Synthesis and evaluation of new benzodioxole-based thiosemicarbazone derivatives as potential antitumor agents

Article abstract of DOI:10.3390/molecules21111598

New benzodioxole-based thiosemicarbazone derivatives were synthesized and evaluated for their cytotoxic effects on A549 human lung adenocarcinoma, C6 rat glioma and NIH/3T3 mouse embryonic fibroblast cells. In order to examine the correlation between anti

Full text of DOI:10.3390/molecules21111598

molecules
Article
Synthesis and Evaluation of New
Benzodioxole-Based Thiosemicarbazone Derivatives
as Potential Antitumor Agents
Mehlika Dilek Altıntop 1,*, Halide Edip Temel 2, Belgin Sever 1, Güls¸en Akalın Çiftçi 2
and Zafer Asım Kaplancıklı 1
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University,
26470 Eskis¸ehir, Turkey; belginsever@anadolu.edu.tr (B.S.); zakaplan@anadolu.edu.tr (Z.A.K.)
Department of Biochemistry, Faculty of Pharmacy, Anadolu University, 26470 Eskis¸ehir, Turkey;
2
heincedal@anadolu.edu.tr (H.E.T.); gakalin@anadolu.edu.tr (G.A.Ç.)
*
Correspondence: mdaltintop@anadolu.edu.tr; Tel.: +90-222-335-0580 (ext. 3772); Fax: +90-222-335-0750
Academic Editor: Derek J. McPhee
Received: 22 July 2016; Accepted: 18 November 2016; Published: 22 November 2016
Abstract: New benzodioxole-based thiosemicarbazone derivatives were synthesized and evaluated
for their cytotoxic effects on A549 human lung adenocarcinoma, C6 rat glioma and NIH/3T3
mouse embryonic fibroblast cells. In order to examine the correlation between anticancer activity
and cholinesterases, the compounds were evaluated for their inhibitory effects on AChE and
BuChE. The most effective anticancer agents were investigated for their effects on DNA synthesis,
apoptosis and mitochondrial membrane potential. 4-(1,3-Benzodioxol-5-yl)-1-([1,10-biphenyl]-4-
ylmethylene)thiosemicarbazide (
and A549 cell lines due to its inhibitory effects on C6 and A549 cells and low toxicity to NIH/3T3
cells. Compound increased early and late apoptosis in A549 and C6 cells. Compound also caused
disturbance on mitochondrial membrane potential and showed DNA synthesis inhibitory activity in
A549 and C6 cells. Compound was investigated for SIRT1 inhibitory activity to provide mechanistic
insight and for that purpose docking studies were also performed for this compound on SIRT1.
On the other hand, compound did not show any inhibitory activity against AChE and BuChE.
5) was identified as the most promising anticancer agent against C6
5
5
5
5
This outcome pointed out that there is no relationship between anticancer activity of compound
and cholinesterases.
5
Keywords: thiosemicarbazone; benzodioxole; cancer; apoptosis; mitochondrial membrane potential;
SIRT1; docking studies
1. Introduction
Cancer is an abnormal cell division caused by some alteration in the expression of plentiful genes
in the nucleus or in the mitochondria of cells. These alterations lead to a disorder in the balance
between cell reproduction and cell death. Indeed, cancer is not a single disease, but rather it is
a group of more than a hundred distinct diseases affecting different organs and systems of the body.
Up to now, many chemotherapeutic drugs have been developed as anticancer agents, but most of
them are insufficient for cancer treatment because of their lack of specificity towards cancer cells and
correspondingly their high risk of toxicity to normal cells. Consequently, pharmaceutical research has
focused on the discovery of more selective and less toxic anticancer agents [1–6].
Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are abnormally expressed in
various pathological conditions, such as tumorigenesis. In recent years, AChE has attracted a great
deal of interest as a potential therapeutic target for the treatment of cancer due to the involvement of
this enzyme in apoptosis and cell adhesion, differentiation, and proliferation [7–9].
Molecules 2016, 21, 1598; doi:10.3390/molecules21111598
www.mdpi.com/journal/molecules

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The mammalian sirtuins are a family of NAD+-dependent enzymes that have been connected
to a great variety of biological activities encompassing cellular stress resistance, genomic stability,
tumorigenesis and energy metabolism. Among them, sirtuin-1 (SIRT1) which belongs to the class III
histone deacetylases (HDACs), is associated with a wide range of human diseases and is an outstanding
potential therapeutic target. Overexpression of SIRT1 has been reported to increase growth and survival
rates of some cancer cell lines and inhibit apoptosis [10–13]. However, the role of sirtuins as tumor
suppressor or tumor promoter in some types of cancer is more diverse and complex than previously
anticipated due to cellular context or targets in specific signaling pathways [14].
Thiosemicarbazones have been the subject of considerable research due to their wide range of
biological activities such as antitumor, antibacterial, antifungal and antiviral effects. Since the 1950s,
their anticancer activity has been continually investigated and some of them were explored as
antileukemic agents. Thiosemicarbazones have iron coordination capacity and the complexation
with most of the transition metal ions has increased their use as potential antitumor agents.
The antitumor activity of thiosemicarbazones has mainly been associated with their inhibition capacity
of ribonucleotide reductase, which is the rate limiting enzyme in the synthesis and repair of DNA.
The ability of thiosemicarbazones to chelate metal ions has been defined as a major mechanism
in the inhibition of ribonucleotide reductase. For this purpose, 3-aminopyridine-2-carboxaldehyde
thiosemicarbazone (3-AP or Triapine®), a promising potent molecule, was studied in several clinical
phase I and II trials where it was found to be ineffective against a variety of solid tumors but very
promising against hematologic malignancies such as leukemia. Triapine® was 1000 times more potent
than the clinically used ribonucleotide reductase inhibitor hydroxyurea in both enzyme and cell assays.
Recent studies have also showed that thiosemicarbazones inhibit topoisomerase-II
α enzyme, which
is highly expressed in many cancer cells acting by stabilization of the cleavable products formed by
topoisomerase-II and DNA through a thiol alkylation. In the recent times, thiosemicarbazones were
reported to inhibit ATP binding cassette (ABC) transporters, which are involved in the development of
multidrug resistance [15–23]. On the other hand, 1,3-benzodioxole is found in a variety of anticancer
agents such as podophyllotoxin, steganacin, and combretastatin A-2 showing good bioavailability and
low cytotoxicity [24].
On the basis of aforementioned findings, in the present investigation we report the synthesis and
evaluation of a new series of benzodioxole-based thiosemicarbazone derivatives as anticancer agents
against A549 human lung adenocarcinoma, C6 rat glioma and NIH/3T3 mouse embryonic fibroblast
(healthy) cell lines. The synthesized compounds were evaluated for their inhibitory effects on AChE
and BuChE to examine the correlation between anticancer activity and cholinesterases. Furthermore,
the most effective anticancer agents were also evaluated for their effects on DNA synthesis, apoptosis
and mitochondrial membrane potential. The SIRT1 inhibitory action of compound
5, the most
promising anticancer agent, was investigated to provide mechanistic insight and suggest a therapeutic
role for this compound. Docking studies were also performed for compound 5 on SIRT1 (PDB Id: 4IG9).
2. Results and Discussion
2.1. Chemistry
The synthesis of thiosemicarbazone derivatives
depicted in Scheme 1. In the initial step, 4-(1,3-benzodioxole-5-yl)thiosemicarbazide (
synthesized via the reaction of 1,3-benzodioxole-5-yl isothiocyanate with hydrazine hydrate.
The reaction of 4-(1,3-benzodioxole-5-yl)thiosemicarbazide ( ) with aromatic aldehydes afforded new
thiosemicarbazone derivatives
10. The IR, 1H-NMR, 13C-NMR, mass spectral data and elemental
1–
10 was carried out according to the steps
A
) was
A
1–
analyses were in agreement with the proposed structures of the compounds
Figures for further details).
1–10 (see Supplementary

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Scheme 1. The synthetic route for the preparation of the thiosemicarbazone derivatives
Reagents and conditions: (i) NH2NH2·H2O, ethanol, rt, 4 h; (ii) Ar-CHO, ethanol, reflux, 8 h.
1–10.
2.2. Biochemistry
2.2.1. Cytotoxicity
MTT assays were carried out to determine the cytotoxic effects of the compounds on A549
human lung adenocarcinoma and C6 rat glioma cell lines. According to assay results, the tested
compounds, except compound
cells. The most effective cytotoxic agent against A549 cancer cell line was found as compound
(IC50 = 10.67 ± 1.53 µM) followed by compounds 10 and (IC50 values were 24.0 3.46,
28.0 ± 1.0, 29.67 5.51 and 51.5 4.95 M, respectively). Furthermore, the most effective
cytotoxic agent against the C6 glioma cell line was found as compound (IC50 = 4.33 1.04 M),
followed by compounds 10 and (IC50 values were 12.33 4.93, 23.33 2.08, 25.33 1.53,
and 49.33 ± 1.15 µM, respectively, Table 1).
3, showed more potent inhibitory effects on C6 cells than A549
5
2
,
3,
9
±
±
±
µ
5
±
µ
,
2,
9
3
±
±
±
Table 1. IC50 values of the compounds against A549, C6 and NIH/3T3 cells for 24 h.
IC50 (µM)
Compound
A549 Cell Line
C6 Cell Line
NIH/3T3 Cell Line
1
2
3
4
>167.0
24.0 ± 3.46
28.0 ± 1.0
>146.0
>167.0
23.33 ± 2.08
49.33 ± 1.15
>146.0
>167.0
>145.2
>154.1
>146.0
5
6
7
8
9
10
10.67 ± 1.53
4.33 ± 1.04
21.33 ± 5.77
2.87 ± 0.05
9.50 ± 2.50
9.16 ± 2.47
>135.8
>157.6
>157.6
>149.7
>132.2
51.5 ± 4.95
29.67 ± 5.51
3.0 ± 0.88
>149.7
>132.2
25.33 ± 1.53
12.33 ± 4.93
6.83 ± 0.28
38.33 ± 2.08
Cisplatin
ND
ND: Not determined.
On the other hand, compounds
concentrations used in both cancer cell lines. The cytotoxicity of compounds
NIH/3T3 cells was lower, whereas the cytotoxicity of compounds and was higher compared to the
toxicity on cancer cells (Table 1). These results indicated that the substituents on the benzene ring had
1,
4,
6
,
7
and
8
showed no significant cytotoxicity at the
1, 2, 3, 4, 5, 9
and 10 against
6,
7
8

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considerable influence on the cytotoxic effects of the compounds. Considering the anticancer effects of
compound , it can be concluded that biphenyl substituent increased the anticancer activity against
5
A549 human lung adenocarcinoma and C6 rat glioma cell lines. The increased anticancer activity can
be attributed to the increased lipophilicity of the compound due to the presence of biphenyl group.
2.2.2. Evaluation of DNA Synthesis Inhibition
The immunostaining procedure was carried out with specific anti-BrdU antibodies in the S-phase
of the cell cycle [25]. DNA synthesis inhibitory effects of all compounds were evaluated for 24 h.
A549 and C6 cell lines were incubated with three different concentrations of the compounds that
were determined according to their IC50 values. Cisplatin was used as a positive control. The tested
compounds showed DNA synthesis inhibitory activity in a dose-dependent manner.
(A)
(B)
Figure 1. DNA Synthesis inhibitory effects of compounds
2, 3, 5, 9, 10 and cisplatin on A549 (A) and
C6 ( ) cells. Mean percent absorbance of untreated control cells were assumed 0%. Three different
B
concentrations (a: IC50/2, b: IC50, c: 3/2 IC50) of test compounds and cisplatin were given. Data points
represent means for two independent experiments ± SD of four independent wells. p < 0.05.
The most cytotoxic compounds (compounds 2, 3, 5, 9 and 10) against A549 and C6 cells were
chosen for DNA synthesis inhibition assay. DNA synthesis inhibitory activity of the compounds
on A549 and C6 cell lines is presented in Figure 1. These results showed that compounds tested in
this assay caused DNA synthesis inhibition. Among these compounds, the most potent inhibitors
of DNA synthesis against A549 and C6 cell lines were found as compounds
synthesis inhibition caused by the concentration of IC50 for each compound tested in A549 cell line
revealed the following potency order: Compound > Compound > Compound 10 > Compound
Compound . A significant inhibition of A549 proliferation by cisplatin (positive control) at its IC50
2, 5 and 10. DNA
2
5
3 >
9

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value (3.0 ± 0.88 µM) was observed (55.73%). DNA synthesis inhibition percents of compounds
10 and were 59.85%, 57.79%, 56.38%, 53.24%, 27.3%, respectively (Figure 1A). On the other hand,
DNA synthesis inhibition caused by the concentration of IC50 for each compound tested in C6 cell line
revealed the following potency order: Compound > Compound > Compound > Compound 10
Compound . DNA synthesis inhibition percents of compounds 10 and were 63.34%, 62.98%,
54.99%, 53.96% and 37.71%, respectively, while the inhibition percent of cisplatin was 53.95% at its IC50
value (6.83 0.28 M) (Figure 1B). Consequently, the compounds showed similar antiproliferative
activities against both cell lines. Compounds and 10 were the most cytotoxic agents and exhibited
2, 5,
,
3
9
2
5
3
>
9
2
,
5
,
3,
9
±
µ
2, 5
more inhibitory effects on DNA synthesis. In this regard, we investigated possible mechanism of
anticancer activity of these compounds.
2.2.3. Evaluation of Flow Cytometric Analyses
Apoptosis
After 24 h incubation period, the apoptotic effects of compounds 2, 5 and 10 which were analyzed
for A549 human lung adenocarcinoma and C6 rat glioma cells based on Annexin V-PI binding capacities
in flow cytometry are depicted in Figures 2 and 3, respectively.
Figure 2. Flow cytometric analysis of A549 cells treated with IC50 values of compounds 2, 5, 10 and
cisplatin. A549 cells were cultured for 24 h in medium with IC50 values of the compounds. At least
10,000 cells were analyzed per sample, and quadrant analysis was performed.

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Figure 3. Flow cytometric analysis of C6 cells treated with IC50 values of compounds 2, 5, 10 and
cisplatin. C6 cells were cultured for 24 h in medium with IC50 values of the compounds. At least
10,000 cells were analyzed per sample, and quadrant analysis was performed.
Following flow cytometric analyses, early and late apoptotic effects of compounds 2, 5, and 10
(for IC50 doses) were determined as percentage of 23.7, 12.11 and 6.2, respectively, while early and
late apoptotic effects of control cells were determined as percentage of 3.1 on A549 cell line (Figure 2,
Table 2).
Table 2. The percents of typical quadrant analysis of annexin V-FITC/propidium iodide flow cytometry
of A549 and C6 cells treated with the compounds.
Early and Late Apoptotic Cells %
Viability %
Groups
C6 Cells
A549 Cells
C6 Cells
A549 Cells
Control (untreated)
Compound 2 treated cells
Compound 5 treated cells
Compound 10 treated cells
Cisplatin treated cells
4.5
5.4
25.0
15.8
16.3
3.1
23.7
12.11
6.2
83.9
94.5
60.2
69.6
69.0
94.3
75.2
81.8
81.2
60.5
32.4
C6 and A549 cells were cultured for 24 h in medium with IC50 values of the compounds. At least 10,000 cells
were analyzed per sample, and quadrant analysis was performed.

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On the other hand, early and late apoptotic effects of compounds
2
,
5, and 10 (for IC50 doses)
were determined as percentage of 5.4, 25.0, and 15.8, respectively, while early and late apoptotic effects
of control cells were determined as percentage of 4.5 on C6 cell line (Figure 3, Table 2). According to
these findings, compound
5 was the most effective apoptotic compound (25.0%) on C6 cells compared
to cisplatin (16.3%). On the other hand, the compounds were not effective as cisplatin (32.4%) on
A549 cells.
Mitochondrial Membrane Potential (JC-1)
During apoptosis, several key events occur in mitochondria including the release of cytochrome
c, changes in electron transport, and loss of mitochondrial transmembrane potential (∆Ψm). For this
reason, ∆Ψm is an important parameter of mitochondrial function in apoptosis [26]. In order to
investigate effects of compounds 2, 5 and 10 on mitochondrial membrane potential of A549 and
C6 cells, the cells were incubated by IC50 concentrations of these compounds for 24 h (Figures 4 and 5
and Table 3, respectively).
Figure 4. The reduction of the mitochondrial membrane potential in A549 cell line by the compounds.
The cells treated or untreated with the IC50 doses of the compounds for 24 h were stained with the
mitochondrial-selectivite JC-1 dye and analyzed by flow cytometry. P2: mitochondrial membrane
polarized cells, P3: mitochondrial membrane depolarized cells.

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Figure 5. The reduction of the mitochondrial membrane potential in C6 cell line by the compounds.
The cells treated or untreated with the IC50 doses of the compounds for 24 h were stained with the
mitochondrial-selectivite JC-1 dye and analyzed by flow cytometry. P2: mitochondrial membrane
polarized cells, P3: mitochondrial membrane depolarized cells.
Following flow cytometric analyses, mitochondrial membrane polarized cell percentages of
compounds
while mitochondrial membrane depolarized cell percentages of compounds
(for IC50 doses) were determined as 9.8, 24.6, 57.9 and 54.0 respectively, on A549 cell line. On the other
hand, mitochondrial membrane polarized cell percentages of compounds 10 and cisplatin (for IC50
2
,
5,
10 and cisplatin (for IC50 doses) were determined as 4.5, 52.7, 21.9 and 41.4 respectively,
2, 5,
10 and cisplatin
2, 5,
doses) were determined as 44.8, 49.8, 35.1 and 46.6 respectively, while mitochondrial membrane
depolarized cell percentages of compounds 2, 5, 10 and cisplatin (for IC50 doses) were determined as
21.1, 45.0, 63.7 and 48.4 respectively, on C6 cell line. According to these findings, compound 10 was the
most effective agent for depolarization of mitochondrial membrane on both cell lines. Furthermore,
compound 5 showed the similar effects on C6 glioma cell line compared to cisplatin.

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Table 3. The effects of the compounds on mitochondrial membrane potential of A549 and C6 cells for
24 h.
P2 (%)
P3 (%)
Groups
C6 Cells
A549 Cells
C6 Cells
A549 Cells
Control (untreated)
Compound 2 treated cells
Compound 5 treated cells
Compound 10 treated cells
Cisplatin treated cells
98.3
44.8
49.8
35.1
46.6
82.6
4.5
52.7
21.9
41.4
1.7
13.1
9.8
24.6
57.9
54.0
21.1
45.0
63.7
48.4
2.2.4. Evaluation of AChE and BuChE Inhibition
The anticholinesterase effects of all compounds on AChE and BuChE were determined by
a modification of Ellman’s spectrophotometric method (Table 4). The tested compounds were more
effective against AChE than BuChE. Among these compounds, the most effective AChE inhibitor
was found as compound 10 (IC50 = 108.0
when compared with galantamine (IC50 = 1.83
substituted 1,3-benzodioxole ring increased the inhibitory activity against AChE. Compounds
did not show any inhibitory activity against AChE, whereas other derivatives showed weak inhibition
on AChE. On the other hand, compounds and 10 showed weak inhibition on BuChE, whilst other
±
3.53
µ
M) followed by compound 9 (IC50 = 205.0 ± 5.0 µM)
0.21 M). This outcome indicated that bromo
and
±
µ
3
5
7, 8
compounds were found to be inactive against BuChE.
Considering the difference between IC50 values of compounds 7 and 9, the introduction of chlorine
atom into the 3th position of benzene ring increased anticholinesterase activity against AChE, whereas
the introduction of chlorine atom into the 3th position of benzene ring decreased anticholinesterase
activity against BuChE.
Compound 5, the most effective apoptotic derivative in this series against C6 rat glioma cells,
did not show any ChE inhibitory activity. On the other hand, compound 10, the most potent
anticholinesterase derivative in this series, did not exhibit significant anticancer activity against
A549 and C6 cell lines. In the current work, there is no direct relationship between anticancer activity
and cholinesterases.
Table 4. The anticholinesterase activity of the compounds.
IC50 (µM)
Compound
AChE
BuChE
1
2
3
>267.25
>232.32
- - -
- - -
- - -
- - -
4
5
>233.68
- - -
- - -
- - -
6
7
8
9
10
>252.10
>239.67
>211.51
205.0 ± 5.0
108.0 ± 3.53
1.83 ± 0.21
- - -
>239.67
>211.51
- - -
>189.46
18.17 ± 0.76
Galantamine
- - -: No inhibition.
2.2.5. Evaluation of Sirtuin-1 Activity
The effects of compound on sirtuin-1 activity were determined by an ELISA Sirtuin-1 activity kit
(Table 5, Figure 6). According to the assay, compound decreased SIRT1 levels in C6 rat glioma cells at
IC50/2 concentration. However, increasing the concentration of compound caused elevated SIRT1
levels compared to control. These results showed that the effects of compound on SIRT1 levels were
dose dependent in C6 glioma cells. In different studies, inhibition of SIRT1 activity leads to elevated
5
5
5
5

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p53 acetylation and transactivation resulting in enhanced apoptosis and cytostasis. Hence, inhibition
of SIRT1 levels seemed as beneficial for cancer treatment [14].
Table 5. The effects of compound 5 on SIRT1 Activity.
Compound
SIRT1 Concentration (ng/mL)
Control
3.12 ± 0.03
0.67 ± 0.22
3.82 ± 0.08
4.53 ± 1.37
3.32 ± 1.41
Compound 5 a
Compound 5 b
Cisplatin a
Cisplatin b
a: IC50/2 concentration; b: IC50 concentration.
Figure 6. The effects of compound
5 on SIRT1 levels in C6 rat glioma cell line. a: IC50/2 concentration;
b: IC50 concentration; * significance from control group p < 0.05.
2.2.6. Docking Studies
Docking studies were also performed for compound
5 on SIRT1 (PDB code: 4IG9). Compound 5
showed good affinity close to active site of the enzyme which was previously defined [12]. In Figure 7,
the best docking position of compound
are shown and described.
5 on SIRT1 and all interactions between compound 5 and SIRT1
Figure 7. The docking position of compound
5 on SIRT1 and the interactions between compound 5
and SIRT1. (Green line: Pi-pi stacking; Red line: Salt bridge). Surface representation with overlaid
electrostatic surface potentials (Red: Negative; Blue: Positive).

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3. Materials and Methods
3.1. General Information
All reagents were purchased from commercial suppliers and were used without further
purification. Melting points (M.p.) were determined on an Electrothermal 9100 melting point
apparatus (Weiss-Gallenkamp, Loughborough, UK) and are uncorrected. IR spectra were recorded on
1
an IRPrestige-21 Fourier Transform Infrared spectrophotometer (Shimadzu, Tokyo, Japan). H-NMR
and 13C-NMR spectra were recorded on a Varian Mercury-400 FT-NMR spectrometer (Agilent,
Palo Alto, CA, USA). Mass spectra were recorded on an Agilent LC-MSD-Trap-SL Mass spectrometer
(Agilent Technologies, Palo Alto, CA, USA). Elemental analyses were performed on a Perkin Elmer
EAL 240 elemental analyzer (Perkin-Elmer, Norwalk, CT, USA) and the results were within
the theoretical values.
±
0.4% of
3.2. General Procedures for the Synthesis of the Compounds
3.2.1. 4-(1,3-Benzodioxol-5-yl)thiosemicarbazide (A)
A mixture of 1,3-benzodioxol-5-yl isothiocyanate (0.1 mol) and hydrazine hydrate (0.2 mol) in
ethanol (30 mL) was stirred at room temperature for 4 h and then filtered. The residue was crystallized
from ethanol.
3.2.2. 4-(1,3-Benzodioxol-5-yl)-1-[(aryl)methylene]thiosemicarbazides 1–10
A mixture of 4-(1,3-benzodioxol-5-yl)thiosemicarbazide (
A) (0.01 mol) and aromatic aldehydes
(0.01 mol) was refluxed in ethanol for 8 h, filtered and crystallized from ethanol.
4-(1,3-Benzodioxole-5-yl)-1-(benzylidene)thiosemicarbazide (1 ν
). Yield: 85%. Mp 181.7 ◦C. IR max (cm−1):
3317.56 (N-H stretching), 3153.61 (aromatic C-H stretching), 2989.66 (aliphatic C-H stretching),
2883.58 (O-CH2 stretching), 1535.34, 1487.12 (C=N, C=C stretching and N-H bending), 1446.61,
1398.39, 1350.17 (C-H bending), 1278.81, 1188.15, 1126.43, 1103.28, 1062.78, 1035.77 (C-N stretching,
C=S stretching and aromatic C-H in plane bending), 931.62, 891.11, 854.47, 812.03, 763.81, 750.31,
1
692.44, 661.58, 624.94 (aromatic C-H out of plane bending and C-S stretching). H-NMR (400 MHz,
DMSO-d6): 6.04 (s, 2H, O-CH2-O), 6.90–6.97 (m, 2H, benzodioxole), 7.19 (s, 1H, benzodioxole),
7.40–7.45 (m, 3H, phenyl), 7.90–7.92 (m, 2H, phenyl), 8.18 (s, 1H, N=CH), 10.02 (s, 1H, NH), 11.82
(s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6): 101.26 (O-C-O), 107.36 (benzodioxole C4), 107.97
(benzodioxole C7), 119.40 (benzodioxole C6), 127.60 (phenyl C3 and C5), 128.62 (phenyl C2 and
C6), 129.99 (benzodioxole C5), 133.10 (phenyl C4), 134.04 (phenyl C1), 142.78 (benzodioxole C7a), 144.87
(N=C), 146.59 (benzodioxole C3a), 176.40 (C=S). MS (ESI) (m/z): [M + H]+ 300. Anal. Calcd. for
C15H13N3O2S: C, 60.19; H, 4.38; N, 14.04. Found: C, 60.39; H, 4.35; N, 14.30.
4-(1,3-Benzodioxole-5-yl)-1-(4-nitrobenzylidene)thiosemicarbazide (2 ν
). Yield: 95%. Mp 233.1 ◦C. IR
max
(cm−1): 3327.21 (N-H stretching), 3153.61 (aromatic C-H stretching), 2995.45 (aliphatic C-H stretching),
2885.51 (O-CH2 stretching), 1585.49, 1334.74 (NO2 stretching), 1539.20, 1496.76 (C=N, C=C stretching
and N-H bending), 1436.97 (C-H stretching), 1274.95, 1257.59, 1234.44, 1186.22, 1130.29, 1076.28, 1033.85
(C-N stretching, C=S stretching and aromatic C-H in plane bending), 935.48, 898.83, 854.47, 835.18,
808.17, 744.52, 690.52, 599.86 (aromatic C-H out of plane bending and C-S stretching). 1H-NMR
(400 MHz, DMSO-d6): 6.06 (s, 2H, O-CH2-O), 6.93 (s, 2H, benzodioxole), 7.15 (s, 1H, benzodioxole),
8.17–8.25 (m, 5H, phenyl and N=CH), 10.21 (s, 1H, NH), 12.06 (s, 1H, NH). 13C-NMR (100 MHz,
DMSO-d6): 101.32 (O-C-O), 107.41 (benzodioxole C4), 108.10 (benzodioxole C7), 119.62 (benzodioxole
C6), 123.70 (phenyl C3 and C5), 128.40 (phenyl C2 and C6), 132.92 (benzodioxole C5), 140.01 (phenyl
C1), 140.55 (benzodioxole C7a), 145.06 (N=C), 146.66 (benzodioxole C3a), 147.63 (phenyl C4), 176.83

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(C=S). MS (ESI) (m/z): [M + H]+ 345. Anal. Calcd. for C15H12N4O4S: C, 52.32; H, 3.51; N, 16.27. Found:
C, 52.22; H, 3.46; N, 16.08.
4-(1,3-Benzodioxole-5-yl)-1-(4-cyanobenzylidene)thiosemicarbazide (
(cm-1): 3309.85 (N-H stretching), 3142.04 (aromatic C-H stretching), 2981.95 (aliphatic C-H stretching),
2891.30 (O-CH2 stretching), 2223.92 (C N stretching), 1541.12, 1498.69, 1485.19 (C=N, C=C stretching
3 ν
). Yield: 90%. Mp 224.1 ◦C. IR
max
≡
and N-H bending), 1433.11, 1411.89 (C-H bending), 1298.09, 1253.73, 1215.15, 1180.44, 1138.00,
1082.07, 1031.92 (C-N stretching, C=S stretching and aromatic C-H in plane bending), 923.90, 889.18,
854.47, 862.18, 827.46, 792.74, 731.02, 661.58, 694.37, 599.86 (aromatic C-H out of plane bending and
1
C-S stretching). H-NMR (400 MHz, DMSO-d6): 6.06 (s, 2H, O-CH2-O), 6.93 (s, 2H, benzodioxole),
7.17 (s, 1H, benzodioxole), 7.87 (d, J = 8.4 Hz, 2H, phenyl), 8.11 (d, J = 8.8 Hz, 2H, phenyl), 8.19
(s, 1H, N=CH), 10.17 (s, 1H, NH), 12.03 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6): 101.32 (O-C-O),
107.40 (benzodioxole C4), 108.12 (benzodioxole C7), 111.67 (C N), 118.79 (benzodioxole C6), 119.60
≡
(phenyl C3 and C5), 128.06 (phenyl C2 and C6), 132.41 (benzodioxole C5), 132.93 (phenyl C1), 138.59
(benzodioxole C7a), 140.54 (N=C), 145.05 (benzodioxole C3a), 146.66 (phenyl C4), 176.75 (C=S). MS (ESI)
(m/z): [M + H]+ 325. Anal. Calcd. for C16H12N4O2S: C, 59.25; H, 3.73; N, 17.27. Found: C, 60.41; H, 3.75;
N, 17.41.
4-(1,3-Benzodioxole-5-yl)-1-(4-dimethylaminobenzylidene)thiosemicarbazide (
IR
max (cm−1): 3282.84 (N-H stretching), 3140.11 (aromatic C-H stretching), 2987.74 (aliphatic C-H
4
). Yield: 80%. Mp 208.6 ◦C.
ν
stretching), 2881.65 (O-CH2 stretching), 1597.06, 1550.77, 1523.76, 1498.69, 1487.12 (C=N, C=C stretching
and N-H bending), 1452.40, 1431.18, 1365.60, 1350.17 (C-H bending), 1294.24, 1257.59, 1219.01, 1184.29,
1165.00, 1132.21, 1033.85 (C-N stretching, C=S stretching and aromatic C-H in plane bending), 933.55,
842.89, 813.96, 794.67, 752.24, 709.80, 653.87 (aromatic C-H out of plane bending and C-S stretching).
1H-NMR (400 MHz, DMSO-d6): 2.95 (s, 6H, N(CH3)2), 6.03 (s, 2H, O-CH2-O), 6.71 (d, J = 8.8 Hz, 2H,
phenyl), 6.89 (d, J = 8.4 Hz, 1H, benzodioxole), 6.96 (dd, J1 = 8.0 Hz and J2 = 2 Hz, 1H, benzodioxole),
7.22 (d, J = 1.6 Hz, 1H, benzodioxole), 7.69 (d, J = 8.8 Hz, 2H, phenyl), 8.05 (s, 1H, N=CH), 9.81
(s, 1H, NH), 11.56 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6): 39.74 (2CH3), 101.19 (O-C-O), 107.30
(benzodioxole C4), 107.66 (benzodioxole C7), 111.59 (phenyl C3 and C5), 118.99 (benzodioxole C6),
121.18 (phenyl C1), 128.98 (phenyl C2 and C6), 133.27 (benzodioxole C5), 143.87 (benzodioxole C7a),
144.61 (N=C), 146.51 (benzodioxole C3a), 151.47 (phenyl C4), 175.27 (C=S). MS (ESI) (m/z): [M + H]+ 343.
Anal. Calcd. for C17H18N4O2S: C, 59.63; H, 5.30; N, 16.36. Found: C, 59.60; H, 5.27; N, 16.36.
◦
4-(1,3-Benzodioxole-5-yl)-1-[(biphenyl-4-yl)methylene]thiosemicarbazide (
IR
5
). Yield: 90%. Mp 195.7 C.
ν
max (cm−1): 3321.42 (N-H stretching), 3130.47 (aromatic C-H stretching), 2980.02 (aliphatic C-H
stretching), 2887.44 (O-CH2 stretching), 1600.92, 1531.48, 1502.55, 1485.19 (C=N, C=C stretching and
N-H bending), 1440.83, 1354.03 (C-H bending), 1273.02, 1246.02, 1209.37, 1184.29, 1122.57, 1066.64,
1029.99 (C-N stretching, C=S stretching and aromatic C-H in plane bending), 921.97, 840.96, 771.53,
1
727.16, 702.09, 638.44 (aromatic C-H out of plane bending and C-S stretching). H-NMR (400 MHz,
DMSO-d6): 6.06 (s, 2H, O-CH2-O), 6.93 (d, J = 8.4 Hz, 1H, benzodioxole), 7.00 (dd, J1 = 8.0 Hz,
J2 = 1.6 Hz, 1H, benzodioxole), 7.24 (d, J = 1.6 Hz, 1H, benzodioxole), 7.39 (t, J = 7.2 Hz, 1H, phenyl),
7.48 (t, J = 7.6 Hz, 2H, phenyl), 7.74 (t, J = 7.6 Hz, 4H, phenyl), 8.02 (d, J = 8.4 Hz, 2H, phenyl), 8.26 (s, 1H,
N=CH), 10.11 (s, 1H, NH), 11.92 (s, 1H, NH). 13C-NMR (100 MHz) (DMSO-d6)
δ (ppm): 101.68 (CH2),
107.79 (CH), 108.49 (CH), 119.93 (CH), 127.13 (2CH), 127.25 (CH), 128.29 (2CH), 129.44 (2CH), 133.53
(CH), 133.61 (CH), 139.79 (2C), 141.85 (CH), 142.71 (C), 145.28 (2C), 146.97 (C), 176.72 (C). MS (ESI)
(m/z): [M + H]+ 376. Anal. Calcd. for C21H17N3O2S: C, 67.18; H, 4.56; N, 11.19. Found: C, 67.35; H, 4.59;
N, 11.05.
4-(1,3-Benzodioxole-5-yl)-1-(4-fluorobenzylidene)thiosemicarbazide (6 ν
). Yield: 90%. Mp 196.8 ◦C. IR
max
(cm−1): 3219.19 (N-H stretching), 3118.90 (aromatic C-H stretching), 2981.95 (aliphatic C-H stretching),
2900.94 (O-CH2 stretching), 1598.99, 1537.27, 1502.55, 1489.05 (C=N, C=C stretching and N-H bending),

Molecules 2016, 21, 1598
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1444.68, 1355.96 (C-H bending), 1228.66, 1211.30, 1188.15, 1157.29, 1072.42, 1037.70 (C-N stretching,
C=S stretching and aromatic C-H in plane bending), 937.40, 856.39, 835.18, 810.10, 790.81, 738.74, 711.73,
1
651.94, 596.00 (aromatic C-H out of plane bending and C-S stretching). H-NMR (400 MHz, DMSO-d6):
6.05 (s, 2H, O-CH2-O), 6.90-6.95 (m, 2H, benzodioxole), 7.17 (s, 1H, benzodioxole), 7.26 (t, J = 8.8 Hz,
2H, phenyl), 7.99 (m, 2H, phenyl), 8.16 (s, 1H, N=CH), 10.04 (s, 1H, NH), 11.81 (s, 1H, NH). 13C-NMR
(100 MHz, DMSO-d6): 101.26 (O-C-O), 107.35 (benzodioxole C4), 108.03 (benzodioxole C7), 115.64
(d, J = 21.3 Hz, phenyl C3 and C5), 119.45 (benzodioxole C6), 129.81 (d, J = 8.4 Hz, phenyl C2 and C6),
130.68 (d, J = 3.0 Hz, benzodioxole C5), 133.09 (phenyl C1), 141.57 (benzodioxole C7a), 144.89 (N=C),
146.59 (benzodioxole C3a), 164.36 (phenyl C4), 176.41 (C=S). MS (ESI) (m/z): [M + H]+ 318. Anal. Calcd.
for C15H12FN3O2S: C, 56.77; H, 3.81; N, 13.24. Found: C, 56.76; H, 3.42; N, 5.98.
4-(1,3-Benzodioxole-5-yl)-1-(4-chlorobenzylidene)thiosemicarbazide (7 ν
). Yield: 80%. Mp 201.4 ◦C. IR
max
(cm−1): 3344.57 (N-H stretching), 3138.18 (aromatic C-H stretching), 2981.95 (aliphatic C-H stretching),
2895.15 (O-CH2 stretching), 1595.13, 1539.20, 1508.33, 1485.19 (C=N, C=C stretching and N-H bending),
1446.61, 1402.25, 1352.10 (C-H bending), 1278.81, 1253.73, 1228.66, 1186.22, 1083.99, 1035.77, 1010.70
(C-N stretching, C=S stretching and aromatic C-H in plane bending), 933.55, 893.04, 846.75, 812.03,
1
752.24, 659.66, 605.65 (aromatic C-H out of plane bending and C-S stretching). H-NMR (400 MHz,
DMSO-d6): 6.05 (s, 2H, O-CH2-O), 6.90–6.96 (m, 2H, benzodioxole), 7.16 (s, 1H, benzodioxole), 7.48
(d, J = 8.4 Hz, 2H, phenyl), 7.95 (d, J = 8.8 Hz, 2H, phenyl), 8.14 (s, 1H, N=CH), 10.07 (s, 1H, NH),
11.86 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6): 101.26 (O-C-O), 107.35 (benzodioxole C4), 108.04
(benzodioxole C7), 119.48 (benzodioxole C6), 128.65 (phenyl C3 and C5), 129.21 (phenyl C2 and C6),
133.02 (benzodioxole C5), 133.05 (phenyl C1), 134.43 (phenyl C4), 141.34 (benzodioxole C7a), 144.91
(N=C), 146.59 (benzodioxole C3a), 176.48 (C=S). MS (ESI) (m/z): [M + H]+ 334. Anal. Calcd. for
C15H12ClN3O2S: C, 53.98; H, 3.62; N, 12.59: C, 53.82; H, 3.50; N, 10.75.
4-(1,3-Benzodioxole-5-yl)-1-(4-bromobenzylidene)thiosemicarbazide (8 ν
). Yield: 82%. Mp 196.3 ◦C. IR
max
(cm−1): 3340.71 (N-H stretching), 3138.18 (aromatic C-H stretching), 2981.95 (aliphatic C-H stretching),
2895.15 (O-CH2 stretching), 1589.34, 1537.27, 1485.19 (C=N, C=C stretching and N-H bending), 1446.61,
1398.39, 1354.03 (C-H bending), 1280.73, 1253.73, 1236.37, 1186.22, 1126.43, 1066.64, 1035.77, 1004.91
(C-N stretching, C=S stretching and aromatic C-H in plane bending), 933.55, 893.04, 864.11, 844.82,
812.03, 752.24, 659.66, 601.79 (aromatic C-H out of plane bending and C-S stretching). 1H-NMR
(400 MHz, DMSO-d6): 6.05 (s, 2H, O-CH2-O), 6.91 (s, 2H, benzodioxole), 7.13 (s, 1H, benzodioxole),
7.61 (d, J = 8.8 Hz, 2H, phenyl), 7.87 (d, J = 8.8 Hz, 2H, phenyl), 8.11 (s, 1H, N=CH), 10.06 (s, 1H,
NH), 11.84 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6): 101.24 (O-C-O), 107.34 (benzodioxole C4),
108.04 (benzodioxole C7), 119.50 (benzodioxole C6), 123.22 (phenyl C4), 129.46 (phenyl C3 and C5),
131.55 (phenyl C2 and C6), 133.03 (benzodioxole C5), 133.37 (phenyl C1), 141.40 (benzodioxole C7a),
144.89 (N=C), 146.56 (benzodioxole C3a), 176.47 (C=S). MS (ESI) (m/z): [M + H]+ 379. Anal. Calcd. for
C15H12BrN3O2S: C, 47.63; H, 3.20; N, 11.11. Found: C, 47.06; H, 3.37; N, 11.29.
◦
4-(1,3-Benzodioxole-5-yl)-1-(3,4-dichlorobenzylidene)thiosemicarbazide (
9
). Yield: 83%. Mp 213.2 C. IR
ν
max
(cm−1): 3228.84 (N-H stretching), 3128.54 (aromatic C-H stretching), 2981.95 (aliphatic C-H stretching),
2889.37 (O-CH2 stretching), 1510.26, 1489.05, 1463.97 (C=N, C=C stretching and N-H bending), 1438.90,
1398.39 (C-H bending), 1249.87, 1222.87, 1186.22, 1128.36, 1076.28, 1037.70 (C-N stretching, C=S
stretching and aromatic C-H in plane bending), 937.40, 866.04, 815.89, 721.38, 661.58, 601.79 (aromatic
1
C-H out of plane bending and C-S stretching). H-NMR (400 MHz, DMSO-d6): 6.06 (s, 2H, O-CH2-O),
6.90–6.95 (m, 2H, benzodioxole), 7.13 (d, J = 1.6 Hz, 1H, benzodioxole), 7.66 (d, J = 8.8 Hz, 1H, phenyl),
7.80 (dd, J = 8.4 Hz and J = 2 Hz, 1H, phenyl), 8.11 (s, 1H, N=CH), 8.34 (d, J = 2 Hz, 1H, phenyl), 10.16
(s, 1H, NH), 11.93 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6): 101.28 (O-C-O), 107.37 (benzodioxole
C4), 108.34 (benzodioxole C7), 119.85 (benzodioxole C6), 128.03 (phenyl C5), 128.32 (phenyl C3), 130.69
(phenyl C6), 131.80 (phenyl C2), 132.03 (benzodioxole C5), 132.99 (phenyl C1), 134.91 (phenyl C4),
139.95 (benzodioxole C7a), 145.05 (N=C), 146.62 (benzodioxole C3a), 176.73 (C=S). MS (ESI) (m/z):

Molecules 2016, 21, 1598
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[M + H]+ 369. Anal. Calcd. for C15H11Cl2N3O2S: C, 48.93; H, 3.01; N, 11.41. Found: C, 48.48; H, 3.00;
N, 11.51.
4-(1,3-Benzodioxole-5-yl)-1-(6-bromo-1,3-benzodioxole-5-yl)methylene)thiosemicarbazide (10). Yield: 85%.
Mp 219.1 ◦C. IR
ν
(cm−1): 3224.98 (N-H stretching), 3161.33, 3030.17 (aromatic C-H stretching),
max
2908.65 (O-CH2 stretching), 1531.48, 1504.48, 1469.76 (C=N, C=C stretching and N-H bending), 1444.68,
1419.61 (C-H bending), 1247.94, 1220.94, 1209.37, 1178.51, 1120.64, 1074.35, 1033.85 (C-N stretching,
C=S stretching and aromatic C-H in plane bending), 933.55, 879.54, 848.68, 798.53, 746.45, 721.38, 682.80,
1
632.65, 605.65 (aromatic C-H out of plane bending and C-S stretching). H-NMR (400 MHz, DMSO-d6):
6.05 (s, 2H, O-CH2-O), 6.13 (s, 2H, O-CH2-O), 6.80-6.95 (m, 2H, benzodioxole), 7.11 (d, J = 2 Hz, 1H,
benzodioxole), 7.24 (s, 1H, benzodioxole), 8.10 (s, 1H, N=CH), 8.45 (s, 1H, benzodioxole), 10.08 (s, 1H,
NH), 11.87 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6): 101.25 (O-C-O), 102.42 (O-C-O), 106.73
(benzodioxole C4), 107.30 (benzodioxole C4), 108.26 (benzodioxole C7), 112.32 (benzodioxole C7),
115.93 (benzodioxole C6), 119.71 (benzodioxole C6), 126.58 (benzodioxole C5), 133.05 (benzodioxole
C5), 141.25 (benzodioxole C7a), 144.92 (N=C), 146.55 (benzodioxole C3a), 147.78 (benzodioxole C3a),
149.78 (benzodioxole C7a), 176.37 (C=S). MS (ESI) (m/z): [M + H]+ 423. Anal. Calcd. for C16H12BrN3O4S:
C, 45.51; H, 2.86; N, 9.95. Found: C, 45.84; H, 2.79; N, 9.72.
3.3. Biochemistry
3.3.1. Cell Culture and Drug Treatment
C6 Rat glioma and NIH/3T3 mouse embryonic fibroblast cells were incubated in Dulbecco’s
Modified Eagle’s Medium (DMEM) (Sigma, Deisenhofen, Germany) supplemented with 10% fetal calf
serum (Gibco, Paisley, UK). A549 Human lung adenocarcinoma cells were incubated in 90% RPMI
supplemented with 10% fetal bovine serum (Gibco). All media were supplemented with 100 IU/mL
penicillin-streptomycin (Gibco) and cells were incubated at 37 ◦C in a humidified atmosphere of 95%
air and 5% CO2. Exponentially growing cells were plated at 2
×
104 cells/mL into 96-well microtiter
tissue culture plates (Nunc, Roskilde, Denmark) and incubated for 24 h before the addition of the
drugs (the optimum cell number for cytotoxicity assays was determined in preliminary experiments).
The stock solutions of the compounds were prepared in dimethyl sulfoxide (DMSO; Sigma Aldrich,
Poole, UK) and further dilutions were made with fresh culture medium (the concentration of DMSO in
the final culture medium was <0.1% which had no effect on the cell viability).
3.3.2. MTT Assay
The level of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma)
reduction was quantified as previously described in the literature with small modifications [27
After 24 h of preincubation, the tested compounds and cisplatin (positive control) were added to give
final concentration in the range 1.03–167 M and the cells were incubated for 24 h. At the end of this
,28].
µ
period, MTT was added to a final concentration of 0.5 mg/mL and the cells were incubated for 4 h
at 37 ◦C. After the medium was removed, the formazan crystals formed by MTT metabolism were
solubilized by addition of 200 µL DMSO to each well and absorbance was read at 540 nm with a
microtiter plate spectrophotometer (Bio-Tek Plate Reader, Winooski, VT, USA). Every concentration was
repeated in three wells. IC50 values were defined as the drug concentrations that reduced absorbance
to 50% of control values.
3.3.3. Analysis of DNA Synthesis
Analysis of DNA synthesis was measured by a Cell Proliferation ELISA, BrdU (colorimetric)
kit (Roche, Mannheim, Germany). This immunostaining procedure is based on measuring the
incorporation of bromodeoxyuridine (BrdU) into nuclear DNA in place of thymidine during the
S-phase of the cell cycle using specific anti-BrdU antibodies [25]. Hence, such method provides

Molecules 2016, 21, 1598
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a colorimetric measurement for DNA synthesis inhibition ratio of the carcinogenic cells. Firstly cells
were seeded into 96 well flat-bottomed microtiter plates at a density of 2
103. The tumor cells were
10 and cisplatin. Microtiter plates
×
cultured in the prese◦nce of various doses of compounds
2, 3, 5, 9,
were incubated at 37 C in a 5% CO2/95% air humidified atmosphere for 24 h. Cells were labeled with
10 µL BrdU solution for 2 h and then fixed. Anti-BrdU-POD (100 µL) was added and incubated for
90 min. Finally, wells were washed with BPS and cells were incubated with substrate. Absorbance of
the samples was measured with an ELX808-IU Bio-Tek apparatus at 492 nm. All experiments were
repeated two times. For each compound dose, dublicate wells were used.
3.3.4. Flow Cytometric Analyses of Apoptosis
After the cells were incubated with compounds
2, 5 and 10 at IC50 concentrations,
phosphatidylserine externalization, which indicates early apoptosis, was measured by Annexin V-PI
(BD Pharmingen, San Jose, CA, USA) on a BD FACSCaliburTM flow cytometer for 24 h. Annexin V
staining protocol was applied according to the manufacturer’s instructions (BD Pharmingen). The cells
were then briefly washed with cold phosphate buffer saline (PBS) and suspended in a binding buffer
at a concentration of 1
transferred to a 5 mL test tube. After 5
15 min at room temperature in the dark. Then 400
×
106 cells/mL. Then, 100
L of Annexin-V and PI was added, the cells were incubated for
L of 1x Binding Buffer was added to each tube and
µ
L of this solution containing 1
×
105 cells was
µ
µ
the cells were processed for data acquisition, and analyzed on a Becton–Dickinson FACS Aria using
FACSDIVA Version 6.1.1. Software (BD Biosciences, San Jose, CA, USA).
3.3.5. Analysis of Mitochondrial Membrane Potential (JC-1) by Flow Cytometry
The cells were seeded in six-well plates at a density of 105 cells/mL, and the IC50 dose of
compounds
2
,
5
and 10 was added to cells. The cells were incubated in 5% CO2 air-conditioned
◦
atmosphere at 37 C. After 48 h of incubation, the cells were trypsinized, washed with PBS, and
centrifuged at 400
iodide (JC-1) dye solution (1
solution was prepared by dissolving DMSO. Then the samples were incubated at a temperature of
37 ◦C for 10–15 min. After incubation, the cells were washed twice with an assay buffer and analyzed
by BD FACS Aria Cell Sorter Software version 6.1.1 flow cytometry (BD Biosciences). The cells showing
mitochondrial membrane potential disruption were determined as a percentage of all cells.
×
g for 5 min. 5,50,6,60-Tetrachloro-1,10,3,30-tetraethylbenzimidazolylcarbocyanine
assay buffer + JC-1 stock solution) was added to the cells. The stock
×
3.3.6. AChE and BuChE Inhibitory Activity
AChE and BuChE inhibitory effects of the compounds were determined by Ellman’s method
with minor modifications (Electric eel acetylcholinesterase enzyme was used instead of bovine
acetylcholinesterase enzyme and buffer was added 2.4 mL instead of 3 mL) [29]. The compounds
were dissolved in DMSO and tested at final concentration range 5–80 µg/mL. 20 µL of enzyme (AChE
or BuChE, 1 U/mL), 10
15 min. After 15 min incubation, 50
µ
L sample added to 2.4 mL buffer, the mixture was incubated at 37 ◦C for
0
µL of 0.01 M 5,5 -dithiobis(2-nitrobenzoic acid) (DTNB) and 20
µL
of 75 mM acetylthiocholine iodide (ATCI) or 25 mM butyrylthiocholine iodide (BTCI) were added, and
the final mixture was incubated at room temperature for 30 min. Blank was prepared using 10 L of
µ
DMSO instead of the test sample, with all other procedures similar to those used in the case of the
sample mixture. Absorbances were measured at 412 nm and 37 ◦C using polystyrene cuvettes with
spectrophotometer (UV-1700, Shimadzu). Experiment was done in triplicate. Galantamine was used
as a positive control. Data are expressed as mean ± standard deviation (SD). The inhibition (percent)
of AChE or BuChE was calculated using the following equation:
I (%) = 100 − (ODsample/ODcontrol) × 100

Molecules 2016, 21, 1598
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3.3.7. Sirtuin-1 Activity Detection by ELISA
C6 glioma cells were administrated by IC50 or IC50/2 concentrations of compound
5
and cisplatin
for 24 h. Cells were detached with trysin and then collected by centrifugation. After cells were
◦
washed by 1
×
PBS for three times, cell lysates were prepared by frozen cells at ≤−20 C. Freeze/thaw
cycles were repeated for three times. Then cells were centrifuged at 1500
×
g for 10 min at 2–8 ◦C to
remove cellular debris. Rat Sirtuin-1 activity protocol was applied according to the manufacturer’s
instructions (USCN, Life Science Inc., Wuhan, China). Briefly, all standards, reagents and samples
were prepared. 100
100
were washed 3 times. Then 100
Solutions were aspirated and washed 5 times. Then 90
15–25 min at 37 ◦C. After 50
L stop solution was added, plate was read at 450 nm with a microtiter
µ
L standard or sample were added to each well and incubated for 2 h at 37 ◦C.
◦
µ
L detection reagent A added and incubated 1 h at 37 C. Then solutions were aspirated and wells
µ
L detection reagent B was added and incubated for 30 min at 37 ◦C.
µ
L substrate solution added and incubated
µ
plate spectrophotometer (Bio-Tek Plate Reader). Every concentration was repeated in double wells.
The results were given as ng/mL.
3.3.8. Statistical Analyses
Statistical Package for the Social Sciences (SPSS, Chicago, IL, USA) for Windows 15.0 was used
for statistical analysis. Data was expressed as Mean
±
SD. Comparisons were performed by one way
ANOVA test for normally distributed continuous variables and post hoc analyses of group differences
were expressed by the Tukey test. Also, Date were compared between two groups using Student’s
t-test. The p < 0.05 was considered as statistically significant in this study.
3.3.9. Docking Studies
Compound
pH = 7.4) at the protonation step and crystal structure of SIRT1 was retrieved from Protein Data Bank
server, (PDB code: 4IG9). The structure of compound was submitted in protein preparation module
of Schrodinger’s Maestro molecular modeling package. In molecular docking simulations: Glide/XP
docking protocols were applied for the prediction of topologies of compound at the active site of
5 was docked to the active site of 4IG9. Ligand was set to the physiological pH
(
5
5
target structure. The formation of active site in the enzyme was determined from previous studies [12].
4. Conclusions
In the present paper, new benzodioxole-based thiosemicarbazone derivatives were synthesized
and evaluated for their cytotoxicity against on A549 human lung adenocarcinoma, C6 rat glioma
and NIH/3T3 mouse embryonic fibroblast cell lines. In order to examine the correlation between
anticancer activity with cholinesterases, each derivative was investigated for its ability to inhibit AChE
and BuChE. The most effective compounds were also investigated for their effects on DNA synthesis,
apoptosis and mitochondrial membrane potential.
Among these compounds, compound
agent against C6 (IC50 = 4.33 1.04 M) and A549 (IC50 = 10.67
to NIH/3T3 (IC50 = 21.33 5.77 M) cells. Compound showed its anticancer activity against C6
cell line via the induction of apoptotic pathway, but this effect of compound was independent of
ChE inhibitory activity. The effects of compound on SIRT1 activity were evaluated for underlying
mechanism of apoptosis. The results indicated that compound decreased SIRT1 levels in C6 rat
glioma cells at IC50/2 concentration, whereas compound caused elevated SIRT1 levels compared to
control at increased concentrations. This outcome pointed out that the effects of compound on SIRT1
levels were dose dependent in C6 glioma cells. Docking studies were also carried out for compound
on SIRT1 to enlighten the exact position and interactions of this compound on the active site of enzyme.
5
can be considered as the most promising anticancer
±
µ
±
1.53 M) cells and low toxicity
µ
±
µ
5
5
5
5
5
5
5

Molecules 2016, 21, 1598
17 of 19
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/21/11/1598/s1.
Acknowledgments: This study was supported by Anadolu University Scientific Research Projects Commission
under the grant no: 1407S336 and 1605S318.
Author Contributions: M.D.A. and Z.A.K. designed the research; M.D.A. and B.S. performed the synthetic work
and mainly wrote the manuscript; B.S. also carried out the docking studies. H.E.T and G.A.Ç. were responsible
for the whole biochemistry part of the manuscript. M.D.A. was also responsible for the correspondence of the
manuscript. All authors discussed, edited and approved the final version.
Conflicts of Interest: The authors declare no conflict of interest.
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