Synthesis, cytotoxic activity and DNA-interaction studies of novel anthraquinone-thiosemicarbazones with tautomerizable methylene group

Article abstract of DOI:10.1016/j.ejmech.2013.03.071

A series of novel anthraquinone-thiosemicarbazone derivatives in a tautomerizable keto-imine form was synthesized and tested for their in vitro cytotoxic activity against human cancer cells (HeLa, MDA-MB-361, MDA-MB-453, K562, A549) and human normal MRC-5

Full text of DOI:10.1016/j.ejmech.2013.03.071

European Journal of Medicinal Chemistry 64 (2013) 228e238
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, cytotoxic activity and DNA-interaction studies of novel
anthraquinoneethiosemicarbazones with tautomerizable methylene
group
b
ꢀ a
ꢀ
ꢀ a
ꢀ b
ꢁ
Violeta Markovic , Ana Janicijevic , Tatjana Stanojkovic , Branka Kolundzija ,
ꢁ
ꢀ c
ꢁ ꢀ d
ꢀ c
ꢀ a
Dusan Sladic , Miroslava Vujcic , Barbara Janovic , Ljubinka Joksovic ,
ꢀ a
ꢀ d
ꢁ
ꢀ c
ꢀ a
,
*
Predrag T. Djurdjevic , Nina Todorovic , Snezana Trifunovic , Milan D. Joksovic
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 Faculty of Chemistry, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia
d
ꢁ
Institute for Chemistry, Technology and Metallurgy, University of Belgrade, Njegoseva 12, 11000 Belgrade, Serbia
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel anthraquinoneethiosemicarbazone derivatives in a tautomerizable keto-imine form
was synthesized and tested for their in vitro cytotoxic activity against human cancer cells (HeLa, MDA-
MB-361, MDA-MB-453, K562, A549) and human normal MRC-5 cells. Several compounds efficiently
inhibited cancer cell growth at micromolar concentrations, especially against K562 and HeLa cells. As
determined by flow cytometric analysis, anthraquinoneethiosemicarbazone caused significant increase
in the number of sub-G1 phase of HeLa cells and apoptosis in a concentration-dependent manner. Also,
inhibition of caspase-3, -8, and -9 with specific caspase inhibitors reduced the apoptosis mediated by the
tested compounds in HeLa cells. All anthraquinoneethiosemicarbazones exhibit calf thymus DNA-
binding activity, but no cleavage of plasmid DNA was observed.
Received 16 January 2013
Received in revised form
27 March 2013
Accepted 31 March 2013
Available online 9 April 2013
Keywords:
Anthraquinone
Thiosemicarbazone
Tautomerism
Ó 2013 Elsevier Masson SAS. All rights reserved.
Cytotoxicity
DNA-binding studies
1. Introduction
Certainly, the antitumor activity of anthraquinone derivatives is
strongly affected by the introduction of the different substituents
Many natural and synthetic anthraquinones represent an
important class of compounds with a great biological utility,
especially in the role of antitumor agents [1e6]. Among them, the
anthracyclines, like doxorubicin and daunorubicin have been
widely applied as chemotherapeutic drugs with broad clinical in-
dications as anticancer compounds [7], but with limited use due to
their cardiotoxicity [8,9]. Anthraquinones generally act as inter-
calators [10,11] and inhibitors of telomerase [12e14] and DNA
topoisomerase through stabilization of the cleavable complex be-
tween topoisomerase II and DNA formed in the catalytic cycle of the
enzyme [15e18]. These compounds are also involved in a redox
cycle system by the presence of quinone component and the gen-
eration of reactive oxygen species (ROS) which may have influence
on cell proliferation in multistage carcinogenesis [19,20].
into aromatic planar system. These side groups can stabilize elec-
trostatic interactions with phosphate backbone of the poly-
nucleotide chain and meet requirements for effective inhibition of
topoisomerase II [21]. Thiosemicarbazones belong to a group of
antitumor pharmacophores [22,23] that could stabilize the cleav-
able complex between topoisomerase II and DNA through alkyl-
ation of thiol on the topo IIeDNA complex [24]. In addition,
thiosemicarbazones can inhibit iron-containing enzyme ribonu-
cleotide reductase by metal ion chelation [25] because iron has
been shown to be an essential molecular target in proliferation of
cancer cells [26] and generation of reactive oxygen species [27].
Besides thiosemicarbazone substituent, the presence of an
additional tautomerizable group linked to the pharmacophoric
anthraquinone unit might be very useful in terms of introduction of
specific oncogene recognition elements into structure of compound
with an antitumor potential. For example, curcumin exhibits anti-
tumor activity through inhibition of glyoxalase 1 (Glo 1). The pos-
sibility of existence of this polyphenol in two tautomeric forms
* Corresponding author.
ꢀ
E-mail address: mjoksovic@kg.ac.rs (M.D. Joksovic).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.03.071

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V. Markovic et al. / European Journal of Medicinal Chemistry 64 (2013) 228e238
229
suggests that keto-enol structural segment directly interacts with
the active site of Glo 1 and appears to be crucial for antitumor ac-
tivity [28,29].
On the basis of above mentioned facts, we supposed that
combining anthraquinone and thiosemicarbazone pharmacophore
that keto carbonyl group is linked directly to the anthraquinone
moiety.
Eight AQ-thiosemicarbazones (AQ denotes anthraquinone
moiety substituted in position 1) have been characterized in two
isomer types, Z and E, with respect to the azomethine CaN bond,
whereas 5a exists only in the form of E isomer. The stereochemistry
of these compounds was confirmed by a NOESY experiment which
indicated a correlation between hydrogens of tautomerizable
methylene group and N2 hydrogens from the thiosemicarbazone
moiety. The ratio of Z and E geometrical isomers is time-dependent
and all values presented in Supplementary material are obtained by
recording NMR spectra of freshly prepared solutions in DMSO-d6.
Also, NMR spectra showed that all synthesized AQ-
thiosemicarbazones exist exclusively in keto-imine tautomeric
form in DMSO-d6 solution (Fig. 1).
with
a tautomerism possibility could result in compounds
with favorable biological potential. Consequently, in this study we
designed and synthesized a series of new tautomerizable anthra-
quinoneethiosemicarbazone conjugate to evaluate their anti-
proliferative activity against different cancer cell lines.
2. Results and discussion
2.1. Chemistry
The preparation of target conjugates is presented in Scheme 1.
The synthesis started with commercially available anthracene 1
which was acylated with acetyl chloride to 1-acetylanthracene 2
according to known literature procedure [30]. The compound 2
was then oxidized by chromium(VI) oxide in glacial acetic acid
yielding 1-acetylanthraquinone 3 [31]. After recrystallization
from ethanol, 1-acetylanthraquinone 3 was directly condensed
with diethyl oxalate in the presence of sodium ethoxide affording
anthraquinone ester 4 in an excellent yield. This compound was
used in the next step without further purification and reacted
with a series of substituted thiosemicarbazides to give the final
compounds 5aei in moderate to good yields. The exact structure
of all compounds was confirmed by means of 1H, 13C, NOESY,
COSY, HSQC, HMBC, 1H-15N HSQC and 1H-15N HMBC spectra using
compound 5a as an illustrative example (see Supplementary
material, Figs. S4eS12). The position of ketone carbonyl group
was determined according to the HMBC spectrum which displays
a three-bond correlation between its carbon atom and the
nearest proton of the aromatic core at 7.78 ppm. This indicates
This form is confirmed by the presence of a singlet at 4.35e
4.44 ppm (integral ¼ 2) assigned to the methylene group protons. It
is interesting that 1H NMR spectrum of AQ-ester
4 (see
Supplementary material, Figs. S1 and S2) unambiguously indicates
keto-enol II form which can be easily confirmed by a singlet for
olefinic proton at 6.54 ppm (integral ¼ 1) and a broad singlet at
13.61 ppm attributed to proton of the hydroxyl group. An imine
derivative 6 with poor antiproliferative activity (IC50w100
mM)
obtained by condensation of 4 and aniline exists only in a form of
keto-enamine tautomer. However, phenylhydrazone derivative 7
existing in the keto-imine tautomeric form showed significantly
better cytotoxic activity (IC50 ¼ 40e50
mM) against all cells in
comparison with compound 6. Although the participation of thio-
semicarbazone moiety is crucial for biological potential, it seems
that keto-imine form is another important prerequisite for good
cytotoxicity of the tested anthraquinone compounds. The same
tautomeric forms for all compounds are also observed in CDCl3
indicating that the applied deutero-solvent has no influence on
their tautomeric behavior.
Scheme 1. Reagents and conditions: a) CH3COCl, AlCl3, CH2Cl2, 2 h, 0 ꢀC, 5 M HCl; b) CrO3, CH3COOH, 5 min reflux, H2O; c) (COOEt)2, NaOEt, dioxane, 1 h, stirring, r.t., 2 M HCl; d)
H2NHNC(aS)NHR, EtOH, 4 h, reflux.

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V. Markovic et al. / European Journal of Medicinal Chemistry 64 (2013) 228e238
Fig. 1. The possible tautomeric forms of the compounds 5aei, 6 and 7. AQ denotes anthraquinone core substituted in position 1.
2.2. Biology
activity against all five cancer cell lines. The IC50 values of these
compounds against all cancer cell lines are in a micromolar range
similar or better to that of the antitumor drug cisplatin. Compound
5h exhibits the highest activity against A549 cancer cell lines and
5c against HeLa cancer cell line. Selectivity was observed for 5c
2.2.1. In vitro cytotoxicity
The in vitro cytotoxicity of the AQ-thiosemicarbazones 5aei
against five human cancer cell lines, cervix adenocarcinoma cell
line (HeLa), human breast cancer (MDA-MB-361 and MDA-MB-
453), chronic myelogenous leukemia (K562), non-small cell lung
carcinoma (A549) and a non-cancerous cell line, MRC-5 (human
embryonic lung fibroblast) was investigated by a tetrazolium based
(MTT) colorimetric assay and compared with those of cisplatin (an
anticancer drug used widely in the clinic) as positive control. The
results of cytotoxic activity in vitro are expressed as IC50, the con-
against resistant MDA-MB-361 cell lines (IC50 value of 4.45
while 5g exhibited an excellent selectivity against K562 cell line
(IC50 value of 2.17 M). Compounds 5c, 5d, 5f and 5h can be
mM)
m
considered as agents with potential antitumor activity, and there-
fore as candidates for further stages of screening in vitro and/or
in vivo. In the non-cancerous lung fibroblasts (MRC-5), most of the
compounds were slightly less cytotoxic than cisplatin. The IC50 of
centration of compound (in
by 50% as compared to control untreated cells (Table 1).
m
M) that inhibits a survival of the cells
cisplatin in this cell line was 14.44
from 11.79 to 42.13 M. The cytotoxicities of 5c and 5f in the lung
fibroblasts were marked by IC50 values of 42.13 and 24.35 M,
mM and compounds 5aei ranged
m
Generally, the compounds demonstrated good cytotoxic activity,
m
IC50 values range from 2.17 to 50.54
m
M against all the tested cell
respectively, indicating that they are several times less cytotoxic in
the healthy cells compared to almost all the tested cancerous cells.
lines. The K562 cell line is much more sensitive to the compounds
than the other cell lines; in this case, the activity of compounds can
even be compared with the activity of cisplatin (IC50 values range
2.2.2. Cell-cycle analysis
from 2.17 to 7.99
m
M). This opens the way for further investigations
To determine whether treatment with compounds 5aei could
affect the cell cycle of HeLa cells, the cells treated for 24 h and the
control cells were stained with propidium iodide (PI) and analyzed
of these compounds as potential drug candidates in the treatment
of leukemia. Compounds 5c, 5d, 5f and 5h exhibit the strongest
Table 1
Concentrations of compounds 5aei that induced a 50% decrease in HeLa, MDA-MB-361, MDA-MB-453, K562, A549 and MRC-5 cell survival (expressed as IC50
compounds were incubated with cells for 72 h.
(mM)). All
Compound
IC50 (
HeLa
m
M)a
MDA-MB-361
MDA-MB-453
K562
A549
MRC-5
5a
5b
5c
5d
5e
5f
5g
5h
14.40 ꢁ 1.82
10.87 ꢁ 1.53
7.66 ꢁ 2.44
9.15 ꢁ 1.97
18.96 ꢁ 0.63
8.25 ꢁ 1.98
18.09 ꢁ 1.5
8.19 ꢁ 1.93
21.01 ꢁ 1.01
2.1 ꢁ 0.20
30.66 ꢁ 1.35
30.83 ꢁ 1.11
4.45 ꢁ 0.68
32.63 ꢁ 8.31
47.75 ꢁ 0.21
25.39 ꢁ 1.58
50.54 ꢁ 1.87
6.61 ꢁ 1.51
32.61 ꢁ 2.01
14.74 ꢁ 0.36
13.61 ꢁ 4.10
11.51 ꢁ 0.51
19.55 ꢁ 3.92
4.64 ꢁ 1.77
19.36 ꢁ 1.45
13.47 ꢁ 2.39
17.68 ꢁ 1.03
6.91 ꢁ 0.56
11.31 ꢁ 0.49
3.75 ꢁ 0.12
7.99 ꢁ 2.33
3.80 ꢁ 1.04
5.86 ꢁ 3.05
2.35 ꢁ 0.08
2.63 ꢁ 0.22
2.80 ꢁ 0.26
2.17 ꢁ 0.02
3.59 ꢁ 1.67
3.97 ꢁ 0.24
5.54 ꢁ 1.03
28.96 ꢁ 5.26
19.56 ꢁ 0.55
43.62 ꢁ 2.08
13.87 ꢁ 4.49
22.62 ꢁ 2.74
12.88 ꢁ 7.57
23.39 ꢁ 7.74
9.89 ꢁ 1.94
22.51 ꢁ 0.09
11.92 ꢁ 2.19
36.19 ꢁ 3.67
21.14 ꢁ 0.80
42.13 ꢁ 4.92
13.60 ꢁ 5.90
38.57 ꢁ 9.19
24.35 ꢁ 0.05
13.60 ꢁ 7.65
11.79 ꢁ 0.36
30.27 ꢁ 7.99
14.44 ꢁ 1.90
5i
Cisplatin
a
IC50 values were expressed as the mean ꢁ SD determined from the results of MTT assay in three independent experiments.

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V. Markovic et al. / European Journal of Medicinal Chemistry 64 (2013) 228e238
231
by flow cytometry. Fig. 2 shows a representative cell-cycle distri-
bution of HeLa cells incubated in the absence or presence of 5c, 5f
and 5h for 24 h, the approximate doubling time of this cell line.
The obtained DNA histograms indicate that treatment with IC50
or 2 ꢂ IC50 for 24 h leads to a significant increase in the number of
sub-G1 phase cells and a decrease in the number of G2/M of HeLa
cells after treatment with these compounds. Treatment with 5c, 5f
and 5h at 2 ꢂ IC50 resulted in the highest percentage of cells
(37.32%, 36.27% and 23.74%, respectively) in sub-G1 phase of the
cell cycle. Obviously, the sub-G1 phase cell population of the
compound-treated cells increased in a dose-dependent manner.
Therefore, it has been shown that the activity of these compounds
could be related to apoptotic cell death. Similar results were also
found when HeLa cells were exposed to all the other compounds
5aei (data not shown).
and -9 might be involved in apoptosis induced by compounds
5aei in HeLa cells.
2.2.4. Antioxidant activity
Reactive oxygen species (ROS) are able to induce cell death and
DNA damage leading to increased tumor proliferation [32]. The
antioxidants which scavenge intracellular ROS may inhibit carci-
nogenesis by affecting the molecular events in the initiation, pro-
motion and progression stages [33]. Antioxidant activity of the
synthesized AQ-thiosemicarbazones has been based on scavenging
activity of stable DPPH radicals, as presented in Table 2.
If antioxidant potential cause cytotoxic behavior, bioreducible
groups of anthraquinone might be responsible for anticancer ac-
tivity. However, if the cytotoxicity depends only on intercalative
capacity of the planar anthraquinone scaffold, it might be affected
by substituents that enhance the binding affinity to DNA. Although
all tested AQ-thiosemicarbazones show good to excellent antioxi-
dant activity, it is difficult to establish a relationship between their
antioxidant and cytotoxic potential as a consequence of different
mechanisms of anticancer action. Nevertheless, by comparing the
antioxidant activities of two compounds of similar structures (5h
and 5i), it can be observed that 5h with much better scavenging
potential exhibits much stronger cytotoxicity.
2.2.3. Apoptosis induced by AQ-thiosemicarbazones is mediated by
a caspase dependent pathway
Fig. 3 shows that the apoptosis induced by compounds 5aei was
mediated by stimulating caspase-3, -8, and -9 activities.
To investigate whether compounds 5aei induced apoptosis
through the caspase cascades-dependent pathway, cells were
individually pretreated with inhibitors of caspase-3, -8, and -9
before the treatment with the compounds concentrations cor-
responding to IC90 values for 24 h. Fig. 3 shows that these specific
inhibitors significantly suppressed the caspase activities and
increased the cell viability in analyzed HeLa cells. As shown in
Fig. 3, compound 5f does not show the expected response to
caspase inhibitors; this indicates that the induction of apoptosis
in this compound involved other mechanisms. On the other
hand, the inhibitors of caspase-3, and -9 exerted almost no in-
fluence on the activity of compound 5g. We conclude that
compound 5g induces apoptotic cell death mainly via mito-
chondrial pathway through activation of caspase-8 in HeLa cells.
Finally, our results suggest that the activation of caspases-3, -8,
2.2.5. DNA-interaction studies
In general, non-covalent binding of small molecules with DNA
occurs in three modes: intercalation binding, minor or major
groove binding and electrostatic binding. Anthraquinone and its
derivatives as good intercalators of DNA have been frequently used
as anticancer drugs [11]. To deduce the binding mode of anthra-
quinone derivatives with thiosemicarbazone substituents which
were used for investigation, the UVeVis spectrometry analysis was
performed. Electronic absorption spectra of all compounds recor-
ded at different concentrations without or with fixed concentration
of CT-DNA are shown in Fig. 4. It was found that the spectra of all
Fig. 2. Cell-cycle distribution after 24 h of continuous action of (IC50 and 2 ꢂ IC50) compounds 5c, 5f and 5h in HeLa cell lines. Representative histograms of cell-cycle distribution of
HeLa measured by flow cytometric analysis of DNA content after treatment with these compounds.

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V. Markovic et al. / European Journal of Medicinal Chemistry 64 (2013) 228e238
232
Fig. 3. Effects of 5aei compounds on caspase inhibitors. HeLa cells were exposed to IC90 of compounds 5aei for 24 h in the presence of specific caspase inhibitors (final con-
centration e 40 M) as described under Experimental.
m
anthraquinones exhibit peaks at 255 nm and 278 nm. The parent
compound and the derivatives with alkyl substituents on thio-
semicarbazone residue exhibit a third peak positioned at 300 nm,
while aromatic substituents and cyclohexyl derivatives had a
shoulder at 311e350 nm. The peaks at 278 nm disappeared after
interaction of the anthraquinones with CT-DNA, followed by an
increase of absorption maximum at 255 nm and 311 nm indicating
the formation of an anthraquinoneeCT-DNA complex. Hypo-
chromism observed with lower concentrations of the compounds
indicates intercalation as the most probable interaction. With in-
crease of concentration hyperchromism occurs as the result of
possible non-covalent or electrostatic interactions between
anthraquinone moiety and double helix of DNA. These additional
interactions were most prominent with the compounds with aro-
matic and cyclic moieties.
for each concentration of DNA. The binding constant KB was
determined using the equation (1) [34]
½DNAꢃꢂðεA ꢄεFÞꢄ1 ¼ ½DNAꢃꢂðεB ꢄεFÞꢄ1 þKBꢄ1 ꢂðεB ꢄεFÞꢄ1 (1)
where εA, εF, εB are absorbance/[compound], extinction coefficient
of the free compound and the extinction coefficient of the bound
compound, respectively. The intrinsic binding constant KB (Fig. 5) of
5aeDNA, 5heDNA and 5ieDNA complexes were: 0.55 ꢂ 109 Mꢄ1
,
9.3 ꢂ 109 Mꢄ1 and 2.91 ꢂ 109
M
ꢄ1, respectively. The values are
higher than the values of KB described in the literature for classical
intercalators, for example, ethidiumeDNA, 7 ꢂ 107 Mꢄ1 [35]. Since
the phenyl derivative 5h was always the most active compound of
these three derivatives, it is very likely that the cytotoxic activity is
the consequence of DNA binding.
In order to obtain information on affinity of the anthraquinonee
CT-DNA complex, spectroscopic titrations of the solution of 5ae
DNA, 5heDNA and 5ieDNA with increasing concentration of CT-
DNA were performed. The absorbance at 255 nm was monitored
The ability of the anthraquinone derivatives to cleave DNA was
tested using pUC18 plasmid, which might undergo transformation
into relaxed circular form (FII) from supercoiled form (FI). Under
applied conditions, i.e. 2 mM anthraquinone and 15.4 nM DNA, the
results of agarose gel electrophoresis showed that there was no
significant cleavage with any of synthesized derivatives (Fig. 6).
Since DNA damage by quinone/hydroquinone compounds is
often mediated by oxygen radicals, which can be generated by
reduced forms (semiquinone or hydroquinone), strand scission
ability was tested under reducing conditions, i.e. in the presence of
iron(II). Iron(II) per se is involved in the damage of DNA [36], and
pUC18 was no exception to that (Fig. 7, lanes P þ Fe(II)). When
anthraquinone compounds and iron were used in mole ratio 1:1,
the anthraquinones abolished the effects of iron (Fig. 7a) probably
by complex formation with the thiosemicarbazone moiety. When
the mole ratio quinoneeiron was 1:2, DNA damage by iron could be
detected (Fig. 7b). Iron(III) had no effect under any conditions.
These results indicate that DNA cleavage could not be the cause of
cytotoxicity.
Table 2
IC50 values of tested AQ-thiosemicarbazones, and two
standard compounds on DPPH free radical inhibition.
Compound
IC50 (mM)
5a
5b
5c
5d
5e
5f
5g
5h
20.5
25.8
26.1
16.4
35.8
66.5
46.9
26.2
41.0
15.9
33.9
5i
Ascorbic acid
BHT

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V. Markovic et al. / European Journal of Medicinal Chemistry 64 (2013) 228e238
233
Fig. 4. Absorption spectra of 5aei (20
line).(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
m
M, 40
m
M, 60
m
M and 80
m
M) without CT-DNA (1.2 ꢂ 10ꢄ4 M, red line); with CT-DNA (1.2 ꢂ 10ꢄ4 M, blue line); CT-DNA (1.2 ꢂ 10ꢄ4 M, green
3. Conclusion
4.2. General procedure for the preparation of 5aei
The novel tautomerizable anthraquinoneethiosemicarbazone
derivatives have been prepared and shown to be active against
several cancer cell lines, especially K562 cells with a better cyto-
toxic potential than cisplatin as a widely used chemotherapy drug.
We demonstrated that newly synthesized compounds inhibited
proliferation leading to an increase in sub-G1 phase and promoted
apoptosis by a caspase dependent manner in cervix adenocarci-
noma HeLa cells. The cytotoxic activity is at least in part a conse-
quence of DNA binding.
To the mixture of 1-acetylanthraquinone 3 (0.50 g, 2.00 mmol)
and diethyl oxalate (1.17 g, 8.00 mmol) in dioxane (20.0 mL), so-
dium ethoxide 21 wt.% solution in denatured ethanol (3.1 mL,
8.20 mmol) was slowly added. The resulting dark mixture was then
stirred for 1 h at room temperature. Afterward, the solution was
diluted with H2O (500 mL) and 2 M HCl was added until pH 2 was
reached, followed with a formation of precipitate. The mixture was
left standing at the room temperature for 1 h and compound 4 was
filtered off, washed with H2O and dried over CaCl2. Compound 4
can be further purified by recrystallization from EtOH.
4. Experimental
A mixture of 4 (0.20 g, 0.57 mmol) and substituted thio-
semicarbazide (0.58 mmol) in EtOH was refluxed for 4 h. After
cooling the flask the amount of the precipitate formed during the
reflux increases. Then, the precipitate (5aei) was filtered off,
washed with a small amount of EtOH and dried over CaCl2.
4.1. Physical measurements and methods
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. IR
spectra were recorded on a Perkin Elmer Spectrum One FT-IR
spectrometer with a KBr disc. 1H and 13C NMR spectra were
recorded on a Bruker Avance III 500 MHz spectrometer. The full
assignments of all reported NMR signals were made by use of 1D
and 2D NMR experiments. A Rayleigh UV-1800 UV/VIS Spectro-
photometer was used for determination of antioxidant activity.
Five thiosemicarbazides used for the synthesis of 5ee5i were
synthesized according to the reported procedure [37], while other
three used for the synthesis of 5be5d were prepared according to
Ref. [38].
4.2.1. Ethyl 4-(9,10-dioxo-9,10-dihydroanthracen-1-yl)-2-hydroxy-
4-oxobut-2-enoate (4)
Light orange powder; yield: 0.62 g (89%); mp: 157e158 ꢀC (Dec.);
1H NMR (500 MHz, DMSO-d6): 1.37, (t, 3H, J ¼ 7.0 Hz, CH3); 4.36, (q,
2H, J ¼ 7.0 Hz, OCH2); 6.54, (s,1H, olefinic); 7.65, (dd,1H at C2, J ¼ 7.5
and 1.0 Hz); 7.83, (m, 2H at C6 and C7); 7.89, (t, 1H at C3, J ¼ 7.5 Hz);
8.23, (m,1H at C5); 8.32, (m,1H at C8); 8.46, (dd,1H at C4, J ¼ 7.5 and
1.0 Hz); 13C NMR (125 MHz, DMSO-d6): 14.03, (CH3); 62.52, (OCH2);
103.95, (olefinic); 127.35, (Ar-H); 127.53, (Ar-H); 129.09, (Ar-H);
130.81, (Ar-H); 132.20, (Ar-H); 132.83, (Ar-H); 133.05, (Ar-H);
134.03, (Ar-H); 134.10, (Ar-H); 134.54, (Ar-H); 134.63, (Ar-H); 140.49,
(Ar-H); 161.95, (COO); 162.00, (olefinic); 182.17, (CaO, AQ); 182.72,

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V. Markovic et al. / European Journal of Medicinal Chemistry 64 (2013) 228e238
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Fig. 6. Agarose gel electrophoresis of the supercoiled form FI and the open circular
form FII of plasmid pUC18 (15.4 nM) (lanes P) after incubation with anthraquinones
(2 mM): 5aei. Lane DMSO denotes the control. The arrows show the changes of the
bands.
4.24, (q, 2H, J ¼ 7.0 Hz, OCH2); 4.36, (s, 2H, CH2); 7.78, (dd, 1H at C2,
J ¼ 7.5 and 1.5 Hz); 7.82, (bs, 1H, NH2); 7.97, (m, 2H at C6 and C7);
8.04, (t, 1H at C3, J ¼ 7.5 Hz); 8.23, (m, 1H at C5); 8.32, (m, 1H at C8);
8.32, (dd, 1H at C4, J ¼ 7.5 and 1.5 Hz); 8.84, (s, 1H, NH2); 10.81, (s,
1H, NH); 13C NMR (125 MHz, DMSO-d6): 13.99, (CH3); 40.88, (CH2);
61.34, (OCH2); 126.79, (Ar-H); 127.50, (Ar-H); 127.99, (Ar-H);
130.23, (Ar-H); 131.49, (Ar-H); 132.69, (Ar-H); 132.74, (Ar-H);
133.25, (Ar-H); 134.57, (CaN); 134.68, (Ar-H); 134.81, (Ar-H);
135.08, (Ar-H); 141.95, (Ar-H); 163.91, (COO); 179.77, (CaS); 181.95,
(CaO, AQ); 183.25, (CaO, AQ); 199.80, (CaO); IR (KBr, cmꢄ1): 3457;
3341; 3315; 1712; 1697; 1673; 1659; 1600; 1287; 706; Anal. Calcd.
For C21H17N3O5S (423.45 g/mol): C, 59.57; H, 4.05; N, 9.92; S, 7.57;
Found: C, 59.53; H, 4.10; N, 9.93; S, 7.27.
4.2.3. Ethyl 4-(9,10-dioxo-9,10-dihydroanthracen-1-yl)-4-oxo-2-
(2-(butylcarbamothioyl) hydrazono)-butanoate ꢂ H2O (5b, main
isomer)
Brown powder; yield: 0.20 g (70%); mp: 135e136 ꢀC (Dec.); 1H
NMR (500 MHz, DMSO-d6): 0.92, (t, 3H, J ¼ 7.0 Hz, CH3, Bu); 1.27, (t,
3H, J ¼ 7.0 Hz, CH3); 1.31, (m, 2H, CH2, Bu); 1.58, (m, 2H, CH2, Bu);
3.60, (m, 2H, NHeCH2); 4.24, (q, 2H, J ¼ 7.0 Hz, OCH2); 4.35, (s, 2H,
CH2); 7.77, (d, 1H at C2, J ¼ 7.5 Hz); 7.96, (m, 2H at C6 and C7); 8.03,
(t, 1H at C3, J ¼ 7.5 Hz); 8.21, (m, 1H at C5); 8.30, (m, 1H at C8); 8.31,
(d,1H at C4, J ¼ 7.5 Hz); 8.46, (t,1H, J ¼ 6.0 Hz, NHeBu); 10.81, (s,1H,
NH); 13C NMR (125 MHz, DMSO-d6): 13.72, (CH3, Bu); 13.99, (CH3);
19.56, (CH2, Bu); 30.43, (CH2, Bu); 40.91, (CH2); 43.61, (CH2, Bu);
61.33, (OCH2); 126.79, (Ar-H); 127.47, (Ar-H); 127.99, (Ar-H); 130.21,
(Ar-H); 131.53, (Ar-H); 132.68, (Ar-H); 132.72, (Ar-H); 133.23, (Ar-
H); 134.12, (CaN); 134.67, (Ar-H); 134.81, (Ar-H); 135.09, (Ar-H);
141.92, (Ar-H); 163.85, (COO); 178.04, (CaS); 181.93, (CaO, AQ);
183.23, (CaO, AQ); 199.85, (CaO); IR (KBr, cmꢄ1): 3303; 2957; 1715;
1704; 1674; 1663; 1287; 709; Anal. Calcd. For C25H25N3O5S ꢂ H2O
Fig. 5. Determination of binding constants by absorption titration of 5a (40 mM), 5h
(40 mM) and 5i (40 mM) with CT-DNA at different concentrations (0.2, 0.4, 0.6, 0.8, 1.0,
1.2 and 1.6) ꢂ 10ꢄ4 M. The arrows show the changes in absorbance upon increasing
amounts of CT-DNA. Insets: plot of [DNA]/(εA ꢄ εF) versus [DNA].
(CaO, AQ); 200.08, (CaO); IR (KBr, cmꢄ1): 3435; 1736; 1725; 1672;
1641; 1324; 1288; 1266; 710; Anal. Calcd. For C20H14O6 (350.33 g/
mol): C, 68.57; H, 4.03; Found: C, 68.04; H, 4.13.
4.2.2. Ethyl 4-(9,10-dioxo-9,10-dihydroanthracen-1-yl)-4-oxo-2-
(2-carbamothioylhydrazono)- butanoate (5a)
Light yellow powder; yield: 0.55 g (52%); mp: 199e200 ꢀC
(Dec.); 1H NMR (500 MHz, DMSO-d6): 1.28, (t, 3H, J ¼ 7.0 Hz, CH3);

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235
Fig. 7. Effect of Fe concentration on AQ-thiosemicarbazone (A) induced pUC18 DNA damage (a) 5c quinoneeiron mole ratio 1:1; (b) 5c quinoneeiron mole ratio 1:2.
(497.57 g/mol): C, 60.35; H, 5.47; N, 8.45; S, 6.44; Found: C, 60.65;
H, 5.34; N, 8.67; S, 6.61.
(m, 2H at C6 and C7); 8.03, (t, 1H at C3, J ¼ 7.5 Hz); 8.23, (m, 1H at
C5); 8.30, (m, 1H at C8); 8.32, (dd, 1H at C4, J ¼ 7.5 and 1.0 Hz); 8.78,
(t, 1H, J ¼ 5.5 Hz, NHeCH2eFur); 10.98, (s, 1H, NH); 13C NMR
(125 MHz, DMSO-d6): 14.01, (CH3); 40.67, (CH2eFur); 40.99, (CH2);
61.38, (OCH2); 107.73, (Fur); 110.55, (Fur); 126.80, (Ar-H); 127.45,
(Ar-H); 128.00, (Ar-H); 130.22, (Ar-H); 131.53, (Ar-H); 132.68, (Ar-
H); 132.72, (Ar-H); 133.24, (Ar-H); 134.68, (CaN); 134.80, (Ar-H);
134.89, (Ar-H); 135.09, (Ar-H); 141.91, (Ar-H); 142.22, (Fur);
151.17, (Fur); 163.76, (COO); 178.57, (CaS); 181.94, (CaO, AQ);
183.22, (CaO, AQ); 199.77, (CaO); IR (KBr, cmꢄ1): 3307; 3148;
1716; 1702; 1674; 1662; 1288; 1165; 709; Anal. Calcd. For
4.2.4. Ethyl 4-(9,10-dioxo-9,10-dihydroanthracen-1-yl)-4-oxo-2-
(2-(hexylcarbamothioyl) hydrazono)- butanoate ꢂ H2O (5c, main
isomer)
Beige powder; yield: 0.19 g (63%); mp: 154e155 ꢀC (Dec.); 1H
NMR (500 MHz, DMSO-d6): 0.87, (t, 3H, J ¼ 6.5 Hz, CH3, Hx); 1.27, (t,
3H, J ¼ 7.0 Hz, CH3); 1.29, (m, 6H, CH2, Hx); 1.59, (m, 2H, CH2, Hx);
3.59, (m, 2H, CH2, Hx); 4.23, (q, 2H, J ¼ 7.0 Hz, OCH2); 4.36, (s, 2H,
CH2); 7.77, (dd, 1H at C2, J ¼ 7.5 and 1.0 Hz); 7.96, (m, 2H at C6 and
C7); 8.03, (t, 1H at C3, J ¼ 7.5 Hz); 8.22, (m, 1H at C5); 8.30, (m, 1H at
C8); 8.32, (dd, 1H at C4, J ¼ 7.5 and 1.0 Hz); 8.46, (t, 1H, J ¼ 6.0 Hz,
NHeHx); 10.81, (s, 1H, NH); 13C NMR (125 MHz, DMSO-d6): 13.90,
(CH3, Hx); 13.99, (CH3); 22.05, (CH2, Hx); 25.99, (CH2, Hx); 28.23,
(CH2, Hx); 30.96, (CH2, Hx); 40.90, (CH2); 43.88, (CH2, Hx); 61.32,
(OCH2); 126.79, (Ar-H); 127.48, (Ar-H); 127.99, (Ar-H); 130.21, (Ar-
H); 131.54, (Ar-H); 132.67, (Ar-H); 132.71, (Ar-H); 133.22, (Ar-H);
134.10, (CaN); 134.67, (Ar-H); 134.81, (Ar-H); 135.09, (Ar-H);
141.92, (Ar-H); 163.85, (COO); 178.02, (CaS); 181.92, (CaO, AQ);
183.23, (CaO, AQ); 199.84, (CaO); IR (KBr, cmꢄ1): 3311; 3231;
2927; 1717; 1698; 1677; 1664; 1288; 1171; 709; Anal. Calcd. For
C
26H21N3O6S ꢂ 0.5H2O (512.54 g/mol): C, 60.93; H, 4.33; N, 8.20; S,
6.26; Found: C, 60.80; H, 4.39; N, 8.24; S, 6.30.
4.2.7. Ethyl 4-(9,10-dioxo-9,10-dihydroanthracen-1-yl)-4-oxo-2-(2-
(thiophen-2-ylmethyl carbamothioyl)hydrazono)-butanoate ꢂ H2O
(5f, main isomer)
Grey powder; yield: 0.19 g (62%); mp: 208e209 ꢀC (Dec.); 1H
NMR (500 MHz, DMSO-d6): 1.26, (t, 3H, J ¼ 7.0 Hz, CH3); 4.24, (q, 2H,
J ¼ 7.0 Hz, CH2); 4.37, (s, 2H, CH2); 5.02, (d, 2H, J ¼ 6.0 Hz, CH2e
Thio); 6.99, (dd, 1H, J ¼ 5.0 and 3.5 Hz, Thio); 7.10, (d, 1H, J ¼ 3.5 Hz,
Thio); 7.41, (dd, 1H, J ¼ 5.0 and 1.5 Hz, Thio); 7.78, (dd, 1H at C2,
J ¼ 7.5 and 1.0 Hz); 7.97, (m, 2H at C6 and C7); 8.03, (t, 1H at C3,
J ¼ 7.5 Hz); 8.23, (m, 1H at C5); 8.30, (m, 1H at C8); 8.31, (dd, 1H at
C4, J ¼ 7.5 and 1.0 Hz); 8.98, (t,1H, J ¼ 6.0 Hz, NHeCH2eThio); 10.94,
(s, 1H, NH); 13C NMR (125 MHz, DMSO-d6): 14.01, (CH3); 41.02,
(CH2); 42.22, (CH2eThio); 61.37, (OCH2); 125.46, (Thio); 126.42,
(Thio); 126.54, (Thio); 126.80, (Ar-H); 127.48, (Ar-H); 128.01, (Ar-
H); 130.22, (Ar-H); 131.53, (Ar-H); 132.70, (Ar-H); 132.72, (Ar-H);
133.25, (Ar-H); 134.69, (CaN); 134.81, (Ar-H); 134.96, (Ar-H);
135.10, (Ar-H); 140.90, (Thio); 141.91, (Ar-H); 163.76, (COO); 178.29,
(CaS); 181.95, (CaO, AQ); 183.24, (CaO, AQ); 199.78, (CaO); IR (KBr,
cmꢄ1): 3303; 1711; 1698; 1674; 1659; 1288; 1176; 709; Anal. Calcd.
For C26H21N3O5S2 ꢂ H2O (537.62 g/mol): C, 58.09; H, 4.31; N, 7.82; S,
11.93; Found: C, 58.17; H, 4.40; N, 7.92; S, 12.12.
C
27H29N3O5S ꢂ H2O (525.63 g/mol): C, 61.70; H, 5.94; N, 7.99; S,
6.10; Found: C, 61.84; H, 6.22; N, 8.09; S, 6.17.
4.2.5. Ethyl 4-(9,10-dioxo-9,10-dihydroanthracen-1-yl)-4-oxo-2-
(2-(cyclohexylcarbamothioyl) hydrazono)-butanoate ꢂ 0.5H2O (5d)
Light yellow powder; yield: 0.16 g (54%); mp: 189e190 ꢀC
(Dec.); 1H NMR (500 MHz, DMSO-d6): 1.26, (m, 1H, Cy); 1.26, (t, 3H,
J ¼ 7.0 Hz, CH3); 1.38, (m, 4H, Cy); 1.57, (m, 1H, Cy); 1.68, (m, 2H,
Cy); 1.91, (m, 2H, Cy); 4.21, (m, 1H, Cy); 4.23, (q, 2H, J ¼ 7.0 Hz,
OCH2); 4.35, (s, 2H, CH2); 7.78, (dd, 1H at C2, J ¼ 7.5 and 1.0 Hz);
7.97, (m, 2H at C6 and C7); 8.03, (t, 1H at C3, J ¼ 7.5 Hz); 8.10, (d, 1H,
J ¼ 8.0 Hz, NHeCy); 8.23, (m, 1H at C5); 8.29, (m, 1H at C8); 8.32,
(dd, 1H at C4, J ¼ 7.5 and 1.0 Hz); 10.89, (s, 1H, NH); 13C NMR
(125 MHz, DMSO-d6): 13.94, (CH3); 24.31, (Cy); 24.99, (Cy); 31.32,
(Cy); 40.86, (CH2); 52.40, (Cy); 61.35, (OCH2); 126.80, (Ar-H);
127.44, (Ar-H); 128.00, (Ar-H); 130.22, (Ar-H); 131.57, (Ar-H);
132.69, (Ar-H); 132.72, (Ar-H); 133.23, (Ar-H); 133.97, (CaN);
134.68, (Ar-H); 134.79, (Ar-H); 135.09, (Ar-H); 141.90, (Ar-H);
163.74, (COO); 176.89, (CaS); 181.94, (CaO, AQ); 183.20, (CaO, AQ);
199.82, (CaO); IR (KBr, cmꢄ1): 3297; 3159; 2930; 1734; 1707; 1675;
1663; 1287; 709; Anal. Calcd. For C27H27N3O5S ꢂ 0.5H2O (514.60 g/
mol): C, 63.02; H, 5.48; N, 8.17; S, 6.23; Found: C, 63.19; H, 5.62; N,
8.23; S, 6.26.
4.2.8. Ethyl 4-(9,10-dioxo-9,10-dihydroanthracen-1-yl)-4-oxo-2-(2-
(pyridin-3-ylmethylcarbamo thioyl)hydrazono)-butanoate ꢂ H2O
(5g, main isomer)
Light brown powder; yield: 0.18 g (59%); mp: 137e138 ꢀC (Dec.);
1H NMR (500 MHz, DMSO-d6): 1.26, (t, 3H, J ¼ 7.0 Hz, CH3); 4.24, (q,
2H, J ¼ 7.0 Hz, CH2); 4.39, (s, 2H, CH2); 4.89, (d, 2H, J ¼ 6.0 Hz, CH2e
Py); 7.39, (dd,1H, J ¼ 7.5 and 4.5 Hz, Py); 7.78, (d, 2H, J ¼ 7.5 Hz, C2 and
Py); 7.96, (m, 2H at C6 and C7); 8.03, (t,1H at C3, J ¼ 7.5 Hz); 8.23, (m,
1H at C5); 8.30, (m, 1H at C8); 8.32, (d, 1H at C4, J ¼ 7.5 Hz); 8.48, (d,
1H, J ¼ 4.5 Hz, Py); 8.59, (s,1H, Py); 9.15, (t,1H, J ¼ 6.0 Hz, NHeCH2e
Py); 10.95, (s, 1H, NH); 13C NMR (125 MHz, DMSO-d6): 14.02, (CH3);
41.08, (CH2); 44.80, (CH2ePy); 61.40, (OCH2); 123.47, (Py); 126.81,
(Ar-H); 127.46, (Ar-H); 128.01, (Ar-H); 130.22, (Ar-H); 131.54, (Ar-H);
132.69, (Ar-H); 132.71, (Ar-H); 133.24, (Ar-H); 134.32, (Py); 134.68,
(CaN); 134.81, (Ar-H); 135.11, (Ar-H); 135.16, (Ar-H); 135.44, (Py);
141.90, (Ar-H); 148.06, (Py); 148.88, (Py); 163.80, (COO); 178.95,
(CaS); 181.94, (CaO, AQ); 183.25, (CaO, AQ); 199.81, (CaO); IR (KBr,
cmꢄ1): 3302; 1709; 1699; 1674; 1661; 1288; 1166; 709; Anal. Calcd.
4.2.6. Ethyl 4-(9,10-dioxo-9,10-dihydroanthracen-1-yl)-4-oxo-2-(2-
(furan-2-ylmethylcarbamo thioyl)hydrazono)-butanoate ꢂ 0.5H2O
(5e, main isomer)
Light yellow powder; yield: 0.16 g (55%); mp: 162e163 ꢀC (Dec.);
1H NMR (500 MHz, DMSO-d6): 1.26, (t, 3H, J ¼ 7.0 Hz, CH3); 4.24, (q,
2H, J ¼ 7.0 Hz, CH2); 4.85, (d, 2H, J ¼ 5.5 Hz, CH2eFur); 6.34, (d, 1H,
J ¼ 3.0 Hz, Fur); 6.43, (dd, 1H, J ¼ 3.0 and 2.0 Hz, Fur); 7.61, (dd, 1H,
J ¼ 2.0 and 1.0 Hz, Fur); 7.78, (dd, 1H at C2, J ¼ 7.5 and 1.0 Hz); 7.97,

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V. Markovic et al. / European Journal of Medicinal Chemistry 64 (2013) 228e238
236
For C27H22N4O5S ꢂ H2O (532.58 g/mol): C, 60.89; H, 4.54; N,10.52; S,
measuring of Asample. The radical scavenging activities of the sam-
ples were expressed in terms of IC50 (concentration required for a
50% decrease in absorbance of DPPH radical) by plotting the ob-
tained IC values against concentration of the tested sample using
OriginPro8 statistical software [40].
6.02; Found: C, 60.93; H, 4.73; N, 10.32; S, 5.92.
4.2.9. Ethyl 4-(9,10-dioxo-9,10-dihydroanthracen-1-yl)-4-oxo-2-
(2-(phenylcarbamothioyl) hydrazono)-butanoate ꢂ H2O (5h, main
isomer)
Beige powder; yield: 0.39 g (66%); mp: 165e166 ꢀC (Dec.); 1H
NMR (500 MHz, DMSO-d6): 1.30, (t, 3H, J ¼ 7.0 Hz, CH3); 4.26, (q, 2H,
J ¼ 7.0 Hz, CH2); 4.44, (s, 2H, CH2); 7.25, (m, 1H, Ph); 7.41, (t, 2H,
J ¼ 7.5 Hz, Ph); 7.60, (dd, 2H, J ¼ 7.5 and 1.0 Hz, Ph); 7.82, (dd, 1H at
C2, J ¼ 7.5 and 1.0 Hz); 7.96, (m, 2H at C6 and C7); 8.04, (t, 1H at C3,
J ¼ 7.5 Hz); 8.23, (m, 1H at C5); 8.31, (m, 1H at C8); 8.34, (dd, 1H at
4.4. Biological experiments
4.4.1. Compounds and solutions
Stock solutions of investigated compounds, were prepared in
DMSO at concentrations of 20 mM and afterward they were diluted
with complete nutrient medium (RPMI-1640 without phenol red)
C4, J ¼ 7.5 and 1.0 Hz); 10.15, (s, 1H, NHePh); 11.13, (s, 1H, NH); 13
C
supplemented with 3 mM L-glutamine, 100 mg/mL streptomycin,
NMR (125 MHz, DMSO-d6): 14.01, (CH3); 41.09, (CH2); 61.44,
(OCH2); 125.14, (Ph); 125.77, (Ph); 126.81, (Ar-H); 127.46, (Ar-H);
128.03, (Ar-H); 128.38, (Ph); 130.27, (Ar-H); 131.58, (Ar-H); 132.71,
(Ar-H); 132.74, (Ar-H); 133.26, (Ar-H); 134.68, (CaN); 134.81, (Ar-
H); 134.92, (Ar-H); 135.11, (Ar-H); 138.53, (Ph); 141.91, (Ar-H);
163.90, (COO); 177.41, (CaS); 181.96, (CaO, AQ); 183.25, (CaO, AQ);
199.81, (CaO); IR (KBr, cmꢄ1): 3284; 1704; 1673; 1655; 1159; 707;
Anal. Calcd. For C27H21N3O5S ꢂ H2O (517.56 g/mol): C, 62.66; H,
4.48; N, 8.12; S, 6.20; Found: C, 62.96; H, 4.54; N, 8.26; S, 6.32.
100 IU/mL penicillin, 10% heat inactivated fetal bovine serum (FBS),
and 25 mM: 2-[4-(2-hydroxyethyl)piperazinyl]ethanesulfonic acid
(HEPES) adjusted to pH 7.2 by bicarbonate solution. The MTT (3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was
dissolved (5 mg/mL) in phosphate buffer saline pH 7.2 and filtered
(0.22 mm) before use.
RPMI-1640, FBS, HEPES, MTT and
Sigma Chemical Co., St. Louis, MO.
L-glutamine were products of
4.4.2. Cell culture
4.2.10. Ethyl 4-(9,10-dioxo-9,10-dihydroanthracen-1-yl)-4-oxo-2-
(2-(p-tolylcarbamothioyl) hydrazono)-butanoate (5i, main isomer)
Beige powder; yield: 0.33 g (56%); mp: 200e201 ꢀC (Dec.); 1H
NMR (500 MHz, DMSO-d6): 1.31, (t, 3H, J ¼ 7.0 Hz, CH3); 2.32, (s, 3H,
CH3ePh); 4.26, (q, 2H, J ¼ 7.0 Hz, CH2); 4.43, (s, 2H, CH2); 7.20, (d,
2H, J ¼ 8.5 Hz, Ph); 7.46, (d, 2H, J ¼ 8.5 Hz, Ph); 7.81, (dd, 1H at C2,
J ¼ 7.5 and 1.0 Hz); 7.98, (m, 2H at C6 and C7); 8.05, (t, 1H at C3,
J ¼ 7.5 Hz); 8.24, (m, 1H at C5); 8.31, (m, 1H at C8); 8.33, (dd, 1H at
Cervix adenocarcinoma cell line (HeLa), human breast cancer
(MDA-MB-361) and (MDA-MB-453) cell lines, human chronic my-
elogenous leukemia (K562) cells, non-small cell lung carcinoma
(A549) and a non-cancerous cell line, MRC-5 (human embryonic
lung fibroblast) were grown in RPMI-1640 medium (Sigma). Media
were supplemented with 10% fetal bovine serum, L-glutamine, and
penicillin-streptomycin (Sigma). CLL lymphocytes were separated
from the heparinized blood samples of patients with chronic
lymphocytic leukemia and resuspended in RPMI-1640 supple-
mented with 10% fetal bovine serum (FBS).
C4, J ¼ 7.5 and 1.0 Hz); 10.06, (s, 1H, NHePh); 11.08, (s, 1H, NH); 13
C
NMR (125 MHz, DMSO-d6): 14.01, (CH3); 20.61, (CH3ePh); 41.07,
(CH2); 61.44, (OCH2); 122.60, (Ph); 125.09, (Ph); 126.82, (Ar-H);
127.47, (Ar-H); 128.03, (Ar-H); 128.84, (Ph); 130.27, (Ar-H); 131.58,
(Ar-H); 132.72, (Ar-H); 132.75, (Ar-H); 133.27, (Ar-H); 134.69,
(CaN); 134.82, (Ar-H); 134.95, (Ar-H); 135.10, (Ar-H); 135.96, (Ph);
141.92, (Ar-H); 163.91, (COO); 177.39, (CaS); 181.98, (CaO, AQ);
183.26, (CaO, AQ); 199.84, (CaO); IR (KBr, cmꢄ1): 3301; 1731; 1703;
4.4.3. Treatment of cell lines
Target cells HeLa (2000 cells per well), MDA-MB-361 (7000 cells
per well), MDA-MB-453 (3000 cells per well), K562 (5000 cells per
well), A549 (5000 cells per well) and non-cancerous MRC-5 (5000
cells per well) were seeded into wells of a 96-well flat-bottomed
microtitre plate. Twenty-four hours later, after the cell adherence,
different concentrations of investigated compounds were added to
the wells, except for the control cells to which a nutrient medium
only was added. Final concentrations achieved in treated wells
1671; 1659; 1287; 1153; 710; Anal. Calcd. For
C28H23N3O5S
(513.57 g/mol): C, 65.48; H, 4.51; N, 8.18; S, 6.24; Found: C, 65.26; H,
4.36; N, 8.23; S, 6.25.
were 5 mM, 10 mM, 25 mM, 50 mM, and 100 mM. The final concen-
4.3. Antioxidant activity
tration of DMSO solvent never exceeded 0.5%, which was non-toxic
to the cells. Especially, compounds were applied to the suspension
of K562 cells 2 h after the cell seeding. All concentrations were set
up in triplicate. Nutrient medium with corresponding concentra-
tions of investigated compounds, but without cells, was used as a
blank, also in triplicate. The cultures were incubated for 72 h.
All the synthesized compounds were evaluated for antioxidant
activity and compared with corresponding standards (ascorbic acid
and BHT). The activity was evaluated using the DPPH method [39].
The 200
tested sample solutions (2 mL each) of different concentrations (8e
1000 M) in methanol. Due to the low solubility, compound 5f was
mM solution of DPPH (2 mL) in methanol was added to
m
4.4.4. Determination of cell survival
dissolved in the mixture of DMSO and MeOH (2:1). The resulting
solution was allowed to react in dark at room temperature for
30 min. Afterward, the absorbance values were measured spec-
trophotometrically at 517 nm and converted into the percentage
antioxidant activity (IC) using formula
The effect of the prepared compounds on cancer cell survival
was determined by the microculture tetrazolium test (MTT) ac-
cording to Mosmann [41] with modification by Ohno and Abe [42]
72 h after addition of the compounds, as described earlier. Briefly,
20 mL of MTT solution (5 mg/mL phosphate-buffered saline) was
added to each well. Samples were incubated for a further 4 h at
hꢀ
ꢁ.
i
37 ꢀC in a humidified atmosphere of 95% air/5% CO2 (v/v). Then
ICð%Þ ¼
ADPPH ꢄ Asample ADPPH ꢂ 100;
100 mL of 100 g/L sodium dodecyl sulfate was added to extract the
insoluble product formazan resulting from conversion of the MTT
dye by viable cells. The number of viable cells in each well was
proportional to the intensity of the absorbance of light, which was
read in an enzyme-linked immunosorbent assay (ELISA) plate
reader at 570 nm. The absorbance (A) at 570 nm was measured 24 h
where ADPPH was the absorbance of DPPH solution and Asample was
the absorbance of sample solution with DPPH. To correct any in-
fluence due to color of the tested compounds blank solutions
(without DPPH) were prepared and used as a reference for the

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V. Markovic et al. / European Journal of Medicinal Chemistry 64 (2013) 228e238
237
later. To determine cell survival (%), the A of a sample with cells
grown in the presence of various concentrations of the investigated
compounds was divided by the control optical density (the A of
control cells grown only in nutrient medium) and multiplied by
100. It was implied that the A of the blank was always subtracted
from the A of the corresponding sample with target cells. IC50 was
defined as the concentration of an agent inhibiting cell survival by
50% compared with a vehicle-treated control. As positive controls,
cis-diaminedichloroplatinum (cis-DDP) and doxorubicin were used.
All experiments were done in triplicate.
200 to 800 nm in 1.0 cm quartz cells. Primary spectra of all spec-
trometric measurements were imported into OriginPro 8.0.
The absorbance titrations were performed at a fixed concen-
tration of anthraquinone compound and varying the concentration
of double stranded CT-DNA. For an individual experiment, each
DNA solution (0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5 and 20
mL of CT-DNA
stock solution) was pipetted into a separate vial, to which 4
mL of
a stock solution of compound was added and the volume adjusted
to 1 mL with the bicarbonate buffer and incubated at 37 ꢀC for 1.5 h
with occasional vortexing. UVeVis spectra were recorded as
described above.
4.4.5. Flow cytometry analysis
Cellular DNA content and cell distribution were quantified by
flow cytometry using propidium iodide (PI). Cells (3 ꢂ 105 cells/
well) were seeded in 6-well plates and incubated with or without
IC50 and 2 ꢂ IC50 concentrations of investigated compounds for
24 h. After treatment, the cells were collected by trypsinization, and
fixed in ice-cold 70% ethanol at ꢄ20 ꢀC overnight. After fixation, the
cells were washed in PBS and pellets obtained by centrifugation
4.5.2. DNA cleavage experiments
4.5.2.1. Preparation and purification of plasmid DNA. The plasmid
pUC18 (pUC18, 2686 bp, purchased from SigmaeAldrich, USA) was
prepared by its transformation in chemically competent cells
Escherichia 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 ꢀC [45] and purification was performed using Qiagen
Plasmid plus Maxi kit. Finally, DNA was eluted in 10 mM TriseHCl
buffer and stored at ꢄ20 ꢀC. The concentration of plasmid DNA
were treated with RNase (100
m
g/mL) at 37 ꢀC temperature for
g/mL)
30 min and then incubated with propidium iodide (PI) (40
m
for at least 30 min. DNA content and cell-cycle distribution were
analyzed using a Becton Dickinson FACSCalibur flow cytometer.
Flow cytometry analysis was performed using a CellQuestR (Becton
Dickinson, San Jose, CA, USA), software on a minimum of 10,000
cells per sample [43].
(131 ng/
DNA containing solution at 260 nm. One optical unit corresponds to
50 g/mL of double stranded DNA.
The cleavage reaction of supercoiled pUC18 DNA by AQ-
thiosemicarbazones (2 mM) was investigated by incubation of
mL) was determined by measuring the absorbance of the
m
262 ng of pUC18 in a 20 mL reaction mixture in 40 mM bicarbonate
4.4.6. Determination of target caspases
buffer pH 8.4 at 37 ꢀC, for 90 min. The reaction mixtures were
vortexed from time to time. The reaction was terminated by short
centrifugation at 10,000 rpm and addition of 5 mL of loading buffer
In order to examine the role of caspases involved in the
apoptotic cell death induced by the investigated extracts, the per-
centages of HeLa cells pretreated with caspase inhibitors in sub-G1
phase were determined. HeLa cells were preincubated for 2 h with
(0.25% bromophenol blue, 0.25% xylene cyanol FF and 30% glycerol
in TAE buffer, pH 8.24 (40 mM Triseacetate, 1 mM EDTA)). The
samples were subjected to electrophoresis on 1% agarose gel
(Amersham Pharmacia-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 Mini-gel 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
specific caspase inhibitors (final concentration e 40 mM): Z-DEVD-
FMK (caspase-3 inhibitor), Z-IETD-FMK (caspase-8 inhibitor) and Z-
LEHD-FMK (caspase-9 inhibitor). Caspase inhibitors were pur-
chased from R&D Systems (Minneapolis, USA). Five tested extracts
were applied to target HeLa cells at concentrations which corre-
sponded to IC90 values obtained for 72 h. For each extract, one
sample of HeLa cells was not treated with inhibitor and served as a
referent sample. After 24 h of incubation, cells were harvested,
fixed in 70% ethanol on ice. Samples were stored at ꢄ20 ꢀC for one
week before PI staining. Changes in the percentages of cells in sub-
G1 phase were determined using a FACSCalibur Flow Cytometer
and analyzed using CellQuest Software.
solution (0.5 mg/mL). The stained gel was illuminated under a UV
transilluminator Vilber-Lourmat (France) at 312 nm and photo-
graphed with a Panasonic DMC-LZ5 Lumix Digital Camera through
filter DEEP YELLOW 15 (TIFFEN, USA).
Interactions of the tested compounds with pUC18 in the
presence of Fe(II) and Fe(III) were done as follows. Solutions of
4.5. DNA-interaction studies
2
m
L FeSO4 (40 mM or 20 mM, freshly prepared in a sterile water)
or 2 L FeCl3 (40 mM or 20 mM, freshly prepared in a sterile
water) with 4 L of 10 mM solution of AQ-thiosemicarbazone in
14
L of bicarbonate buffer were incubated at 37 ꢀC, for 60 min,
then 2 L of pUC18 were added to reaction mixtures and incu-
m
4.5.1. DNA-binding experiments
m
Calf thymus DNA (lyophilized, highly polymerized, obtained
from Serva, Heidelberg) (CT-DNA) was dissolved in Tris buffer
(10 mM TriseHCL pH 7.9) overnight at 4 ꢀC. This stock solution was
stored at 4 ꢀC and was stable for several days. A solution of CT-DNA
in water gave a ratio of UV absorbance at 260 and 280 nm, A260/A280
of 1.89e2.01, indicating that DNA was sufficiently free of protein.
The concentration of DNA (2.6 mg/mL) was determined from
the UV absorbance at 260 nm using the extinction coefficient
ε260 ¼ 6600 Mꢄ1cmꢄ1 [44].
m
m
bated further for 90 min. The reaction mixtures were vortexed
from time to time and terminated by short centrifugation at
10,000 rpm and addition of 5
described previously.
mL of loading buffer and analyzed as
Acknowledgments
All AQ-thiosemicarbazone compounds were dissolved in
dimethyl sulfoxide in concentrations of 10 mM and these solutions
were used as stock solutions.
The authors are grateful to the Ministry of Science and Tech-
nological Development of the Republic of Serbia for financial sup-
port (Grant Nos 172016 and 175011).
Reaction mixtures (1 mL in 40 mM bicarbonate buffer, pH 8.4)
consisting of different concentrations of compounds (20
m
M, 40
m
M,
Appendix A. Supplementary information
60 M and 80
m
m
M) and 0.12 mM of CT-DNA were incubated at 37 ꢀC
for 1.5 h with occasional vortexing. UVeVis spectra were recorded
on a UV-1800 Shimadzu UV/Visible spectrometer operating from
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.03.071.

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V. Markovic et al. / European Journal of Medicinal Chemistry 64 (2013) 228e238
238
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[22] I. Ðilovic, M. Rubcic, V. Vrdoljak, S. Kraljevic Pavelic, M. Kralj, I. Piantanida,
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