Novel 1,3,4-thiadiazole-chalcone hybrids containing catechol moiety: synthesis, antioxidant activity, cytotoxicity and DNA interaction studies

Article abstract of DOI:10.1039/c8md00316e

Hybrid compounds that combine the 1,3,4-thiadiazole-containing catechol moiety with a chalcone motif were synthesized and examined for their antioxidant activity, cytotoxicity, and DNA-binding activity. A series of thirteen compounds showed strong antioxi

Full text of DOI:10.1039/c8md00316e

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and V. Markovi, Med. Chem. Commun., 2018, DOI: 10.1039/C8MD00316E.
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DOI: 10.1039/C8MD00316E
Novel 1,3,4-thiadiazole-chalcone hybrids containing catechol moiety:
Synthesis, antioxidant activity, cytotoxicity and DNA interaction studies
Katarina Jakovljević,a Milan D. Joksović,a Ivana Z. Matić,b Nina Petrović,b,c Tatjana
Stanojković,b Dušan Sladić,d Miroslava Vujčić,e Barbara Janović,e Ljubinka Joksović,a
Snežana Trifunovićd and Violeta Marković*a
a Faculty of Science, Department of Chemistry, University of Kragujevac, R. Domanovica 12, 34000
Kragujevac, Serbia
b Institute of Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade, Serbia
c Laboratory for Radiobiology and Molecular Genetics, “Vinča” Institute of Nuclear Sciences, University of
Belgrade, 11000 Belgrade, Serbia
d Faculty of Chemistry, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia
e Institute for Chemistry, Technology and Metallurgy, Njegoševa 12, 11000 Belgrade, Serbia
Abstract Hybrid compounds that combine 1,3,4ꢀthiadiazole containing catechol moiety with chalcone motif
have been synthesized and examined for their antioxidant activity, cytotoxicity and DNAꢀbinding activity. The
series of thirteen compounds showed strong antioxidant and cytotoxic effects on human acute promyelocytic
leukemia HLꢀ60 cells. Several compounds exerted good cytotoxic activities on cervical adenocarcinoma HeLa
cells. The treatment of HeLa cells with IC50 and double IC50 concentrations of the compounds 5a, 5c, 5f and 5m
induced statistically significant increase in the percentage of cells within subG1 cell cycle phase. The examined
compounds caused G2/M cell cycle arrest in HeLa cells. Each of these compounds triggered apoptosis in HeLa
cells through activation of caspaseꢀ3, the main effector caspase, caspaseꢀ8, which is involved in the extrinsic
apoptotic pathway, and caspaseꢀ9, which is involved in the intrinsic apoptotic pathway. All of the examined
compounds decreased the expression levels of MMP2 in HeLa cells and levels of protumorigenic miRꢀ133b.
Compounds 5a and 5m lowered the expression level of oncogenic miRꢀ21 in HeLa cells. In addition,
compounds 5a, 5f and 5m decreased the expression levels of oncogenic miRꢀ155 while the treatment of HeLa
cells with compounds 5a, 5c and 5f increased expression of tumorꢀsuppressive miRꢀ206. The observed effects
of the compounds on expression levels of four examined miRNAs suggest their prominent cancerꢀsuppressive
activity. The investigation by absorption and fluorescence spectroscopy showed more efficient calf thymus
DNA binding activity of the compound 5m in comparison to other tested compounds. Results of pUC19
plasmid cleavage study and comet assay showed DNA damaging activities of compounds 5a and 5c.
Keywords: 1,3,4ꢀthiadiazoleꢀchalcone, antioxidant, apoptosis, gene expression, CTꢀDNA binding
activity
_____________________
∗ Corresponding author. Eꢀmail address: markovicvioleta@kg.ac.rs (V. Marković)
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1. Introduction
Anticancer hybrid molecules incorporate two or more different, covalently linked
pharmacophores with ability to modulate multiple biological targets and improve therapeutic potential
of the designed compounds in comparison to single bioactive precursors. Using molecular
hybridization techniques, it is possible to synthesize numerous hybrids based on known anticancer
scaffolds leading to a more favorable pharmacological profile than the sum of each individual
compounds.1 Chalcones are frequently selected as one of privilege structures because of their
significant anticancer properties and facile preparation, offering major advancements in the field of
hybrid molecules.2,3 Such chalcone hybrid analogues with promising anticancer activity have already
been synthesized by the coupling of chalcones with various bioactive compounds including
coumarin,4,5 1,2,3ꢀtriazole,6 retinoid,7 naphthoquinone,8 βꢀcarboline,9 artemisinin,10 Nꢀ4ꢀpiperazinylꢀ
ciprofloxacin,11 anthraquinone, 12 pyrazoleꢀ5ꢀcarboxamide,13 thiazole14 and isoxazole.15
We selected 1,3,4ꢀthiadiazole as a second bioactive compound for combining with chalcone
unit to generate a new hybrid molecule suitable to simultaneously target several pathogenic
mechanisms. The covalent bond was established by formation of an amide in the reaction of amine
group of heterocycle and previously transformed carboxylic function into acid chloride of chalcone
analogue. 1,3,4ꢀThiadiazoles exhibited a wide spectrum of anticancer activities owing to high
electronꢀdonating ability of nitrogen atoms to form hydrogen bonds or to coordinate metal ions.16,17
Although 1,3,4ꢀthiadiazoles have a promising anticancer potential, their toxicity still remains a major
concern.18 Molecular hybridization may be a way to enhance activity or selectivity and also, overcome
the side effects associated with the single compound.
It is well known that antioxidants (including phenolic ones) block free radicals to induce
damage of biological macromolecules under oxidative stress, preventing their oxidation, DNA
mutations and malignant changes.19 Very recently, we combined bioactive functions of 1,3,4ꢀ
thiadiazole and phenolic acid moiety to obtain novel conjugates with antioxidant and antiproliferative
activity.20 The reported antiproliferative activities of 1,3,4ꢀthiadiazoles and chalcone analogues, as
well as radical scavenging properties of phenolic compounds, led us to rational design and synthesis
of new molecular hybrids with evaluation of their antioxidant, cytotoxic and DNAꢀbinding potential.
2

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2. Results and Discussion
2.1.
Chemistry
The novel 1,3,4ꢀthiadiazoleꢀchalcone hybrids were synthesized in three steps (Scheme 1).
Initially, chalcone analogues 4aꢀm were prepared using the ClaisenꢀSchmidt condensation of 4ꢀ
formylbenzoic acid 3 with various substituted acetophenones in the presence of NaOH, according to
slightly modified literature procedure.21 Subsequently, chalcones 4aꢀm were converted into acyl
chloride by action of SOCl2 and, without isolation, reacted with 4ꢀ(5ꢀaminoꢀ1,3,4ꢀthiadiazolꢀ2ꢀ
yl)benzeneꢀ1,2ꢀdiol 2 in dioxane giving the final hybrid compounds 5a-m in moderate to good yields
(53ꢀ87%). Previously, thiadiazole derivative was synthesized by the reaction of 3,4ꢀdihydroxybenzoic
acid and thiosemicarbazide in phosphoryl chloride.20 Although acid chlorides are very reactive
compounds, the formation of an amide requires long time and high temperature due to poor
nucleophilicity of amino group of 1,3,4ꢀthiadiazole. Almost all compounds still contained a
significant amount of dioxane. The solvent was eliminated by dissolving crude product in DMF or
DMSO and subsequent precipitation with water or by complete evaporation of THF solutions. The
structure of all compounds was confirmed by means of 1H and 13C NMR spectroscopy (see
Supplementary content), IR and elemental analysis. The olefinic protons of the chalcone double bond
in hybrid compounds 5aꢀm, as well as their corresponding precursors 4aꢀm, appeared as an AB
system. On the basis of coupling constant values (J = 15.6−16.0 Hz), the compounds 5a, 5c, 5d, 5f,
5g, and 5i-m were isolated and characterized in Eꢀisomeric form. However, the compounds 5b, 5e
and 5h, and their chalcone precursors (4b, 4e and 4h) having the coupling constant values in the range
from 7.0 to 8.2 Hz correspond to the less stable Zꢀconfiguration.22 This kind of geometry of the
chalcone double bond is not very frequent and since these derivatives contain an orthoꢀsubstituted
acetophenone moiety in their structure, it is probable that Zꢀisomeric form is favored due to the steric
factors. Aromatic protons corresponding to the phenolic group appeared in the range of 6.86ꢀ7.44
ppm, giving a doublet for Hꢀ5, doublet of doublets for Hꢀ6 and another doublet for Hꢀ2. Protons
belonging to the benzoic acid moiety were well resolved in the form of the AB system for most of the
thiadiazoleꢀchalcone derivatives.
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Scheme 1. Reagents and conditions: a) POCl3, rt, H2NHNC(=S)NH2, 1h, reflux; b) substituted
acetophenones, NaOH, MeOH, 2h, reflux, HCl; c) SOCl2, DMF, CH2Cl2, 2h, rt; d) 2, dioxane, 12h,
reflux.
2.2. Biology
2.2.1. Radical scavenging activity and effects of pretreatment of HeLa cells with
1,3,4-thiadiazole-chalcone hybrids on ROS levels
Antioxidant activity of phenolic acids has been well known because of their preventive effect
on malignant changes that are associated with radical species.23 Reactive oxygen species (ROS) in the
form of free radicals (superoxide and hydroxyl radical) and neutral molecules (H2O2) have been
neutralized by stable phenolic antioxidants, diminishing DNA damaging and cancer formation.19
Thus, the compounds showing antioxidant and cytotoxic activity have a great importance and a
combination of phenolic antioxidant moiety with other bioactive pharmacophores could be a good
way to obtain more potent radical scavengers as a result of their synergistic effects.
Generally, all synthesized hybrid molecules exhibited better DPPH radical scavenging
activity than referent ascorbic acid (Table 1).
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Table 1. DPPH scavenging activity of the 1,3,4ꢀthiadiazoleꢀchalcone hybrids 5a-m.a
Compound
IC50 ± SD (ꢁM)
12.03 ± 0.50
11.66 ± 0.41
17.17 ± 0.38
11.81 ± 0.31
9.76 ± 0.75
10.33 ± 0.30
17.86 ± 0.03
13.91 ± 0.32
17.82 ± 0.65
18.04 ± 0.23
14.01 ± 0.34
13.45 ± 0.09
11.06 ± 0.24
20.23 ± 0.14
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
Ascorbic acid
a
Results are mean values
± SD from three measurements.
Unfortunately, all our compounds were practically insoluble in methanol, a common solvent
for DPPH test, and determination of radical scavenging activity was performed in diluted dimethyl
sulfoxide. However, DMSO was found to considerably decrease antiradical capacity of ascorbic acid
due to the formation of a molecular complex with DMSO through intermolecular hydrogen bonds.24
Also, dramatic solvent effects were established on the rates of Hꢀabstraction from phenolic
compounds.25 In accordance with these facts, in separate experiments we observed significantly lower
scavenging activity at higher concentrations of DMSO, suggesting the formation of a molecular
complex between DMSO and our compounds. From this reason we were not able to consider the
influence of chalcone moiety with various electron donating or withdrawing substituents and make a
comparison with other 1,3,4ꢀthiazole compounds containing phenolic hydroxyl groups. Anyway, the
potent radical scavenging potential of our compounds is evident, and for that reason we decided to
explore their effects on ROS levels in human cervical adenocarcinoma HeLa cells. We examined the
effects of four compounds – 5a, 5c, 5f and 5m, which were selected for all further analyses due to
their prominent cytotoxic activity and good selectivity in the antiproliferative action (Table 2). As it
could be seen in Fig. 1 A, significant accumulation of ROS in the H2O2ꢀtreated cells indicates
activation of endogenous ROS production by added H2O2. The pretreatment of HeLa cells with
subtoxic IC20 concentrations of 5a and 5f for 24 h (10 µM for each compound) slightly increased the
ROS levels induced by H2O2, while pretreatment with compounds 5c and 5m did not affect the ROS
levels triggered by H2O2. The radical species (phenoxyl radical etc.) produced by the oxidation of 5a
and 5f with H2O2 are highly reactive and subject to further oxidation giving quinone compounds.
Quinones are still reactive and can be stabilized by interaction with nucleic acid.26 This reaction might
be responsible for the toxic proꢀoxidant effect of 5a and 5f which may damage cellular constituents
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like DNA by induction of oxidative cytotoxic stress in cancer cells, activating programmed cell death
– apoptosis.
In the absence of exogenously added H2O2, the intracellular ROS levels in HeLa cells treated
for 24 h with each of the four compounds were decreased when compared with basal ROS levels in
control. This behavior of all tested compounds, especially 5c, is a result of their radical scavenging
potential to neutralize basal ROS in HeLa cells (Fig. 1, B). Some cancer cells, in advanced stage of
disease, have adapted to oxidative stress due to their antioxidative defense capacity. Therefore, the
cancer cells show resistance to the drugs that induce intracellular ROS production, such as paclitaxel
or doxorubicin.27 Thus, possibility of drugs to reduce antioxidant system in combination with oxidant
agents might be useful in antitumor therapies. A redox modulation of ROS level by production of
sufficient amount of ROS provoking apoptosis or by decrease in antioxidant level could be a good
way to kill cancer cells which are more sensitive to exogenous oxidative stress than normal cells.
Fig. 1. Effects of 24 h pretreatment of HeLa cells with IC20 concentrations of the 1,3,4ꢀthiadiazoleꢀ
chalcone hybrids 5a, 5c, 5f and 5m (10 µM for each compound) on ROS generation induced by
hydrogen peroxide (10 mM) (A) and endogenous ROS levels (B). The results are presented as the
mean ± SD of two independent experiments.
2.2.2. Cytotoxic activity
The cytotoxicity of 1,3,4ꢀthiadiazoleꢀchalcone hybrids containing antioxidant catechol moiety
was evaluated against three human malignant cell lines (cervical carcinoma HeLa, acute
promyelocytic leukemia HLꢀ60, and lung carcinoma A549), and normal human lung fibroblasts
MRCꢀ5 using MTT cell survival test. The obtained IC50 values are shown in Table 2. Generally, all
tested compounds exerted the strongest cytotoxic activity against leukemia HLꢀ60 cells with IC50
values in the range from 6.92 µM to 16.35 µM. The compounds 5a, 5f, 5h, 5l and 5m also showed
strong cytotoxic effects on HeLa cells, with IC50 values from 9.12 µM to 12.72 µM. Lung carcinoma
A549 cells were the least sensitive to the cytotoxic activity of the examined hybrids, while other
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tested derivatives exerted moderate to low cytotoxic activity. As it can be seen in Table 2, there is no
significant difference between the influence of electronꢀdonating and electronꢀwithdrawing groups of
the acetophenone moiety on the cytotoxic action against cancer cells. This indicates that the
thiadiazoleꢀchalcone pharmacophore present in the structure of the tested compounds has the crucial
role in their antiproliferative action. All of the compounds from this series exerted two to four times
higher cytotoxic activity against HeLa and HLꢀ60 malignant cell lines in comparison to their activity
against normal MRCꢀ5 cells, with the exception of 5g, 5j and 5k against HeLa cells. In addition, all
tested derivatives showed lower toxicity against normal MRCꢀ5 cells comparing to cisplatin as a
referent chemotherapeutic.
Table 2. The cytotoxic activity of the investigated 1,3,4ꢀthiadiazoleꢀchalcone hybrids containing
antioxidant phenolic moiety.a
IC50±SD (µM)
Compd.
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
HeLa
HL-60
A549
MRC-5
9.37±0.86
9.12±1.19
9.63±1.26
11.20±2.32
10.22±0.73
9.98±0.99
57.55±7.58
11.08±2.10
17.75±0.17
40.22±3.64
54.76±2.77
11.04±2.41
12.72±3.24
4.91±0.74
8.40±1.44
7.62±1.40
8.39±1.39
8.44±1.52
11.97±1.27
9.92±0.50
16.35±0.87
8.88±1.32
10.63±0.96
9.74±1.54
11.17±0.52
6.92±0.53
15.72±1.66
2.88±0.34
42.75±2.77
21.80±2.55
27.87±4.05
26.35±1.90
50.23±5.35
92.14±6.64
89.10±9.35
46.89±1.71
89.33±2.99
87.74±6.82
127.75±8.37
23.23±1.37
47.85±1.92
13.21±0.89
36.00±1.10
18.56±2.25
34.25±4.88
33.72±3.35
48.01±6.43
42.54±3.88
81.33±9.25
39.58±4.82
45.81±3.38
38.24±4.95
39.44±3.49
21.17±1.23
68.52±7.69
9.35±1.29
5m
cisplatin
a Results are mean values ± SD of three independent experiments.
2.2.3. Effects of the 1,3,4-thiadiazole-chalcone hybrids on cell cycle phase
distribution
With the aim to investigate the mechanisms of the cytotoxic activity of four selected 1,3,4ꢀ
thiadiazoleꢀchalcone hybrids 5a, 5c, 5f and 5m, the changes in cell cycle phase distribution of cervical
adenocarcinoma HeLa cells treated with IC50 and 2IC50 concentrations of these compounds for 24 h
were assessed. The incubation with both tested concentrations of the compounds induced statistically
significant increase in the percentage of HeLa cells within subG1 phase when compared with
untreated, control cell samples (Fig. 2). These results point to ability of the examined compounds to
induce cell death in HeLa cells. Furthermore, compound 5c applied at IC50 concentration and
compound 5m applied at double IC50 concentration caused significant increase in the percentage of
cells within G2/M cell cycle phase in comparison to those percentages in the control cells. The
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incubation of HeLa cells with double IC50 concentrations of the compounds 5a, 5c and 5f also led to
accumulation of cells in G2/M phase, although those differences were not statistically significant. The
obtained data demonstrate that tested compounds cause G2/M cell cycle arrest in HeLa cells, thus
preventing cell entry into mitosis and eventually leading to cell apoptosis. The double IC50
concentrations of compounds 5a and 5m, as well as IC50 and double IC50 concentrations of compound
5c, induced significant decrease in the percentage of treated cells within G1 phase when compared
with control cells, suggesting enhanced sensitivity of cells within G1 phase to the cytotoxic action of
these compounds. Our findings are in accordance with literature data showing that compounds
bearing chalcone bioactive scaffold induce G2/M cell cycle arrest and apoptotic cell death in cancer
cells.28
Fig. 2. Changes in the cell cycle phase distribution of human cervical carcinoma HeLa cells treated
with IC50 (A) and 2IC50 concentrations (B) of the 1,3,4ꢀthiadiazoleꢀchalcone hybrids 5a, 5c, 5f and 5
m after 24 h treatment. The results are presented as the mean ± SD of three independent experiments.
Statistically significant differences between control and treated cell samples are marked with *
(p<0.05).
2.2.4. Effects of the specific caspase inhibitors
To further examine whether the tested compounds could induce apoptotic cell death in treated
HeLa cells, the cells were pretreated with specific peptide inhibitors of caspaseꢀ3, caspaseꢀ8 or
caspaseꢀ9 two hours before addition of compounds and their effects were determined by cell cycle
analysis. As it could be seen in Fig. 3, the notable decrease in the percentage of HeLa cells within
subG1 cell cycle phase was observed in cell samples which were pretreated with caspaseꢀ3 inhibitor,
caspaseꢀ8 inhibitor or caspaseꢀ9 inhibitor, and afterwards exposed to compounds 5a, 5c, 5f or 5m
when compared with HeLa cell samples which were not pretreated with inhibitors before addition of
the compound. The obtained results indicate that examined 1,3,4ꢀthiadiazoleꢀchalcone hybrids (5a, 5c,
5f and 5m) trigger apoptosis in HeLa cells through the activation of main effector caspaseꢀ3,
activation of caspaseꢀ8, implicated in extrinsic pathway of apoptosis and caspaseꢀ9, implicated in
intrinsic pathway of apoptosis.
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Fig. 3. Effects of the specific caspase inhibitors (ZꢀDEVDꢀFMK ꢀ caspaseꢀ3 inhibitor, ZꢀIETDꢀFMK
ꢀcaspaseꢀ8 inhibitor, ZꢀLEHDꢀFMK ꢀ caspaseꢀ9 inhibitor) on the percentages of HeLa cells within
subG1 phase treated with 2IC50 concentrations of the compounds 5a (A), 5c (B), 5f (C), and 5m (D).
Representative graphs are shown.
2.2.5. Effects of 1,3,4-thiadiazole-chalcone hybrids on gene and microRNA expression
levels
Increased levels of matrixꢀmetalloproteinaseꢀ2 (MMP2), matrixꢀmetalloproteinaseꢀ9 (MMP9),
and vascular endothelial growth factor A (VEGFA), and increased levels of typical oncogenic
miRNAs (miRꢀ21 and miRꢀ155) overexpressed in various cancer types,29 and decreased levels of
matrixꢀmetalloproteinase inhibitor 3 (TIMP3)30 are associated with the cell growth, migration,
epithelial mesenchymal transition, invasion, metastasis, and angiogenesis.31 MiRꢀ133b was known to
be downregulated in prostate cancer, but this microRNA exerted protumorigenic activity in cervical
cancer.32 MiRꢀ206 is described to be downregulated in cervical cancer,33 and that overexpression
induces apoptosis.34
To further elucidate the molecular mechanisms of cytotoxic activity of our compounds, we
examined changes in the expression levels of extracellular matrixꢀdegrading proteases MMP2 and
MMP9, TIMP3 which inhibits matrix metalloproteinases and regulates proteolysis of extracellular
matrix, as well as in the expression of inducer of angiogenesis VEGFA, and several microRNAsꢀmiRꢀ
21/133b/155/206 in human cervical adenocarcinoma HeLa cells treated with subtoxic IC20
concentrations of the examined compounds (Fig. 4).
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Fig. 4. Changes in expression levels of MMP2 gene(A), MMP9 gene (B), TIMP3 gene (C), VEGFA
gene (D), miRꢀ21 (E), miRꢀ133b (F), miRꢀ155 (G) and miRꢀ206 (H) in HeLa cells exposed to
subtoxic IC20 concentrations of the compounds 5a, 5c, 5f and 5 m (10 µM for each compound) for 24
h. Representative graphs are shown.
All of the examined compounds (5a, 5c, 5f, and 5m) decreased expression levels of MMP2
and TIMP3, but increased expression levels of MMP9, when compared with those levels in untreated,
control HeLa cells. In addition, compound 5a slightly increased the VEGFA expression level in HeLa
cells, while compounds 5f and 5m slightly decreased the expression of VEGFA in these cells. The
observed downregulation of VEGFA expression in HeLa cells and inhibition of formation of tubular
structures by EA.hy926 cells exerted by compounds 5f and 5m indicate the mild antiangiogenic
activities of these compounds (Fig. 5).
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Fig. 5. Photomicrographs of control EA.hy926 cells and EA.hy926 cells incubated with subtoxic IC20
concentrations of the compounds 5a, 5c, 5f (6.5 µM for each compound) and 5m (8 µM) for 20 h.
The ability of the examined 1,3,4ꢀthiadiazoleꢀchalcone hybrids to downregulate expression levels
of MMP2 in HeLa cells might suggest their suppressive effects on cervical cancer cell invasion and
establishment of metastasis. The investigation of Kato et al. suggested that expression levels of
MMP2 may be closely associated with invasion ability of cervical cancer cells.35 We observed that all
tested compounds remarkably increased MMP9 expression levels in HeLa cells. MMP9 is connected
with increased invasion and metastasis, but besides this protumorigenic role, there is experimental
evidence that MMP9 may exert protective effects on cancer progression and metastasis in addition to
antiꢀangiogenic effect.36,37 The study in a mouse model of multistage skin carcinogenesis caused by
HPV16 oncogene demonstrated that MMP9 deficient transgenic mice had decreased proliferation rate
of keratinocytes and a lower incidence of invasive tumors, which had more aggressive phenotype,
while mice expressing MMP9 developed a larger number of tumors, which had less aggressive
phenotype.37 Furthermore, MMP9 may suppress further progression of malignant tumors through
generation of various antiꢀangiogenic peptides, such as angiostatin and tumstatin.36 Taken together,
these findings might suggest that upregulation of MMP9 expression levels in tumor stroma which
could be induced by our compounds, might contribute to suppression of tumor growth and
progression.
Considering the effects of the compounds on microRNA expression levels in HeLa cells, the
compound 5a decreased levels of three crucial oncogenic miRNAsꢀmiRꢀ21/155 and protumorigenic
miRꢀ133b in cervical cancer, while it slightly increased level of tumor suppressive miRꢀ206, when
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compared with those levels measured in control cells. The compound 5c remarkably increased
expression levels of miRꢀ206 and miRꢀ155 in comparison to control HeLa cells, and decreased
expression of protumorigenic miRꢀ133b. Treatment with compounds 5c and 5f caused mild increase
in miRꢀ21 expression in HeLa cells. Exposure to compound 5f caused decrease in the expression of
miRꢀ133b and miRꢀ155 in addition to increase in the expression level of miRꢀ206. The compound 5m
lowered the expression levels of all four examined miRNAs when compared with controls cells. Our
research showed that compounds 5a and 5m induced remarkable decrease in the expression level of
oncogenic miRꢀ21, that could result in reduced proliferation, migration and invasion of cervical
carcinoma cells. These tumorꢀsuppressive effects had shown to be regulated through phosphatase and
tensin homolog (PTEN).38 The antiproliferative potential of our four compounds is further proved by
their ability to reduce levels of oncogenic miRꢀ133b, which had been reported to be involved in
enhanced proliferation and colony formation of cervical cancer cells, as well as in further progression
and metastasis of cervical cancers, affecting AKT1 and ERK signaling pathways.39 Moreover, the
compounds 5a, 5f and 5m lowered the expression levels of miRꢀ155, which is overexpressed in
cervical cancer tissue, stimulates cell proliferation and exerts oncogenic activity, confirming their
cancerꢀsuppressive properties.40 The proꢀapoptotic effects of compounds 5a, 5c and 5f could be, at
least in part, attributed to the increased expression of tumorꢀsuppressor miRꢀ206 in treated HeLa cells.
The miRꢀ206 had been reported as an inducer of apoptosis in HeLa cells, that was associated with
inhibition of neurogenic locus notch homolog protein 3 (Notch 3).34
The investigated compounds and miRNAs changed the examined gene expression levels. TIMP3 is
a proven target of miRꢀ21, MMP9 is a target of miRꢀ133b, while VEGF mRNA is target of miRꢀ206
according to TargetScan Human 7.0 (http://www.targetscan.org/).41
2.2.6. DNA binding study
As it is well known, the interaction of DNA with a small molecule can give rise to changes in
the absorbance and in the peak position in the absorption spectra. Hyperchromism and hypochromism
are regarded as spectral evidence for DNA doubleꢀhelix structural change when DNA reacts with
other molecules. Hyperchromism originates from the disruption of the DNA duplex secondary
structure and is indicative of the partial or nonꢀintercalative modes42 and hypochromism originates
from the stabilization of the DNA duplex by either the intercalation binding mode or the electrostatic
effect of small molecules.43,44 Electronic absorption spectra of biologically most active 1,3,4ꢀ
thiadiazole–chalcone hybrids recorded at different concentrations without or with fixed concentration
of CTꢀDNA are shown in Fig. 6A. UV–vis spectra of all compounds displayed similar absorption
bands. It was found that the maximum absorption of 5a, 5c, 5f and 5m was centered at 309 nm, 310
nm, 305 nm and 323 nm, respectively. Upon interaction with CTꢀDNA, the formation of a
compound─CTꢀDNA occured with no shift of absorption maximum at 259 nm. Detailed absorption
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changes induced by binding of the compounds to CTꢀDNA were further calculated from the
experimental data, Fig. 6B. The value of the sum of absorbances at 259 nm of a free compound and
free CTꢀDNA was a different from the absorption value of a compound─CTꢀDNA.
Fig. 6A. Changes in UV–Vis absorption spectra of CTꢀDNA (95 µM) after interaction with different
concentrations of 1,3,4ꢀthiadiazoleꢀchalcone hybrids. Panels A, B, C and D: UV–Vis absorption
spectra of 5a (10µM, 20µM and 40µM), 5c (10µM, 20µM and 40µM), 5f (10µM, 20µM and 40µM)
and 5m (10µM, 20µM and 40µM), respectively before and after interaction with CTꢀDNA.
Fig. 6B. Changes in UV–Vis absorption spectra of CTꢀDNA (95 µM) after interaction with different
concentrations of 1,3,4ꢀthiadiazoleꢀchalcone hybrids. Panels E, F, G and H: comparison of absorption
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at 259 nm between the CTꢀDNA─1,3,4ꢀthiadiazole–chalcone compounds and the sum values of CTꢀ
DNA and 1,3,4ꢀthiadiazole–chalcone compounds. The representative absorption assay curves are
shown. Triplicate assays were applied to all compounds and control CTꢀDNA.
The hypochromism of about 13% was observed with lower concentrations of 5a, Fig. 6B
(Panel E) while the decrease in absorption intensity at 259 nm was less pronounced at higher
concentrations. In case of 5c, Fig. 6B (Panel F) the hyperchromism was observed (ꢀ13.7%, ꢀ7.36%
and ꢀ4.9% at 10 µM, 20 µM and 40 µM, respectively) and with 5m, Fig. 6B (Panel H),
hyperchromism was also observed, more pronounced at lower concentrations probably. The DNA
showed the least changes after interaction with 5f, Fig. 6B (Panel G) (this weak hypochromism was
calculated as 2.4% at the lowest concentration of the compound). Previous studies demonstrated that a
significant hypochromic effect with a concomitant red shift upon ligand binding to DNA was the
typical characteristic of the intercalating mode.45,46 In this work, the obtained hypochromic effects of
5a and 5f and hyperchromic effects of 5c and 5m with no red shift probably reflect changes in the
conformation and structure of CTꢀDNA upon binding of the compounds in the minor groove, via
formation of hydrogen bonds between hydroxyl groups of 1,3,4ꢀthiadiazole─chalcone parts of the
compounds and exposed AT base pairs.
The stability of binding between CTꢀDNA and 1,3,4ꢀthiadiazole–chalcone compounds was
determined by a spectroscopic titration. Absorption spectra of 5a, 5c, 5f and 5m without and with CTꢀ
DNA at different concentrations are shown in Fig. 7, Panels A – D). The absorbance at 259 nm was
monitored for each concentration of DNA (insets in Fig. 7, show plots after linearization). Binding
constant Kb of 5a, 5c, 5f and 5m were calculated using equation (2) and values of 1.09×103 M−1,
0.74×103 M−1, 0.71×103 M−1 and 1.01×103 M−1, respectively, are obtained. These values are
comparable with ones previously published for anthraquinoneꢀchalcone hybrids12 and other
structurally different compounds.47,48 However, relatively low correlation coefficients, indicate low
binding strength of interaction of 1,3,4ꢀthiadiazoleꢀchalcone hybrids with helix of CTꢀDNA.
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Fig. 7. The representative absorption titration curves of 5a (A), 5c (B), 5f (C) and 5m (D) at fixed
concentration (40 µM) with increasing concentrations of CTꢀDNA (2.2, 4.4, 6.6, 8.8, 11 and 13 × 10–5
M); Insets: determination of binding constant (Kb) by plot of [DNA]/(εa – εf) vs. [DNA]; The arrow
show the changes in absorbance upon increasing amounts of CTꢀDNA. Triplicate assays were applied
to all compounds and control CTꢀDNA.
In order to provide additional insight into the interactions between the DNA and 1,3,4ꢀ
thiadiazole─chalcone hybrids, the study with the minor groove binder Hoechst 33258 was performed.
Hoechst 33258 (H) binds strongly and selectively with high affinity to doubleꢀstranded BꢀDNA
structure and like other minor groove binders, it recognizes at least four AT base pairs. It binds by
combination of hydrogen bonding, van der Waals contacts with the walls of the minor groove, and
electrostatic interactions between its cationic structure and the DNA.49 Binding of Hoechst 33258 to
CTꢀDNA was followed by excitation at 350 nm with maximum in fluorescence at 444 nm. The
fluorescence intensity of the band at 444 nm of the Hoechst−CTꢀDNA system decreased remarkably
with the increasing concentration of the compounds (Fig. 8). The fluorescence intensities of H−CTꢀ
DNA−compound system were 58%, 60%, 55% and 51% of H−CTꢀDNA system at maximal applied
concentration of 5a, 5c, 5f and 5m, respectively. The observed reduction of fluorescence indicated the
propensity of the 1,3,4ꢀthiadiazole–chalcone hybrids to bind to DNA minor groove. Insets in Fig. 8
show the quenching plots demonstrating that quenching of H bound to CTꢀDNA by these compounds
are in agreement with the linear SternꢀVolmer Eq. (3) for the investigated concentration ranges of the
compounds. The corresponding quenching constants of H−CTꢀDNA system for 5a, 5c, 5f and 5m
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were calculated by linear regression of the plot I0/I versus [compound]/[CTꢀDNA] as K = 3.30, 2.95,
3.95 and 4.66, respectively.
Fig. 8. Displacement of DNAꢀbound Hoechst 33258 (H) by 5a (A), 5c (B), 5f (C) and 5m (D).
Emission spectra (λex = 350 nm) of H (2.8 x 10ꢀ5 M) bound to CTꢀDNA (1 x 10ꢀ4 M, top line) and
quenching of H—CTꢀDNA system by the compounds at increasing concentrations (0 – 2.2 x 10ꢀ5 M,
curves from top to bottom). Insets: Fluorescence curves of EB bound to CTꢀDNA at λmax = 444 nm by
1,3,4ꢀthiadiazoleꢀchalcone compounds; r = [compound]/[CTꢀDNA]. The arrow shows that
fluorescence intensity decreased with increasing concentration of the complex. The representative
fluorescence assay curves are shown. Triplicate assays were applied to all compounds and control CTꢀ
DNA.
Comparing K values for the quenching fluorescence intensity obtained by the displacement
experiments, it may be concluded that 5m with thiophene ring was more efficient as a groove binder
than other tested 1,3,4ꢀthiadiazole–chalcone hybrids with benzene rings. However, considering the
extent of the interaction of the compounds with CTꢀDNA, it can be concluded that these compounds
binds to DNA exhibiting minor groove mode with low binding strength, Also, the results suggested
that the differences in benzoic moiety contribute to binding, as well as the binding in the groove most
likely occurs via forming hydrogen bonds among hydroxyl groups of 1,3,4ꢀthiadiazole─chalcone
parts of the compounds and exposed AT base pairs.
2.2.7. Comet assay
Comet assay50 is a versatile and sensitive method for measuring DNA damage in terms of
single and doubleꢀstrand breaks in DNA. In order to evaluate the extent of DNA damage induced by
selected compounds comet assay was performed on MRCꢀ5 cells which were treated for 24h with
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different concentration of compounds 5a, 5c, 5f and 5m. From the results presented in Fig. 9 it can be
seen that the compounds caused DNA damage, evidenced by the increase in % of DNA in the comet
tail. Each of the tested compounds exhibited as concentrationꢀdependent DNA damage in MRCꢀ5
cells. At the highest concentration tested (25 µM) DNA damage detected was 45% in case of 5a
compound, while in the case of 5c DNA damage was 38%. Lower degree of DNA damage was
detected in case of compounds 5m and 5f, 25% and 20%, respectively.
Fig. 9. Detection of DNA damage (% tail DNA) using the Comet assay with MRCꢀ5 cells after 24h
exposure with compounds 5f, 5m, 5a and 5c (3.125 – 25 µM). PBS was used as negative control, and
H2O2 (50 ꢁM, 5min inꢀgel exposure) was used as positive control. The yꢀaxis shows the mean ± SD of
DNA damage measured through tail intensity parameter. The experiments were conducted in triplicate
and repeated twice.
2.2.8. pUC19 DNA cleavage study
Since a weak DNA damaging activity was defected in cell culture, the abilities of most potent
1,3,4ꢀthiadiazole–chalcone hybrids to cleave doubleꢀstranded plasmid DNA were investigated using
an agarose electrophoretic assay and DMSO as solvent of compounds in the performed range of
concentration (from 0.5 – 20%) had no effects on conformation of plasmid DNA (results not shown).
As shown by a representative agarose gel electrophoresis in Fig. 10 (A), lane 1, control plasmid
pUC19 consisted mainly of supercoiled form FI and nicked form FII, with also a small quantity of
linear form FIII. All of the compounds were tested and the results showed that there were no
significant changes in band mobility, and no strand scission under applied range of concentrations. In
Fig. 10 (A) results obtained with compound 5a are shown. Very similar results were obtained with
compounds 5c, 5f and 5m (not shown). Further, strand scission was tested under reducing conditions,
in presence of iron(II). Iron(II) per se is involved in the damage of DNA51 the results of cleavage
pUC19 by iron(II) shown in Fig. 10 (B), lanes P+Fe(II) (1:1) and P+Fe(II) (1:2). When the compound
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and iron were used in mole ratio 1:1, (Fig. 10 (B), lanes P+5a+Fe(II) (1:1), P+5cFe(II) (1:1),
P+5f+Fe(II) (1:1) and P+5m+Fe(II) (1:1)) and mole ratio 1:2 ((Fig. 10 (B), lanes P+5a+Fe(II) (1:2),
P+5cFe(II) (1:2), P+5f+Fe(II) (1:2) and P+5m+Fe(II) (1:2)), 1,3,4ꢀthiadiazole–chalcone hybrids
abolished the effects of iron, probably by complex formation and/or by antiꢀoxidative action of
catechol moiety. Iron Fe(III) had no effect under any conditions, Fig. 10 (B), lanes P+5m+Fe(III)
(1:2) and P+Fe(III) (1:2). These results indicate that DNA cleavage could not be the cause of
cytotoxicity. The obtained protective activities of 1,3,4ꢀthiadiazole–chalcone hybrids are comparable
to those published for anthraquinoneꢀchalcone hybrids.12
Fig. 10. The representative agarose gel electrophoresis of plasmid pUC19 treated with 1,3,4ꢀ
thiadiazole–chalcone hybrid: (A) plasmid pUC19 (12 nM) without (lane P) and with 5a at
concentration of 0.05 mM, 0.1 mM, 0.15 mM, 0.2 mM, 0.25 mM, 0.3 mM and 0.4 mM, lanes 1, 2, 3,
4, 5, 6, and 7, respectively; (B) effects of 5a, 5c, 5f and 5m on Fe(II) induced pUC19 damage with
compound–iron mole ratio 1:1 and 1:2.
3. Conclusion
Combining two pharmacophores, 1,3,4ꢀthiadiazole derived from protocatechuic acid with
antioxidant properties and a series of chalcones, thirteen novel hybrid compounds were prepared and
evaluated for radical scavenging, cytotoxic and DNAꢀbinding potential. The synthesized 1,3,4ꢀ
thiadiazoleꢀchalcone hybrids containing phenolic moiety exerted a strong antioxidant and moderate to
good cytotoxic activity against HLꢀ60 and HeLa cells. The selected compounds 5a, 5c, 5f and 5m
caused G2/M cell cycle arrest in HeLa cells and showed the ability to trigger apoptosis in HeLa cells
through activation of caspaseꢀ3, caspaseꢀ8, and caspaseꢀ9. All examined compounds remarkably
downregulated the expression levels of MMP2 in treated HeLa cells, suggesting their suppressive
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effects on cervical cancer cell invasion and metastasis. The compounds 5a and 5m induced
remarkable decrease in the expression level of oncogenic miRꢀ21 in HeLa cells while all four
examined compounds lowered levels of protumorigenic miRꢀ133b. In addition, the compounds 5a, 5f
and 5m decreased the expression levels of oncogenic miRꢀ155. The treatment of HeLa cells with
compounds 5a, 5c and 5f elevated expression of tumorꢀsuppressor miRꢀ206. These effects of
compounds on expression levels of four examined miRNAs point to their prominent cancerꢀ
suppressive properties. 1,3,4ꢀThiadiazoleꢀchalcone hybrids bind weakly to the helix of CTꢀDNA in
the minor groove, induce weak DNA damage in cell culture, and protect plasmid DNA from iron(II)–
induced DNA damage, but DNA displacement of fluorescent probes and plasmid DNA damage
activity as well as damage DNA by comet assay indicated their DNA damaging potential. Taken
together, the results of our research indicate the promising antiproliferative properties of four newly
synthesized 1,3,4ꢀthiadiazoleꢀchalcone hybrids containing phenolic moiety.
4. Experimental section
4.1. Chemistry
4.1.1. Physical measurements and methods
Melting points were determined on a MelꢀTemp 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 Technologies, Cary 300 Series UVꢀVis Spectrophotometer. IR spectra were obtained on a
1
Perkin Elmer Spectrum One FTꢀIR spectrometer with a KBr disc. H and 13CꢀNMR spectra were
taken on a Varian Gemini 200 MHz spectrometer.
4.1.2. Procedure for the preparation of 5a-m
Procedure for the synthesis of 2. A mixture of 3,4ꢀdihydroxybenzoic acid, 1 (2.00 mmol,
0.308 g,) and POCl3 (1.0 mL) was stirred for 20 minutes at the room temperature. Then,
thiosemicarbazide (2.50 mmol, 0.228 g) was added and the resulting suspension was refluxed for 1
hour. After cooling the flask in an ice bath, 3.0 ml of distilled water was added carefully, and reflux
was continued for 1 hour. The mixture was then cooled to the room temperature, saturated aqueous
solution of NaOH was added until pH 8.5 was reached and the suspension was stirred for 1 hour at the
room temperature. The formed precipitate of the corresponding 1,3,4ꢀthiadiazole derivative (2) was
then filtrated, dried over CaCl2 and recrystallized from hot 50 % aqueous EtOH.
Procedure for the synthesis of 4. A mixture of 4ꢀformylbenzoic acid (2.3 mmol, 0.345 g), a
corresponding substituted acetophenone (2.00 mmol) and solid NaOH (4.6 mmol, 0.184 g) in dry
methanol (10.0 ml) was refluxed for 2 hours. After completion, the mixture was cooled to the room
temperature, 5.0 ml of distilled water was added and pH of the solution was adjusted to 4.5 with HCl
aqueous solution (2 M), upon which a precipitate was formed. The precipitate (4a-m) was stirred for
30 minutes and then subsequently collected by suction filtration, dried over CaCl2 and recrystallized
from hot MeOH.
Procedure for the synthesis of 5a-m. To the mixture of 4 (1.00 mmol) in dry dichloromethane
(4.0 ml), SOCl2 (4.00 mmol, 0.3 mL) was slowly added, followed by two drops of
dimethylformamide. The resulting mixture was then stirred for 2 h at room temperature. Afterwards,
the solvent was evaporated under reduced pressure, and the excess of SOCl2 was removed by
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azeotropic distillation with toluene. 2ꢀAminoꢀ1,3,4ꢀthiadiazole, 2 (1.00 mmol, 0.227 g) and dry
dioxane were added to the formed acid chloride, without its previous isolation, and the mixture was
then refluxed for 12 hours. After cooling, 20.0 ml of distilled water was slowly added to the solution
with vigorous stirring at room temperature, followed with a formation of precipitate (5a-m). Stirring
was continued for 1 hour and precipitate was then filtrated, washed with water and dried over CaCl2.
All compounds (except 5e) contained a smaller quantity of dioxane which was not removed even after
drying at 110 °C for 12 h. To remove dioxane, final compounds 5j and 5l were dissolved in N,Nꢀ
dimethylformamide and 5k was dissolved in dimethyl sulfoxide, after which distilled water was
added, followed with a formation of precipitate. The formed precipitate was then filtrated, washed
with water and dried over CaCl2. All other final compounds (5a-d, 5f-i and 5m) were dissolved in
tetrahydrofuran and solvent was evaporated to dryness under reduced pressure.
1
4.1.2.1. (E)ꢀ4ꢀ(3ꢀOxoꢀ3ꢀphenylpropꢀ1ꢀenꢀ1ꢀyl)benzoic acid (4a): H NMR (200 MHz, DMSOꢀd6):
7.55ꢀ7.69, (m, 3H, ArꢀH); 7.78, (d, 1H, JAB = 15.8 Hz, CH=); 8.00, (s, 4H, ArꢀH); 8.06, (d, 1H, JBA
=
15.8 Hz, CH=); 8.18, (d, 2H, J = 7.4 Hz, ArꢀH); 13.16, (bs, 1H, OH); 13C NMR (50 MHz, DMSOꢀd6):
124.44, 128.71 (2C), 128.95 (4C), 129.83 (2C), 132.26, 133.40, 137.51, 138.88, 142.63, 166.93,
189.32.
1
4.1.2.2. (Z)ꢀ4ꢀ(3ꢀOxoꢀ3ꢀ(oꢀtolyl)propꢀ1ꢀenꢀ1ꢀyl)benzoic acid (4b): H NMR (200 MHz, DMSOꢀd6):
2.39, (s, 3H, CH3); 7.31ꢀ7.38, (m, 2H, ArꢀH); 7.44, (d, 1H, JAB = 7.0 Hz, CH=); 7.53, (s, 1H, ArꢀH);
7.68, (d, 1H, JBA = 7.0 Hz, CH=); 7.90, (d, 2H, JAB = 8.2 Hz, ArꢀH); 7.97, (d, 2H, JBA = 8.2 Hz, Arꢀ
H); 13.13, (bs, 1H, OH); 13C NMR (50 MHz, DMSOꢀd6): 20.13, 125.88, 128.35, 128.60, 128.85 (2C),
129.84 (2C), 131.03, 131.41, 132.32, 136.76, 138.55, 138.62, 143.24, 166.89, 194.77.
1
4.1.2.3. (E)ꢀ4ꢀ(3ꢀOxoꢀ3ꢀ(mꢀtolyl)propꢀ1ꢀenꢀ1ꢀyl)benzoic acid (4c): H NMR (200 MHz, DMSOꢀd6):
2.42, (s, 3H, CH3); 7.42ꢀ7.51, (m, 2H, ArꢀH); 7.77, (d, 1H, JAB = 15.6 Hz, CH=); 7.96ꢀ8.04, (m, 6H,
ArꢀH); 8.05, (d, 1H, JBA = 7.0 Hz, CH=); 13.81, (bs, 1H, OH); 13C NMR (50 MHz, DMSOꢀd6): 20.99,
124.50, 125.92, 128.79, 128.95 (2C), 129.11, 129.81 (2C), 132.22, 134.01, 137.55, 138.37, 138.91,
142.45, 166.93, 189.30.
1
4.1.2.4. (E)ꢀ4ꢀ(3ꢀOxoꢀ3ꢀ(pꢀtolyl)propꢀ1ꢀenꢀ1ꢀyl)benzoic acid (4d): H NMR (200 MHz, DMSOꢀd6):
2.40, (s, 3H, CH3); 7.38, (d, 2H, JAB = 7.8 Hz, ArꢀH); 7.78, (d, 1H, JAB = 15.6 Hz, CH=); 7.99, (s, 4H,
ArꢀH); 8.03, (d, 1H, JBA = 15.6 Hz, CH=); 8.09, (d, 2H, JBA = 7.8 Hz, ArꢀH); 13.09, (bs, 1H, OH); 13C
NMR (50 MHz, DMSOꢀd6): 21.36, 124.42, 128.92 (2C), 128.98 (2C), 129.55 (2C), 129.88 (2C),
132.21, 135.04, 139.00, 142.32, 143.96, 167.02, 188.70.
1
4.1.2.5. (Z)ꢀ4ꢀ(3ꢀ(2ꢀMethoxyphenyl)ꢀ3ꢀoxopropꢀ1ꢀenꢀ1ꢀyl)benzoic acid (4e): H NMR (200 MHz,
DMSOꢀd6): 3.88, (s, 3H, OCH3); 7.07, (t, 1H, J = 7.2 Hz, ArꢀH); 7.20, (d, 1H, JAB = 8.2 Hz, CH=);
7.46ꢀ7.63, (m, 3H, ArꢀH); 7.53, (d, 1H, JBA = 8.2 Hz, CH=); 7.85, (d, 2H, JAB = 8.2 Hz, ArꢀH); 7.97,
(d, 2H, JBA = 8.2 Hz, ArꢀH); 13.09, (bs, 1H, OH); 13C NMR (50 MHz, DMSOꢀd6): 56.07, 112.60,
120.79, 128.70 (3C), 129.14, 129.85, 130.00 (2C), 132.18, 133.52, 138.94, 141.05, 158.11, 167.00,
192.05.
1
4.1.2.6. (E)ꢀ4ꢀ(3ꢀ(3ꢀMethoxyphenyl)ꢀ3ꢀoxopropꢀ1ꢀenꢀ1ꢀyl)benzoic acid (4f): H NMR (200 MHz,
DMSOꢀd6): 3.85, (s, 3H, OCH3); 7.25, (dd, 1H, J = 8.0 and 2.2 Hz, ArꢀH); 7.50, (t, 1H, J = 8.0 Hz,
ArꢀH); 7.63, (s, 1H, ArꢀH); 7.77, (d, 1H, JAB = 15.6 Hz, CH=); 7.79, (d, 1H, J = 8.0 Hz, ArꢀH); 7.98,
(d, 2H, JAB = 8.6 Hz, ArꢀH); 8.02, (d, 2H, JBA = 8.6 Hz, ArꢀH); 8.04, (d, 1H, JBA = 15.6 Hz, CH=);
13.09, (bs, 1H, OH); 13C NMR (50 MHz, DMSOꢀd6): 55.56, 113.29, 119.53, 121.33, 124.43, 129.09
(2C), 129.89 (2C), 130.12, 132.31, 138.92, 138.97, 142.78, 159.78, 167.03, 189.04.
1
4.1.2.7. (E)ꢀ4ꢀ(3ꢀ(4ꢀMethoxyphenyl)ꢀ3ꢀoxopropꢀ1ꢀenꢀ1ꢀyl)benzoic acid (4g): H NMR (200 MHz,
DMSOꢀd6): 3.87, (s, 3H, OCH3); 7.09, (d, 2H, J = 9.0 Hz, ArꢀH); 7.74, (d, 1H, JAB = 15.6 Hz, CH=);
7.99, (s, 4H, ArꢀH); 8.06, (d, 1H, JBA = 15.6 Hz, CH=); 8.19, (d, 2H, JBA = 9.0 Hz, ArꢀH); 13.11, (bs,
1H, OH); 13C NMR (50 MHz, DMSOꢀd6): 55.75, 114.25 (2C), 124.43, 128.93 (2C), 129.87 (2C),
130.45, 131.21 (2C), 132.10, 139.12, 141.85, 163.56, 167.03, 187.46.
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4.1.2.8. (Z)ꢀ4ꢀ(3ꢀ(2ꢀFluorophenyl)ꢀ3ꢀoxopropꢀ1ꢀenꢀ1ꢀyl)benzoic acid (4h): 1H NMR (200 MHz,
DMSOꢀd6): 7.36, (d, 1H, JAB = 7.6 Hz, CH=); 7.38ꢀ7.44, (m, 1H, ArꢀH); 7.52ꢀ7.74, (m, 2H, ArꢀH);
7.68, (d, 1H, JBA = 7.6 Hz, CH=); 7.81, (t, 1H, J = 7.6 Hz, ArꢀH); 7.91, (d, 2H, JAB = 8.2 Hz, ArꢀH);
7.99, (d, 2H, JBA = 8.2 Hz, ArꢀH); 13.07, (bs, 1H, OH); 13C NMR (50 MHz, DMSOꢀd6): 116.85 (JCF
=
22.2 Hz), 125.05 (JCF = 3.3 Hz), 126.79 (JCF = 12.9 Hz), 127.70 (JCF = 3.7 Hz), 128.99 (2C), 129.98
(2C), 130.74, 132.53, 134.67 (JCF = 8.8 Hz), 138.50, 143.20, 160.50 (JCF = 250.6 Hz), 166.94, 188.91.
4.1.2.9. (E)ꢀ4ꢀ(3ꢀ(4ꢀFluorophenyl)ꢀ3ꢀoxopropꢀ1ꢀenꢀ1ꢀyl)benzoic acid (4i): 1H NMR (200 MHz,
DMSOꢀd6): 7.42, (t, 2H, J = 8.8 Hz, ArꢀH); 7.78, (d, 1H, JAB = 15.6 Hz, CH=); 7.96ꢀ8.01, (m, 4H, Arꢀ
H); 8.08, (d, 1H, JBA = 15.6 Hz, CH=); 8.28, (m, 2H, ArꢀH); 13.15, (bs, 1H, OH); 13C NMR (50 MHz,
DMSOꢀd6): 115.99 (2C, JCF = 21.8 Hz), 124.16, 129.08 (2C), 129.87 (2C), 131.80 (2C, JCF = 9.3 Hz),
132.33, 134.21 (JCF = 2.4 Hz), 138.88, 142.83, 165.33 (JCF = 250.8 Hz), 167.00, 187.80.
4.1.2.10. (E)ꢀ4ꢀ(3ꢀ(4ꢀBromophenyl)ꢀ3ꢀoxopropꢀ1ꢀenꢀ1ꢀyl)benzoic acid (4j): 1H NMR (200 MHz,
DMSOꢀd6): 7.79, (d, 1H, JAB = 15.6 Hz, CH=); 7.80, (d, 2H, JAB = 8.4 Hz, ArꢀH); 7.96ꢀ8.05, (m, 4H,
ArꢀH); 8.05, (d, 1H, JBA = 15.6 Hz, CH=); 8.12, (d, 2H, JBA = 8.4 Hz, ArꢀH); 13.17, (bs, 1H, OH); 13
C
NMR (50 MHz, DMSOꢀd6): 124.01, 127.67, 129.10 (2C), 129.84 (2C), 130.77 (2C), 132.02 (2C),
132.45, 136.47, 138.77, 143.15, 166.97, 188.40.
4.1.2.11. (E)ꢀ4ꢀ(3ꢀ(4ꢀNitrophenyl)ꢀ3ꢀoxopropꢀ1ꢀenꢀ1ꢀyl)benzoic acid (4k): 1H NMR (200 MHz,
DMSOꢀd6): 7.82, (d, 1H, JAB = 15.6 Hz, CH=); 8.00, (s, 4H, ArꢀH); 8.06, (d, 1H, JBA = 15.6 Hz,
CH=); 8.37, (s, 4H, ArꢀH); 13.18, (bs, 1H, OH); 13C NMR (50 MHz, DMSOꢀd6): 123.97 (3C), 129.22
(2C), 129.85 (2C), 130.09 (2C), 132.64, 138.54, 142.17, 144.09, 150.04, 166.93, 188.39.
1
4.1.2.12. 4ꢀ(3ꢀ(Anthracenꢀ1ꢀyl)ꢀ3ꢀoxopropꢀ1ꢀenꢀ1ꢀyl)benzoic acid (4l): H NMR (200 MHz, DMSOꢀ
d6): 7.54ꢀ7.68, (m, 3H, ArꢀHanthracene); 7.74, (d, 1H, JAB = 16.0 Hz, CH=); 7.86, (d, 1H, JBA = 16.0 Hz,
CH=); 7.96, (d, 2H, JAB = 8.4 Hz, ArꢀH); 8.00, (d, 2H, JBA = 8.4 Hz, ArꢀH); 8.10ꢀ8.18, (m, 3H, Arꢀ
Hanthracene); 8.35, (d, 1H, J = 8.6 Hz, ArꢀHanthracene); 8.71, (s, 1H, ArꢀHanthracene); 9.13, (s, 1H, Arꢀ
H
anthracene); 13.17, (s, 1H, OH); 13C NMR (50 MHz, DMSOꢀd6): 124.32, 124.76, 126.43 (2C), 127.25,
127.76, 128.03, 128.54, 128.79, 128.98 (2C), 129.30, 129.92 (2C), 131.31, 131.61, 132.22, 132.35,
133.10, 135.62, 138.80, 143.30, 166.99, 193.52.
4.1.2.13. (E)ꢀ4ꢀ(3ꢀOxoꢀ3ꢀ(thiophenꢀ2ꢀyl)propꢀ1ꢀenꢀ1ꢀyl)benzoic acid (4m): 1H NMR (200 MHz,
DMSOꢀd6): 7.33, (t, 1H, J = 4.4 Hz, ArꢀHthiophene); 7.76, (d, 1H, JAB = 15.6 Hz, CH=); 8.00, (s, 4H, Arꢀ
H); 8.01, (d, 1H, JBA = 15.6 Hz, CH=); 8.09, (d, 1H, J = 4.4 Hz, ArꢀHthiophene); 8.38, (d, 1H, J = 4.4 Hz,
ArꢀHthiophene); 13.15, (s, 1H, OH); 13C NMR (50 MHz, DMSOꢀd6): 124.24, 129.06 (3C), 129.88 (2C),
132.31, 134.20, 136.02, 138.77, 141.87, 145.44, 166.99, 181.66.
4.1.2.14.
(E)ꢀNꢀ(5ꢀ(3,4ꢀDihydroxyphenyl)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀ4ꢀ(3ꢀoxoꢀ3ꢀphenylpropꢀ1ꢀenꢀ1ꢀ
1
yl)benzꢀamide × H2O (5a): Beige powder; yield: 0.31 g (68%); mp: > 250ºC; H NMR (200 MHz,
DMSOꢀd6): 6.87, (d, 1H, J = 8.2 Hz, Hꢀ5phenolic); 7.24, (dd, 1H, J = 8.2 and 1.6 Hz, Hꢀ6phenolic); 7.39,
(d, 1H, J = 1.6 Hz, Hꢀ2phenolic); 7.55ꢀ7.73, (m, 3H, ArꢀH); 7.80, (d, 1H, JAB = 15.6 Hz, CH=); 8.09, (d,
2H, J = 7.4 Hz, ArꢀH); 8.12, (d, 1H, JBA = 15.6 Hz, CH=); 8.18ꢀ8.22, (m, 4H, ArꢀH); 9.45, (s, 1H,
OH); 9.61, (s, 1H, OH); 13.13, (s, 1H, NH); 13C NMR (50 MHz, DMSOꢀd6): 113.86, 116.34, 119.21,
121.58, 124.57, 128.74 (2C), 128.93 (2C), 129.00 (4C), 133.05, 133.41, 137.50, 138.94, 142.48,
146.00, 148.33, 158.58, 162.53, 164.76, 189.26; IR (KBr, cmꢀ1): 3435; 2925; 1666; 1655; 1601; 1533;
1309; 1296; 1219, 747; Anal. Calcd. For C24H17N3O4S×H2O (461.50 g/mol): C, 62.46; H, 4.15; N,
9.10; S, 6.95; Found: C, 62.54; H, 4.14; N, 9.07; S, 6.97.
4.1.2.15.
(Z)ꢀNꢀ(5ꢀ(3,4ꢀDihydroxyphenyl)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀ4ꢀ(3ꢀoxoꢀ3ꢀ(oꢀtolyl)propꢀ1ꢀenꢀ1ꢀ
1
yl)benzꢀamide × 1.5 H2O (5b): Light yellow powder; yield: 0.41 g (85%); mp: > 250ºC; H NMR
(200 MHz, DMSOꢀd6): 2.41, (s, 3H, CH3); 6.86, (d, 1H, J = 8.0 Hz, Hꢀ5phenolic); 7.24, (d, 1H, J = 8.0
Hz, Hꢀ6phenolic); 7.33ꢀ7.38, (m, 2H, ArꢀH and 1H, Hꢀ2phenolic); 7.45, (d, 1H, JAB = 7.0 Hz, CH=); 7.58,
(s, 2H, ArꢀH); 7.72, (d, 1H, JBA = 7.0 Hz, CH=); 7.98, (d, 2H, JAB = 8.0 Hz, ArꢀH); 8.17, (d, 2H, JBA
=
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8.0 Hz, ArꢀH); 9.45, (s, 1H, OH); 9.61, (s, 1H, OH); 13.11, (s, 1H, NH); 13C NMR (50 MHz, DMSOꢀ
d6): 20.18, 113.87, 116.34, 119.21, 121.58, 125.87, 128.47, 128.68, 128.88 (2C), 129.04 (2C), 131.06,
131.43, 133.14, 136.83, 138.52, 138.69, 143.04, 146.00, 148.33, 158.60, 162.54, 164.81, 194.68; IR
(KBr, cmꢀ1): 3466; 3173; 1667; 1630; 1606; 1532; 1450; 1301; 1275; 807; Anal. Calcd. For
C25H19N3O4S×1.5H2O (484.53 g/mol): C, 61.97; H, 4.58; N, 8.67; S, 6.62; Found: C, 61.99; H, 4.59;
N, 8.65; S, 6.63.
4.1.2.16.
(E)ꢀNꢀ(5ꢀ(3,4ꢀDihydroxyphenyl)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀ4ꢀ(3ꢀoxoꢀ3ꢀ(mꢀtolyl)propꢀ1ꢀenꢀ1ꢀ
yl)benzꢀamide (5c): Yellow powder; yield: 0.38 g (82%); mp: > 250ºC; 1H NMR (200 MHz, DMSOꢀ
d6): 2.44, (s, 3H, CH3); 6.88, (d, 1H, J = 8.0 Hz, Hꢀ5phenolic); 7.26, (dd, 1H, J = 8.0 and 2.0 Hz, Hꢀ
6phenolic); 7.40, (d, 1H, J = 2.0 Hz, Hꢀ2phenolic); 7.44ꢀ7.53, (m, 2H, ArꢀH); 7.81, (d, 1H, JAB = 15.6 Hz,
CH=); 7.96ꢀ8.24, (m, 6H, ArꢀH); 8.12, (d, 1H, JBA = 15.6 Hz, CH=), 9.46, (s, 1H, OH); 9.62, (s, 1H,
OH); 13.14, (s, 1H, NH); 13C NMR (50 MHz, DMSOꢀd6): 20.98, 113.84, 116.31, 119.17, 121.54,
124.67, 125.94, 128.79, 128.97 (4C), 129.12, 133.01, 134.03, 137.52, 138.37, 138.97, 142.29, 145.97,
148.30, 158.52, 162.50, 164.74, 189.26; IR (KBr, cmꢀ1): 3426; 3169; 1644; 1653; 1607; 1532; 1309;
1297; 1254; 758; Anal. Calcd. For C25H19N3O4S (457.51 g/mol): C, 65.63; H, 4.19; N, 9.18; S, 7.01;
Found: C, 65.61; H, 4.18; N, 9.20; S, 7.02.
4.1.2.17.
(E)ꢀNꢀ(5ꢀ(3,4ꢀDihydroxyphenyl)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀ4ꢀ(3ꢀoxoꢀ3ꢀ(pꢀtolyl)propꢀ1ꢀenꢀ1ꢀ
yl)benzꢀamide (5d): Yellow powder; yield: 0.38 g (84%); mp: > 250ºC; 1H NMR (200 MHz, DMSOꢀ
d6): 2.71, (s, 3H, CH3); 6.87, (d, 1H, J = 8.2 Hz, Hꢀ5phenolic); 7.24, (dd, 1H, J = 8.2 and 1.8 Hz, Hꢀ
6phenolic); 7.39, (d, 1H, J = 1.8 Hz, Hꢀ2phenolic); 7.39, (d, 2H, JAB = 7.8 Hz, ArꢀH); 7.78, (d, 1H, JAB =
15.6 Hz, CH=); 8.06ꢀ8.13, (m, 4H, ArꢀH); 8.11, (d, 1H, JBA = 15.6 Hz, CH=); 8.20, (d, 2H, JBA = 7.8
Hz, ArꢀH); 9.46, (s, 1H, OH); 9.61, (s, 1H, OH); 13.13, (s, 1H, NH); 13C NMR (50 MHz, DMSOꢀd6):
21.31, 113.88, 116.34, 119.20, 121.59, 124.61, 128.96 (6C), 129.49 (2C), 132.97, 135.01, 139.02,
142.09, 143.92, 146.00, 148.33, 158.59, 162.53, 164.78, 188.67; IR (KBr, cmꢀ1): 3434; 3192; 2920;
1668; 1654; 1609; 1533; 1308; 1296; 1210; 759; Anal. Calcd. For C25H19N3O4S (457.51 g/mol): C,
65.63; H, 4.19; N, 9.18; S, 7.01; Found: C, 65.60; H, 4.20; N, 9.17; S, 7.00.
4.1.2.18. (Z)ꢀNꢀ(5ꢀ(3,4ꢀDihydroxyphenyl)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀ4ꢀ(3ꢀ(2ꢀmethoxyphenyl)ꢀ3ꢀoxopropꢀ1ꢀ
1
enꢀ1ꢀyl)benzamide × 2 H2O (5e): Yellowꢀgreen powder; yield: 0.36 g (70%); mp: > 250ºC; H NMR
(200 MHz, DMSOꢀd6): 3.89, (s, 3H, OCH3); 6.86, (d, 1H, J = 8.2 Hz, Hꢀ5phenolic); 7.08, (t, 1H, J = 7.4
Hz, ArꢀH); 7.22, (d, 1H, JAB = 8.2 Hz, CH=); 7.23, (d, 1H, J = 8.2 Hz, Hꢀ6phenolic); 7.38, (s, 1H, Hꢀ
2phenolic); 7.54, (d, 1H, JBA = 8.2 Hz, CH=); 7.58, (s, 3H, ArꢀH); 7.93, (d, 2H, JAB = 8.2 Hz, ArꢀH);
8.17, (d, 2H, JBA = 8.2 Hz, ArꢀH); 9.45, (s, 1H, OH); 9.61, (s, 1H, OH); 13.11, (s, 1H, NH); 13C NMR
(50 MHz, DMSOꢀd6): 56.03, 112.56, 113.87, 116.34, 119.22, 120.72, 121.60, 128.62 (2C), 128.73,
129.10 (2C), 129.28, 129.76, 132.99, 133.41, 138.95, 140.84, 146.00, 148.33, 158.06, 158.59, 162.51,
164.81, 192.00; IR (KBr, cmꢀ1): 3434; 3236; 2934; 1661; 1607; 1536; 1448; 1315; 1295; 1245; 747;
Anal. Calcd. For C25H19N3O5S×2H2O (509.54 g/mol): C, 58.93; H, 4.55; N, 8.25; S, 6.29; Found: C,
58.95; H, 4.57; N, 8.26; S, 6.27.
4.1.2.19. (E)ꢀNꢀ(5ꢀ(3,4ꢀDihydroxyphenyl)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀ4ꢀ(3ꢀ(3ꢀmethoxyphenyl)ꢀ3ꢀoxopropꢀ
1
1ꢀenꢀ1ꢀyl)benzamide × H2O (5f): Dark yellow powder; yield: 0.38 g (78%); mp: > 250ºC; H NMR
(200 MHz, DMSOꢀd6): 3.86, (s, 3H, OCH3); 6.87, (d, 1H, J = 8.0 Hz, Hꢀ5phenolic); 7.23ꢀ7.27, (m, 1H,
ArꢀH and 1H, Hꢀ6phenolic); 7.39, (s, 1H, Hꢀ2phenolic); 7.51, (t, 1H, J = 8.0 Hz, ArꢀH); 7.65, (s, 1H, ArꢀH);
7.80, (d, 1H, JAB = 15.6 Hz, CH=); 7.82, (d, 1H, J = 8.0 Hz, ArꢀH); 8.09, (d, 2H, JAB = 8.6 Hz, ArꢀH);
8.10, (d, 1H, JBA = 15.6 Hz, CH=); 8.20, (d, 2H, JBA = 8.6 Hz, ArꢀH); 9.45, (s, 1H, OH); 9.61, (s, 1H,
OH); 13.13, (s, 1H, NH); 13C NMR (50 MHz, DMSOꢀd6): 55.54, 113.32, 113.88, 116.35, 119.23,
119.46, 121.30, 121.61, 124.59, 129.04 (4C), 130.06, 133.06, 138.94 (2C), 142.56, 146.01, 148.34,
158.63, 159.74, 162.55, 164.78, 189.00; IR (KBr, cmꢀ1): 3434; 2937; 1663; 1607; 1538; 1448; 1317;
1293; 1259; 759; Anal. Calcd. For C25H19N3O5S×H2O (491.52 g/mol): C, 61.09; H, 4.31; N, 8.55; S,
6.52; Found: C, 61.11; H, 4.29; N, 8.57; S, 6.53.
4.1.2.20. (E)ꢀNꢀ(5ꢀ(3,4ꢀDihydroxyphenyl)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀ4ꢀ(3ꢀ(4ꢀmethoxyphenyl)ꢀ3ꢀoxopropꢀ
1
1ꢀenꢀ1ꢀyl)benzamide × 0.5 H2O (5g): Light yellow powder; yield: 0.34 g (70%); mp: > 250ºC; H
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NMR (200 MHz, DMSOꢀd6): 3.88, (s, 3H, OCH3); 6.87, (d, 1H, J = 8.2 Hz, Hꢀ5phenolic); 7.10, (d, 2H,
JAB = 9.0 Hz ArꢀH); 7.24, (dd, 1H, J = 8.2 and 2.0 Hz, Hꢀ6phenolic); 7.39, (d, 1H, J = 2.0 Hz, Hꢀ2phenolic);
7.76, (d, 1H, JAB = 15.6 Hz, CH=); 8.08, (d, 2H, JAB = 8.4 Hz, ArꢀH); 8.13, (d, 1H, JBA = 15.6 Hz,
CH=); 8.20, (d, 2H, JBA = 8.4 Hz, ArꢀH); 8.21, (d, 2H, JBA = 9.0 Hz, ArꢀH); 9.46, (s, 1H, OH); 9.61,
(s, 1H, OH); 13.12, (s, 1H, NH); 13C NMR (50 MHz, DMSOꢀd6): 55.71, 113.84, 114.20 (2C), 116.32,
119.18, 121.56, 124.62, 128.46, 128.89 (2C), 128.98 (2C), 130.42, 131.17 (2C), 139.14, 141.61,
145.98, 148.31, 158.48, 162.52, 163.53, 164.78, 187.40; IR (KBr, cmꢀ1): 3406; 3173; 2940; 1670;
1654; 1605; 1593; 1533; 1307; 1263; 1172; 765; Anal. Calcd. For C25H19N3O5S×0.5H2O (482.52
g/mol): C, 62.23; H, 4.18; N, 8.71; S, 6.64; Found: C, 62.25; H, 4.19; N, 8.72; S, 6.65.
4.1.2.21. (Z)ꢀNꢀ(5ꢀ(3,4ꢀDihydroxyphenyl)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀ4ꢀ(3ꢀ(2ꢀfluorophenyl)ꢀ3ꢀoxopropꢀ1ꢀ
1
enꢀ1ꢀyl)benzamide × H2O (5h): Yellow powder; yield: 0.26 g (54%); mp: > 250ºC; H NMR (200
MHz, DMSOꢀd6): 6.86, (d, 1H, J = 8.2 Hz, Hꢀ5phenolic); 7.24, (dd, 1H, J = 8.2 and 1.6 Hz, Hꢀ6phenolic);
7.37, (d, 1H, JAB = 7.6 Hz, CH=); 7.39ꢀ7.44, (m, 1H, ArꢀH and 1H, Hꢀ2phenolic); 7.58ꢀ7.80 (m, 2H, Arꢀ
H and 1H, CH=), 7.83 (t, 1H, J = 7.6 Hz, ArꢀH); 7.99, (d, 2H, JAB = 8.2 Hz, ArꢀH); 8.18, (d, 1H, JBA
=
8.2 Hz, ΑrꢀΗ); 9.46, (s, 1H, OH); 9.61, (s, 1H, OH); 13.14, (s, 1H, NH); 13C NMR (50 MHz, DMSOꢀ
d6): 113.86, 116.33, 116.78 (JCF = 22.2 Hz), 119.21, 121.57, 124.97 (JCF = 3.3 Hz), 126.76 (JCF = 12.8
Hz), 127.81 (JCF = 3.7 Hz), 128.92 (2C), 129.10 (2C), 130.70 (JCF = 2.1 Hz), 133.34, 134.59 (JCF = 8.8
Hz), 138.51, 142.97, 146.00, 148.33, 158.64, 160.46 (JCF = 250.8 Hz), 162.51, 164.79, 188.84; IR
(KBr, cmꢀ1): 3405; 2928; 1671; 1662; 1611; 1539; 1451; 1317; 1293; 1268; 755; Anal. Calcd. For
C24H16N3O4SF×H2O (479.49 g/mol): C, 60.11; H, 3.96; N, 8.76; S, 6.69; Found: C, 60.13; H, 3.95; N,
8.78; S, 6.70.
4.1.2.22. (E)ꢀNꢀ(5ꢀ(3,4ꢀDihydroxyphenyl)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀ4ꢀ(3ꢀ(4ꢀfluorophenyl)ꢀ3ꢀoxopropꢀ1ꢀ
1
enꢀ1ꢀyl)benzamide × 2 H2O (5i): Yellow powder; yield: 0.31 g (63%); mp: > 250ºC; H NMR (200
MHz, DMSOꢀd6): 6.87, (d, 1H, J = 8.2 Hz, Hꢀ5phenolic); 7.24, (dd, 1H, J = 8.2 and 1.6 Hz, Hꢀ6phenolic);
7.38, (d, 1H, J = 1.6 Hz, Hꢀ2phenolic); 7.42, (t, 2H, J = 8.8 Hz, ArꢀH); 7.81, (d, 1H, JAB = 15.6 Hz,
CH=); 8.09, (d, 2H, JAB = 8.4 Hz, ArꢀH); 8.14, (d, 1H, JBA =15.6 Hz, CH=); 8.20, (d, 2H, JBA = 8.4
Hz, ArꢀH); 8.31, (m, 2H, ArꢀH); 9.45, (s, 1H, OH); 9.61, (s, 1H, OH); 13.13, (s, 1H, NH); 13C NMR
(50 MHz, DMSOꢀd6): 113.84, 115.93 (2C, JCF = 21.6 Hz), 116.31, 119.18, 121.55, 124.32, 128.99
(2C), 129.02 (2C), 131.76 (2C, JCF = 9.4 Hz), 133.06, 134.17 (JCF = 2.6 Hz), 138.88, 142.61, 145.98,
148.31, 158.50 (JCF = 6.7 Hz), 162.51, 164.73, 165.28 (JCF = 250.8 Hz), 187.75; IR (KBr, cmꢀ1): 3225;
3071; 2951; 1668; 1654; 1603; 1538; 1508; 1308; 1220; 831; Anal. Calcd. For C24H16N3O4SF×2H2O
(497.50 g/mol): C, 57.94; H, 4.05; N, 8.45; S, 6.44; Found: C, 57.92; H, 4.06; N, 8.43; S, 6.45.
4.1.2.23. (E)ꢀNꢀ(5ꢀ(3,4ꢀDihydroxyphenyl)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀ4ꢀ(3ꢀ(4ꢀbromophenyl)ꢀ3ꢀoxopropꢀ1ꢀ
1
enꢀ1ꢀyl)benzamide × 2 H2O (5j): Yellowꢀgreen powder; yield: 0.49 g (87%); mp: > 250ºC; H NMR
(200 MHz, DMSOꢀd6): 6.87, (d, 1H, J = 7.6 Hz, Hꢀ5phenolic); 7.24, (d, 1H, J = 7.6 Hz, Hꢀ6phenolic); 7.39,
(s, 1H, Hꢀ2phenolic); 7.79, (d, 2H, J = 8.4 Hz, ArꢀH); 7.80, (d, 1H, JAB = 15.6 Hz, CH=); 8.05ꢀ8.21, (m,
6H, ArꢀH and 1H, CH=); 9.46, (s, 1H, OH); 9.61, (s, 1H, OH); 13.13, (s, 1H, NH); 13C NMR (50
MHz, DMSOꢀd6): 113.85, 116.32, 119.19, 121.55, 124.20, 127.63, 129.00 (2C), 129.08 (2C), 130.75
(2C), 131.99 (2C), 133.14, 136.46, 138.83, 142.95, 145.99, 148.32, 158.59, 162.53, 164.69, 188.37;
IR (KBr, cmꢀ1): 3406; 3033; 2923; 1662; 1655; 1600; 1584; 1534; 1305; 1214; 759; Anal. Calcd. For
C24H16N3O4SBr×2H2O (558.41 g/mol): C, 51.62; H, 3.61; N, 7.52; S, 5.74; Found: C, 51.64; H, 3.60;
N, 7.54; S, 5.75.
4.1.2.24. (E)ꢀNꢀ(5ꢀ(3,4ꢀDihydroxyphenyl)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀ4ꢀ(3ꢀ(4ꢀnitrophenyl)ꢀ3ꢀoxopropꢀ1ꢀenꢀ
1ꢀyl)benzamide x 4 H2O (5k): Brown powder; yield: 0.30 g (53%); mp: > 250ºC; 1H NMR (200 MHz,
DMSOꢀd6): 6.86, (d, 1H, J = 6.4 Hz, Hꢀ5phenolic); 7.24, (d, 1H, J = 6.4 Hz, Hꢀ6phenolic); 7.38, (s, 1H, Hꢀ
2phenolic); 7.86, (d, 1H, JAB = 15.6 Hz, CH=); 8.09ꢀ8.19, (m, 4H, ArꢀH and 1H, CH=); 8.40, (s, 4H, Arꢀ
H); 9.45, (s, 1H, OH); 9.61, (s, 1H, OH); 13.16, (s, 1H, NH); 13C NMR (50 MHz, DMSOꢀd6): 113.84,
116.31, 119.18, 121.54, 123.95 (2C), 124.22, 129.02 (2C), 129.23 (2C), 130.09 (2C), 133.36, 138.61,
142.18, 143.91, 145.98, 148.33, 150.07, 158.59, 162.51, 164.71, 188.42; IR (KBr, cmꢀ1): 3404; 3165;
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2930; 1664; 1655; 1607; 1590; 1523; 1306; 1298; 1212; 747; Anal. Calcd. For C24H16N4O6S×4H2O
(560.54 g/mol): C, 51.43; H, 4.32; N, 10.00; S, 5.72; Found: C, 51.45; H, 4.31; N, 9.98; S, 5.73.
4.1.2.25.
(E)ꢀ4ꢀ(3ꢀ(Anthracenꢀ1ꢀyl)ꢀ3ꢀoxopropꢀ1ꢀenꢀ1ꢀyl)ꢀNꢀ(5ꢀ(3,4ꢀdihydroxyphenyl)ꢀ1,3,4ꢀ
1
thiadiazolꢀ2ꢀyl)benzamide × H2O (5l): Dark orange powder; yield: 0.43 g (76%); mp: > 250ºC; H
NMR (200 MHz, DMSOꢀd6): 6.87, (d, 1H, J = 8.2 Hz, Hꢀ5phenolic); 7.25, (d, 1H, J = 8.2 Hz, Hꢀ6phenolic);
7.39, (s, 1H, Hꢀ2phenolic); 7.55ꢀ7.70, (m, 3H, ArꢀHanthracene), 7.78, (d, 1H, JAB = 15.8 Hz, CH=); 7.92, (d,
1H, JBA = 15.8 Hz, CH=); 7.98ꢀ8.22, (m, 4H, ArꢀH and 3H, ArꢀHanthracene); 8.37, (d, 1H, J = 8.4 Hz,
ArꢀHanthracene); 8.73, (s, 1H, ArꢀHanthracene); 9.16, (s, 1H, ArꢀHanthracene); 9.46, (s, 1H, OH); 9.62, (s, 1H,
OH); 13.14, (s, 1H, NH); 13C NMR (50 MHz, DMSOꢀd6): 113.90, 116.36, 119.24, 121.62, 124.27,
124.72, 126.38 (2C), 127.21, 127.72, 127.99, 128.65, 128.74 (2C), 128.93 (2C), 129.05, 131.28,
131.58, 132.19, 133.10, 135.57, 138.82, 143.06, 146.02, 148.35, 158.63, 162.54, 164.80, 193.43; IR
(KBr, cmꢀ1): 3405; 3047; 2930; 1655; 1607; 1538; 1510; 1302; 1290; 1255; 1201; 749; Anal. Calcd.
For C32H21N3O4S×H2O (561.62 g/mol): C, 68.43; H, 4.13; N, 7.48; S, 5.71; Found: C, 68.40; H, 4.14;
N, 7.50; S, 5.73.
4.1.2.26. (E)ꢀNꢀ(5ꢀ(3,4ꢀDihydroxyphenyl)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀ4ꢀ(3ꢀoxoꢀ3ꢀ(thiophenꢀ2ꢀyl)propꢀ1ꢀenꢀ
1
1ꢀyl)benzamide (5m): Yellow powder; yield: 0.28 g (62%); mp: > 250ºC; H NMR (200 MHz,
DMSOꢀd6): 6.87, (d, 1H, J = 8.2 Hz, Hꢀ5phenolic); 7.24, (dd, 1H, J = 8.2 and 1.8 Hz, Hꢀ6phenolic); 7.34, (t,
1H, J = 4.4 Hz, ArꢀHthiophene); 7.39, (d, 1H, J = 1.8 Hz, Hꢀ2phenolic); 7.79, (d, 1H, JAB = 15.6 Hz, CH=);
8.06, (d, 1H, JBA = 15.6 Hz, CH=); 8.08, (d, 2H, JAB = 8.4 Hz, ArꢀH); 8.10, (d, 1H, J = 4.4 Hz, Arꢀ
Hthiophene); 8.20, (d, 2H, JBA = 8.4 Hz, ArꢀH); 8.41, (d, 1H, J = 4.4 Hz, ArꢀHthiophene); 9.47, (s, 1H, OH);
9.62, (s, 1H, OH); 13.13, (s, 1H, NH); 13C NMR (50 MHz, DMSOꢀd6): 113.85, 116.33, 119.20,
121.55, 124.44, 129.01 (5C), 133.07, 134.18, 135.99, 138.80, 141.66, 145.39, 145.99, 148.32, 158.55,
162.53, 164.81, 181.63; IR (KBr, cmꢀ1): 3406; 2927; 1671; 1654; 1595; 1540; 1413; 1317; 1292;
1189; 760; Anal. Calcd. For C22H15N3O4S2 (449.51 g/mol): C, 58.78; H, 3.36; N, 9.35; S, 14.26;
Found: C, 58.80; H, 3.34; N, 9.36; S, 14.21.
4.2. Biology
4.2.1. Cytotoxic activity
The cytotoxic activity of the newly synthesized 1,3,4ꢀthiadiazoleꢀchalcone hybrids containing
antioxidant phenolic moiety was evaluated on three human malignant cell lines: cervical
adenocarcinoma HeLa, acute promyelocytic leukemia HLꢀ60, lung carcinoma A549, and human
normal lung fibroblasts MRCꢀ5, as described in our previous studies.20 All tested cell lines were
obtained from the American Type Culture Collection (Manassas, VA, USA). HeLa (2,000 cells per
well), A549 (5,000 cells per well) and MRCꢀ5 cells (5,000 cells per well) were seeded into 96ꢀwell
microtiter plates and after 20 h the five different concentrations of the compounds (ranging from 6.25
µM to 100 µM or 3.125 µM to 50 µM) were added to the cells. The control cell samples were
incubated in nutrient medium only. HLꢀ60 cells (7,000 cells per well), were seeded 2 h before the
addition of solutions of the compounds. Stock solutions of the compounds were made in DMSO at a
concentration of 5 mM. The cell survival was measured by MTT assay after treatment that lasted 72h,
according to the standard procedure firstly described by Mosmann,52 and which was modified by
Ohno and Abe,53 as described previously in more detail.20 The concentrations of compounds dissolved
in DMSO and further diluted in nutrient medium were carefully chosen in order to eliminate any toxic
side effect of DMSO on all tested human cell lines. The maximum final concentrations of DMSO did
not exceed 1%.
The chemotherapy drug cisplatin was used as a positive control. Each of the three independent
experiment was performed in triplicate.
4.2.2. DPPH free radical scavenging assay
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The determination of the DPPH free radical scavenging activity of the studied compounds
was conducted according to method described by Kontogiorgis et al.54 Briefly, 1 mL (0.05 mM) of
DPPH solution in methanol was mixed with an equivalent volume of the tested compound (20 ꢁL of
compound solution in DMSO and 980 ꢁL of methanol). The sample was stored in dark at room
temperature. The absorbance was acquired at 517 nm after incubation period of 30 min. Methanol was
taken as control. IC50 values represent the concentration necessary to obtain 50% of a maximum
scavenging activity. Ascorbic acid was used as positive control. The results are presented as mean ±
SD calculated from the independent triplicate experiments using Microsoft Excel software.
4.2.3. Determination of intracellular ROS levels
Human HeLa cells were incubated with subtoxic IC20 concentrations of the selected
compounds 5a, 5c, 5f and 5m (applied concentration was 10 µM for each compound) for 24 h.
Afterwards, the HeLa cells were collected, washed with phosphate buffered saline (PBS) and
incubated in a solution of 30 µM 2′,7′ꢀdichlorodihydrofluorescein diacetate (Sigma Aldrich) in PBS
for 45 min at 37°C, according to standard experimental procedure which we described previously.55
The cells were then washed with PBS. The part of treated cell samples was analysed immediately,
while the other part of treated cells samples was exposed to 10 mM hydrogen peroxide solution
(H2O2) for 30 min at 37°C. After 30 min, these cells were washed with PBS and collected. The
intensity of green fluorescence emitted by the dichlorofluorescein was determined on a FACSCalibur
flow cytometer (BD Biosciences Franklin Lakes, NJ, USA). The data (20,000 events acquired for
each cell sample) were analysed using CELLQuest software (BD Biosciences).
4.2.4. Cell cycle analysis by flow cytometry
Human cervical carcinoma HeLa cells were incubated with compounds 5a, 5c, 5f and 5m for
24 h (applied concentrations corresponded to IC50 and 2IC50 concentrations which were determined
after 72 h treatment). Following 24 h incubation, the cells were collected, washed with PBS and fixed
in 70% ethanol, according to standard protocol.56 Cell samples were stored at ꢀ20°C for at least one
week before staining. Afterwards, HeLa cells were collected by centrifugation, washed, resuspended
in PBS containing RNase A and incubated for 30 min at 37°C. Subsequently the propidium iodide
staining solution was added to the cells. Percentages of HeLa cells within specific phases of the cell
cycle were assessed using a BD FACSCalibur flow cytometer. The analyses of acquired data (10,000
events collected for each gated cell sample) were performed using a CELLQuest software. Cell cycle
distribution data are presented as mean±S.D. of three independent experiments. The statistical
significance of differences between the control and cell samples exposed to tested compounds was
evaluated using oneꢀway ANOVA with Dunnett’s post test. p values below 0.05 were considered
statistically significant.
4.2.5. Identification of target caspases
To investigate whether the examined compounds could induce apoptosis in treated HeLa cells
the percentages of subG1 cells in the samples pretreated with specific caspase inhibitors were
detyermined, as previously described in our previous research.20 The HeLa cells were pretreated for 2
h with 40 µM concentrations of the specific peptide caspase inhibitors: ZꢀDEVDꢀFMK, a caspaseꢀ3
inhibitor, ZꢀIETDꢀFMK, a caspaseꢀ8 inhibitor and ZꢀLEHDꢀFMK, a caspaseꢀ9 inhibitor (R&D
Systems, Minneapolis, USA).
4.2.6. Gene and microRNA expression analyses
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HeLa cells were seeded into 75 cm2 cell culture flasks (4×106 cells/flask). After 2 h, the cells
were treated with low subtoxic IC20 concentrations of the compounds 5a, 5c, 5f and 5m for 24 h (10
µM for each compound). Control cells were grown in nutrient medium only. After 24h incubation,
the cells were collected, washed and the cell samples were stored at ꢀ80°C until further analyses. Total
RNA was extracted with TriReagent (Sigma), according to manufacturer’s protocol. Gene expression
was quantified by twoꢀstep reverse transcription reaction followed by realꢀtime quantitative PCR (RTꢀ
qPCR) with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems by Thermo Fisher
Scientific, Vilnius, Lithuania). Reaction of cDNA amplification was performed with MMP2
(Hs01548727_m1), MMP9 (Hs00957562_m1), VEGFA (Hs00900055_m1), and TIMP3
(Hs00165949_m1) assays, using TaqMan technology.
Stemꢀloop reverse transcription and RTꢀqPCR were used for miRꢀ21 (ID 000397), miRꢀ133b (ID
002247), miRꢀ155 (ID 002623), and miRꢀ206 (ID 000510) expression analysis.
Gene and microRNA expression values were normalized to GAPDH (Hs02758991_g1), and small
nuclear RNAꢀRNU6 B (ID001093) and calculated by comparative ꢂꢂCt method, with 7500 System
SDS software (Applied Biosystems, Foster City, California, USA).
4.2.7. Endothelial cell tube formation assay
To explore whether antiangiogenic effects of the compounds were explored on human
umbilical vein endothelial EA.hy926 cells.57,58 The EA.hy926 cells seeded on Corning® Matrigel®
basement membrane matrix (Corning: cat. number 356234) were exposed to suboxic IC20
concentrations of the four tested compounds (the compounds 5a, 5c, 5f (6.5 µM for each compound)
and 5m (8 µM) for 20 h. After treatment that lasted 20 h, the photomicrographs of EA.hy926 cells
were captured under the inverted phaseꢀcontrast microscope.
4.2.8. Interaction with DNA
4.2.8.1. Absorption spectral measurements
Calf thymus DNA (lyophilized, highly polymerized, obtained from Serva, Heidelberg) (CTꢀ
DNA) was used for the experiments. The stock solution was prepared by dissolving the DNA in Tris
buffer (10 mM TrisꢀHCl pH 7.9) overnight at 4 °C. This stock solution was stored at 4 °C and was
stable for several days. A solution of CTꢀDNA in water gave a ratio of UV absorbance at 260 and 280
nm, A260/A280 of 1.89–2.01, indicating that DNA was sufficiently free of protein. The concentration of
DNA (3.16 mg mL−1) was determined from the UV absorbance at 260 nm using the extinction
coefficient ε260=6600 M−1 cm−1.59 1,3,4–thiadiazoleꢀchalcone hybrids 5a, 5c, 5f and 5m were
dissolved in dimethyl sulfoxide in concentration of 2 mM. These solutions were used as stock
solutions.
For an UVꢀVis measurement, a small volume of a stock solution of the compound (20 µL) was added
to DNA solution (10 µL of CTꢀDNA) and the volume was adjusted up to 1 mL with 40 mM
bicarbonate buffer, pH 8.4. Reaction mixtures were incubated at 37 °C for 90 min with occasional
vortexing. UV–Vis spectra were recorded on a UVꢀ1800 Shimadzu UV/Visible spectrophotometer
operating from 200 to 800 nm in 1.0 cm quartz cells. Spectra of the compounds of the same
concentrations were also recorded, as well as spectra of CTꢀDNA.
The percentage of hyperchromism or hypocromism was determined from
{[(εDNA + εCOMP) – εB]/ (εDNA + εCOMP)} x 100
(1)
where εDNA is the extinction coefficient of CTꢀDNA, εCOMP is the extinction coefficient of free
compound and εB is the extinction coefficient of the bound compound complex.
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The absorbance titrations were performed at a fixed concentration of the compound and gradually
increasing concentration of double stranded CTꢀDNA. The absorbance at 259 nm was monitored for
each concentration of DNA. The binding constant Kb was determined using the equation (2):60
[DNA] × (εa – εf)−1 = [DNA] × (εb – εf) −1 + Kb−1 × (εb – εf) −1,
(2)
where εa, εf, εb are absorbance/[compound], extinction coefficient of the free compound and the
extinction coefficient of the bound compound, respectively. A plot of [DNA]/(εa – εf) versus [DNA]
gave a slope and an intercept equal to 1/(εa – εf) and (1/Kb)(1/(εb – εf)), respectively. The binding
constant Kb is calculated from the ratio of the slope to the intercept.
4.2.8.2. Fluorescence measurements
The competitive interactions of 1,3,4ꢀthiadiazoleꢀchalcone hybrids 5a, 5c, 5f and 5m and the
fluorescence probe Hoechst 33258 (H), with CTꢀDNA have been studied by measuring the change of
fluorescence intensity of H−DNA solution after addition of the compound. Reaction mixtures
containing 100 µM of CTꢀDNA (calculated per phosphate) in 1 mL of 40 mM bicarbonate solution
(pH 8.4) were pretreated with 1.5 µL of 1% H probe solution (28 µM final concentration) (in separate
experiments) for 20 min and the mixture was analyzed by fluorescence measurement. Then the
increasing concentrations of the compounds were successively added and the changes in the
fluorescence intensity were measured using a Thermo Scientific Lumina Fluorescence spectrometer
(Finland) equipped with a 150 W Xenon lamp. The slits on the excitation and emission beams were
fixed at 10 nm. All measurements were performed by excitation at 350 nm in the range of 390−550
nm. The control was H−CTꢀDNA solution. Compounds 5a, 5c, 5f and 5m, did not have fluorescence
under applied conditions. The obtained fluorescence quenching data were analyzed according to the
Stern–Volmer equation (3):61
I0/I = 1 + Kr
(3)
where I0 and I represent the fluorescence intensities of H−CTꢀDNA in absence and presence of the
compounds, respectively, K is quenching constant and r is ratio of the bound concentration of a probe
to the bound concentration of DNA (r = [compound]/[CTꢀDNA]. The K value is calculated from the
ratio of the slope to the intercept from the plot of I0/I versus r.
Primary spectra of all spectrometric measurements were imported into OriginPro 9.0 and were
processed by this software package.
4.2.8.3. Experiments with plasmid DNA
Plasmid pUC19 (2686 bp in length, purchased from SigmaꢀAldrich, USA) was prepared by its
transformation in chemically competent cells Escherichia coli (E. coli) strain XL1 blue. Amplification
of the clone was done according to the protocol for growing E. coli culture overnight in LB medium at
37 °C62 and purification was performed using Qiagen Plasmid plus Maxi kit. Finally, DNA was eluted
in 10 mM TrisꢀHCl buffer and stored at −20 °C. The concentration of plasmid DNA (0.460 µg/µL)
was determined by measuring the absorbance of the DNAꢀcontaining solution at 260 nm. One optical
unit corresponds to 50 µg mL−1 of double stranded DNA.
Plasmid DNA (1 µL, 460 ng/ µL) was incubated with increasing volumes of 2 mM stock
solution in DMSO of the compound (0.5, 1, 1.5, 2, 2.5, 3 and 4 µL) in a 20 µL reaction mixture in 40
mM bicarbonate buffer (pH 8.4) at 37 °C, for 90 minutes. DMSO had no efects on DNA conformation
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under aplied concentration. The reaction mixtures were vortexed from time to time. The reaction was
terminated by short centrifugation at 10000 rpm and addition of 5 µL of loading buffer (0.25%
bromophenol blue, 0.25% xylene cyanol FF and 30% glycerol in TAE buffer, pH 8.24 (40 mM Trisꢀ
acetate, 1 mM EDTA)), and analyzed by an agarose electrophoresis.
Interactions of the tested compounds with pUC19 in presence of Fe(II) and Fe(III) were done
as follows. Solutions of 20 µL volume consisted of 1 µL or 2 µL FeSO4 (8 mM, freshly prepared in a
sterile water) or 1 µL or 2 µL FeCl3 (8 mM, freshly prepared in a sterile water) with 4 µL of 2 mM
solution of 1,3,4ꢀthiadiazoleꢀchalcone hybrids 5a, 5c, 5f and 5m in bicarbonate buffer were incubated
at 37oC, for 60 minutes, then 1 µL of pUC19 were added to reaction mixtures and incubated further
for 90 minutes. The reaction mixtures were vortexed from time to time and terminated by short
centrifugation at 10000 rpm and addition of 5 µL of loading buffer and analyzed by agarose
electrophoresis.
4.2.8.4. Agarose electrophoresis
The samples were subjected to electrophoresis on 1% agarose gel (Amersham Pharmaciaꢀ
Biotech, Inc) prepared in TAE buffer pH 8.24. The electrophoresis was performed at a constant
voltage (80 V) until bromophenol blue had passed through 75% of the gel. A Submarine Miniꢀgel
Electrophoresis Unit (Hoeffer HE 33) with an EPS 300 power supply was used. After electrophoresis,
the gel was stained for 30 min by soaking it in an aqueous ethidium bromide solution (0.5 µg mL−1).
The stained gel was illuminated under a UV transilluminator VilberꢀLourmat (France) at 312 nm and
photographed with a Nikon Coolpix P340 Digital Camera through filter DEEP YELLOW 15
(TIFFEN, USA).
4.2.8.5. Comet assay
Human normal MRCꢀ5 cells were treated with four concentrations of selected compounds 5a,
5c, 5f and 5m (applied concentrations were 25 µM, 12.5 µM, 6.25 µM and 3.125 µM for each
compound) for 24 h. The IC20 subtoxic concentration was 25 µM for all four compounds, as
determined by MTT test after 24 h treatment. After incubation, the cells were collected, washed with
phosphate buffered saline (PBS), suspended in freezing medium (RPMI with 10% DMSO and 20 %
FCS) and the cell samples were frozen at ꢀ80°C.
The mediumꢀthroughput version of the singleꢀcell gel electrophoresis assay was used to evaluate
DNA damage as previously described63 with some modifications. Frozen MRCꢀ5 cells were thawed
by addition of 1 ml of PBS to a frozen 0.5 mL aliquot and as soon as the sample was thawed the
suspended cells were centrifuged for 10 min at 2000 rpm at 4°C. The pellet was suspended in PBS
and the washing step was repeated. The number of cells was adjusted to 2.5x105 /ml with PBS and
30µL of cell suspension were mixed with 140 ꢁL of 1% LMP agarose at 37°C. Twelve drops (10 ꢁL)
of agaroseꢀcell suspension were placed on an NMP agarose coated slide using template provided on
the metal base of the 12 gel chamber. Cells were lysed by placing the slides in 2.5 M NaCl, 0.1 M
Na2EDTA, 10 mM Tris with 1% Triton Xꢀ100 pH 10 for 1 h at 4°C. The slides were placed in
horizontal gel electrophoresis tank in electrophoresis solution (0.3 M NaOH, 0.001 M Na2EDTA) for
20 min. Electrophoresis was carried out for 30 min at voltage gradient of 1V/cm across the platform at
4° C. Neutralization of the slides was performed by washing them for 10 min in PBS in a staining jar
at 4°C. Slides were than fixed by placing them in 70% ethanol for 10 min followed by 10 min
incubation in absolute ethanol. DNA was stained by immersing the slides in a staining jar with SYBR
Gold (Invitrogen) for 30 min in the dark. Staining solution was prepared according to the
manufacturer’s instructions in 10 mM Tris, 1 mM EDTA buffer, pH 8. Stained slides were rinsed
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twice with water and left to dry in the dark. Scoring of the comets was carried out using semiꢀ
automated image analysis system (Comet Assay IV; Perceptive Instruments). On each gel 50
nucleoids were analyzed and the results were expressed as percentage of tail intensity (% of DNA in
tail). All experiments were performed in triplicate. PBS was used as negative control. The median %
tail DNA for 50 comets was calculated for each of the duplicate gel in the experiments; the mean of
the two median values was then calculated. The mean percentage of DNA in the tail is calculated
from the independent triplicate experiments using Microsoft Excel software.
Conflicts of interest
There are no conflicts to declare.
Acknowledgments
The authors are grateful to the Ministry of Science and Technological Development of the Republic of
Serbia for financial support (Grant Nos 172016, 172055 and 175011).
Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/
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