146.0, 149.6, 156.4, 162.9,
163.7; IR (KBr, cmꢂ1): 3435, 3204; 1615; 1595; 1517; 1491; 1451;
1301; 1253; 746; anal. calcd for C14H10N2O4 ꢀ 0.5H2O (279.25 g
molꢂ1): C, 60.21; H, 3.97; N, 10.03; found: C, 60.23; H, 3.96; N,
10.05.
4.2.2.16. 4-(5-(3,5-Dihydroxyphenyl)-1,3,4-oxadiazol-2-yl)benzene-
1,2-diol ꢀ 2H2O (7h). Brown powder; yield: 0.18 g (57%); mp:
>250 ꢃC; 1H NMR (200 MHz, DMSO-d6): 6.43, (t, 1H, J ¼ 2.0 Hz, Ar-
H); 6.91–6.95, (m, 3H, Ar-H); 7.38, (d, 1H, J ¼ 8.2 and 2.0 Hz, Ar-H);
7.45, (d, 1H, J ¼ 2.0 Hz, Ar-H); 9.58, (s, 1H, OH); 9.81, (s, 3H, OH);
13C NMR (50 MHz, DMSO-d6): 104.57 (2C), 106.0, 113.7, 114.4,
116.4, 119.0, 125.0, 146.0, 149.5, 159.2 (2C), 163.6, 164.2; IR (KBr,
cmꢂ1): 3237, 1605; 1573; 1505; 1453; 1285; 1162; 854; anal. calcd for
C14H10N2O5 ꢀ 2H2O (322.27 g molꢂ1): C, 52.18; H, 4.38; N, 8.69;
found: C, 52.16; H, 4.37; N, 8.70.
4.2.2.9. 2-(5-Phenyl-1,3,4-oxadiazol-2-yl)phenol (7a). White
powder; yield: 0.16 g (66%); mp: 158–159 ꢃC; for 1H and 13C
NMR see ref. 35 or our spectral data placed in ESI.†
4.2.2.10. 3-(5-Phenyl-1,3,4-oxadiazol-2-yl)phenol
ꢀ
0.5H2O
1
ꢃ
(7b). Beige powder; yield: 0.14 g (56%); mp: 179–180 C; for H
and 13C NMR see ref. 34 or our spectral data placed in ESI.†
4.2.2.11. 2-Methoxy-4-(5-phenyl-1,3,4-oxadiazol-2-yl)phenol ꢀ
2.5H2O (7c). Grey powder; yield: 0.17 g (53%); mp: 162–163 ꢃC; 1H
NMR (200 MHz, DMSO-d6): 3.90, (s, 3H, CH3); 6.99, (d, 1H, J ¼
8.8 Hz, Ar-H); 7.58–7.65, (m, 5H, Ar-H); 8.11–8.15, (m, 2H, Ar-H);
9.99, (s, 1H, OH); 13C NMR (50 MHz, DMSO-d6): 56.0, 110.4,
114.4, 116.2, 120.7, 123.6, 126.6 (2C), 129.5 (2C), 131.9, 148.3,
4.3. DPPH free radical scavenging method
The antioxidant activity of synthesized diacylhydrazines and
1,3,4-oxadiazoles was evaluated using DPPH free radical scav-
enging activity method by Kumarasamy et al.37 1 mL of meth-
anol solution of DPPH (80 mg Lꢂ1) was mixed with the same
volume of referent antioxidants and synthesized diacylhy-
drazines and 1,3,4-oxadiazoles dissolved in methanol. The
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absorbance was measured spectrophotometrically at 517 nm
The antioxidant capacity was expressed as IC50 (concentra-
aer incubation for 30 min in the dark. The DPPH radical tion of compounds in mM required for 50% reduction of H2O2).
scavenging activity of newly synthesized compounds was
compared with well – known referent antioxidants, ascorbic
acid and NDGA. DPPH radical scavenging activity was calcu-
lated as:
4.6. F–C reducing capacity assay
The ability of synthesized compounds to reduce Folin–Cio-
calteu's reagent (FC) was measured according to the previously
Ac ꢂ As
described method of Singleton and Rossi.40 The reaction
mixture consisted of 1580 mL H2O, 20 mL sample in DMSO, 100
mL of FC reagent and 300 mL of 20% Na2CO3 solution. Aer 2 h
of incubation at room temperature absorbance was measured at
765 nm and results were expressed as grams of gallic acid
equivalents per gram of tested compound (gGAE per g) using
gallic acid calibration curve 10–1000 mg mLꢂ1 with linearity
range r ¼ 0.993.
% radical scavenging activity ¼
ꢀ 100;
Ac
where Ac is the absorbance of control (DPPH in methanol) and
As is absorbance of the samples. Results were expressed as the
concentration of the compounds providing 50% of scavenging
of DPPH radicals (IC50), which were calculated through the
sigmoidal dose response curve, as mM, using OriginPro8
statistical soware. All radical scavenging assays of DPPH were
analyzed in triplicates.
4.7. Ferric ion-reducing capacity assay
4.4. ABTS radical cation scavenging assay
Reducing power of tested compounds was performed according
to the method of Pownall et al.41 Sample dilutions were
prepared in 50 mM phosphate buffer, pH 7.0 and 500 mL of
dilutions were mixed with 250 mL of 1% potassiumꢃferricyanide
solution followed by incubation for 20 min at 50 C. Aer the
incubation 500 mL of 10% trichloroacetic acid was mixed with
500 mL of the incubated sample, 100 mL of 0.1% ferric chloride
and 500 mL of distilled water. The mixture was le to incubate
for 10 min at room temperature and the absorbance was
immediately measured at 700 nm, against blank, which con-
sisted of phosphate buffer and appropriate volume of solvent.
The results were expressed as absorbance units at 700 nm which
was considered as a measure of reducing power.
For the measurement of total antioxidant capacity of synthe-
sized compounds we have employed automated assay using
stable ABTS radical cation described previously by Erel.38
Reaction mixture consisted of 200 mL of Reagent 1 (acetate
buffer 0.4 M, pH 5.8) containing 5 mL of different sample dilu-
tions prepared in DMSO was mixed with 20 mL of Reagent 2
(10 mM ABTSc+, 2 mM H2O2 in 30 mM acetate buffer pH 3.6.
Solution should be prepared 12–16 h prior to analysis). The rst
absorbance measurement at 660 nm was taken before mixing
Reagent 1 and Reagent 2 and it served as sample blank, the last
absorbance measurement was taken aer the incubation period
of 5 min aer the mixing two reagents. The control sample
consisted of the same reagents with 5 mL of DMSO instead of the
sample in Reagent 1. Results were calculated as follows:
4.8. Cell culture
ABTSc+ scavenging% ¼ [A660 nm control ꢂ A660 nm sample
/A660 nm control ꢀ 100
]
Human normal lung broblasts MRC-5 were cultured as
monolayers at 37 C in an atmosphere of 5% CO2 and humid-
ꢃ
ied air. The cell line was purchased from the American Type
Culture Collection (Manassas, VA, USA). The nutrient medium
was RPMI-1640 supplemented with 2 mM L-glutamine, 100 mg
mLꢂ1 streptomycin, 100 IU mLꢂ1 penicillin, 10% heat-
inactivated (56 ꢃC) fetal bovine serum and 25 mM HEPES,
adjusted to pH 7.2 with a bicarbonate solution. RPMI-1640, fetal
bovine serum and HEPES were products of Sigma Aldrich (Saint
Louis, MO, USA).
The antioxidant capacity was expressed as IC50 (concentra-
tion of compounds in mM required for 50% reduction of ABTS
radicals).
4.5. H2O2 scavenging assay
For the detection of hydrogen peroxide scavenging activity
previously described method of Ruch et al. was employed.39
Reaction mixture contained 0.6 mL 40 mM H2O2 in 50 mM
phosphate buffer pH 7.4 which was added to 3.4 mL of the same
buffer containing different concentrations of test samples.
Upon the addition of peroxide, mixture was shaken vigorously
and le to incubate at room temperature for 10 min, than the
absorbance was recorded at 230 nm. Control sample consisted
of phosphate buffer and H2O2 without samples. Percent of H2O2
scavenging was calculated according to the following equation:
4.9. MTT cell survival assay
MRC-5 normal lung broblasts (5000 cells per well) were seeded
in 96-well microtiter plates with at bottom and 20 h later, aer
the cell adherence, ve concentrations of the investigated
compounds were added to the cells (range from 12.5 mM to 200
mM). Nutrient medium only was added to the cells in the control
wells. Stock solutions of tested compounds were made in
dimethyl sulfoxide (DMSO) at a concentration of 10 mM. Aer
72 h treatment, the survival of MRC-5 cells was assessed by MTT
cell survival test according to the method of Mosmann42 and
modied by Ohno and Abe,43 as described elsewhere by our
research group.44
H2O2 scavenging% ¼ [A230 nm control ꢂ A230 nm sample
/A230 nm control ꢀ 100
]
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All experiments were done in triplicate. A chemotherapy
RSC Advances
Superoxide dismutase (EC 1.15.1.1) activity was measured
drug cisplatin was used as a positive control. The used chem- according to Beuchamp and Fridovich.48 Protein extract (50 mL)
icals were products of Sigma Aldrich.
was added to the reaction mixture (1.5 mL) containing 50 mM
potassium phosphate buffer (pH 7.0), 0.1 mM EDTA, 13 mM
methionine, 2 mM riboavin and 75 mM nitro blue tetrazolium
(NBT). Riboavin was added last and the tubes were shaken.
The reaction was started by exposing the mixture to a cool white
uorescent light. Aer 15 min the light was switched off, the
tubes were mixed and the absorbance measured at 560 nm. One
unit of enzyme activity was measured as the amount of SOD
capable of inhibiting 50% of nitrite formation under the assay
conditions.
4.10. Measurement of intracellular ROS levels
Aer 24 h exposure to non-toxic concentrations of investigated
diacylhydrazines and 1,3,4-oxadiazoles (values were determined
using MTT test for the 24 h treatment with compounds), the
treated and control MRC-5 cells were loaded with a 30 mM 20,70-
dichlorodihydrouorescein diacetate (Sigma Aldrich, catalog
number D6883) in a phosphate buffered saline (PBS) for 45 min
ꢃ
at 37 C. The cells were then washed with PBS and exposed to
2.5 mM hydrogen peroxide solution (H2O2) for 30 min at 37 ꢃC
to induce generation of ROS. Following incubation with H2O2,
the cell samples were washed with PBS, collected and the
intensity of green uorescence emitted by the generated probe
dichlorouorescein was measured on a FACSCalibur ow
cytometer (BD Biosciences Franklin Lakes, NJ, USA). The data
(20 000 events acquired for each cell sample) were analysed
using CELLQuest soware (BD Biosciences). Applied non-toxic
concentrations of the investigated compounds were: 50 mM
for the compounds 6d, 6e, 6g and 6h, and 20 mM for the
compounds 7d, 7e, 7g and 7h.
4.12. Statistical evaluations
The results of DPPH scavenging activity are expressed as mean
of three experiments ꢁ standard deviation (SD). The IC50 was
calculated using OriginPro8 statistical soware. The results
were statistically analyzed by one way analysis of variance
(ANOVA) using the SPSS statistical soware package
(version13.0). Comparison between IC50 values was performed
with T3 Dunnett test or with Bonferroni test. The results were
considered to be statistically signicant at p < 0.05.
4.11. Antioxidative enzymes assay
Acknowledgements
MRC-5 cells (control and cell samples exposed for 24 h to non-
toxic concentrations of investigated compounds) were har-
vested from culture asks, washed with PBS and centrifuged at
1500 ꢀ g for 10 min at 4 ꢃC. Cell pellets were re-suspended in 0.5
mL of PBS and then sonicated on ice three times, for 30 s each.
The total extract was centrifuged at 3000 ꢀ g for 15 min at 4 ꢃC.
Aliquots of the supernatant were used for enzyme assays.
Protein concentration in the extracts was determined according
to Bradford,45 using a BioRad assay kit with bovine serum
albumin as standard.
Glutathione reductase (EC 1.6.4.2) was measured according
to Foyer and Halliwell.46 The assay medium contained
0.025 mM phosphate buffer pH 7.8, 0.5 mM GSSG, 0.12 mM
NADPH-Na4 and 50 mL of protein extract. NADPH oxidation was
determined by recording the decrease of absorbance at 340 nm.
The activity of this enzyme was expressed as mU mgꢂ1, using
extinction co-efficient of NADPH (E) ¼ 6.2 mMꢂ1 cmꢂ1. One unit
of GR activity has been dened as one mM of NADPH per minute
under standard conditions.
The authors are grateful to the Ministry of Education, Science
and Technological Development of the Republic of Serbia for
nancial support (Projects 172016, 175011 and 173005).
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