Synthesis, characterization, antiproliferative of pyrimidine based ligand and its Ni(II) and Pd(II) complexes and effectiveness of electroporation

Article abstract of DOI:10.1080/07391102.2020.1852965

In the study, a new Schiff base (ligand) was obtained using 4-aminopyrimidine-2(1H)-one, the starting material, and 2,3,4-trimethoxy benzaldehyde. Ni(II) and Pd(II) complexes were obtained from the reaction of the ligand and NiCl₂·6H₂O, PdCl₂(CH₃CN)₂ (1:1 ratio). These compounds were characterized using the elemental and mass analysis, ¹H, ¹³C-NMR, FT-IR, UV-Vis, magnetic susceptibility, thermal analysis, and the X-ray diffraction analyses. The antiproliferative activities of the synthesized ligand, Ni(II) and Pd(II) complexes were identified on the HepG2 (human liver cancer cells) cell line and their biocompatibility was tested on the L-929 (fibroblast cells) cell line by the MTT analysis method. Furthermore, the effects of electroporation (EP) on the cytotoxic activities of synthesized compounds were investigated in HepG2 cancer cells. According to the MTT findings of the study, the ligand did not exhibit an antiproliferative activity while its Ni(II) and Pd(II) complexes exhibited an antiproliferative activity. Moreover, it was observed that the antiproliferative activity of the Pd(II) complex was stronger than that of the Ni(II) complex. The combined application of EP + compounds is much more effective than the usage of the compounds alone in the treatment of HepG2 cancer cells. The EP increased the cytotoxicity of the Ni(II) and Pd(II) complexes by 1.66, and 2.54 times, respectively. It was concluded that Ni(II) and Pd(II) complexes may contribute as potential anti-cancer agents for the treatment of hepatocellular carcinoma and yield promising results in the case of being used in ECT. Communicated by Ramaswamy H. Sarma.

Full text of DOI:10.1080/07391102.2020.1852965

Journal of Biomolecular Structure and Dynamics
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/tbsd20
Synthesis, characterization, antiproliferative of
pyrimidine based ligand and its Ni(II) and Pd(II)
complexes and effectiveness of electroporation
Mehmet Eşref Alkış , Kenan Buldurun , Nevin Turan , Yusuf Alan , Ünzile
Keleştemur Yılmaz & Asim Mantarcı
To cite this article: Mehmet Eşref Alkış , Kenan Buldurun , Nevin Turan , Yusuf Alan , Ünzile
Keleştemur Yılmaz & Asim Mantarcı (2020): Synthesis, characterization, antiproliferative of
pyrimidine based ligand and its Ni(II) and Pd(II) complexes and effectiveness of electroporation,
Journal of Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2020.1852965
To link to this article: https://doi.org/10.1080/07391102.2020.1852965
Published online: 30 Nov 2020.
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JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
https://doi.org/10.1080/07391102.2020.1852965
Synthesis, characterization, antiproliferative of pyrimidine based ligand and its
Ni(II) and Pd(II) complexes and effectiveness of electroporation
a
b
c
d
a
€
Mehmet E s¸ ref Alkı s¸
and Asim Mantarcı
, Kenan Buldurun
, Nevin Turan
, Yusuf Alan
, Unzile Kele s¸ temur Yılmaz
e
a
b
Department of Occupational Health and Safety, Faculty of Health Sciences, Mu s¸ Alparslan University, Mu s¸ , Turkey; Department of Food
c
Processing, Technical Sciences Vocational School, Mu s¸ Alparslan University, Mu s¸ , Turkey; Department of Chemistry, Faculty of Arts and
Sciences, Mu s¸ Alparslan University, Mu s¸ , Turkey; Department of Primary Education, Education Faculty, Mu s¸ Alparslan University, Mu s¸ ,
Turkey; Department of Physics, Faculty of Arts and Sciences, Mu s¸ Alparslan University, Mu s¸ , Turkey
d
e
Communicated by Ramaswamy H. Sarma
ABSTRACT
ARTICLE HISTORY
In the study, a new Schiff base (ligand) was obtained using 4-aminopyrimidine-2(1H)-one, the starting
material, and 2,3,4-trimethoxy benzaldehyde. Ni(II) and Pd(II) complexes were obtained from the reac-
tion of the ligand and NiCl ꢀ6H O, PdCl (CH CN) (1:1 ratio). These compounds were characterized
Received 21 August 2020
Accepted 15 November 2020
2
2
2
3
2
1
13
KEYWORDS
using the elemental and mass analysis, H, C-NMR, FT-IR, UV-Vis, magnetic susceptibility, thermal
analysis, and the X-ray diffraction analyses. The antiproliferative activities of the synthesized ligand,
Ni(II) and Pd(II) complexes were identified on the HepG2 (human liver cancer cells) cell line and their
biocompatibility was tested on the L-929 (fibroblast cells) cell line by the MTT analysis method.
Furthermore, the effects of electroporation (EP) on the cytotoxic activities of synthesized compounds
were investigated in HepG2 cancer cells. According to the MTT findings of the study, the ligand did
not exhibit an antiproliferative activity while its Ni(II) and Pd(II) complexes exhibited an antiprolifera-
tive activity. Moreover, it was observed that the antiproliferative activity of the Pd(II) complex was
stronger than that of the Ni(II) complex. The combined application of EP þ compounds is much more
effective than the usage of the compounds alone in the treatment of HepG2 cancer cells. The EP
increased the cytotoxicity of the Ni(II) and Pd(II) complexes by 1.66, and 2.54 times, respectively. It was
concluded that Ni(II) and Pd(II) complexes may contribute as potential anti-cancer agents for the treat-
ment of hepatocellular carcinoma and yield promising results in the case of being used in ECT.
Antiproliferative; cytotox-
icity; electroporation; metal
complexes; Schiff base
1
. Introduction
transition metal complexes have potential biomedicine and
biochemistry applications due to their successful biological
system models and unique properties (Bernadette Amali
et al., 2019; Zoubi, 2013). Schiff bases and their complexes
can be easily obtained using mild reaction conditions and
short reaction times, as well as thermodynamically and wea-
ther resistant compounds that exhibit interesting electronic
and/or magnetic properties (Fetoh et al., 2019; Qin et al.,
Schiff bases exhibited significant effects in medical and
pharmaceutical fields due to various biological activities (e.g.
anti-inflammatory, analgesic, antimicrobial, anticonvulsant,
antituberculosis, anticancer, antioxidant, anthelmintic) (Al-
Humaidi, 2019; Buldurun et al., 2020). Schiff bases are highly
preferred ligands in transition metal chemistry due to their
ability to stabilize metals in different oxidation states
2
013). Recently, many pyrimidine-containing compounds and
(
Alothman et al., 2020; Fekri et al., 2019). The most important
their derivatives are reported to exhibit anticancer activity
through interaction with different enzymes and receptors
source of biological effects is undoubtedly (CH ¼ N) azome-
thine group. The nitrogen atom of the azomethine group
plays an important role in the formation of hydrogen bonds
with the active centers of the cell components and interfere
with normal cell processes (Bing o€ l & Turan, 2020; Vashi &
(Rahmouni et al., 2016; Rashad et al., 2011).
Hepatocellular carcinoma is the sixth most common can-
cer diagnosed worldwide (Emam et al., 2019) and ranks the
third in the mortal cancer cases (Jemal et al., 2011). In gen-
Naik, 2004). In addition to biological activities, Schiff bases eral, radiotherapy, surgery, and chemotherapy drug treat-
are used as catalysts, intermediates in organic synthesis, dyes ment are the conventional methods used in the treatment of
and in various material science fields (e.g. imaging systems cancer (Levitsky & Dembitsky, 2015). Chemotherapy is a
and optical memory devices) (Dehghani-Firouzabadi
&
treatment method in which the cytotoxic drugs are used to
Firouzmandi, 2016; Geeta et al., 2010; Jain et al., 2020; Tahir prevent or eliminate the proliferation of cancer cells.
et al., 2019; Ye gꢀ iner et al., 2017). Schiff bases and their Hepatocellular carcinoma, by its very nature, resists most of
CONTACT Mehmet E s¸ ref Alkı s¸
me.alkis@alparslan.edu.tr
Department of Occupational Health and Safety, Faculty of Health Sciences, Mu s¸ Alparslan
University, Mu s¸ 49250, Turkey
Supplemental data for this article can be accessed online at https://doi.org/10.1080/07391102.2020.1852965.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
M. E. ALKIŞ ET AL.
the chemotherapeutic agents. Therefore, high-dose cytotoxic 2. Materials and methods
drugs with many side effects are being used in the treatment
2
,3,4-trimethoxy benzaldehyde, 4-aminopyrimidine-2-(1H)-one
of hepatocellular carcinoma. Because of these reasons, there
is a need for developing different therapeutic drugs and
treatment methods for hepatocellular carcinoma.
(
gemcitabine), NiCl ꢀ6H O, PdCl (CH CN) and ethanol,
2
2
2
3
2
dimethyl sulphoxide (DMSO), dichloromethane, acetone,
dimethylformamide (DMF), chloroform, methanol, diethyl
ether used in this study were purchased from Sigma-Aldrich
and used without purification. FT-IR spectra were performed
on a Perkin Elmer (FT-IR model 65 spectrophotometer in the
Electroporation (EP) is a physical method in which the
short-duration and high-strength electric pulses are applied
to the cells or tissues and nanometer-sized transient pores
are formed on the cell membranes. Thanks to the increase of
cell membrane permeability poor permeable or nonperme-
able molecules (e.g. DNA, RNA, drugs, dyes and antibodies)
enter into the cell (Gehl, 2003; Neumann et al., 1982). This
method was initially used for the gene transfer into cells;
however, recently, it has been started to be applied in the
transfer of various anti-cancer drugs into the cell (Cemazar &
Sersa, 2019). The chemotherapy treatment carried out using
the EP technique is called Electrochemotherapy (ECT). This
treatment technique is efficient for cutaneous and subcuta-
neous cancers in humans and is now being used in the treat-
ment of internal tumors (V ꢁa squez et al., 2015). ECT makes it
possible to provide treatment with a lower dose of the che-
motherapeutic drugs by means of facilitating the delivery of
anti-cancer drugs to the cell, thereby reducing the side
effects of the drugs (Michel et al., 2018). The EP efficiency
varies depending on the conditions such as electric field
strength, cell concentration, type and duration of the current,
temperature, and electroporation fluid (Batista Napotnik &
Miklav ꢂc i ꢂc , 2018). The optimum EP conditions were deter-
mined for the HepG2 cell line examined in this study by
applying electric currents at different voltages. EP is more
effective on the cancer cells than the healthy cells in terms
of triggering cell death. The possible reason for this is the
factors that differentiate the cancer cells from the healthy
ones, such as different membrane compositions, membrane
repair capacities, and energy reserves (Frandsen et al., 2016;
Zielichowska et al., 2016).
ꢁ
1
1
region 4000–400 cm using KBr pellets). The NMR ( H and
13
C) spectra were mentioned on a Bruker 300 MHz spectrom-
eter in DMSO-d as the solvent. The microanalysis (C, H, N)
was performed by using a Vario III CHN analyzer. Mass spec-
tra were performed on an Agilent technologies AB Sciex
6
3
200 QTrap spectrometer. X-ray diffraction analysis was per-
formed by an UltimaIV X-ray diffractometer with Rigaku
ꢂ
(
CuKa ¼ 1.540562 A ). UV-Visible spectra were recorded in
EtOH by used Shimadzu UV-1800 spectrophotometer.
Thermogravimetric analysis (TGA) was monitored through a
ꢂ
Universal TGA Q50 instrument (a heating rate of 10 C/min
between 50 and 900 C).
The followings were used in the chemotherapy and ECT:
Dimethylsulfoxide (DMSO), ethanol, fetal bovine serum (FBS)
ꢂ
[PAN-Biotech, Europe], MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyl-2H-tetrazolium bromide] (AppliChem, Germany),
Penicillin-Streptomycin (Sigma, USA), RPMI-1640 medium
(Sigma, USA), Trypan blue (Sigma, USA), Trypsin-EDTA
(Biological Industries, Israel), and Propidium iodide (PI). In all the
experiments carried out on the HepG2 and L-929 cells, the cells
between passages 8 and 12 were used. The cells used in the
experiments were obtained from the Department of
Biochemistry, Faculty of Arts and Science, Inonu University. The
cells were incubated in a 5% CO incubator (Esco, Singapore) at
a temperature of 37 C and a humidity of 95%. The cell culture
work was carried out using a biosafety cabinet (ESCO, USA)
under sterile conditions. Gene Pulser Xcell
CA, USA), 4 mm EP cuvettes (Bio-Rad, Hercules, CA, USA), and
flow cytometry (Becton-Dickinson) were used for ECT.
2
ꢂ
TM
(Bio-Rad, Hercules,
Many researchers have carried out studies on different
Schiff bases and metal complexes to develop new, safe,
and effective chemotherapeutic agents (Arafath et al.,
2
.1. Synthesis and characterization of Schiff base (4-(2,3,
-trimethoxy benzylidene)amino)pyrimidine-2-(1H)-
on) (L)
2
017; Mohamed et al., 2019). However, although they
4
examined the anti-cancer properties of the Schiff base and
metal complexes they synthesized, they did not examine
the effectiveness of the methods that would facilitate the
delivery of molecules to the cancer cells, such as ECT. One
of the main purposes of this study is to examine the effect
of EP on the antiproliferative activities of synthesized com-
pounds. A new type of pyrimidine Schiff base ligand and
its Ni(II) and Pd(II) complexes were successfully synthesized
and characterized in this work. Ligand and its metal com-
The Schiff base (L) was prepared through refluxing methanol
solution of 4-aminopyrimidine-2-(1H)-one (1.0 g; 1.0 mmol)
with 2,3,4-trimethoxy benzaldehyde (1.76 g; 1.0 mmol) in
methanol for 4 h and then allowed to cool. The white color
crude product was washed methanol and dried at room
temperature.
ꢂ
Schiff base (L): White solid, yield 80%, m.p.: 270 C. Anal.
plexes were characterized using different spectral methods calc. for C H N O (FW: 289.29 g/mol) (%): C; 58.07, H; 5.18,
1
4 15 3 4
1
13
(
e.g. H NMR, C NMR, FT-IR, UV–Vis TGA, X-ray diffraction N; 14.51. Found: C; 58.16, H; 5.30, N; 14.62. FT-IR (KBr, v max
ꢁ
1
analysis, mass spectra and microanalysis). The cytotoxic (cm )): 3176 (NH), 3097 (Ar–CH), 2899 (Al–CH), 1732 (C ¼ O),
activities of the synthesized compounds were studied 1662 (CH ¼ N), 1632 (C ¼ N), 1541, 1518, 1478 (C ¼ C), 1100
1
against the cancer cell line (HepG2) and a normal cell line (OCH ). H-NMR (300 MHz, DMSO-d , ppm) ¼ 8.10 (s, H, NH),
3
6
(
L-929). Finally, the effects of EP on the cytotoxic activities 8.48 (s, H, N ¼ CH), 7.93–6.50 (m, 4H, Ar-H), 3.80–3.60 (s, 9H,
1
3
of synthesized compounds were investigated in HepG2 OCH ).
cancer cells.
C-NMR (100 MHz, DMSO-d , ppm)
¼
162.45
3
6
(CH ¼ N), 158.59 (C ¼ N), 157.90 (C ¼ O), 151.65–103.51 (Ar-C),

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
3
6
1.63–56.00 (OCH ) . UV-Vis. (k max/nm): 210, 220, 232, 249, 1640) at the concentrations of 100, 200, 300, 400, and
3 3
2
66, 274, 282, 295, 302, 360.
500 mM were added to each well, and then the cells were
again left for incubation for 24 h in an environment contain-
ꢂ
ing 5% CO at 37 C. The same concentrations were used for
2
2.2. Synthesis of nickel and palladium complexes
all samples in the MTT tests. Only 100 mL of medium (RPMI-
1
640) was added to the cells in the control wells (Aktas¸
In a 100 mL round bottom flask, the Schiff base ligand
et al., 2018; Sarı et al., 2020; Tan et al., 2020).
(
0.40 g, 1.38 mmol) was dissolved in methanol (20 mL). A
solution of NiCl ꢀ6H O and PdCl (CH CN) (0.33 g, 1.38 mmol
2
2
2
3
2
and 0.35 g, 1.38 mmol) in 20 mL of methanol was added to
the mixture, the reaction mixture was refluxed for 6 h. The
completion of the complexing reaction was checked by TLC
and then let it be cool. The precipitate formed was filtrated.
2
.3.3. Determining the optimum electroporation conditions
It is of prime importance to monitor the permeability of the
cells before proceeding to the electroporation application
(Emam et al., 2019). In this study, propidium iodide (PI) was
The product was washed with diethyl ether and dried.
used to determine the cell electropermeabilization. 10 mL PI
and 90 mL cell suspension (1 ꢃ 10 cell/mL) were added to
ꢂ
[
NiCl L(H O)]ꢀ2H O: Green solid, yield 72%, m.p.: >270 C.
6
2
2
2
meff (B.M.): 2.78. Anal. calc. for C H N O NiCl (FW: 472.82 g/
14
21
3
7
2
the 4 mm EP cuvettes (Bio-Rad, Hercules, CA, USA) and then
the cuvettes were placed in the EP device for the electrical
pulse application. 8 square-wave pulse trains having various
electric field strengths (375, 625, 875, 1125, and 1250 V/cm)
with a duration of 100 ms and a frequency of 1 Hz were
applied to the cell suspensions in order to determine the
optimum electroporation conditions for the HepG2 cells to
be used in the study. The control cells were also placed in
the cuvettes under the same conditions, but the electric field
was not applied to them. Following the EP, the cells were incu-
bated with PI for 15min at room temperature and then the
percentage of permeabilized cells was measured using the flow
cytometry (Becton-Dickinson). At least 10,000 cells were meas-
ured from each sample. The percentage of stained cells was
found by making a comparison with the non-electroporated
control cells. The electropermeabilization alone is not sufficient
to determine the most appropriate electric field to be used in
ECT. In addition to the best permeability, it is also important to
determine the voltage causing the least cell death. For this pur-
mol): C; 35.53, H; 4.44, N; 8.88. Found: C; 35.60, H; 4.56, N;
ꢁ
1
8
.94. FT-IR (KBr, v max (cm )): 3320 (OH), 3176 (NH), 3097
(
(
5
2
4
Ar–CH), 2982 (Al–CH), 1730 (C ¼ O), 1656 (CH ¼ N), 1636
C ¼ N), 1566, 1501, 1446 (C ¼ C)Ar, 1094 (OCH ), 800 (H O),
3
2
99, 546, 529 (M–O), 461 (M–N). UV–Vis. (k max/nm): 219,
27, 237, 249, 278, 287, 357, 369, 484, 516. MS [ESI]: m/z
þ
73.82 (Calc.), 473.39 (Found) [M þ H] .
[
PdL(H O)]ꢀ0.5H OꢀCl : Cream solid, yield 74%, m.p.:
2
2
2
ꢂ
270 C. m (B.M.): Dia, Anal. calc. for C H N O5.5PdCl2
eff 14 18 3
>
(
FW: 493.32 g/mol): C; 34.05, H; 3.64, N; 8.51. Found: C; 34.15,
ꢁ1
H; 3.60, N; 8.54. FT-IR (KBr, v max (cm )): 3424, 3310 (OH),
3
178 (NH), 3096 (Ar–CH), 2886 (Al–CH), 1717 (C ¼ O), 1670
(
(
CH ¼ N), 1633 (C ¼ N), 1534, 1503, 1473 (C ¼ C) , 1120
Ar
1
OCH ), 885 (H O), 579, 525 (M–O), 451 (M–N). H-NMR (300
3
2
Mz, DMSO-d ): d (ppm) ¼ 8.10 (s, H, NH), 8.32 (s, H, N ¼ CH),
6
13
7
.85–6.39 (m, 4H, Ar-H), 3.80–3.45 (s, 9H, OCH ). C-NMR
3
(
1
100 MHz, DMSO-d , ppm): 163.50 (CH ¼ N), 159.28 (C ¼ N),
6
53.43 (C ¼ O), 151.18–103.03 (Ar-C), 61.90–52.18 (OCH ) .
3
3
UV–Vis. (k max/nm): 219, 228, 235, 249, 256, 275, 387, 426.
MS [ESI]: m/z 494.32 (Calc.), 494.96 (Found) [M þ H] .
6
pose, 400 mL cell suspension (1ꢃ 10 cell/mL) were placed in
þ
the 4 mm EP cuvettes without adding any agent or PI, and
then the same voltages as in the electropermeabilization were
applied. The HepG2 cells were examined in terms of both
short-term (20-minute incubation after EP) and long-term (24 h
incubation after EP) cell viability. The electroporation parame-
2.3. Antiproliferative activity
2
.3.1. Cell culture experiments
The HepG2 and L-929 cells were used as models in the cell ters were optimized for high permeability and low cellular mor-
culture experiments. The development of the cells was exam- tality based on the MTT and PI analyses.
ined under an inverted microscope daily. The cells were fed
with the RPMI-1640 medium which was prepared by adding
2
2.3.4. Electrochemotherapy (ECT)
When the HepG2 cells incubated to be used in the study
reached a confluency of 80–90%, they were removed using
1
0% FBS and 1% penicillin-streptomycin in 25 cm culture
flasks. The cells were incubated in a 5% CO incubator at a
2
ꢂ
temperature of 37 C and a humidity of 95%.
1
X Trypsin-EDTA, and then the cell solution was transferred
into falcon tubes and sedimented by centrifugation at
2
.3.2. Chemotherapy
1000 rpm for 5 min. 400 mL cell suspension at a density of
6
The cells were counted using a cell counting device, and the 1 ꢃ 10 cell/mL was transferred to the 4 mm EP cuvettes in a
viability was measured using the Trypan Blue Solution way that the concentration of each compound was at
(
0.4%). The experiments were not started in case the viability 200 mM. The cuvettes were placed in the Gene Pulser Xcell
TM
rate was below 90%. In the experiments, the cells were
(Bio-Rad, Hercules, CA, USA) for the electric field applica-
4
seeded in the 96-well plates at a density of 1 ꢃ 10 cells per tion. Eight square-wave pulse trains having a strength of
well. The seeded cells were incubated for 24 h in an environ- 1125 V/cm with a duration of 100 ms and a frequency of
ꢂ
ment containing 5% CO at 37 C. After the medium liquids 1 Hz, which correspond to the parameters used in ECT clin-
2
were removed from the incubated cells, 100 mL of the syn- ical practice (Campana et al., 2019), were applied to the cell
thesized substance solutions prepared in medium (RPMI- suspensions. Likewise, the control cells were placed in the

4
M. E. ALKIŞ ET AL.
cuvettes under the same conditions, but the electric field different frequencies for the two complexes showing the
was not applied to them. The experiments were repeated involvement of the CH ¼ N group in the complexation. In the
ꢁ
1
three times. 10–15 min after the EP, the cells were seeded in IR spectrum of the ligand, the band observed at 3176 cm
the 24-well cell culture plates and left for incubation. After was attributed to the ꢀ(NH) group. The ꢀ(NH) band appeared
4 h incubation, the viability of the experimented cells was in the same position for the complexes and it showed that
evaluated using the MTT analysis method.
this group was not involved in complexing. The band
2
ꢁ
1
showed at 1632 cm was due to the ꢀ(C ¼ N) structure of
the pyrimidine group of the ligand. This group did not
2
.3.5. MTT test (cytotoxicity test)
change in Pd(II) complex. The band of ꢀ(OCH ) of the ligand
3
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
ꢁ
1
ꢁ1
ꢁ1
seen at 1100 cm was shifted to 1094 cm and 1120 cm
bromide] test was carried out to determine the cytotoxicity
of the solutions of the synthesized substances prepared at
different concentrations. For this purpose, the solution with a
concentration of 5 mg/mL (60 mg MTT powder in 12 mL PBS)
for the Ni(II), Pd(II) complexes, respectively. This indicated
that OCH3 group was involved in chelation for the com-
plexes. These observations were supported by new bands
ꢁ
1
ꢁ1
ꢂ
that appeared at 525–599 cm and 451–461 cm assigned
to ꢀ(M–O) and ꢀ(M–N), respectively. All complexes exhibited
was covered with aluminum foil and left at þ4 C until the
time it would be used. The plates, which were left for incu-
bation after adding the substances, were taken and the
medium in the wells was removed. Then, 10 mL MTT solution
and 90 mL RPMI-1640 were added to each well and the plates
ꢁ
1
a wide absorption band in the ranges of 3424–3310 cm
ꢁ
1
and 885–800 cm relatives to the of hydration water mole-
cules and the coordination water attached to metal(II) ions
(Ali, 2017; Gomathi & Gopalakrishnan, 2016; Turan et al.,
ꢂ
were left for incubation for 4 h at 37 C in an incubator con-
taining 5% CO . After the 4 h of incubation, the medium con-
2
019; Ye gꢀ iner et al., 2017) (Figure 1).
2
taining MTT was removed and 100 ml DMSO was added to
each well to dissolve the formazan crystals formed due to
MTT reduction, and their optical density (OD) was measured
at the wavelength of 570 nm (Mahajan et al., 2012) using a
microplate reader. Only the fresh medium was used in the
control group. The control wells were read and then the
absorbance values obtained by reading were averaged. This
value was accepted as 100% viable cell. The absorbance val-
1
3
.2. H-NMR
The signal at 8.48 ppm corresponding to the CH ¼ N reson-
1
ance of the azomethine group in the H NMR spectrum of
the ligand shifted to 8.38 ppm in the spectra of the Pd(II)
complex. This shift confirmed that the Schiff base was
coordinated to Pd(II) ion as a chelating ligand. Signals
ues read from the wells, to which the solutions of the syn- observed in the ligand at 3.80–3.60 ppm were attributed to
thesized substances were added, were proportioned to the the OCH group. These signals shifted to 3.80–3.45 ppm in
absorbance value of the control group and calculated as per- the spectra of the Pd(II) complex. Shifting at signals to the
3
cent viability using the following formula.
high area for Pd(II) complex is evidence for the coordin-
ation of nitrogen atom of imine (CH ¼ N) group and oxy-
%
Viability ¼ ðOD of the group being examined=
OD of the control groupÞ ꢃ 100
gen atom of methoxy (OCH ) group to metal centers (Ali,
3
2
014; Kargar et al., 2020).
The substance concentration yielding 50% inhibition (IC )
50
was determined by the calculation carried out on the graph of
the percentage of inhibition plotted against the substance con-
3
.3. UV-visible spectroscopy
centration. The lower the IC50 value, the higher the anti-cancer The electronic spectrum of the Schiff base ligand in ethanol
capacity. All the analyzes were carried out in three parallel repli- is due to bands p ! p
cates, and each parallel experiment was repeated 6–8 times. 282 nm. It showed a shoulder at 360 nm and a strong band
ꢄ transition of the non-common
ꢄ transitions at 220, 249, 266 and
The results were expressed as mean and standard deviation.
at 302 nm due to the n! p
electrons found in the nitrogen of the azomethine group on
ꢄ
ꢄ transitions were shifted
the Schiff base. The p ! p , n! p
2
.3.6. Statistical analysis
after the metal ions were coordinated towards the Schiff
base ligand, this change supports that the nitrogen and oxy-
gen atoms of the azomethine, carbonyl and pyrimidine
group in the ring participate in coordination towards the
metal ions. Spectral bands appearing at 227, 237, 249, 278,
Statistical analyzes were performed by Graph Pad Prism soft-
ware (Graph Pad Software Inc., San Diego, USA). Data were
analyzed by one-way ANOVA followed by Dunnett’s multiple
comparisons test, or by Unpaired Parametric two-tailed t test
where it was appropriate. p < 0.05 was considered to be stat-
istically significant.
2
87, 357, 369, 484 and 516 nm can be ascribed to the p!
ꢄ
ꢄ
p , n! p and MLCT transitions that support octahedral
geometry, respectively (Saad, 2014; Shah et al., 2016; Ye gꢀ iner
et al., 2017). The magnetic moment of the Ni(II) complex was
found as 2.78 B.M. Palladium complex originates from two
absorption bands d ! d transitions observed at 387 nm and
3
. Result and discussion
3.1. IR spectra
The FT-IR spectrum of ligand refers to the band ꢀ(CH ¼ N) 426 nm in the UV spectrum. Electronic spectral results pro-
ꢁ
1
group assigned at 1662 cm . This band was altered to posed that the Pd(II) complex had diamagnetic behavior and

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
5
Figure 1. The FTIR spectrum of the compounds.
square planar geometry (Ali, 2017; Kumari et al., 2012; 3.5. Powder X-ray diffraction analyses
Ye gꢀ iner et al., 2017).
UltimaIV X-ray diffractometer (Rigaku model with
CuK ¼0.1540562 nm) was operated to measure the crystal
a
structure of the complexes. XRD working conditions can be
ꢂ
ꢂ
3
.4. TGA analyses
listed as follows; scan step: 0.02 , scanning range: 10–90 ,
scanning mode: 2Theta/Theta, scanning type: continuous
scanning, operating current: 30 mA, operating voltage: 40 kV.
XRD spectrums of Ni(II) and Pd(II) complexes are shown in
Figure 2. Estimated crystal parameters (FWHM, grain size,
detected diffraction angle) of these complexes are displayed
in Table 2.
The result of TGA analyses showed that spectral analyses were
coherent with the theoretical formulae. [NiCl L(H O)]ꢀ2H O com-
2
2
2
plex decomposed in four stages. Its thermogram indicated a
ꢂ
weight loss of 14.80% between 50 and 180 C. This indicated
the loss of three coordinated/hydration water molecules and
one O atom of the carbonyl group. The second and third stages
showed a weight loss of 48.20% (34.66% and 13.54%) in the
ꢂ
80–600 C proving to the loss of C H N Cl , C H O organic 3.5.1. XRD results of Ni(II) complex
4 3 3 2 2 6 2
1
moiety. The remaining part of the complex decomposed in the XRD phase behavior of the Ni(II) complex was given in
ꢂ
Figure 2. The peak having high intensity was observed from
fourth step between 600 and 790 C with a loss of 24.95% leav-
XRD measurement. The peak detected diffraction angle and
ing behind Ni metal ion. The [PdL(H O)]ꢀ0.5H OꢀCl complex
2
2
2
ꢂ
related intensity were 28.19 and ꢅ357 (a.u), respectively.
decomposes in three stages. Its thermogram showed a weight
ꢂ
The orientation of the observed peaks cannot be given
because the complex is synthesized for the first time and is
not available in the literature. Some crystallographic parame-
ters of a similar complex are referred to (Buldurun et al.,
loss of 5.47% in the 50–220 C region confirming the loss of
three coordinated/hydration water molecules. The second and
third stages showed a weight loss of 69.70% (7.18% and
2.52%) in the 220–700 C confirming the loss of Cl,
ꢂ
6
2
019; Nithya et al., 2018). We estimated grain sizes of the
complex with Scherer’s equation;
rk
FCosh
C H N O Cl organic moiety. The rest of the ligand decom-
14 15 3 3
posed at the fourth step for Pd(II) complex and metal oxide
PdO) remained as a residue (Ali, 2014; Buldurun et al., 2019;
Gaber et al., 2019). The proposed stepwise decomposition pat-
(
K ¼
(1)
tern of [NiCl L(H O)]ꢀ2H O and [PdL(H O)]ꢀ0.5H OꢀCl complexes k shows X-ray wavelength, h shows Bragg’s diffraction angle,
2
2
2
2
2
2
at different steps were due to the loss of different organic moi- r ¼ 0.94 shows constant, F shows full width of half maximum
eties with respect to temperature, which was showed in Table 1. intensity. The average grain sizes (K) of the complex were

6
M. E. ALKIŞ ET AL.
Table 1. Thermal analysis of Ni(II) and Pd(II) complexes.
ꢂ
Compounds
NiCl L(H
O)]ꢀ2H
Dissoc. stage
Decomp. temp. ( C)
Weight loss[%] calc. (found)
Decomp. assign.
3H O, O atom
Cl
[
2
2
2
O
1
2
3
4
50–180
180–450
450–600
600–790
>790
50–220
220–320
320–700
>800
14.86
34.05
13.57
25.03
12.49
5.38
14.80
34.66
13.54
24.95
12.41
5.47
2
4 3 3 2
C H N
2 6 2
C H O
C H O
8 6
Ni metal
Residue
PdL(H
O)]ꢀ0.5H
[
2
2
OꢀCl
2
1
2
3
2
1.5H O
7.05
7.18
Cl
61.25
26.32
62.52
24.83
14 15 3 3
C H N O Cl
Residue
PdO
Figure 2. XRD behavior of Ni(II) and Pd(II) complexes.
ꢂ
Table 2. Grain sizes of the Pd(II) and Ni(II) complexes.
peaks at about 40 . It may be due to the diffraction peaks
Grain size (K)(nm) corresponding to the precursors.
ꢂ
ꢂ
Compounds
2h ( )
Full width half max. (F) ( )
Ni(II) complex
Pd(II) complex
28.19
11.8
0.49
0.26
0.16
0.39
0.48
0.34
0.15
17.06
31.95
51.93
21.32
17.33
24.48
55.59
1
1
1
1
2
2.4
5
6.1
7.7
2.7
3
.6. Antiproliferative activity
The solutions of the synthesized ligand and its Ni(II) and
Pd(II) complexes having five different concentrations (100,
2
00, 300, 400, and 500 mM) were treated with the HepG2 and
L-929 cells for 24 h and their spectrophotometric absorban-
ces were measured. The percent inhibition values of the
HepG2 and L-929 cells were calculated with the measured
absorbances. The inhibition occurring in the cell proliferation
was determined by the MTT test. The IC50 values of the test
substances were calculated in order to find out whether they
calculated from the highest peak intensity. Grain size values
of the Ni(II) complex given in Table 2. The grain size of the
related peak of the complex was found to be 17.06 nm.
3
.5.2. XRD results of Pd(II) complex
XRD phase behavior of the Pd(II) complex was given in had cytotoxic effects on the hepatocellular carcinoma and
Figure 2. Several peaks having high intensities were detected the mouse fibroblast cells (Table 3).
ꢂ
from XRD. These peaks can be listed as follows; 11.8 (inten-
In this study, the IC50 values of the chemical test agents
ꢂ
ꢂ
sity 279 (a.u)), 12.4 (intensity 247 (a.u)), 15 (intensity 226 applied in vitro to the HepG2 and L-929 cells were analyzed.
a.u)), 16.1 (intensity 206 (a.u)), 17.7 (intensity 222 (a.u)), The higher the IC50 value, the lower the cytotoxicity. When
2.7 (intensity 118 (a.u)). The highest intensity was found at the parameters measured in this study are analyzed, it can
the peak of 11.8 , while the lowest intensity was found at be asserted that the synthesized Ni(II) and Pd(II) complexes
ꢂ
ꢂ
(
2
ꢂ
ꢂ
ꢂ
the peak of 22.7 . XRD measurement confirmed that this have the anti-cancer properties and the cytotoxicity of the
complex has a polycrystalline phase. The orientation of the Pd(II) complex is higher than the Ni(II) complex (Gaber et al.,
detected peaks cannot be given because the complex is syn- 2015). The geometry of the complex can affect its DNA bind-
thesized for the first time and is not available in the litera- ing and activity (Indumathy et al., 2008). The possible reason
ture. Grain sizes of the complex were calculated with for the Ni(II) complex to exhibit a lower activity than the
Scherer’s equation (Equation (1)). Grain size values of the Pd(II) complex is the fact that its geometry is not planar.
Pd(II) complex can be given in Table 2. Our results gave that Furthermore, as can be understood from the IC50 values in
ꢂ
ꢂ
the highest grain size was obtained at a 22.7 diffraction Table 3, although the Pd(II) complex displayed a very high
angle; the lowest one was obtained at a 16.1 diffraction cytotoxic effect on the HepG2 cells, it displayed a very low
angle. From XRD measurement, it was also detected weaker cytotoxic effect on the L-929 cells.

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
7
Scheme 1. Structure of Schiff base and its Ni(II), Pd(II) complexes.
Several studies have shown that Schiff base ligands, Ni(II)
Table 3. The IC50 values of the ligand and its Ni(II), Pd(II) complexes in the
and Pd(II) complexes exhibit cytotoxicity in various cancer HepG2 and L-929 cells.
cells and tissues. Arafath et al. (2017) reported that the car- Compounds
Hep G2 IC50 (mM)
L-929 IC50 (mM)
bothioamide Schiff base ligand and its Ni(II), Pd(II) and Pt(II)
complexes have strong cytotoxic property and selectivity
against human cervical and colorectal tumor cells. Therefore,
further studies are recommended as complexes derived from
carbothioamide Schiff base could be promising chemothera-
peutic agents against human malignancy. In another study,
the cytotoxicity of chitosan, chitosan-Schiff base and Cu(II),
Ni(II) and Zn(II) complexes was tested against K562 chronic
myelogenous leukemia (CML) and MG-63 (osteosarcoma can-
cer) cell lines by the MTT assay. It is shown that the anti-
L
989.62
454.62
387.5
866.57
1622.73
1224.44
Ni(II) complex
Pd(II) complex
the cells also increased. However, drastic changes were
observed in the permeability of the cells at 1125 V/cm. It was
observed that the permeability of the HepG2 cells at this
electric field strength was found to be approximately 59%.
Moreover, the HepG2 cells were analyzed in terms of both
short-term (20 min incubation after EP) and long-term (24 h
cancer activity of Schiff base and complexes is much better incubation after EP) cell viability by means of applying the
than that of pure Chitosan against MG63 cancer cell line same electric fields used in the electropermeabilization.
(
(
Malekshah et al., 2020). In a study conducted by S¸ ahin et al.
While the short-term cell viability was measured using a
2018) the anti-cancer activity of the synthesized ligand, cell counting device (Thermo scientific), the long-term cell
Pd(II) and Pt(II) complexes was evaluated in vitro on MCF-7 viability was measured using the MTT test. As can be seen in
human breast adenocarcinoma), LS174T (human colon car- the Figure 4 above, as the strength of the electric field
(
cinoma) and LNCAP (human prostate adenocarcinoma) can- pulses increased, a decrease was observed in the viability of
cer cell lines using the MTT assay. Based on the activity the cells. After 1125 V/cm, sharp declines were observed in
results, the Pd(II) complex has been shown to have higher the cell viability. 20 min and 24 h after the electroporation,
anti-tumor effect than the ligand and its Pt(II) complex the viabilities of the cells were measured to be 52.5% and
against the tested human breast adenocarcinoma, human 69.57%, respectively, at 1125 V/cm. When the electropermea-
prostate adenocarcinoma and human colon carcinoma cell bilization and short- and long-term cell viability analyzes
lines. The findings obtained in our study are consistent with were evaluated together, it was found that the electric field
the results of the studies mentioned above.
strength of 1125 V/cm (100 ls, 1 Hz, 8 square waves) was
appropriate for the electroporation as it exhibited a high per-
meability and low cellular mortality.
3.7. Electropermeabilization and viability of the cells
The electropermeabilization of the HepG2 cells was meas-
ured by flow cytometry using PI. The permeability percen-
tages of the cell membrane depending on various electric
3
.8. Efficacy of electroporation in treatment
field strengths (0, 375, 625, 875, 1125, and 1250 V/cm) were 8 square-wave pulse trains having a strength of 1125 V/cm with
given in Figure 3.
a duration of 100 ms and a repetition frequency of 1 Hz were
As can be seen in the Figure 3, as the magnitude of the used in the ECT. After the 24 h incubation, the cell viability per-
electric fields increased, the permeabilization percentages of centages in the compound-alone and compound þ EP groups

8
M. E. ALKIŞ ET AL.
Figure 3. The permeabilization percentage of the HepG2 cells 20 min after the electroporation treatment. The data is given as the mean ± SD of three independent
experiments.
Figure 4. Cell viability of electroporated HepG2 cells at 20 min and 24 h after
the electroporation. The data were taken from three independent experiments
and presented as mean ± SD.
were measured using the MTT analysis, and the data were
given in the Figure 5.
There was a statistically significant decrease in MCF-7 can-
Figure 5. Cell viability of HepG2 cells at 24 h under different treatment condi-
cer cells viability (%) of all treatment (Ni(II), Pd(II), Ni(II)þEP tions: Control, Ni(II) complex only, Pd(II) complex only, Ni(II) complex þ EP and
Pd(II) complex þ EP treatment. (A) The treatment groups were compared with
and Pd(II)þEP) groups compared to control group (p < 0.05).
the control group using One Way ANOVA, Dunnett’s multiple comparisons test.
When the chemotherapy (Ni(II), Pd (II)) and electrochemo-
(
B) Ni(II), Ni(II)þEP and Pd(II), Pd(II)þEP were compared among themselves
therapy (Ni(II) þ EP, Pd(II) þ EP) groups were compared, the using Unpaired Parametric two-tailed t test. A significant difference was found
ꢄ
ꢄꢄ
between groups at different levels ( p < 0.05, significant; p < 0.01, very sig-
difference between them was found to be statistically signifi-
cant (p < 0.05) (Figure 5B). The antiproliferative activities of
the Ni(II) and Pd(II) complexes significantly increased with
the EP. When the two complexes were compared, it was
found that the Pd(II) complex highly reduced the number of
viable cells and so, displayed the best antiproliferative effect.
As can be understood from the Figure 5, the synthesized
ꢄꢄꢄ
ꢄꢄꢄꢄ
nificant;
p < 0.001 and
p < 0.0001, extremely significant). The data
were taken from three independent experiments and presented as mean ± SD.
that have large molecular structures and are otherwise diffi-
cult to deliver, such as nucleic acids, cytotoxic drugs, pro-
teins, peptides, and ions, can be delivered across the
membrane (Campana et al., 2019; Zhang et al., 2010). The
compounds in general exhibited a more antiproliferative possible main reason why the complexes synthesized in this
effect in the ECT than the standard chemotherapy. When the study displayed a better antiproliferative activity with the
electric field pulses are applied to a cell, a transmembrane help of EP is the fact that the EP increases the uptake of
voltage is induced on the cell membrane (Zhang et al., these complexes to the cell.
2
010). If the voltage induced on the cell membrane exceeds
After the electric field exposure is applied at an appropri-
a certain value, it causes a significant increase in the perme- ate amplitude (<1500 V/cm) and for an appropriate duration,
ability and electrical conductivity of the membrane. With the the membrane returns to its normal state (reversible EP). On
help of the increased membrane permeability, the molecules the other hand, if the amplitude of the electric field is too

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
9
high (1500–3000 V/cm) or if the duration of the electric field expected that the results of this study contribute to new
exposure is too long, the membrane does not close after the therapeutic studies for hepatocellular carcinoma.
exposure and this causes cell death (irreversible EP) (Bharti
et al., 2010). The reversible EP is being used for increasing
the delivery of cytotoxic drugs in the local tumor treatment
Acknowledgements
as well as being used in many fields such as DNA vaccination This work is supported by the Scientific Research Project Fund of Mus¸
Alparslan University under the project number BAP-18-MMF-4901-02.
and gene therapy (Romeo et al., 2018). The optimum EP con-
ditions (1125 V/cm, 100 ms, 1 Hz, 8 pulses) used in this study
were determined based on the electropermeabilization ana-
lysis and the analyses carried out on the HepG2 cells to find
out their electric field sensitivity at various electric
Disclosure statement
The authors declare that no conflicts of interest.
field strengths.
ECT is highly effective even when used with low-dose
chemotherapeutic agents, and it has become a standard
treatment method for the therapy of the deep-seated tumors
and the local cutaneous and subcutaneous metastatic tumors
ORCID
Mehmet E ¸s ref Alkı ¸s
Kenan Buldurun
Nevin Turan
http://orcid.org/0000-0002-3321-2873
http://orcid.org/0000-0002-2462-7006
http://orcid.org/0000-0001-6740-6812
http://orcid.org/0000-0003-0007-0212
in different parts of the body. Recently, it has been applied Yusuf Alan
€
Unzile Kele ¸s temur Yılmaz
http://orcid.org/0000-0003-4531-6378
in many clinics in Europe (Cadossi et al., 2014; Miklav ꢂc i ꢂc
et al., 2012). Today, ECT is being developed and evaluated
for the treatment of subcutaneous tumors or internal organ
Asim Mantarcı http://orcid.org/0000-0001-8369-3559
tumors (Schneider et al., 1990). Mali et al. (2013) examined References
4 studies on the effectiveness of ECT in the treatment of
4
€
€
ꢀ
Akta s¸ , A., Kele s¸ temur, U., Gok, Y., Balcıoglu, S., Ate s¸ , B., & Ayg u€ n, M.
malignant melanomas, breast cancer, and head and neck
tumors. They found that ECT yielded an objective response rate
of 84.1% and a complete remission rate of 59.4%, regardless of
tumor type. On the other hand, Esmekaya et al. (2016) reported
that the electroporation increased the delivery of the anti-can-
cer drugs to the cells and decreased the minimum dosage
required for treatment. Our findings are in parallel with the
findings of these studies. It was found that, in the HepG2 cell
(
2018). 2-Morpholinoethyl-substituted N-heterocyclic carbene (NHC)
precursors and their silver(I)NHC complexes: Synthesis, crystal struc-
ture and in vitro anticancer properties. Journal of the Iranian Chemical
Society, 15(1), 131–139. https://doi.org/10.1007/s13738-017-1216-8
Al-Humaidi, J. Y. (2019). In situ alkaline media: Synthesis, spectroscopic,
morphology and anticancer assignments of some transition metal ion
complexes of 1-((2-aminophenylimino) methyl) naphthalen-2-ol Schiff
base. Journal of Molecular Structure, 1183, 190–201. https://doi.org/10.
1016/j.molstruc.2019.01.083
line, the EP increased the cytotoxicity of the Ni(II) and Pd(II) Ali, O. A. M. (2014). Synthesis, spectroscopic, fluorescence properties and
biological evaluation of novel Pd(II) and Cd(II) complexes of NOON
complexes by 1.66, and 2.54 times, respectively, compared to
tetradentate Schiff bases. Spectrochimica Acta. Part A, Molecular and
applying only the metal complexes.
Biomolecular Spectroscopy, 121, 188–195. https://doi.org/10.1016/j.saa.
2013.10.015
Ali, O. A. M. (2017). Characterization, thermal and fluorescence study of
Mn(II) and Pd(II) Schiff base complexes. Journal of Thermal Analysis
and Calorimetry, 128(3), 1579–1590. https://doi.org/10.1007/s10973-
4
. Conclusion
A new type of pyrimidine bases Schiff base ligand and its
Ni(II) and Pd(II) complexes were successfully synthesized and
characterized in this study. Ligand and its metal complexes
were characterized using the elemental and mass analysis,
016-6055-9
Alothman, A. A., Al-Farraj, E. S., Al-Onazi, W. A., Almarhoon, Z. M., & Al-
Mohaimeed, A. M. (2020). Spectral characterization, electrochemical, anti-
microbial and cytotoxic studies on new metal(II) complexes containing
1
13
H-NMR, C-NMR, FT-IR, UV-VIS, magnetic susceptibility, ther-
N O
2 4
donor hexadentate Schiff base ligand. Arabian Journal of Chemistry,
1
3(2), 3889–3902. https://doi.org/10.1016/j.arabjc.2019.02.003
mal analysis. In vitro antiproliferative activities of the com-
pounds on the HepG2 human cancer cell line was performed
by MTT assay. Furthermore, the effects of electroporation on
the cytotoxic activities of synthesized compounds were
investigated in HepG2 cancer cells. The results showed that
the ligand did not exhibit an antiproliferative activity while
its Ni(II) and Pd(II) complexes showed an antiproliferative
activity and Pd(II) had stronger antiproliferative activity than
Ni(II) against HepG2 cancer cells. The combined application
of EP þ compounds was much more effective than the usage
of the compounds alone in the treatment of HepG2 cancer
cells. EP increased the cytotoxicity of the compounds, there-
fore lower doses of compounds treatment with EP were as
effective as higher doses for compounds treatment without
EP. It was observed that the efficacy of ECT in treatment was
quite high compared to the standard chemotherapy. It is
Arafath, M. A., Adam, F., Razali, M. R., Ahmed Hassan, L. E., Ahamed,
M. B. K., & Majid, A. M. S. A. (2017). Synthesis, characterization and
anticancer studies of Ni(II), Pd(II) and Pt(II) complexes with Schiff base
derived from N-methylhydrazinecarbothioamide and 2-hydroxy-5-
methoxy-3-nitrobenzaldehyde. Journal of Molecular Structure, 1130,
791–798. https://doi.org/10.1016/j.molstruc.2016.10.099
Batista Napotnik, T., & Miklav ꢂc i ꢂc , D. (2018). In vitro electroporation detection
methods - An overview. Bioelectrochemistry (Amsterdam, Netherlands), 120,
166–182. https://doi.org/10.1016/j.bioelechem.2017.12.005
Bernadette Amali, I., Kesavan, M. P., Vijayakumar, V., Indra Gandhi, N.,
Rajesh, J., & Rajagopal, G. (2019). Structural analysis, antimicrobial and
2 2
cytotoxic studies on new metal(II) complexes containing N O donor
Schiff base ligand. Journal of Molecular Structure, 1183, 342–350.
https://doi.org/10.1016/j.molstruc.2019.02.005
Bharti, S. K., Nath, G., Tilak, R., & Singh, S. K. (2010). Synthesis, anti-bac-
terial and anti-fungal activities of some novel Schiff bases containing
2
,4-disubstituted thiazole ring. European Journal of Medicinal
Chemistry, 45(2), 651–660. https://doi.org/10.1016/j.ejmech.2009.11.008

1
0
M. E. ALKIŞ ET AL.
Bing o€ l, M., & Turan, N. (2020). Schiff base and metal(II) complexes con- Gehl, J. (2003). Electroporation: Theory and methods, perspectives for
taining thiophene-3-carboxylate: Synthesis, characterization and anti-
oxidant activities. Journal of Molecular Structure, 1205, 127542. https://
doi.org/10.1016/j.molstruc.2019.127542
drug delivery, gene therapy and research. Acta Physiologica
Scandinavica, 177(4), 437–447. https://doi.org/10.1046/j.1365-201X.
2003.01093.x
Buldurun, K., Turan, N., Aras, A., Mantarcı, A., Turkan, F., & Bursal, E. Gomathi, G., & Gopalakrishnan, R. (2016). A hydrazone Schiff base single
(
2019). Spectroscopic and structural characterization, enzyme inhibi-
crystal (E)-methyl N(’)-(3,4,5-trimethoxybenzylidene) hydrazine carb-
oxylate: Physicochemical, in vitro investigation of antimicrobial activ-
ities and molecular docking with DNA gyrase protein. Materials
Science & Engineering. C, Materials for Biological Applications, 64,
133–138. https://doi.org/10.1016/j.msec.2016.03.084
tions, and antioxidant effects of new Ru(II) and Ni(II) complexes of
Schiff base. Chemistry & Biodiversity, 16(8), e1900243. https://doi.org/
1
Buldurun, K., Turan, N., Bursal, E., Aras, A., Mantarcı, A., C¸ olak, N., T u€ rkan, F.,
&
analysis, thermal stability, antioxidant properties and enzyme inhibitions
of M(II)-Schiff base ligand complexes. Journal of Biomolecular Structure
and Dynamics. https://doi.org/10.1080/07391102.2020.1802340
Cadossi, R., Ronchetti, M., & Cadossi, M. (2014). Locally enhanced chemo-
therapy by electroporation: Clinical experiences and perspective of
use of electrochemotherapy. Future Oncology, 10(5), 877–890. https://
doi.org/10.2217/fon.13.235
0.1002/cbdv.201900243
G u€ l c¸ in, I. (2020). Synthesis, characterization, powder X-ray diffraction
_
Indumathy, R., Kanthimathi, M., Weyhermuller, T., & Nair, B. U. (2008).
Cobalt complexes of terpyridine ligands: Crystal structure and nucle-
ase activity. Polyhedron, 27(17), 3443–3450. https://doi.org/10.1016/j.
poly.2008.08.003
Jain, P., Kumar, D., Chandra, S., & Misra, N. (2020). Experimental and theoret-
ical studies of Mn(II) and Co(II) metal complexes of a tridentate Schiff’s
base ligand and their biological activities. Applied Organometallic
Chemistry, 34(2), e5371. https://doi.org/10.1002/aoc.5371
Jemal, A., Bray, F., Center, M. M., Ferlay, J., Ward, E., & Forman, D. (2011).
Global cancer statistics. CA: A Cancer Journal for Clinicians, 61(2),
Campana, L. G., Miklav ꢂc i ꢂc , D., Bertino, G., Marconato, R., Valpione, S.,
Imarisio, I., Dieci, M. V., Granziera, E., Cemazar, M., Alaibac, M., & Sersa,
G. (2019). Electrochemotherapy of superficial tumors - Current status:
Basic principles, operating procedures, shared indications, and emerg-
ing applications. Seminars in Oncology, 46(2), 173–191. https://doi.org/
69–90. https://doi.org/10.3322/caac.20107
Kargar, H., Torabi, V., Akbari, A., Behjatmanesh-Ardakani, R., Sahraei, A., &
Tahir, M. N. (2020). Pd(II) and Ni(II) complexes containing an asymmet-
ric Schiff base ligand: Synthesis, x-ray crystal structure, spectroscopic
investigations and computational studies. Journal of Molecular
Structure, 1205, 127642. https://doi.org/10.1016/j.molstruc.2019.127642
Kumari, N., Yadav, V. K., Zalis, S., & Mishra, L. (2012). ChemInform
abstract: Pd(II) catalyzed transformation of Schiff bases in complexes
1
0.1053/j.seminoncol.2019.04.002
Cemazar, M., & Sersa, G. (2019). Recent advances in electrochemotherapy.
Bioelectricity, 1(4), 204–213. https://doi.org/10.1089/bioe.2019.0028
Dehghani-Firouzabadi, A., & Firouzmandi, S. (2016). Synthesis and charac-
terization of a new unsymmetrical potentially pentadentate Schiff
base ligand and related complexes with manganese(II), nickel(II),
copper(II), zinc(II) and cadmium(II). Journal of the Brazilian Chemical
Society, 28, 768–774. https://doi.org/10.21577/0103-5053.20160226
Emam, M. A., Khattab, H. I., & Hegazy, M. G. (2019). Assessment of anti-
cancer activity of pulicaria undulata on hepatocellular carcinoma
HepG2 cell line. Tumour Biology, 41(10), 1010428319880080. https://
doi.org/10.1177/1010428319880080
Esmekaya, M. A., Kayhan, H., Coskun, A., & Canseven, A. G. (2016). Effects
of cisplatin electrochemotherapy on human neuroblastoma cells. The
Journal of Membrane Biology, 249(5), 601–610. https://doi.org/10.1007/
s00232-016-9891-4
Fekri, R., Salehi, M., Asadi, A., & Kubicki, M. (2019). Synthesis, character-
ization, anticancer and antibacterial evaluation of Schiff base ligands
derived from hydrazone and their transition metal complexes.
Inorganica Chimica Acta, 484, 245–254. https://doi.org/10.1016/j.ica.
2 2 2 3
of the type trans-[PdCl (NH Ar-X) ] (X: H, CH , Cl). ChemInform, 43(34),
554-563. https://doi.org/10.1002/chin.201234032
Levitsky, D. O., & Dembitsky, V. M. (2015). Anti-breast cancer agents
derived from plants. Natural Products and Bioprospecting, 5(1), 1–16.
https://doi.org/10.1007/s13659-014-0048-9
Mahajan, S. D., Law, W.-C., Aalinkeel, R., Reynolds, J., Nair, B. B., Yong, K.-T.,
Roy, I., Prasad, P. N., & Schwartz, S. A. (2012). Nanoparticle-mediated tar-
geted delivery of antiretrovirals to the brain. Methods in Enzymology, 509,
41–60. https://doi.org/10.1016/B978-0-12-391858-1.00003-4
Malekshah, R. E., Shakeri, F., Khaleghian, A., Salehi, M. (2020).
&
Developing a biopolymeric chitosan supported Schiff-base and Cu(II),
Ni(II) and Zn(II) complexes and biological evaluation as pro-drug.
International Journal of Biological Macromolecules, 152, 846–861.
https://doi.org/10.1016/j.ijbiomac.2020.02.245
Mali, B., Jarm, T., Snoj, M., Sersa, G., & Miklavcic, D. (2013). Antitumor
effectiveness of electro chemotherapy: A systematic review and meta-
analysis. European Journal of Surgical Oncology, 39(1), 4–16. https://
doi.org/10.1016/j.ejso.2012.08.016
Michel, O., Kulbacka, J., Saczko, J., M a˛ czy nꢁ ska, J., Błasiak, P., Rossowska,
J., & Rzechonek, A. (2018). Electroporation with cisplatin against meta-
static pancreatic Cancer: In vitrostudy on human primary cell culture.
BioMed Research International, 2018, 1–12. https://doi.org/10.1155/
2
018.09.022
Fetoh, A., Asla, K. A., El-Sherif, A. A., El-Didamony, H., & Abu El-Reash,
G. M. (2019). Synthesis, structural characterization, thermogravimetric,
molecular modelling and biological studies of Co(II) and Ni(II) Schiff
bases complexes. Journal of Molecular Structure, 1178, 524–537.
https://doi.org/10.1016/j.molstruc.2018.10.066
Frandsen, S. K., McNeil, A. K., Novak, I., McNeil, P. L., & Gehl, J. (2016).
Difference in membrane repair capacity between cancer cell lines and
a normal cell line. The Journal of Membrane Biology, 249(4), 569–576.
https://doi.org/10.1007/s00232-016-9910-5
Gaber, M., El-Ghamry, H. A., & Fathalla, S. K. (2015). Ni(II), Pd(II) and Pt(II)
complexes of (1H-1,2,4-triazole-3-ylimino)methyl]naphthalene-2-ol.
Structural, spectroscopic, biological, cytotoxicity, antioxidant and DNA
binding. Spectrochimica Acta. Part A, Molecular and Biomolecular
Spectroscopy, 139, 396–404. https://doi.org/10.1016/j.saa.2014.12.057
Gaber, M., El-Wakiel, N., El-Baradie, K., & Hafez, S. (2019). Chromone
Schiff base complexes: Synthesis, structural elucidation, molecular
modeling, antitumor, antimicrobial, and DNA studies of Co(II), Ni(II),
2
018/7364539
Miklav ꢂc i ꢂc , D., Ser ꢂs a, G., Brecelj, E., Gehl, J., Soden, D., Bianchi, G.,
Ruggieri, P., Rossi, C. R., Campana, L. G., Jarm, T. (2012).
&
Electrochemotherapy: Technological advancements for efficient elec-
troporation-based treatment of internal tumors. Medical & Biological
Engineering & Computing, 50(12), 1213–1225. https://doi.org/10.1007/
s11517-012-0991-8
Mohamed, R. G., Elantabli, F. M., Abdel Aziz, A. A., Moustafa, H., & El-Medani,
S. M. (2019). Synthesis, characterization, NLO properties, antimicrobial, CT-
DNA binding and DFT modeling of Ni(II), Pd(II), Pt(II), Mo(IV) and Ru(I)
complexes with NOS Schiff base. Journal of Molecular Structure, 1176,
501–514. https://doi.org/10.1016/j.molstruc.2018.08.113
and Cu(II) complexes. Journal of the Iranian Chemical Society, 16(1), Neumann, E., Schaefer-Ridder, M., Wang, Y., & Hofschneider, P. H. (1982).
1
69–182. https://doi.org/10.1007/s13738-018-1494-9
Gene transfer into mouse lyoma cells by electroporation in high elec-
tric fields. The EMBO Journal, 1(7), 841–845. https://doi.org/10.1002/j.
1460-2075.1982.tb01257.x
Geeta, B., Shravankumar, K., Reddy, P. M., Ravikrishna, E., Sarangapani,
M., Reddy, K. K., & Ravinder, V. (2010). Binuclear cobalt(II), nickel(II),
copper(II) and palladium(II) complexes of a new Schiff-base as ligand: Nithya, P., Rajamanikandan, R., Simpson, J., Ilanchelian, M.,
&
Synthesis, structural characterization, and antibacterial activity.
Govindarajan, S. (2018). Solvent assisted synthesis, structural charac-
Spectrochimica Acta. Part A, Molecular and Biomolecular Spectroscopy,
terization and biological evaluation of cobalt(II) and nickel(II) com-
plexes of Schiff bases generated from benzyl carbazate and cyclic
7
7(4), 911–915. https://doi.org/10.1016/j.saa.2010.08.004

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
11
ketones. Polyhedron, 145, 200–217. https://doi.org/10.1016/j.poly.2018.
2.008
Qin, W., Long, S., Panunzio, M., & Biondi, S. (2013). Schiff bases: A short
modeling, drug-likeness, and spectral. Journal of Thermal Analysis and
Calorimetry, 123(1), 731–743. https://doi.org/10.1007/s10973-015-4838-
z
0
survey on an evergreen chemistry tool. Molecules (Basel, Switzerland), Tahir, M., Sirajuddin, M., Haider, A., Ali, S., Nadhman, A., & Rizzoli, C.
1
8(10), 12264–12289. https://doi.org/10.3390/molecules181012264
(2019). Synthesis, spectroscopic characterization, crystal structure,
interaction with DNA, CTAB as well as evaluation of biological
potency, docking and molecular dynamics studies of N-(3,4,5-trime-
thoxybenzylidene)-2,3-dimethylbenzenamine. Journal of Molecular
Structure, 1178, 29–38. https://doi.org/10.1016/j.molstruc.2018.10.014
Tan, A., Kizilkaya, S., Kelestemur, U., Akdemir, A., & Kara, Y. (2020). The
synthesis, anticancer activity, structure–activity relationships and
molecular modelling studies of novel isoindole-1,3(2H)-dione com-
pounds containing different functional groups. Anti-Cancer Agents in
Medicinal Chemistry, 20(11), 1368–1378. https://doi.org/10.2174/
1871520620666200410080648
Rahmouni, A., Souiei, S., Belkacem, M. A., Romdhane, A., Bouajila, J., &
Ben Jannet, H. (2016). Synthesis and biological evaluation of novel
pyrazolopyrimidines derivatives as anticancer and anti-5-lipoxygenase
agents. Bioorganic Chemistry, 66, 160–168. https://doi.org/10.1016/j.
bioorg.2016.05.001
Rashad, A. E., Mahmoud, A. E., & Ali, M. M. (2011). Synthesis and anti-
cancer effects of some novel pyrazolo[3,4-d]pyrimidine derivatives by
generating reactive oxygen species in human breast adenocarcinoma
cells. European Journal of Medicinal Chemistry, 46(4), 1019–1026.
https://doi.org/10.1016/j.ejmech.2011.01.013
Romeo, S., Sannino, A., Scarf ꢃı , M. R., Vernier, P. T., Cadossi, R., Gehl, J., & Turan, N., Buldurun, K., Alan, Y., Savci, A., C¸ olak, N., & Mantarcı, A. (2019).
Zeni, O. (2018). ESOPE-equivalent pulsing protocols for calcium elec-
troporation: An in vitro optimization study on 2 cancer cell models.
Technology in Cancer Research & Treatment, 17, 153303381878807.
https://doi.org/10.1177/1533033818788072
Synthesis, characterization, antioxidant, antimicrobial and DNA bind-
ing properties of ruthenium(II), cobalt(II) and nickel(II) complexes of
Schiff base containing o-vanillin. Research on Chemical Intermediates,
45, 3525–3540. https://doi.org/10.1007/s11164-019-03806-3
Saad, F. A. (2014). Synthesis, spectral, electrochemical and X-ray single Vashi, K., & Naik, H. B. (2004). Synthesis of novel Schiff base and azetidi-
crystal studies on Ni(II) and Co(II) complexes derived from 1-benzoyl-
-(4-methylpyridin-2-yl) thiourea. Spectrochimica Acta. Part A,
none derivatives and their antibacterial activity. E-Journal of
Chemistry, 1(5), 272–276. https://doi.org/10.1155/2004/158924
3
Molecular and Biomolecular Spectroscopy, 128, 386–392. doi:0.1016/ V ꢁa squez, J. L., Ibsen, P., Lindberg, H., & Gehl, J. (2015). In vitro and
j.saa.2014.02.189 https://doi.org/10.1016/j.saa.2014.02.189
S¸ ahin, O., Ozdemir, U. O., Sefero gꢀ lu, N., Genc, Z. K., Kaya, K., Aydıner, B.,
Tekin, S., & Sefero gꢀ lu, Z. (2018). New platinum (II) and palladium (II)
in vivo experiments on electro chemotherapy for bladder cancer.
Journal of Urology, 193(3), 1009–1015. https://doi.org/10.1016/j.juro.
2014.09.039
€
€
€
€
€
€
€
€
ꢀ
complexes of coumarin-thiazole Schiff base with a fluorescent chemo- Ye gꢀ iner, G., G u€ lcan, M., I s¸ ık, S., Urut, G. O., Ozdemir, S., & Kurtoglu, M.
sensor properties: Synthesis, spectroscopic characterization, X-ray
structure determination, in vitro anticancer activity on various human
carcinoma cell lines and computational studies. Journal of
Photochemistry and Photobiology. B, Biology, 178, 428–439. https://doi.
org/10.1016/j.jphotobiol.2017.11.030
(2017). Transition metal (II) complexes with a novel azo-azomethine Schiff
base ligand: Synthesis, structural and spectroscopic characterization, ther-
mal properties and biological applications. Journal of Fluorescence, 27(6),
2239–2251. https://doi.org/10.1007/s10985-017-2166-3
Zhang, Y., Zhang, L., Liu, L., Guo, J., Wu, D., Xu, G., Wang, X., & Jia, D.
(2010). Anticancer activity, structure, and theoretical calculation of N-
(1-phenyl-3-methyl-4-propyl-pyrazolone-5)-salicylidene hydrazone and
its copper(II) complex. Inorganica Chimica Acta, 363(2), 289–293.
https://doi.org/10.1016/j.ica.2009.08.017
Zielichowska, A., Daczewska, M., Saczko, J., Michel, O., & Kulbacka, J.
(2016). Applications of calcium electroporation to effective apoptosis
induction in fibrosarcoma cells and stimulation of normal muscle
cells. Bioelectrochemistry (Amsterdam, Netherlands), 109, 70–78. https://
doi.org/10.1016/j.bioelechem.2016.01.005
€
Sarı, Y., G u€ rses, C., Celepci, D. B., Kele s¸ temur, U., Akta s¸ , A., Y u€ ksel, S¸ .,
Ate s¸ , B., & G o€ k, Y. (2020). 4-Vinylbenzyl and 2-morpholinoethyl substi-
tuted ruthenium(II) complexes: Design, synthesis, and biological
evaluation. Journal of Molecular Structure, 1202, 127355. https://doi.
org/10.1016/j.molstruc.2019.127355
Schneider, E., Hsiang, Y.-H., & Liu, L. F. (1990). DNA topoisomerases as
anticancer drug targets. Advances in Pharmacology, 21, 149–183.
https://doi.org/10.1016/S1054-3589(08)60342-7
Shah, R. K., Abou-Melha, K. S., Saad, F. A., Yousef, T., Al-Hazmi, G. A. A.,
Elghalban, M. G., Khedr, A. M., & El-Metwaly, N. (2016). Elaborated Zoubi, W. A. (2013). Biological activities of schiff bases and their com-
studies on nano-sized homo-binuclear Mn(II), Fe(III), Co(II), Ni(II), and
Cu(II) complexes derived from N Schiff base, thermal, molecular
plexes: A review of recent works. International Journal of Organic
Chemistry, 03(03), 73–95. https://doi.org/10.4236/ijoc.2013.33A008
2 2
O