Synthesis and antitumor, antioxidant effects studies of N-ethylpiperazine substitute thiourea ligands and their copper(II) complexes

Article abstract of DOI:10.1080/00387010.2012.720338

A series of N-ethylpiperazine substitute thioureas [C₆N ₂H1₃NHCSNHR], where R = -C₃H₅ (L₁), -C₁₀H₇ (L₂), and -C ₇H₇ (L₃), and their copper (II) complexes have been synthesized. These ligands and complexes have been characterized by elemental analyses, IR, ¹H and ¹³C-NMR spectra, UV-Vis, magnetic susceptibility, thermogravimetrical analyses, and MALDI-TOF MS. In vitro antitumor activity of ligands and their complexes has been screened toward several tumor cell lines. The effects on these complexes of the growth of L1210 and MCF7 were studied comparatively with that of free ligands. Antioxidant and radical scavenging activities of synthesized compounds were determined by various in vitro assays including 1,1-diphenyl-2-picryl-hydrazyl free radicals (DPPH), 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid radicals (ABTS+), and ferrous ion (Fe²⁺) chelating activities. Moreover, these activities were compared to synthetic and standard antioxidant trolox. The results showed that the synthesized compounds had effective antioxidant power.

Full text of DOI:10.1080/00387010.2012.720338

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Synthesis and Antitumor, Antioxidant Effects Studies
of N-Ethylpiperazine Substitute Thiourea Ligands and
Their Copper(II) Complexes
Zuhal Karagoz a , Murat Genc b , Engin Yılmaz c & Serhat Keser a
a Department of Chemistry, Faculty of Science , University of Firat , Elazig , Turkey
b Department of Chemistry, Faculty of Science and Arts , Adiyaman University , Adiyaman ,
Turkey
c Department of Chemistry, Faculty of Science and Arts , University of Bitlis Eren , Bitlis ,
Turkey
Accepted author version posted online: 12 Sep 2012.Published online: 04 Feb 2013.
To cite this article: Zuhal Karagoz , Murat Genc , Engin Yılmaz & Serhat Keser (2013) Synthesis and Antitumor, Antioxidant
Effects Studies of N-Ethylpiperazine Substitute Thiourea Ligands and Their Copper(II) Complexes, Spectroscopy Letters: An
International Journal for Rapid Communication, 46:3, 182-190, DOI: 10.1080/00387010.2012.720338
To link to this article: http://dx.doi.org/10.1080/00387010.2012.720338
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Spectroscopy Letters, 46:182–190, 2013
Copyright # Taylor & Francis Group, LLC
ISSN: 0038-7010 print=1532-2289 online
DOI: 10.1080/00387010.2012.720338
Synthesis and Antitumor, Antioxidant
Effects Studies of N-Ethylpiperazine
Substitute Thiourea Ligands and
Their Copper(II) Complexes
Zuhal Karagoz1,
Murat Genc2,
ABSTRACT A series of N-ethylpiperazine substitute thioureas
[C6N2H13NHCSNHR], where R ¼ -C3H5 (L1), -C10H7 (L2), and -C7H7 (L3),
Engin Yılmaz3,
and Serhat Keser1
and their copper (II) complexes have been synthesized. These ligands
1Department of Chemistry,
1
and complexes have been characterized by elemental analyses, IR, H and
13C-NMR spectra, UV-Vis, magnetic susceptibility, thermogravimetrical
analyses, and MALDI-TOF MS. In vitro antitumor activity of ligands and their
complexes has been screened toward several tumor cell lines. The effects on
these complexes of the growth of L1210 and MCF7 were studied compara-
tively with that of free ligands. Antioxidant and radical scavenging activities
of synthesized compounds were determined by various in vitro assays
including 1,1-diphenyl-2-picryl-hydrazyl free radicals (DPPH), 2,20-azino-
bis(3-ethylbenzthiazoline-6-sulfonic acid radicals (ABTSþ), and ferrous ion
(Fe2þ) chelating activities. Moreover, these activities were compared to
synthetic and standard antioxidant trolox. The results showed that the
synthesized compounds had effective antioxidant power.
Faculty of Science,
University of Firat, Elazig, Turkey
2Department of Chemistry,
Faculty of Science and Arts,
Adiyaman University, Adiyaman,
Turkey
3Department of Chemistry,
Faculty of Science and Arts,
University of Bitlis Eren, Bitlis,
Turkey
KEYWORDS antioxidant, antitumor, spectroscopy, thiourea
INTRODUCTION
Aliphatic and aromatic thiourea, triazole, and thiadiazine compounds
have been demonstrated to be antimicrobial agents able to control different
microorganisms. Thioureas have strong antifungal activities comparable to
the activity observed for the common antifungal antibiotic ketoconazole,[1,2]
and their antimicrobial and insecticidal properties have been documented
for more than 50 years.[3] Not only can thioureas be used in the control of
plant pathogenic fungi,[4,5] but also they have been shown to possess
antitubercular, antithyroid, anthelmintic, antibacterial, insecticidal, and
rodenticidal properties.[6–9]
Received 9 June 2012;
accepted 8 August 2012.
Address correspondence to
Murat Genc, Department of
Chemistry, Faculty of Science and
Arts, Adiyaman University,
Adiyaman 02040, Turkey. E-mail:
mgenc23@gmail.com
The finding that metal ions are capable of binding to nucleic acids brings a
new, promising approach to cancer treatment using metal-based drugs.[10–13]
Copper is an essential trace metal and has been used for centuries either as
182

copper ions or in complexes to disinfect liquids, solids,
and human tissues.[14] Accumulation of high concentra-
tions of copper is observed to be a physiological
feature of many tumor tissues and cells, as was demon-
strated in many types of human cancers including
breast, prostate, colon, lung, and brain.[15–17] However,
some studies support the idea of copper control as an
anticancer strategy.[18,19]
formation with Cu(II) ions and their spectral character-
istics, composition, and structure have been studied. In
addition, in vitro antitumor activity and antioxidant
effects of the ligands and their Cu(II) complexes have
been studied.
RESULTS AND DISCUSSION
The IR spectra of compounds (L1, L2, L3) show
absorption bands of tmax=cmꢀ1 3295–3258 (N-H
stretching), 1150–1107 (C-S stretching), and
=
1250–1269 (HN-C S stretching). The addition of
=
amine to the C N double bond takes place by attack-
There are several organic metal compounds that
actively and specifically inhibit the chymotrypsinlike
activity of the proteasome in vitro and in human
tumor-cell cultures.[20] This is because the ubiquitin=
proteasome system plays an important role in the
degradation of cellular proteins and organo-copper
compounds inhibit the proteasome activity in tumor
cells in 15 min.[21,22] The redox cycling properties
between Cu2þ and Cuþ can initiate the production
of highly reactive hydroxyl radicals, which can sub-
sequently damage biomolecular species like lipids,
proteins, and DNA.[23–26] DNA is one of the most
affected cell components that is damaged by the
metal-generated hydroxyl radical, because it contains
the genetic vital material of a cell. The utilization of
sulfur compounds like cysteine, glutathione, and
their derivatives as antioxidants has been
reported,[27] which may ameliorate oxidative damage
caused by reactive oxygen species (OH, Oꢀ2 ). Copper
complexes of sulphur-containing ligands, in addition
to their nonbiological applications, have been tested
for their cytotoxic=antiproliferative activity against
various types of tumors.[28–30]
ing the carbon atom in the isothiocyanate group,
resulting in the formation of the desired compounds.
After complete reactions, the strong band at around
2100 cmꢀ1 (N C ¼ S stretching) in the isothiocyanate
=
disappears. The band around tmax=cmꢀ1 2550–2610
corresponding to the -SH is not found, which indi-
=
cates the absence of the N C-SH tautomeric form in
all compounds.[10,31] A comparative absorption pat-
tern of complexes with the values of the free ligand
demonstrates that the coordination of thiourea
ligands to copper atoms has an insignificant effect
on t(N-H), t(C-N-C), and t(HNCSNH) frequencies.
=
The stretching frequency of C S present in the ligand
and the coordinated compounds remains the same,
which ruled out the involvement of the thiocarbonyl
group in coordination.
The spectrum of [Cu2L1(AcO)4 ꢁ (H2O)4]ꢁ 2H2O and
[Cu2L32(AcO)4] ꢁ H2O shows another two bands due to
ts and tas of an acetate group at 1385–1415 cmꢀ1 and
1385–1419 cmꢀ1, respectively. The Dt ¼ 30–34 cmꢀ1
can be taken as evidence for the existence of a brid-
ging bidendate acetate group in these complexes
In the present work, a number of chelating thiourea
derivatives, 1-allyl-3-(2-(piperazin-1-yl)ethyl)thiourea
(L1),
1-(naphthalen-1-yl)-3-(2-(piperazin-1-yl)ethyl)
thiourea (L2), and 1-(2-(piperazin-1-yl)ethyl)-3-
(p-tolyl)thiourea (L3) (Scheme 1), for complex
FIGURE 1 Suggested structure of the square-planar Cu(II)
SCHEME 1 Synthesis of N-ethylpiperazine substitute thiourea.
complex of the ligand (L1).
183
N-Ethylpiperazine Substitute Thiourea Ligands and Their Cu(II) Complexes

FIGURE 2 Suggested structure of the square-planar Cu(II)
complex of the ligand (L2).
FIGURE 3 Suggested structure of the square-planar Cu(II)
complex of the ligand (L3).
(Fig. 1).[32,33] The spectrum of [CuL2(AcO)2 ꢁ (H2O)2]
displays bands at 1641 and 1363 cmꢀ1 due to nas(OCO)
and ns(OCO), respectively, of the acetato ligand. The
wavenumber separation value between these two
bands, Dt ¼ 278 cmꢀ1, is characteristic of a monoden-
tate acetato ligand in this complex (Fig. 2).[31,33,34]
The magnetic moment of the Cu(II) complexes
recorded at room temperature lies in the range
1.17–1.30 BM (Table 1), corresponding to one
unpaired electron, which is lower than the spin-only
value, that is, 1.73 BM for one unpaired electron for
L1 and L3 complexes (Fig. 3). This reveals that these
complexes are dimeric in nature and also shows the
presence of metal–metal interaction along the axial
positions, whereas the L2-type complex shows mag-
netic moment at 1.98 BM, which indicates the
absence of metal–metal interaction.
2
2B1g ! Eg (dx2ꢀy ! dxz dyz) in order of increasing
energy.
The electronic spectra of complexes having the
molecular formulas [Cu2L1(AcO)4 ꢁ (H2O)4]. 2H2O
and [CuL2(AcO)2 ꢁ (H2O)2] show broad bands in the
range of 16.667–11.778 cmꢀ1, assigned to the
2
2B1g ! Eg transition, and a more intense band at
28.570 cmꢀ1 is assigned to the LMCT transition.[30]
These band positions are in agreement with
those generally observed for octahedral copper
(II) complexes. The electronic spectrum of
[Cu2L32(AcO)4] ꢁ H2O exhibits two bands: an asymmet-
ric broad band at 15.870 cmꢀ1 is assigned to the
2
3T2g ! Eg transition and a more intense band at
28.570 cmꢀ1 is assigned to the LMCT transition.[35]
These band positions are in agreement with those
generally observed for square-planar copper (II)
complexes.[32,35]
Electronic spectra of six-coordinate Cu(II) com-
plexes have either D4h or C4v symmetry and eg and
2
t2g levels of the D free ion term will split into B1g,
The mass spectrum of [Cu2L32(AcO)4] ꢁ H2O showed
that the molecular ion peak coincides with its molecu-
lar formula (Calcd. 936.09; found 937.08), indicating its
[M-Hþ]. On the other hand, the spectrums of [Cu2L1
(AcO)4 ꢁ (H2O)4] ꢁ 2H2O and [CuL2(AcO)2 ꢁ (H2O)2]
showed peaks at 597.23 and 561.62, corresponding to
[M-2Hþ] of the complexes’ molecules.
A1g, B2g, and Eg levels, respectively. Thus, three
spin-allowed transitions are expected in the visible
and near-IR region. But only a few complexes
known, in which such bands are resolved by single
crystal polarization studies.[35] These bands may be
2
2
assigned to the following transitions: B1g ! A1g
2
(d2xꢀy ! d2z),
2B1g ! B2g(d2xꢀy ! dxy),
and
TABLE 1 Elemental Analyses of the Ligands and their Compounds
Elemental analyses= (%) calculated (found)
FW
Yield
meff
(BM)
Compounds
(g=mol) mp (ꢂC)
(%)
C
H
N
S
C10N4SH20 (L1)
228
315
278
67
64
56
64
42
69
—
—
—
52.63 (53.68) 8.77 (8.44) 24.56 (24.44) 14.04 (14.44)
64.76 (65.22) 6.98 (5.99) 17.78 (17.01) 10.16 (11.01)
60.43 (59.38) 7.91 (7.18) 20.14 (20.22) 11.51 (11.22)
C
C
17N4SH22 (L2)
14N4SH22 (L3)
[Cu2L1(CH3COO)43H2O] ꢁ 2H2O 699.08
360<
360<
360<
1.17 30.90 (31.59) 6.29 (6.16)
8.01 (8.14)
4.58 (5.14)
6.02 (6.92)
6.83 (7.02)
[CuL2(CH3COO)2 ꢁ (H2O)2]
[Cu2L32(CH3COO)4] ꢁ H2O
532.5
1.30 47.36 (48.89) 6.02 (6.30) 10.52 (10.92)
1.98 38.46 (39.24) 5.89 (5.93) 11.95 (11.01)
937.08
Z. Karagoz et al.
184

The thermogravimetric results obtained are in
good agreement with the theoretical formulae
suggested from elemental analyses (Table 1) and
mass spectra. The [Cu2L1(AcO)4 ꢁ (H2O)4] ꢁ 2H2O and
[Cu2L32(AcO)4] ꢁ H2O complexes have four decompo-
sition steps. These complexes show the first mass
loss step, attributed to dehydration, in the tempera-
ture range 100–122ꢂC. In the second decomposition
step of L1 and L2 complexes, abrupt mass losses
occur at 145–220ꢂC and 135–200ꢂC, respectively,
which may represent the new removal of coordi-
nated water associated with the other products of
the second decomposition step. The third and fourth
decomposition steps are in the range of 210–701ꢂC
and 210–765ꢂC, respectively. The fourth step ends
with the formation of copper metal. In case of the
copper complex or the corresponding oxides, the
[CuL2(AcO)2 ꢁ (H2O)2] complex, there are three
decomposition steps. The first mass loss step starts
at 103ꢂC, which could be due to the removal of coor-
dination water. The second step starts at 242ꢂC,
whereas the third one is in the range of 427–670ꢂC
and comprises the formation of CuO. The amounts
of residue are in good agreement with the calculated
values.
different from the control in both MCF-7 and L1210
cells.
Ligands and their metal complexes produced
dose- and time-dependent antiproliferative activity
in both MCF-7 and L1210 cell cultures. It was seen
that cytotoxicity of the ligands and their complexes
on L1210 cells was greater than that of the MCF-7
cells.[36]
It has been reported that the structure and confor-
mation of ligand have an influence on the redox
potential of central atoms in coordination com-
pounds. The changes (coordination sphere) in metal
ions are related to the change of diverse biological
function of compound. The fundamental knowledge
of these laws can be used to synthesize more active
complexes or contribute to our understanding of bio-
logical properties of natural biocoordinative com-
pounds. Our results appear to confirm these
explanations; the activity is affected strongly by the
structure of the ligands. Furthermore, the activities
of the L2 ligand were found to be higher than that
of the other ligands.[10] The L2 Cu complex exhibits
antiproliferative activity against the cancer cell lines
used, comparable to that of the less active L1 Cu
and L3 Cu complexes.
Inhibition of Cell Proliferation
Antioxidant Activities
Cytotoxicity assays were carried out by using the
trypan blue test. Growing cultures of each cell line
were exposed to an increasing test compound con-
The antioxidant and radical scavenging effects of
the synthesized compounds were determined
in vitro with different bioanalytical methodologies.
The antioxidant and radical scavenging activities of
the compounds were compared with Trolox. These
comparisons were performed using a series of
in vitro tests including DPPH, ABTS radical scaveng-
centration (7.5–30 mM) for 24 and 48 hr.
A
dose-dependent decrease in cell proliferation was
clearly observed in the two cell lines studied
(Tables 2 and 3). Treated samples were significantly
TABLE 2 Dose- and Time-dependent Cell Viability Results in MCF-7 Cells after Exposure to Ligands and their Complexes
Groups (n ¼ 7)
24 hr 7.5 mM
24 hr 15 mM
24 hr 30 mM
48 hr 7.5 mM
48 hr 15 mM
48 hr 30 mM
Control
L1
98.33 ꢃ 2.87
80.50 ꢃ 3.27
66.00 ꢃ 2.83
78.33 ꢃ 2.80
66.00 ꢃ 7.72
82.00 ꢃ 8.92c
63.50 ꢃ 8.19
98.33 ꢃ 2.87
67.00 ꢃ 4.24a
57.33 ꢃ 5.72c
64.17 ꢃ 2.04
73.17 ꢃ 5.23
66.00 ꢃ 4.69
60.00 ꢃ 6.81b
98.33 ꢃ 2.87
47.36 ꢃ 2.86c
48.67 ꢃ 4.27c
41.83 ꢃ 7.57
51.17 ꢃ 6.08c
51.33 ꢃ 5.16c
46.83 ꢃ 4.99c
96.17 ꢃ 1.83
64.50 ꢃ 4.23
58.00 ꢃ 4.98b
54.00 ꢃ 7.72c
59.00 ꢃ 4.34a
63.33 ꢃ 5.61
51.00 ꢃ 5.66c
96.17 ꢃ 1.83
49.00 ꢃ 3.79c
47.50 ꢃ 3.21c
39.83 ꢃ 5.49c
46.67 ꢃ 5.35c
46.00 ꢃ 3.79c
47.83 ꢃ 4.45c
96.33 ꢃ 2.87
37.36 ꢃ 2.86c
28.67 ꢃ 4.27c
10.83 ꢃ 7.57
11.17 ꢃ 6.08c
31.33 ꢃ 5.16c
36.83 ꢃ 4.99c
L1-Cu
L2
L2-Cu
L3
L3-Cu
ap < 0.05.
bp < 0.01.
cp < 0.001.
185
N-Ethylpiperazine Substitute Thiourea Ligands and Their Cu(II) Complexes

TABLE 3 Dose- and Time-dependent Cell Viability Results in L1210 Cells after Exposure to Ligands and their Complexes
Groups(N ¼ 7)
24 hr 7.5 mM
24 hr 15 mM
24 hr 30 mM
48 hr 7.5 mM
48 hr 15 mM
48 hr 30 mM
Control
L1
96.17 ꢃ 2.14
63.83 ꢃ 7.93a
58.00 ꢃ 2.76a
37.50 ꢃ 6.75a
41.00 ꢃ 3.35a
56.67 ꢃ 4.45a
47.00 ꢃ 5.93a
96.17 ꢃ 2.14
53.83 ꢃ 4.99a
49.17 ꢃ 5.95a
26.17 ꢃ 4.83a
20.33 ꢃ 6.98a
46.00 ꢃ 2.37a
37.50 ꢃ 4.68a
97.67 ꢃ 1.03
32.33 ꢃ 2.73a
31.83 ꢃ 5.12a
30.33 ꢃ 6.71a
15.17 ꢃ 4.62a
26.33 ꢃ 4.80a
22.00 ꢃ 4.52a
96.17 ꢃ 2.14
46.67 ꢃ 5.61a
40.00 ꢃ 4.89a
22.33 ꢃ 4.59a
16.33 ꢃ 3.27a
38.00 ꢃ 2.97a
13.00 ꢃ 1.89a
97.67 ꢃ 1.03
39.67 ꢃ 14.54a
33.00 ꢃ 3.89a
20.00 ꢃ 7.07a
14.00 ꢃ 2.45a
32.67 ꢃ 4.55a
12.17 ꢃ 5.23a
95.33 ꢃ 2.87
27.36 ꢃ 2.86a
18.67 ꢃ 4.27a
11.83 ꢃ 7.57
7.17 ꢃ 6.08a
11.33 ꢃ 5.16a
16.83 ꢃ 4.99a
L1-Cu
L2
L2-Cu
L3
L3-Cu
ap < 0.001.
ing activities, and metal chelating on ferrous ion
(Fe2þ) activities.
peroxidation chain reactions. Assays based on the
use of DPPH and ABTS radicals are among the most
popular spectrophotometric methods to determine
the antiradical capacity of molecules. These radicals
may directly react with antioxidants. Additionally,
these radical scavenging assays have been used to
evaluate the antioxidant activity of compounds due
to their simple, rapid, and sensitive nature. These
assays are standard tests in antioxidant activity stu-
dies.[37] As can be seen in Table 4, DPPH and ABTS
radical scavenging activities of the synthesized com-
pounds were evaluated. From this perspective, all
synthesized compounds had marked high radical
scavenging ability on DPPH and ABTS radicals. The
highest DPPH scavenging effect was detected in
compound P-N-Cu. The ABTS scavenging effect
was more highly detected in compound P-N than
Trolox.
Ferrous ions generate the most effective
pro-oxidants in biological systems. The good chelat-
ing effect is proposed to be more beneficial. Obvi-
ation of free iron ions from the cycle is a hopeful
approach to prevent oxidative stress-induced dis-
eases. By being chelated, the iron ion may lose its
pro-oxidant attributes. Iron can be found as ferrous
(Fe2þ) or ferric ions (Fe3þ) in nature. Ferrous chela-
tion exposes important antioxidative effects by
delaying metal-catalyzed oxidation.[37] Ferrous ion
chelating activities of synthesized compounds and
Trolox are shown in Table 4. The synthesized com-
pounds also exhibited considerable Fe2þ chelating
activity. Metal chelating activities of synthesized
compounds are usually close to or higher than those
used in reference antioxidants.
The free radical chain reaction is a common mech-
anism of lipid peroxidation. Antiradical compounds
(synthetic or natural) can immediately react with
radicals. They quench radicals to terminate the
EXPERIMENTAL
Instrumentation and Chemicals
All reagents were of commercial quality and sol-
vents were used without further purification. The
human breast carcinoma MCF-7 cell line and murine
leukemia L1210 cell line were obtained from the
American Type Culture Collection (ATCC). Dulbec-
co’s modified Eagle’s medium (DMEM) and newborn
calf serum were purchased from Hyclone (Waltham,
MA, USA); trypsin, penicillin, and streptomycin were
from Sigma Chemical Co. (St. Louis, MO, USA). Infra-
red (IR) spectrum was determined on a Perkin-Elmer
Spectrum One Fourier transform-infrared (FT-IR)
spectrometer. Electronic spectral studies were con-
ducted on a Shimadzu model UV-1700 spectrophot-
ometer in the wavelength 1000–200 nm. NMR
spectra were recorded on a Bruker 300-MHz
TABLE 4 ABTS, DPPH Scavenging, and Metal Chelating
Activity Results of Synthesized Compounds and Trolox
% ABTS
scavenging
activity
% Metal
chelating
activities
% DPPH.
scavenging
activity
Samples
(50 mg=mL)
(250 mg=mL)
(250 mg=mL)
Control
L1
0
0
0
93.65
70.75
95.26
93.29
92.29
73.81
78.41
51.18
94.59
95.65
85.65
89.65
78.12
92.00
30.38
79.17
9.65
L1-Cu
L2
L2-Cu
L3
L3-Cu, P-N
Trolox
89.54
7.86
86.14
91.39
Z. Karagoz et al.
186

spectrometer. Thermo analyses (TGA and DTA) of
compounds were carried out in a nitrogen atmos-
phere with a heating rate of 20ꢂC=min using a
Shimadzu DTG-60 AH (Shimadzu DSC 60 A) thermal
analyzer. MALDI-MS analyses were performed on a
Bruker Micro TOF-ESI=MS system. Elemental analy-
ses were performed on a LECO model 932 instru-
ment. Melting point (uncorrected) was determined
with the Stuart SMP 30 apparatus. The cells were
incubated under 5% CO2=air at 37ꢂC conditions in
a Nuaire humidified carbon dioxide incubator
(Playmouth, MN, USA). Cell state was checked by
using an inverted microscope (Soif Optical Inc.,
China). Results are expressed as mean ꢃ SD. Statisti-
cal analysis and comparison between mean values
for cytotoxicity were performed by Tukey variance
analysis (SPSS 10.0 for Windows; Chicago, IL, USA).
1-(Naphthalen-1-yl)-3-(2-(piperazin-1-yl)
ethyl)thiourea (L2)
Colorless; mp: 287ꢂC; IR (near): tmax: 3258–3160
(N-H), 3054 (aromatic C-H), 2928–2813 (aliphatic
1
C-H), 1594 (C C), 1520 (N-H), 1111 (C-S); H-NMR
=
(300 MHz, CHCl3-d): 1.81 (Br, 1H, piperazine NH),
2.03 (m, 4H, piperazine N-CH2), 2.28 (m, 6H, pipera-
zine NH-CH2 and CH2), 3.57 (q, 2H, J: 4.2 Hz,
N-CH2), 6.69 (Br, 1H, NH), 7.41–7.56 (m, 4H, ArH),
7.85–8.01 (m, 3H, Ar-H), 8.35 (Br, 1H, NH);
13C-NMR (75 MHz, CHCl3-d): 41, 45, 53, 55, 122,
125, 126, 127, 128, 130, 132, 134, 182; Anal. calcd.
for C17H22N4S (314.45): C, 64.76; H, 6.98; N, 17.78;
S, 10.16. Found: C, 65.22; H, 5.99; N, 17.01; S, 11.01
(yield 92%).
Preparation of 1-(2-(piperazin-1-yl)ethyl)-
3-(p-tolyl)thiourea (L3)
A solution of 2-aminoethyl piperazine (0.01 mol)
in THF (10 mL) was added to a solution of 1-naphthyl
isothiocyanate (0.01 mol) in THF (10 mL) with stirring
at room temperature. The reaction mixture was
maintained at 50ꢂC for 2 hr. Then the mixture was
cooled and added to ice-cold water. The resulting
solid was washed with water, dried, and recrystal-
lized from ethanol.
The Preparations of Ligands
Preparation of 1-allyl-3-(2-(piperazin-1-yl)
ethyl)thiourea (L1) and 1-(naphthalen-1-yl)-
3-(2-(piperazin-1-yl)ethyl)thiourea (L2)
A solution of 2-aminoethyl piperazine (0.01 mol)
in acetonitrile (15 mL) was added to a solution of
1-allyl isothiocyanate (4-methylphenyl isothiocya-
nate) (0.01 mol) in acetonitrile (15 mL) with stirring
at room temperature. The reaction mixture was
maintained at 50ꢂC for 4 hr. Then the mixture was
cooled and added to ice-cold water. The resulting
solid was washed with water, dried, and recrystal-
lized from ethanol.
1-(2-(Piperazin-1-yl)ethyl)-3-(p-tolyl)thiourea
Colorless; mp: 218ꢂC; IR (near): tmax: 3285–3177
(N-H), 3042 (aromatic C-H), 2945–2913 (aliphatic
1
C-H), 1595 (C C), 1527 (N-H), 1140 (C-S); H-NMR
=
(300 MHz, CHCl3-d): 2.28 (m, 7H, piperazine N-CH2
and CH3), 2.55 (m, 4H, piperazine NH-CH2), 2.66
(s, 2H, N-CH2), 3.60 (m, 2H, HN-CH2), 3.86 (Br,
1H, piperazine NH), 7.29–7.09 (m, 4H, ArH), 9.25
(Br, 1H, NH), 9.67 (Br, 1H, NH); 13C-NMR (75 MHz,
CHCl3-d): 45, 48, 54, 56, 125, 128, 129, 133, 134,
138, 181; Anal. calcd. for C14H22N4S (278): C, 60.43;
H, 7.91; N, 20.14; S, 11.51. Found: C, 59.38; H, 7.18;
N, 20.22; S, 11.22 (yield 96%).
1-Allyl-3-(2-(piperazin-1-yl)ethyl)thiourea
Colorless; mp: 234ꢂC; IR (near): tmax: 3295–3255
=
(N-H), 3059 (C C-H), 2937–2824 (aliphatic C-H),
=
1646 (C C), 1555 (N-H), 1140 (C-S); 1H-NMR
(300 MHz, CHCl3-d): 2.56 (t, 4H, J: 5.1 Hz, piperazine
N-CH2), 2.61 (t, 4H, J: 5.7 Hz piperazine NH-CH2),
3.55 (Br, 1H, piperazine NH), 3.84 (t, 2H, J: 5.1 Hz
N-CH2), 4.08 (Br, 1H, NH), 4.34 (t, 2H, J: 6.6 Hz,
The Preparations of Complexes
=
HN-CH2), 5.28 (m, 2H, C CH2), 5.60 (s, 2H,
=
2HC-C ¼), 5.93 (m,1H, HC CH2), 6.46 (Br, 1H, NH);
Twenty milliliters of ethanolic solution of [Cu(CH3-
COO)2] ꢁ 2H2O (0.435 g, 2 mmol) acidified with 3-mL
concentrate acetic acid was mixed with 20-mL hot
DMF solution of ligand L1(0.228 g, 1 mmol),
L2(0.63 g, 2 mmol), and L3(0.556 g, 2 mmol). The
13C-NMR (75 MHz, CHCl3-d) 43, 45, 48, 54, 56, 115,
134, 181; Anal. calcd. for C10H20N4S (228.14): C,
52.63; H, 8.77; N, 24.56; S, 14.04. Found: C, 53.68;
H, 8.44; N, 24.44; S, 14.44 (yield 90%).
187
N-Ethylpiperazine Substitute Thiourea Ligands and Their Cu(II) Complexes

reaction mixture was refluxed for 3 hr with
occasional stirring. After cooling to room tempera-
ture, the precipitated product was filtered off,
washed with DMF followed by EtOH, and dried
(yields: 47%, 65%, 55%).
absorbance of 0.750 ꢃ 0.025 at 734 nm in 0.1 M
sodium phosphate buffer (pH 7.4). Then, 3 mL of
þ
ABTS. solution was added to 50 mL of synthesized
compound solutions. The absorbance was recorded
for 0.5 hr after the mixing and the percentage of rad-
ical scavenging was calculated for each concen-
tration relative to a blank containing no scavenger.
The extent of decolorization is calculated as percent-
age reduction of absorbance. The scavenging capa-
bility of test compounds was calculated by the
following equation:[41]
Cell Culture and Treatment
L1210 cells were grown in RPMI 1640 medium (pH
7–8) supplemented with 10% fetal calf serum (FCS),
1% L-glutamine (200 mM), and 1% penicillin=strepto-
mycin and incubated in an air-humidified incubator
at 37ꢂC at 5% CO2. MCF-7 cancer cell lines were
grown as a monolayer culture in a high glucose
(4.5 g=L) DMEM medium supplemented with 10%
fetal calf serum (FCS), 1% L-glutamine (200 mM),
and 1% of penicillin=streptomycin incubated at
37ꢂC in an air-humidified incubator at 5% CO2.
ABTSꢅþ scavengingeffectð%Þ¼½ðA0 ꢀA1Þ=A0ꢆꢄ100
where A0 is the absorbance of control and A1 is the
absorbance in the presence of the sample of synthe-
sized compounds or Trolox.
.
DPPH Radical Scavenging Capacity
Cell Viability
The free radical scavenging capacity of synthe-
sized compounds was measured by 2,2-diphenyl-1-
picryl-hydrazil (DPPH.).[42] Briefly, 0.1 mM solution
of DPPH. in ethanol was prepared and 4 mL of this
solution was added to 0.25 mL of synthesized com-
pound solution. Absorbance at 517 nm was deter-
For the cytotoxicity analysis of test compounds,
cells were seeded at 1 ꢄ 106 cell=mL for MCF-7 cells
and 1 ꢄ 105 cell=mL for L1210 cells, per Eppendorf
tubes in six-replicates.
The test compounds were dissolved in DMSO.
Proliferative MCF-7 and L1210 cells were seeded in
flasks and incubated for 24 hr. After preincubation,
the cell-culture medium was replaced with fresh
medium. Each test compound was added to the
medium within the range of 7.5 and 30 mM, and the
cells were incubated under 5% CO2=air at 37ꢂC
conditions. After 24 and 48 hr of incubation, cell
viability was measured using the trypan blue
exclusion method.[38,39]
mined after 0.5 hr against
a
blank solution
containing the ethanol. Lower absorbance of the
reaction mixture indicates the higher free radical
scavenging activity. When a hydrogen atom or elec-
tron was transferred to the odd electron in DPPH.,
the absorbance at 517 nm was decreased proportion-
ally to the increase of nonradical forms of DPPH. The
capability to scavenge the DPPH. radical was calcu-
lated by the following equation:[41]
Vehicle-treated tubes served as controls. The final
concentration of DMSO in culture medium was never
>0.5%. This concentration of DMSO by itself
produced no observable toxic effects. The concen-
tration of DMSO by itself produced no observable
effects on metabolic status.
DPPHꢅ scavengingeffectð%Þ¼½ðA0 ꢀA1Þ=A0ꢆꢄ100
where A0 is the absorbance of control reaction and
A1 is the absorbance in the presence of synthesized
compounds.
ABTS.þ Radical Scavenging Capacity
Metal Chelating Activity
The spectrophotometric analysis of ABTS.þ radical
scavenging capacity was determined according to
The chelating of ferrous ions by the synthesized
compounds and Trolox was estimated by the
method of Dinis et al.[43] Briefly, synthesized com-
pounds 250 mg=mL were added to a solution of
2 mM FeCl2 (0.05 mL). The reaction was started by
þ
the method of Re et al.[40] ABTS. was produced by
reacting 2 mM ABTS in H2O with 2.45 mM K2S2O8
and stored for 12 hr at room temperature in the
þ
dark. The ABTS. solution was diluted to give an
Z. Karagoz et al.
188

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synthesized. All compounds were tested for their
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ACKNOWLEDGMENTS
We are indebted to the Adiyaman University
Research Foundation (FEFBAP2010=004) for financial
support of this work.
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