Synthesis, characterization, and antiproliferative activity of Cu 2+, V(IV)O2+, Co2+, Mn2+, and Ni2+ complexes with 3-(2-(4-methoxyphenylcarbamothioyl)hydrazinyl)-3- OXO-N-(Thiazol-2-yl)propanamide against human breast adenocarcinoma cells

Article abstract of DOI:10.1080/10426507.2013.855764

(Graph present)3-(2-(4-methoxyphenylcarbamothioyl)hydrazinyl)-3-oxo-N- (thiazol-2-yl)propan-amide (H₄L) has been synthesized and its structure has been confirmed by elemental analysis, IR, mass, and ¹H and ¹³C NMR spectroscopy. This ligand has been used to synthesize complexes with Cu²⁺, V(IV)O²⁺, Co²⁺, Mn ²⁺, and Ni²⁺. The structures of these complexes have been verified by elemental analyses, molar conductivities, magnetic measurements as well as UV-VIS, IR, ¹H-NMR spectroscopy. The IR spectra showed that H₄L acts as a uni-negative tetradentate or bidentate ligand. The molar conductance measurements proved that all complexes are nonelectrolytes except complexes 2 and 3. Moreover, the metal complexes geometrical arrangements were square planar, tetrahedral, square-pyramidal, or octahedral. The structures are consistent with the IR, UV-VIS, ESR, as well as conductivity measurements. The antiproliferative activity of the synthesized complexes against human breast adenocarcinoma MCF-7 cell line showed exploited potent to moderate growth inhibitory activity, in particular complex 4 which exhibited superior potency to the reference drug cisplatin. The antitumor activity of these compounds was accompanied by significant increase in the activity of superoxide dismutase with concomitant decrease in the activities of catalase and glutathione peroxidase and reduced glutathione level. The overproduction of free radicals allowed reactive oxygen species-mediated tumor cells death. 2014 Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/10426507.2013.855764

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Phosphorus, Sulfur, and Silicon and the
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Synthesis, Characterization,
and Antiproliferative Activity of
Cu2+, V(IV)O2+, Co2+, Mn2+, and
Ni2+ Complexes with 3-(2-(4-
Methoxyphenylcarbamothioyl)Hydrazinyl)-3-
OXO-N-(Thiazol-2-yl)Propanamide
against Human Breast Adenocarcinoma
Cells
Eman A. El-Zahanya, Mamdouh M. Alib, Sayed A. Drweesha, Ahmed
M. A. El-Seidya, Bakr F. Abdel-Wahabc & Nabil S. Youssefa
a Inorganic Chemistry Department, National Research Centre, P.O.
12622 Dokki, Cairo, Egypt
b Biochemistry Department, National Research Centre, P.O. 12622
Dokki, Cairo, Egypt
c Applied Organic Chemistry Department, National Research Centre,
P.O. 12622 Dokki, Cairo, Egypt
Accepted author version posted online: 21 Feb 2014.Published
online: 12 Jun 2014.
To cite this article: Eman A. El-Zahany, Mamdouh M. Ali, Sayed A. Drweesh, Ahmed M. A.
El-Seidy, Bakr F. Abdel-Wahab & Nabil S. Youssef (2014) Synthesis, Characterization, and
Antiproliferative Activity of Cu2+, V(IV)O2+, Co2+, Mn2+, and Ni2+ Complexes with 3-(2-(4-
Methoxyphenylcarbamothioyl)Hydrazinyl)-3-OXO-N-(Thiazol-2-yl)Propanamide against Human Breast
Adenocarcinoma Cells, Phosphorus, Sulfur, and Silicon and the Related Elements, 189:6, 762-777, DOI:
10.1080/10426507.2013.855764
To link to this article: http://dx.doi.org/10.1080/10426507.2013.855764
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Phosphorus, Sulfur, and Silicon, 189:762–777, 2014
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426507.2013.855764
SYNTHESIS, CHARACTERIZATION,
AND ANTIPROLIFERATIVE ACTIVITY OF Cu2+, V(IV)O2+,
Co2+, Mn2+, AND Ni2+ COMPLEXES WITH 3-(2-(4-
METHOXYPHENYLCARBAMOTHIOYL)HYDRAZINYL)-3-
OXO-N-(THIAZOL-2-YL)PROPANAMIDE AGAINST HUMAN
BREAST ADENOCARCINOMA CELLS
Eman A. El-Zahany,1 Mamdouh M. Ali,2 Sayed A. Drweesh,1
Ahmed M. A. El-Seidy,1 Bakr F. Abdel-Wahab,3
and Nabil S. Youssef1
1Inorganic Chemistry Department, National Research Centre, P.O. 12622 Dokki,
Cairo, Egypt
2Biochemistry Department, National Research Centre, P.O. 12622 Dokki, Cairo,
Egypt
3Applied Organic Chemistry Department, National Research Centre, P.O. 12622
Dokki, Cairo, Egypt
GRAPHICAL ABSTRACT
Abstract 3-(2-(4-methoxyphenylcarbamothioyl)hydrazinyl)-3-oxo-N-(thiazol-2-yl)propan-
amide (H4L) has been synthesized and its structure has been confirmed by elemental anal-
ysis, IR, mass, and 1H and 13C NMR spectroscopy. This ligand has been used to synthesize
complexes with Cu2+, V(IV)O2+, Co2+, Mn2+, and Ni2+. The structures of these complexes
have been verified by elemental analyses, molar conductivities, magnetic measurements as
well as UV–VIS, IR, 1H-NMR spectroscopy. The IR spectra showed that H4L acts as a uni-
negative tetradentate or bidentate ligand. The molar conductance measurements proved that
Received 11 April 2013; accepted 20 September 2013.
Address correspondence to Nabil S. Youssef, Inorganic Chemistry Department, National Research Centre,
P.O. 12622 Dokki, Cairo, Egypt. Tel.: +20233371718; Fax: +20233370931. E-mail: nabilyoussef@hotmail.com
762

ACTIVITY OF Cu2+, V(IV)O2+, Co2+, Mn2+, AND Ni2+ COMPLEXES
763
all complexes are nonelectrolytes except complexes 2 and 3. Moreover, the metal complexes
geometrical arrangements were square planar, tetrahedral, square-pyramidal, or octahedral.
The structures are consistent with the IR, UV–VIS, ESR, as well as conductivity measurements.
The antiproliferative activity of the synthesized complexes against human breast adenocar-
cinoma MCF-7 cell line showed exploited potent to moderate growth inhibitory activity, in
particular complex 4 which exhibited superior potency to the reference drug cisplatin. The
antitumor activity of these compounds was accompanied by significant increase in the activity
of superoxide dismutase with concomitant decrease in the activities of catalase and glutathione
peroxidase and reduced glutathione level. The overproduction of free radicals allowed reactive
oxygen species-mediated tumor cells death.
Keywords Thiazol derivatives; Complexes; Cytotoxic activity; Breast cancer
INTRODUCTION
One of the major applications of the transition metal complexes has been the testing
of their antibacterial and antitumor activities with the aim to find an effective and safe
therapeutic regimen for the treatment of cancer and infections. The coordination chem-
istry of nitrogen–sulfur donor ligands such as thiazol derivatives is well documented in
the literature.1 In addition to the antifungal and antibacterial properties, some of these
transition metal complexes of nitrogen–sulfur donor ligands also exhibit antitumor activity
against a variety of tumor cells.2 Many thiazoles were found to be associated with various
biological activities, such as antimicrobial,3 antifungal,4 anti-inflammatory,5 analgesic,6
antitubercular,7 antitumor,8 and antiprotozoal9 properties. The mechanism of metal com-
plexes as anticancer agents occurs via binding with the tumor cell, DNA resulting in the
inhibition of its growth. An understanding of DNA binding mode and cleavage is needed
in order to develop new and efficient antitumor drugs. This depends on the effective bind-
ing mode and their affinity toward the DNA.10 The metal complexes can bind to DNA in
noncovalent mode; for example, electrostatic, intercalative, and groove binding.11 Reactive
oxygen species (ROS), such as superoxide anions and hydrogen peroxide (H2O2) are nor-
mal byproducts of cellular aerobic metabolism; the superoxide anions (O2−) are known to
be converted to H2O2 by the superoxide dismutase (SOD) as secondary steps. H2O2 has a
dual role in cellular homeostasis; low levels of intracellular H2O2 stimulate cell growth.12
On the other hand, high levels of H2O2 lead to cell senescence and apoptosis.13 Several
anticancer agents, such as 5-fluorouracil,14 platinum,15 and anthracyclines16 increase the
levels of ROS. Simultaneously tumor cells are known to have resulting high levels of reac-
tion toward oxygen species in comparison to those of the normal cells. This leads to high
sensitivity to the additional oxidative stress generated by the anticancer agents. In addition,
cells contain a variety of free radical scavenging systems that protect them from the effects
of drugs that generate oxygen free radicals such as catalase and glutathione peroxidase.
ROS causes oxidative damage to cells leading to age related degenerative diseases, cancer
and a wide range of different human diseases.17 A simple effective way of preventing dis-
eases is the inhibition of oxidative damage. Many natural as well as synthetic antioxidants
are used for the treatment of different diseases.18 The major action of antioxidants in the
human diseases is to prevent damage caused by the action of ROS. As a result of many
extensive studies in the field of anticancer drugs, different types of anticancer chemicals
with promising effects have been synthesized such as anthracylines, cisplatin, and other
alkylating agents that induce apoptotic cell death in cancer cells via generation of active
oxygen species.19 In continuation to our investigations,20 a number of newly synthesized

764
E. A. EL-ZAHANY ET AL.
Scheme 1 Synthesis of the ligand. H4L.
biologically active thiazole complexes with anticancer profiles on human breast adenocarci-
noma cell line MCF-7 have been synthesized. A special attention to the causal relationship
of ROS induction in the cells have been studied.
RESULTS AND DISCUSSION
Synthesis of the Ligand
3-(2-(4-methoxyphenylcarbamothioyl)hydrazinyl)-3-oxo-N-(thiazol-2-yl)propanamide,
H4L was synthesized (Scheme 1) by heating a mixture of 3-hydrazino-3-oxo-N-thiazol-2-
yl-propanamide (A) and 1-isothiocyanato-4-methoxybenzene (B) under reflux in absolute
ethanol. Compound A was synthesized as described before.21
Elemental Analysis
The elemental and physical data of the ligand H4L (Figure 1) and its complexes
(Table 1) showed that the stoichiometry of the complexes obtained is either 1:1 or 1:2 M/L.
Mass Spectra of the Ligand
The mass spectra of the ligand H4L revealed the molecular ion peak at m/e 401.0
which is coincident with the formulae weight 401.4, for this ligand and supports the identity
of its structure. The pathway fragmentation pattern of the mass spectra of this ligand is
depicted in Scheme S1.
32
H
17
23
O
3
O
29
H
28
H
27
H
N16
33
H
14
N 5
N 7
H 26
18
C
21
N
4
N
20
2
1
6
13
12
22
37
S
9
H
19
H 38
36
34H
H35
10
H
H
S
31
30
8
24 H
O
11
15
H
39
H
25
Figure 1 Structure of H4L Ligand.

765

766
E. A. EL-ZAHANY ET AL.
Conductivity Measurements
All metal complexes are stable in air and insoluble in common organic solvents but
easily soluble in DMSO. The molar conductivity of the complexes in DMSO (10−3 M) are
listed in Table 1. All complexes showed a nonelectrolyte nature22 except 2 and 3 behaved
as electrolytes.23
Infrared Spectra
The IR spectra of the free ligand and its metal complexes were carried out in the
range of 4000–400 cm−1 (Table 2). The infrared spectrum of H4L displays character-
istic bands at 1682, 1668, 1576, 1038, 744, 3254, and 3170 cm−1 assigned to ν(C
O)1, ν(C O)2, ν(C N)ring, ν(N–N), ν(C S), ν(NH) of (NH–(C O)1), and ν(NH) of
(NH–(C S)1) vibrations, respectively.24,25 A comparison of the main vibrational bands
of H4L has been carried out to establish its ligating behavior in the complexes. In
all complexes, a negative shift or weakness in the ν(C S) and ν(NH) adjacent to
(C O)1 with the disappearance of ν(NH–(C S)1) band has been observed. This sug-
gests loss of a proton from HN(C S)1 group after thioenolization beside participation of
the –NH group attached to (C O)1 and sulfur atom of the ligand in bonding to the metal
ion.25 This is further supported by the negative shift or the weakness of the ν(N N)26
and the disappearance of ν(NH) adjacent to thione with simultaneous appearance of the
new bands at 1605–1615 cm−1 assignable to the new ν(C N∗)1 in all complexes. The
ν(C O)1 vibration remains at the same position in all complexes (Table 2) indicating
that this group does not participate in coordination. The IR spectra of complexes 4, 5,
and 6 reflect that ν(C O)2 and ν(C N)ring vibrations remain at more or less the same
position (Table 2) which indicated that they do not participate in coordination to the metal
ion. Hence, H4L acts as a monoanionic bidentate ligand coordinating via –NH groups
attached to ν(C O)1 and ν(C S) upon thioenolization in these complexes. On the other
hand, the IR spectra of complexes 2, 3, and 7–9 showed a negative shift of ν(C O)2
and ν(C N)ring indicating the involvement of these groups in bonding. Thus, H4L be-
haves as uni-negative tetradentate S, O, N, N donor in these complexes. Moreover, the
new band shown around 957 cm−1 is due to ν(V O) stretching in case of complex 5.
The two new bands appeared at 1380 cm−1 and 822 cm−1 show that the nitrate group
acts as ionic nitrate in complex 3.27 Also, the new bands observed in the 518–521 cm−1
and 460–470 cm−1 regions in all complexes may be assignable to ν(M O) and ν(M N),
respectively.
NMR Studies
1
The H-NMR spectrum of the H4L ligand showed characteristic bands supporting
the proposed structure. The signals at 12.30(s, 1H), 10.21(s, 1H), 9.71(s, 1H), and 9.55(s,
1H) ppm were assigned to H(31), H(30), H(29), and H(28), respectively.28,29 The aromatic
protons gave signals at 7.45(psd, J = 8.4, 2H, H(25) and H(26)) and 6.74(psd, J = 8.4,
2H, H(24) and H(27)), while the thiazole protons showed two signals at 7.53(d, J = 7.5,
1H, H(32)) and 7.29 (d, J = 7.5, 1H, H(33)). The signals at 3.72 and 2.46 ppm correspond
to the –OCH3 and –CH2 protons, respectively.28,30 The 13C-NMR spectrum of this ligand,
H4L, was assigned as: C(6) 183.2, C(2) 181.9, C(6) 171.2, C(22) 165.0, C(20) 162.1, C(12)
159.7, C(17) 133.1, C(14, 10) 126.1, C(13, 11) 114.9, C(18) 112.8, C(36) 54.5, C(1) 45.1

767

768
E. A. EL-ZAHANY ET AL.
Table 3 1H-NMR spectral data and assignments of the ligand H4Land some of its metal complexes
No. Ligand/Complex H(31)
H(29)
H(30)
H(32)
H(33)
H(28) H(25), H(26) H(24), H(27)
1
2
4
H4L
[Ni2(H3L)2]Cl2
Ni(H3L)2
12.27
12.38
12.24
11.97
—
—
10.21
10.31
10.30
9.67
9.62
9.64
9.52
8.58
9.51
7.45
7.40
7.48
7.30
7.30
7.30
6.88
6.84
6.88
The 1H-NMR spectra of the diamagnetic Ni(II) complexes 2 and 4 in DMSO-d6 have
been interpreted by comparing their spectra with that of H4L. In both complexes, the signal
corresponding to H(29) disappeared indicating deprotonation of the thioamide proton prior
to complex formation. In case of complex 4 the signal for H(31) is less affected on complex
formation than that of complex 2. The shift in the signals spectra in case of both complexes
from those of the ligand is a further support complex formation.24,25
Electronic Spectra and Magnetic Measurements
The electronic spectra and magnetic moments of the metal complexes are listed in
Table 4. Generally, the absorption bands due to π→π∗ and n→π∗ transitions observed
in the spectrum of the free ligand lower than 400 nm have shifted to higher frequencies
due to the coordination of the ligand to the metal ions. The electronic spectrum of the
Ni(II) complexes 2 and 4 showed that these complexes are diamagnetic and of low-spin
1
1
with 1A1g→ A2g, 1A1g→ Eg transitions, respectively. These complexes have square–planar
geometry because they are diamagnetic and there is no band seen below 1000 nm.31 The
electronic spectrum of V(IV) with square–pyramidal geometry has two bands in the visible
2
2
2
2
region assigned to B2→ E and B2→ B1 transitions, respectively in accordance with
the Ballhausen–Gray scheme. In complex 5, the first band appeared at 720 nm due to
2
2
2B2→ E transition, while the other band due to 2B2→ B1 transition disappeared due to the
broadness of the first band.32 Also, the magnetic moment value of this complex is 1.72 BM
which confirms that vanadium ions are in V(IV) state with square–pyramidal geometry.
The electronic spectra of the copper complexes 6 and 9 showed one broad absorption band.
The 2B1g(G)← Ag transition in complex 6 lies at 640 nm suggesting distorted octahedral
2
geometry33 as expected from the Jahn–Teller distortion in hexa-coordinated d9 metal ion.
2
2
The electronic spectrum of complex 9 showed a band at 680 nm due to T2g(G)← Eg
Table 4 The electronic absorption spectral bands (nm) and magnetic moment (B.M.) for the ligand H4Land its
metal complexes
No.
Ligand/complex
λmax (nm)
d–d bands
Geometry
μeff in BM
1
2
3
4
5
6
7
8
9
H4L
[Ni2(H3L)2]Cl2
[Co2(H3L)2](NO3)2 H2O
Ni(H3L)2
VO(H3L)2
294, 287, 280, 373
310, 395, 415
315, 396, 420
310, 390, 420
310, 380
313, 397, 465
303, 394
310
310, 397, 428
—
—
621
—
720
640
470
448, 600
680
—
Diamagnetic
4.1
Diamagnetic
1.72
Square planar
Tetrahedral
Square planar
Square pyramidal
Octahedral
Octahedral
Octahedral
Distorted octahedral
Cu(H3L)2(H2O)2
1.90
5.35
4.4
1.55
Mn2(H3L)2(H2O)2Cl2
Co2(H3L)2(H2O)2Cl2
[Cu2(H3L)2(H2O)2Cl2]. 2H2O

ACTIVITY OF Cu2+, V(IV)O2+, Co2+, Mn2+, AND Ni2+ COMPLEXES
769
transition in agreement with octahedral arrangement.34 The magnetic moments of copper
complexes 6 and 9 is (1.90, 1.55 BM), respectively, indicate the presence of an unpaired
electron on Cu(II) ion in an octahedral geometry environment.35 The lower magnetic
moment value of complex 9 may be attributed to the exchange existing between the metal
ions.36,37 In the tetrahedral environment, cobalt(II) complex is reported to consist of only
4
4
one transition, due to A2(F)→ T1(F).38 In the spectrum of complex 3, only one band
is observed in the visible region at 621 nm. This indicates tetrahedral geometry for this
cobalt(II) complex.39 The electronic spectrum of the Co(II) complex 8 showed that it has an
octahedral geometry. Generally, in Co(II) octahedral complexes the spectra usually consist
4
of three bands.33 The first band, due to 4T2g(F)← T1g(F) transition, has not been observed
due to the fact that it occurs in the near-infrared region, and out of the range of the used
instrument. The other two bands have been observed in the visible region, the former
4
4
one due to A2g(F)← T1g(F) transition is observed at 600 nm. The second band due to
4
4T1g(P)← T1g(F) transition appeared at 448 nm. The magnetic moment value of the cobalt
complexes 3, 8 have lower value than expected for the tetrahedral and octahedral geometry
(4.1 and 4.4 B.M.) respectively, indicating antiferromagnetic exchange existing between the
metal ions.36 The electronic spectra of Mn(II) complex 7 showed abroad band at 470 nm.
This band, attributable to the spin forbidden d–d transition, is responsible for the color of
the compound. The magnetic moment of this complex is 5.35 B.M., which is lower than
the normal value 5.95 B.M., corresponding to high-spin configuration due to five unpaired
electrons in ground-state 6S (6A1) in which orbital contribution to the magnetic moment is
quenched.40
ESR
The ESR spectra of the polycrystalline samples of the copper(II) complexes were
recorded at room temperature. The experimental ESR parameters are presented in Table 5.
The ESR spectrum of these complexes is of axial shape with g > g characteristic of
ꢁ
⊥
complexes with 2B1(dx2−y2) ground state and has a large line width typical of dipolar
interactions. The average g-values were calculated according to the equation gav = 1/3[gꢁ
+2g⊥]. All complexes exhibit gꢁ < 2.3, suggesting covalent characters of the copper–ligand
bonding in these complexes. The values of gꢁ/Aꢁ may be considered41,42 as diagnostic of
the stereochemistry. It has been suggested that this quotient may be used as an empirical
index of geometry.43 The range reported for square–planar complexes is 105–135 cm and
for tetrahedrally distorted complexes is 150–250 cm.43 The gꢁ/Aꢁ values for the compound
under consideration lie just in the range expected for tetrahedral distorted complexes. The
g-values are related by the expression G = (gꢁ−2)/(g⊥−2).44 If G > 4.0, then the local
tetragonal axes are aligned parallel or only slightly misaligned, If G < 4.0, significant
exchange coupling is present. For current complexes the G-value is greater than 4.0, which
is in agreement with local tetragonal geometry.
Table 5 ESR parameters for copper(II) complexes
No.
Complexes
g//
g
g///A//
G
gav.
⊥
6
9
Cu(H3L)2(H2O)2
[Cu2(H3L)2(H2O)2Cl2]. 2H2O
2.20
2.12
2.04
2.03
200
203
5.2
4.25
2.09
2.06

770
E. A. EL-ZAHANY ET AL.
Figure 2 The proposed structures of H4L complexes.
All the proposed structures of the H4L ligand complexes are shown in Figure 2.
BIOLOGICAL ACTIVITIES
In Vitro Antiproliferative Activity
In the present study, we search for new antitumor agents from newly synthesized
complexes. Most of the clinicals used antitumor agents possessing significant cytotoxicity
activity in cell culture systems as cytotoxicity is one of the chemotherapeutic targets of
antitumor activity.45 The antiproliferative activity of the newly synthesized complexes was
evaluated against human mammary carcinoma cell line (MCF-7) using sulfo-rhodamine-B
(SRB) colorimetric assay, in comparison with Cisplatin as a reference drug.46 The antipro-
liferative activities are expressed by median growth inhibitory concentration (IC50) and
provided in Table S1 and shown in Figure S1 (Supplementary Materials). From the results,
it is evident that all the tested complexes displayed potent to moderate growth inhibitory
activity; in particular, complex 4 (IC50 = 4.00 μg/ml) was found to be more potent and

ACTIVITY OF Cu2+, V(IV)O2+, Co2+, Mn2+, AND Ni2+ COMPLEXES
771
efficacious than Cisplatin (IC50 value 4.50 μg/ml). The tumor cell line showed normal
growth in our culture system and DMSO did not seem to have any noticeable effect on
cellular growth. A gradual decrease in viability of cancer cells was observed with increasing
concentration of the tested complexes, in a dose-dependent inhibitory effect. The median
growth inhibitory concentration (IC50) after 24 h for compounds 1--2, 4, and 6–9 was
18.00, 15.00, 4.00, 8.00, 11.00, 10.00, and 16.50 μg/mL, respectively. It is clear from the
data that the comparison of the cytotoxicity against MCF-7 cell line (Table S2) of the tested
complexes has shown that the cell killing potency follows the order, 4 > 6 > 8 > 7 > 2
> 9 > 1, while complexes 3 and 5 did not show any antiproliferative activity even at high
doses. Complex 4 was the best complex exerting a significant cytotoxic effect on MCF-7
cells compared to cisplatin (the commonly used anticancer drug). Generally, the study has
shown that complexation of metals to the ligand improves the anticancer activities of the
ligands. This may be considered in the light of Overtone’s concept47 and Tweedy’s chelation
theory.40,48,49 According to Overtone’s concept of cell permeability, the lipid membrane that
surrounds the cell favors the passage of only the lipid soluble materials whose liposolubility
is an important factor, which controls the antimicrobial activity. On chelation, the polarity
of the metal ion is reduced to a great extent due to the overlap of the ligand orbital and partial
sharing of the positive charge of the metal ion with donor groups. Further, it increases the de-
localization of π-electrons over the whole chelate ring and enhances the lipophilicity of the
complexes. This increased lipophilicity enhances the penetration of the complexes into the
lipid membranes and blocking of the metal binding sites in the enzymes of microorganisms.
These complexes also disturb the respiration process of the cell and thus block the synthesis
of proteins, which restricts further growth of the organism. Moreover, there is an equilibrium
between a free radical (FR)/ROS formation and endogenous antioxidant defense mecha-
nisms, but if this balance is disturbed, it can produce oxidative stress. This state of oxidative
stress can result in injury to all the important cellular components like proteins, DNA, and
membrane lipids which can cause cell death. In the recent years, increasing experimental
and clinical data have provided compelling evidences for the involvement of oxidative
stress in large number of pathological states including carcinogenesis.50 Oxidative stress,
however, is not always deterimental but selective oxidative stress sometimes is desirable
and can be utilized therapeutically also. There are numerous drugs which are known to act
by the mechanism of oxidative stress and can be utilized therapeutically such as chloroquin,
quinine, mefloquine, primaquin, artemisinin,51 and cisplatin,52 etc. Recently, new therapeu-
tic strategies that take advantage of increased ROS or inhibition of endogenous antioxidant
defense, hence producing a state of oxidative stress selectively in cancer cells have gained
importance.
Biochemical Assays
To elucidate the mechanism by which the new complexes exert their antitumor
activities, six complexes of various levels of antiproliferative activity were selected to
estimate their ability to induce oxidative stress in cancer cells. We estimated the activities
of the free-radical-metabolizing enzymes including superoxide dismutase (SOD), catalase
(CAT), and glutathione peroxidase (GSH-Px) as well as the levels of the oxidative stress
parameters including hydrogen peroxide (H2O2), nitric oxide (NO), and reduced glutathione
(GSH) in MCF-7 cells treated with the synthesized complexes. In addition, the effect of these
complexes on the levels of total protein and nucleic acids was determined. As shown in Table

772
E. A. EL-ZAHANY ET AL.
S2 (Supplementary Materials), in general treatment of the cells with different complexes
or cisplatin (at the 1/10 of IC50 values)53 resulted in a significant increase in the activity
of SOD and level of H2O2 higher than those of control, accompanied with a significant
decrease in the activity of CAT and GSH-Px, and depletion in GSH level. This means that
the antitumor activity of these complexes was accompanied with high activity of SOD with
subsequent increase in H2O2 production. The produced H2O2 should be rapidly removed
through the activation of CAT and GSH-Px. The present results showed that activities of
CAT and GSH-Px and the level of reduced GSH were lowered in groups treated with the
synthesized complexes compared to control cells. Consequently, the excess H2O2 produced
in tumor cells with the complexes cannot be removed. In other words, the accumulation of
H2O2 and other free radicals in tumor cells should be partly the cause of tumor cell killing.
It should be mentioned that the changes in the activities of the free-radical-metabolizing
enzymes and oxidative stress parameters were in the order of 4>6>8>7>2>9>1 which
is in accordance with the order of antiproliferative activity of the tested complexes. The
highest activity was found for the most potent antitumor complex 4, which resulted in the
highest SOD activity and H2O2 and low activities of CAT and GSH-Px as well as GSH
level than the other tested complexes. The consistency between antiproliferative activity and
biochemical assay results indicated that the antitumor effect of the present complexes may
be exerted at least partly by production of ROS. Moreover, results in Table S3 illustrated
that treatment with these complexes led to significant increase in the level of NO. There is
a growing body of evidence indicating that NO is able to induce apoptosis by helping to
dissipate the membrane potential of mitochondria and therefore make it more permeable.54
In addition, the elevated level of NO was accompanied with depletion in the levels of total
protein and nucleic acids compared to control. This can be explained by several cytotoxic
effects that include reaction of NO with proteins and nucleic acids. The main targets of
NO in proteins are the thiol group55 and iron of active sites.56 In the nucleus, NO has been
shown to cause gene mutation57 to inhibit DNA repair enzymes58 and to mediate DNA
strand breaks.59 Moreover, it has been reported60 that most chemotherapeutic agents cause
cells to overgenerate ROS and thus, are capable of inducing apoptosis, and causing oxidative
damage to DNA and proteins. The cascade of signals mediating apoptosis often involves a
ROS intermediate messenger, and ROS can short circuit the pathway, by passing the need
for upstream signals for cell suicide. Latter, Huang et al.61 reported that regulation of free
radical-producing agents may also have important clinical applications. This mechanism
for the effects of ROS generating anticancer agents is only beginning to be understood,
as the mechanism of most anticancer agents was previously believed to be due mainly
to direct interaction with DNA and interference with DNA regulatory machinery (e.g.,
topoisomerases, helicases) and to the initiation of DNA damage via production of ROS.62
BIOLOGICAL EVALUATION
Cell Culture
The MCF-7 human breast cell line was obtained from the American Type Culture
Collection (Rockville, MD, USA). The tumor cells were maintained in Dulbecco’s mod-
ified Eagle’s medium (DMEM) supplemented with 10% heat inactivated fetal calf serum
◦
(GIBCO), penicillin (100 U/mL) and streptomycin (100 μg/mL) at 37 C in humidified
atmosphere containing 5% CO2. MCF-7 cells at a concentration of 0.50 × 106 were grown
in a 25 cm3 flask in 5 mL of complete culture medium.

ACTIVITY OF Cu2+, V(IV)O2+, Co2+, Mn2+, AND Ni2+ COMPLEXES
773
In Vitro Antiproliferative Assay
The antiproliferative activity was measured in vitro using the sulfo-rhodamine-B stain
(SRB) assay by the method of Skehan.61 Cells were inoculated in 96-well microtiter plate
(104 cells/well) for 24 h before treatment with the complex(s) to allow attachment of cell to
the wall of the plate. The tested complexes were dissolved in DMSO and diluted with saline
to the appropriate volume. Different concentrations of the complex under test (2, 5, 10,
and 20 μg/mL) were added to the cell monolayer. Triplicate wells were prepared for each
individual dose. Monolayer cells were incubated with each complex for 48 h at 37 ◦C and
in atmosphere of 5% CO2. After 48 h, the cells were fixed, washed, and stained for 30 min
with 0.4% (w/v) SRB dissolved in 1% acetic acid. The unbound dye was removed by four
washes with 1% acetic acid, and attached stain was recovered with Tris-EDTA buffer. Color
intensity was measured in an ELISA reader length 540 nm. The relation between surviving
fraction and drug concentration is plotted to get the survival curve for breast tumor cell
line after the specified time. The concentration required for 50% inhibition of cell viability
(IC50) was calculated and the results are given in Table S1.
Biochemical Assays
The cells in culture medium were treated with 20 μL of 1/10 of IC50 values of the
complexes or the standard reference drug, Cisplatin, then incubated for 24 h at 37 ◦C, in a hu-
midified 5% CO2 atmosphere. The MCF-7 cells were harvested and the homogenates were
prepared in saline using a tight pestle homogenizer until complete cell disruption for further
biochemical analysis. The supernatant obtained after centrifugation of cell homogenates
was used for determination of enzymes activities superoxide dismutase (SOD), catalase
(CAT), glutathione peroxidase (GSH-Px) as described by Paglia and Valentine,64 Aebi,65
and Marklund and Marklund,66 respectively. The levels of hydrogen peroxide (H2O2),
nitric oxide (NO), and reduced glutathione (GSH) were determined by the methods of
Wolf,67 Montgomery and Dymock,68 and Ellman.69 respectively. Nucleic acids (DNA and
RNA) and total protein were precipitated and measured in cell homogenates. Total DNA
was extracted and assayed according to the method described by Zhou et al.70 Total RNA
was extracted and assayed according to the method adopted from the method provided by
Hybaid/AGS (Germany), and total cellular protein was assayed by the method of Lowry
et al.71
Statistical Analysis
The results are reported as mean
standard error (S.E.) for at least four
times experiments. Statistical differences were analyzed by one-way ANOVA test
followed by Student’s t-test wherein the differences were considered to be significant at
p < 0.05.
CONCLUSIONS
The new thiazole ligand (H4L) and its metal complexes of Cu2+, (VO)2+, Co2+, Mn2+
,
and Ni2+ have been synthesized and structurally characterized. The analytical data show
that the metal–ligand stoichiometry in these complexes is either 1:1 or 1:2. On the basis of
elemental and spectral studies, 4-coordinated or 6-coordinated geometry was assigned to

774
E. A. EL-ZAHANY ET AL.
these complexes. In the light of these results, it is suggested that all these complexes are
assigned to be in square planar, tetrahedral, square–pyramidal, or octahedral geometries.
The complexes 1–9 were tested in vitro against the human breast cancer cell line (MCF-7).
The present results suggest that the synthesized complexes possess significant antitumor
activity comparable to the activity of commonly used anticancer drug, cisplatin, and they
exert their antitumor activities by modulating free radicals production by increasing the
activity of superoxide dismutase and depletion of intracellular reduced glutathione level,
catalase, glutathione peroxidase activities, accompanied with highly production of hydrogen
peroxide, nitric oxide, and other free radicals causing tumor cells death, as monitoring by
reduction in the synthesis of protein and nucleic acids.
EXPERIMENTAL
Materials and Methods
All the reagents and solvents employed for the preparation of the ligand and its
complexes were of the best grade available and were used without further purification.
Physical Measurements
The ligand and its metal complexes were analyzed for C, H, N, and S contents
at the microanalytical laboratory of the National Research Centre, Dokki, Cairo, Egypt.
Analytical and physical data of the ligand and its metal complexes are reported in Table 1.
The metal ion contents of the complexes were also determined by the previously reported
methods.63 IR spectra of the ligand and its metal complexes were recorded using KBr disks
with a Jasco FT/IR 300E Fourier transform infrared spectrophotometer covering the range
400–4000 cm−1 and in the 500–100 cm−1 region using polyethylene-sandwiched Nujol
1
mulls on a Perkin-Elmer FT-IR 1650 spectrophotometer. H and 13C-NMR spectra were
obtained on BruckerAvance 300-DRX or Avance 400-DRX spectrometers and Chemical
shifts (ppm) are reported relative to TMS. The electronic spectra of the ligand and its
complexes were obtained in Nujol mulls using a Shimadzu UV-240 UV–VIS recording
spectrophotometer. Molar conductivities of the metal complexes in DMSO (10−3 M) were
measured using a dip cell and a Bibbyconductimeter MC1 at room temperature. The
resistance measured in ohms and the molar conductivities were calculated according to the
equation: ꢀ = V × K × Mw/g × ꢁ, where ꢀ, molar conductivity (ohm−1 cm2 mol−1);
V, volume of the complex solution (mL); K, cell constant 0.92 cm−1; Mw, molecular
weight of the complex; g, weight of the complex; and ꢁ, resistance measured in ohms.
Magnetic moments at 298 K were determined using the Gouy method with Hg[Co(SCN)4]
as calibrant. Mass spectra of the solid ligand were recorded using JEUL JMS-AX-500 mass
spectrometer. The solid ESR spectra of the complexes were recorded with ELEXSYS E500
Brucker spectrometer in 3-mm Pyrex Tubes at 298 K. Diphenylpicrylhydrazide (DPPH)
was used as a g-marker for the calibration of the spectra.
Synthesis of the Ligand
3-(2-(4-methoxyphenylcarbamothioyl)hydrazinyl)-3-oxo-N-(thiazol-2-yl)propanamide,
H4L was synthesized by heating a mixture of 3-hydrazino-3-oxo-N-thiazol-2-yl-
propanamide (5 mmol, 1.00 g) and 1-isothiocyanato-4-methoxybenzene (5 mmol, 0.80 g)

ACTIVITY OF Cu2+, V(IV)O2+, Co2+, Mn2+, AND Ni2+ COMPLEXES
775
under reflux in absolute ethanol (50 mL) for 1 h. The white crystals formed were filtered
off, washed with ethanol, and dried under vacuum over anhydrous CaCl2 (60% yield).
Synthesis of the Metal Complexes
The metal complexes of the ligand H4L were prepared by mixing a hot methanolic
methanol solution (25 mL) of the metal salt with the required amount of a hot methanol
solution (25 mL) of the H4L ligand to form 1:1 or 1:2 M/L (metal/ligand), as shown in
Table S4. The mixture was left under reflux with continuous stirring for 2 h and the solid
complexes precipitated. The resulting solid was filtered off, washed several times with hot
methanol, then with diethyl ether and dried under vacuum at 50◦C for 5 h.
SUPPLEMENTAL MATERIAL
Supplementary data of this article can be accessed on the publisher’s website,
www.tandfonline.com/gpss
REFERENCES
1. Jaishree, V.; Ramdas, N.; Sachin, J.; Ramesh, B. J. Saudi Chem. Soc. 2012, 16, 371-376.
2. Shrivastav, A.; Singh, N. K.; Singh, S. M. Bioorg. Med. Chem. 2002, 10, 887-895.
3. Pattan, S. R.; Dighe, N. S.; Nirmal, S. A.; Marekar, A. N.; Laware, R. B.; Shinde, H. V.; Musade,
D. S. Asian J. Res. Chem. 2009, 2, 196-201.
4. Sarkar, B. S.; Kalyan, R. S. Asian J. Chem. 2008, 20, 6600-6602.
5. Karbasanagouda, T.; Ariody, V. A.; Ramgopal, D.; Parmeshwarappa, G. Ind. J. Chem. B. 2008,
47, 144-152.
6. Kalkhambkar, R. G.; Kulkarni, G. M.; Shivkumar, H.; Rao, R. N. Eur. J. Med. Chem. 2007, 42,
1272-1276.
7. UshaRani, P.; Sekhar, B. V. V. N. C.; Rameshkumar, N.; Mahendra, R. S.; Kalian, C. A. Asian J.
Chem. 2008, 20, 718-722.
8. El-Subbagh, H. E.; Al-Obaid, A. A. Eur. J. Med. Chem. 1996, 31, 1017-1021.
9. Ricardo, A. T.; Luz, A.; Carlos, D. P. Bioorg. Med. Chem. 2003, 11, 2175-2182.
10. Friedman, A. E.; Kumar, C. V.; Turro, N. J.; Barton, J. K. Nucl. Acids Res. 1991, 19, 2595-2602.
11. Tiwari, A. D.; Mishra, A. K.; Mishra, S. B.; Mamba, B. B.; Maji, B.; Bhattacharya, S. Spectrochim.
Acta Part A. 2011, 79, 1050-1056.
12. Sundaresan, M.; Yu, Z. X.; Ferrans, V. J.; Irani, K.; Finkel, T. Science 1995, 270, 296-299.
13. Bernard, D.; Gosselin, K.; Monte, D.; Vercamer, C.; Bouali, F.; Pourtier, A.; Vandenbunder, B.;
Abbadie, C. Cancer Res. 2004, 64, 472-481.
14. Hwang, P. M.; Bunz, F.; Yu, J.; Rago, C.; Chan, T. A.; Murphy, M. P.; Kelso, G. F.; Smith, R.
A.; Kinzler, K. W.; Vogelstein, B. Nat. Med. 2001, 7, 1111-1117.
15. Laurent, A.; Nicco, C.; Chereau, C.; Goulvestre, C.; Alexandre, J.; Alves, A.; Levy, E.; Gold-
wasser, F.; Panis, Y.; Soubrane, O.; Weill, B.; Batteux, F. Cancer Res. 2005, 65, 948-956.
16. Hussain, S. P.; Amstad, P.; He, P.; Robles, A.; Lupold, S.; Kaneko, I.; Ichimiya, M.; Sengupta, S.;
Mechanic, L.; Okamura, S.; Hofseth, L. J.; Moake, M.; Nagashima, M.; Forrester, K. S.; Harris,
C. C. Cancer Res. 2004, 64, 2350-2356.
17. Amarowicz, R.; Estrella, I.; Herna´ndez, T.; Robredo, S.; ska, A. T.; ska, A. K.; Pegg, R. B. Food
Chem. 2010, 121, 705-711.
18. Hadjipavlou-Litina, D.; Magoulas, G. E.; Krokidis, M.; Papaioannou, D. Eur. J. Med. Chem.
2010, 45, 298-310.
19. Das, K. A text Book on Medicinal Aspects of Bio-Inorganic Chemistry; CBS: India, 1990.

776
E. A. EL-ZAHANY ET AL.
20. El-Seidy, A. M. A.; El-Zahany, E.; Barakat, A. S.; Youssef, N. S.; Galal, S. A.; Drweesh, S. A.
Synth. React. Inorg. Met.-Org. Nano-Met. Chem. 2013, 43, 46-56.
21. Abdel-Wahab, B. F.; Mohamed, S. F.; Amr, A. E.-G. E.; Abdalla, M. M. Monatsh. Chem. 2008,
139, 1083-1090.
22. Refat, M. S.; Moussa, M. A. A.; Mohamed, S. F. J. Mol. Str. 2011, 994, 194-201.
23. Ilhan, S.; Temel, H.; Yilmaz, I.; S¸ekerc, M. Polyhedron 2007, 26, 2795-2802.
24. Zaky, R. R.; Yousef, T. A. J. Mol. Str. 2011, 1002, 76-85.
25. Singh, M.; Aggarwal, V.; Singh, U. P.; Singh, N. K. Polyhedron. 2009, 28, 107-112.
26. Saydam, S.; Yilmaz, E. Spectrochim. Acta Part A. 2006, 63, 506-510.
27. Youssef, N. S.; Zahany, E. A. E.; Anwar, M. M.; Hassan, S. A. Phosphorus, Sulfur, Silicon Relat.
Elem. 2009, 184, 103-125.
28. Labisbal, E., Rodr´ıguez, L.; Sousa-Pedrares, A.; Alonso, M.; Vizoso, A.; Romero, Garc´ıa-
Va´zquez, J.; J. A.; Sousa, A. J. Organomet. Chem. 2006, 691, 1321-1332.
29. Tascioglu, S.; Yalcin, B.; Nasrullayeva, T. M.; Andac, O.; Buyukgungor, O.; Aydin, A.; Medjidov,
A. A. Polyhedron 2006, 25, 1279-1286.
30. Yin, H. D.; Chen, S. W. Inorg. Chim. Acta 2006, 359, 3330-3338.
31. Zayed, M. A.; Abdallah, S. M. Spectrochim. Acta Part A 2005, 61, 2231-2238.
32. Boghaei, D. M.; Bezaatpour, A.; Behzad, M. J. Mol. Cat. A 2006, 245, 12-16.
33. Emara, A. A. A.; Abou-Hussen, A. A. A. Spectrochim. Acta Part A 2006, 64, 1010-1024.
34. Swamy, S. J.; Pola, S. Spectrochim. Acta Part A 2008, 70, 929-933.
35. Rosu, T.; Negoiu, M.; Pasculescu, S.; Pahontu, E.; Poirier, D.; Gulea, A. Eur. J. Med. Chem.
2010, 45, 774-781.
36. Youssef, N. S.; El-Zahany, E. A.; El-Seidy, A. M. A.; Caselli, A.; Cenini, S. J. Mol. Catal. A
2009, 308, 159-168.
37. Youssef, N. S.; El-Zahany, E.; El-Seidy, A. M. A.; Caselli, A.; Fantauzzi, S.; Cenini, S. Inorg.
Chim. Acta 2009, 362, 2006-2014.
38. Lever, A. B. P. Inorganic Electronic Spectroscopy; Elsevier: Amsterdam, 1984.
39. Joseyphus, R. S.; Dhanaraj, C. J.; Nair, M. S. Trans. Met. Chem. 2006, 31, 699-702.
40. Youssef, N. S.; El-Zahany, E. A.; Ali, M. M. Phosph Sulfur Silicon Relat. Elem. 2010, 185,
2171-2181.
41. Salaun, J. Chem. Rev. 1989, 89, 1247-1270.
42. Brown, K.; Tegoni, M.; Prudencio, M.; Pereira, A. S.; Besson, S.; Moura, J. J.; Moura, I.;
Cambillau, C. Nat. Struct. Biol. 2000, 7, 191-195.
43. Haltia, T.; Brown, K.; Tegoni, M.; Cambillau, C.; Saraste, M.; Mattila, K.; Djinovic-Carugo, K.
Biochem. J. 2003, 369, 77-88.
44. Oganesyan, V. S.; Rasmussen, T.; Fairhurst, S.; Thomson, A. J. Chem. Soc. Dalton Trans. 2004,
996-1002.
45. Suffness, M.; Pezzuto, J. M. Methods in Plant Biochemistry; Academic Press: New York, 1991.
46. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; Mahon, J.; Vistica, D.; Warren, J. T.; Bokesch,
H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1107-1112.
47. Tu¨mer, M.; Ko¨ksal, H.; Sener, M. K.; Serin, S. Trans. Met. Chem. 1999, 24, 414-420.
48. Creaven, B. S.; Duff, B.; Egan, D. A.; Kavanagh, K.; Rosair, G.; Thangella, V. R.; Walsh, M.
Inorg. Chim. Acta 2010, 363, 4048-4058.
49. Tweedy, B. G. Phytopathology 1964, 55, 910-914.
50. Pillai, C. K.; Pillai, K. S. Ind. J. Physiol. Pharmacol. 2002, 46, 1-5.
51. Tracy, J. W.; Webster, L. T. In: J. G. Hardman, L. E. Limbird, A. G. Gilman (Eds.), Drug Used in
Chemotherapy of Protozoal Infection,10th ed.; McGraw-Hill: New York, 2001; pp. 1069-1079.
52. Huang, H.-L.; Fang, L.-W.; Lu, S.-P.; Chou, C.-K.; Luh, T.-Y.; Lai, M.-Z. Oncogene 2003, 22,
8168-8177.
53. Ali, M. M.; Mahmoud, A. E.; Rashad, A. E. Asian J. Pharm. Clin. Res. 2011, 4, 118-122.
54. Bru¨ne, B. Cell Death Differ 2003, 10, 864-869.

ACTIVITY OF Cu2+, V(IV)O2+, Co2+, Mn2+, AND Ni2+ COMPLEXES
777
55. Molina, Y.; Vedia, L.; McDonald, B.; Reep, B.; Brne, B.; Di Silvio, M.; Billiar, T. R.; Lapetina,
E. G. J. Biol. Chem. 1992, 267, 24929-24932.
56. Hibbs, J. J.; Taintor, R. R.; Vavrin, Z.; Rachlin, E. M. Biophys. Res. Commun. 1988, 157, 87-94.
57. Juedes, M. J.; Wogan, G. N. Mutat. Res. 1996, 349, 51-61.
58. Lepoivre, M.; Fieschi, F.; Coves, J.; Thelander, L.; Fontecave, M. Biochem. Biophys. Res.
Commun. 1991, 179, 442-448.
59. Fehsel, K.; Jalowy, A.; Qi, S.; Burkart, V.; Hartmann, B.; Kolb, H. Diabetes 1993, 42, 496-500.
60. Bienvenu, P.; Caron, L.; Gasparutto, D.; Kergonou, J. F. Experiment. Suppl. 1992, 62, 257-265.
61. Huang, P.; Feng, L.; Oldham, E. A.; Keating, M. J.; Plunkett, W. Nature Protocols 2000, 407,
390-395.
62. Gewirtz, D. A. Biochem. Pharmacol. 1999, 57, 727-741.
63. Vogel, A. I. A Text Book of Quantitative Inorganic Analysis; Longmans: London, 1978.
64. Paglia, E. D.; Valentine, W. N. J. Lab. Clin. Med. 1967, 70, 158-169.
65. Aebi, H. Catalase In: Method of Enzymatic Analysis; Academic Press: New York, 1984.
66. Marklund, S.; Marklund, G. Eur. J. Biochem. 1974, 47, 469-474.
67. Wolff, S. P. Meth. Enzymol. 1994, 233, 182-189.
68. Montgomery, H. A. C.; Dymock, J. F. Analyst 1961, 86, 414-416.
69. Ellman, G. L. Arch. Biochem. Biophys. 1959, 82, 70-77.
70. Zhou, T.; Zhou, G.; Song, W.; Eguchi, N.; Lu, W.; Lundin, E.; Jin, T.; Nordberg, G. Toxicol.
1999, 142, 1-13.
71. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193, 265-275.