Zinc(II) complexes of 2-acetyl pyridine 1-(4-fluorophenyl)-piperazinyl thiosemicarbazone: Synthesis, spectroscopic study and crystal structures - Potential anticancer drugs

Article abstract of DOI:10.1016/j.jinorgbio.2009.12.021

2-Acetyl pyridine thiosemicarbazone containing an 1-(4-fluorophenyl)-piperazinyl ring incorporated at N(4)-position, HAcPipPheF (1) and the zinc(II) complexes [Zn(AcPipPheF)₂] (2) and [Zn(OAc)(AcPipPheF)]₂ (3) have been prepared and structurally characterized by means of vibrational and NMR (¹H and ¹³C) spectroscopy. The crystal structures of the compounds 1-3 have been determined by X-ray crystallography. The metal coordination geometry of [Zn(AcPipPheF)₂] is described as distorted octahedral configuration in a trans-N-cis-N-cis-S configuration. In [Zn(OAc)(AcPipPheF)]₂ one of the acetato group exhibits monoatomic bridge and the other bridges in a bidentate manner. The zinc(1) metal ion is coordinated in a distorted octahedral configuration while the metal coordination of Zn(2) is described as distorted square pyramidal. Biomedical studies revealed that, compounds 1-3 displayed potent anticancer activity. The antiproliferative activity of 1-3 was found to be considerably stronger than that of cis-platin. The IC₅₀ values range from 26 to 90nM, against all cell lines tested, while for cis-platin the IC₅₀ values range from 2 to 17μM and for the zinc salt, ZnCl₂, the IC₅₀ values range from 81 to 93μM. The complex 3 shows the highest activity against all four cancer cell lines and the highest selectivity against K562 and MDA-MB-453 cancer cell lines. The compounds inhibited tumor cell proliferation by arresting the cell cycle progression at the S phase.

Full text of DOI:10.1016/j.jinorgbio.2009.12.021

Journal of Inorganic Biochemistry 104 (2010) 467–476
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Zinc(II) complexes of 2-acetyl pyridine 1-(4-fluorophenyl)-piperazinyl
thiosemicarbazone: Synthesis, spectroscopic study and crystal
structures – Potential anticancer drugs
a
c
Tatjana P. Stanojkovic b, Dimitra Kovala-Demertzi a, , Alexandra Primikyri , Isabel Garcia-Santos ,
*
Alfonso Castineiras c, Zorica Juranic b, Mavroudis A. Demertzis a
a Inorganic and Analytical Chemistry, Department of Chemistry, University of Ioannina, 45100 Ioannina, Greece
b Institute for Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade, Serbia
c
´
Departamento de Qu´ımica Inorganica, Universidad de Santiago de Compostela, E-15706 Santiago de Compostela, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 June 2009
Received in revised form 30 December 2009
Accepted 31 December 2009
Available online 7 January 2010
2-Acetyl pyridine thiosemicarbazone containing an 1-(4-fluorophenyl)-piperazinyl ring incorporated at
N(4)-position, HAcPipPheF (1) and the zinc(II) complexes [Zn(AcPipPheF)2] (2) and [Zn(OAc)(AcPip-
PheF)]2 (3) have been prepared and structurally characterized by means of vibrational and NMR (1H
and 13C) spectroscopy. The crystal structures of the compounds 1–3 have been determined by X-ray crys-
tallography. The metal coordination geometry of [Zn(AcPipPheF)2] is described as distorted octahedral
configuration in a trans-N–cis-N–cis-S configuration. In [Zn(OAc)(AcPipPheF)]2 one of the acetato group
exhibits monoatomic bridge and the other bridges in a bidentate manner. The zinc(1) metal ion is coor-
dinated in a distorted octahedral configuration while the metal coordination of Zn(2) is described as dis-
torted square pyramidal. Biomedical studies revealed that, compounds 1–3 displayed potent anticancer
activity. The antiproliferative activity of 1–3 was found to be considerably stronger than that of cis-platin.
The IC50 values range from 26 to 90 nM, against all cell lines tested, while for cis-platin the IC50 values
Keywords:
Zn(II)
Complexes
Thiosemicarbazone
Crystal structure
In vitro antitumor activity
Biological evaluation
range from 2 to 17 lM and for the zinc salt, ZnCl2, the IC50 values range from 81 to 93 lM. The complex
3 shows the highest activity against all four cancer cell lines and the highest selectivity against K562 and
MDA-MB-453 cancer cell lines. The compounds inhibited tumor cell proliferation by arresting the cell
cycle progression at the S phase.
Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction
cytoprotective and suppresses apoptotic pathways. Zinc plays a
role in brain, where it has a specific function as a neuromodulator
Thiosemicarbazones are versatile molecules not only because of
their broad profile in pharmacological activity, but also because
can act as ligands in coordination chemistry in different ways. It
has been demonstrated in previous publications that thiosemicar-
bazones afford a diverse variety of compounds with different activ-
ities [1–7]. The mechanism of action of TSCs, is due to its ability to
inhibit the biosynthesis of DNA, possibly blocking the enzyme ribo-
nucleotide diphosphate reductase; binding to the nitrogen bases of
DNA or RNA, hindering or blocking base replication; creation of le-
sions in DNA strands by oxidative rupture [1].
Zinc the second most prominent trace metal in the human body
after iron is essential for growth, development and plays an impor-
tant role in various biological systems. It is a vital component an
essential cofactor, critical for numerous cellular processes and
may be a major regulatory ion in the metabolism of cells. Zinc is
in addition to its other typical cellular functions [7,8]. A structural
review of main group metal complexes of semicarbazones and
thiosemicarbazones shows that TSCs are very versatile coordina-
tion agents with these acceptors [9].
Given the potential biological activity of thiosemicarbazones
and the involvement of the Zn(II) in the metabolism of cells and
our previous results on Zn(II) TSCs complexes [10–12], we thought
it would be of interest to explore this chemistry. In order to widen
the scope of investigations on the coordination behaviour of TSCs,
towards Zn(II), we carried out systematic studies with the final
goal to develop new biologically active pharmaceuticals. With
metallopharmaceuticals playing a significant role in therapeutic
and diagnostic medicine, the discovery and development of new
metallodrugs remain an ever-growing area of research in medici-
nal inorganic chemistry.
The present paper includes synthesis, spectral characterization
of the novel prepared zinc(II) complexes with 2-acetylpyridine
N(4)-(4-fluorophenyl)-piperazine thiosemicarbazone, HAcPipPheF,
* Corresponding author. Tel.: +30 2651098425; fax: +30 2651098791.
E-mail address: dkovala@cc.uoi.gr (D. Kovala-Demertzi).
0162-0134/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2009.12.021

468
T.P. Stanojkovic et al. / Journal of Inorganic Biochemistry 104 (2010) 467–476
1 (Scheme 1) and the crystal structure of the ligand and the com-
plexes [Zn(AcPipPheF)2] (2) and [Zn(OAc)(AcPipPheF)]2 (3). The
compounds 1–3 were tested for their antiproliferative activity
in vitro against the cells of four human cancer cell lines: HeLa (cer-
vix adenocarcinoma cell line), K562 (chronic myelogenous leukae-
mia), MDA-MB-361 and MDA-MB-453 (breast cancer cell lines).
v(C@C) v(C@N); 1416s, 1363s, 1297s,
m(NCS); 1223s, (Ar–F);
1088m, (N–N); 931s,
m
m
(CS); 650m, d(py). 1H NMR (DMSO-d6) d
(ppm): 9.35 (s, 1H, N(3)H); 8.71 (d, 1H, J = 4.4 Hz, C(1)H); 7.49 (t,
1H, J = 5.7 Hz, C(2)H); 7.92–7.95 (m, 1H, C(3)H); 7.95–7.97 (m,
1H, C(4)H); 2.57 (s, 3H, C(7)H); 4.14 (t, 4H, C(9,11)H); 3.19 (t, 4H,
C(10,12)H); 6.98–7.04 (m, 2H C(14,18)H); 7.05–7.13 (m, 2H,
C(15,17)H); 13C NMR d (ppm): 150.25 C(1); 125.71 C(2); 138.48
C(3); 122.34 C(4); 155.28 C(5); 148.48 C(6); 14.10 C(7); 184.37
C(8); 50.02 C(9,11); 49.61 C(10); 48.03 C(12); 148.51 C(13);
118.35 C(14); 116.12 C(15); 159.04 C(16); 116.47 C(17); 118.47
C(18); Mass spectrum, MS (electrospray Ionization, ESI, m/z): 358
[1+H]+, 380 [1+Na]+, 326 [1ꢀS]+. Elemental analysis are consistent
with C18H20N5SF found: C, 60.3; H, 5.6; N, 19,3; S, 8.9; Calc.: C,
60.5; H, 5.5; N, 19.6; S, 9.0. Suitable crystals for X-ray study were
obtained by crystallization from a fresh solution of CHCl3/C6H6.
2. Experimental
2.1. General and instrumental
All reagents were commercially available (Aldrich or Merck) and
used as supplied. Solvents were purified according to standard pro-
cedures. The MTT (3-(4,5-di-methyl-thiazol-2-yl)-2,5-diphenyl
tetrazolium bromide) was dissolved (5 mg/mL) in phosphate buffer
saline pH 7.2 and filtered (0.22 lm) before use. The RPMI-1640 cell
2.2.2. Preparation of bis(2-acetylpyridine-N4-1-(4-fluorophenyl)-
piperazinyl thiosemicarbazonato)zinc(II), [Zn(AcPipPheF)2]; (2)
A solution of ZnCl2 (0.068 g, 0.5 mM) in 10 mL of EtOH was
added to a solution of HAcPipPheF (0.393 g, 1.1 mM) in 15 mL of
EtOH. The mixture was stirred and some drops of Et3N were added.
The apparent pH value was 8. The solution was refluxed for 2 h in
80 °C and was left in the fridge overnight. The yellow precipitate
was filtered off, washed with cold EtOH and dried in vacuo over sil-
ica gel. M.p. 275–277 °C. Yield: 48.0%. IR (cmꢀ1): 2828s, v(CH);
1591s, 1548w, 1507s, v(C@C) v(C@N); 1417s, 1366s, 1299s,
culture medium, fetal calf serum, MTT, ethidium bromide and acri-
dine orange were purchased from Sigma Chemical Company, USA.
Melting points were determined in open capillaries and are uncor-
rected. Infrared and far-infrared spectra were recorded on a Perkin
Elmer Spectrum GX Fourier transform spectrophotometer using
KBr pellets (4000–400 cmꢀ1) and Nujol mulls dispersed between
polyethylene disks (400–40 cmꢀ1). The intensity of reported IR sig-
nals is defined as m = medium, mw = medium weak, s = strong,
ms = medium strong. NVR spectra were recorded on a Bruker
AV-400 spectrometer operating at 400 and 100 MHz for 1H and
13C acquisition, respectively, or on a Bruker AV-250 spectrometer
operating at 250.13 and 62.90 MHz for 1H and 13C acquisition
respectively. The splitting of proton resonances in the reported
1H NMR spectra is defined as s = singlet, d = doublet, t = triplet,
and m = multiplet. The spectra were acquired at room temperature
(298 K). The chemical shifts are reported in ppm for 1H and 13C
NMR. Samples were dissolved in dimethylsulfoxide-d6 and spectra
were obtained at room temperature with the signal of free dimeth-
ylsulfoxide-d6 (at 2.50 ppm 1H NMR, 39.5 ppm 13C NMR) as a ref-
erence. Mass spectra were recorded on an Agilent LC/MSD Trap
SL spectrometer. Elemental analyses, C, H, N and S were performed
on a Carlo Erba EA (model 1108).
m
(NCS); 1220s, v(Ar–F); 1155s,
d(py); 433m, 419m, v(Zn–N); 382m, 369m, v(Zn–S); 255m,
239m, v(Zn–Npyr) cmꢀ1 1H NMR (DMSO-d6) d (ppm): 8.65 (d, 1H,
m(N–N); 928s, 869m, m(CS); 662w,
.
C(1)H); 7.33 (t, 1H, J = 6.2 Hz, C(2)H); 7.84 (t, 1H, J = 4.5 Hz,
C(3)H); 7.94 (t, 1H, J = 7.5 Hz, C(4)H); 2.66 (s, 3H, C(7)H); 4.10 (t,
4H, C(9,11)H); 3.16 (t, 4H, C(10,12)H); 7.02–7.08 (m, 2H,
C(14,18)H); 7.10–7.14 (m, 2H, C(15,17)H); 13C NMR d (ppm):
150.63 C(1); 124.65 C(2); 139.66 C(3); 121.99 C(4); 154.90 C(5);
146.01 C(6); 14.12 C(7); 181.43 C(8); 49.81 C(9,11); 46.85
C(10,12); 148.51 C(13); 118.13 C(14); 116.07 C(15); 158.62
C(16); 115.72 C(17); 118.01 C(18). MS (ESI, m/z): 779 [2+H]+. Ele-
mental analysis are consistent with ZnC36H38N10S2F2 found: C,
55.3; H, 5.1; N, 17.8; S, 8.0; Zn, 8.10. Calc.: C, 55.6; H, 4.9; N,
18.0; S, 8.2; Zn, 8.40. Suitable crystals for X-ray study were ob-
tained by crystallization from a fresh solution of EtOH/C6H6.
2.2. Synthesis of the ligand and the complexes
2.2.1. Preparation of 2-acetylpyridine-N4-1-(4-
2.2.3. Preparation of bis(l-acetato(2-acetylpyridine-N4-1-
fluorophenyl)piperazinyl thiosemicarbazone, HAcPipPheF; (1)
4-Methyl-4-phenyl-3-thiosemicarbazide was prepared accord-
ing to the method described by Scovill et al. [13]. The crude prod-
uct, 4-methyl-4-phenyl-3-thiosemicarbazide, was recrystallized
from a mixture of EtOH and distilled water (3:1). 1-(4-fluoro-
phenyl)-piperazine (0.5407 g, 3 mM) and 2-acetylpyridine
(0.363 mL, 3 mM) were added to a solution of 4-methyl-4-phe-
nyl-3-thiosemicarbazide (0.543 g, 3 mM) in CH3CN (4 mL). The
mixture was stirred and refluxed for 30 min. The resulting yellow
precipitate was filtered off, washed with cold CH3CN and dried in
vacuo over silica gel. M.p. 155–157 °C. Yield: 56%. IR (cmꢀ1):
(4-fluorophenyl)-piperazinyl thiosemicarbazonato)zinc(II),
[Zn(HAcPipPheF)(OAc)]; (3)
A solution of [Zn(CH3COO)2(H2O)2] (0.241 g, 1.1 mM) in 10 mL
of EtOH was added to a solution of HAcPipPheF (0.357 g, 1 mM)
in 15 mL of EtOH. The mixture was stirred and some drops of
Et3N were added. The apparent pH value was 8. The solution was
refluxed for 2 h and then it was left in the fridge overnight. The yel-
low precipitate was filtered off, washed with cold EtOH and dried
in vacuo over silica gel. Yield: 77.3%. M.p. 280–282 °C. IR (cmꢀ1):
2812s, v(CH); 1580s, 1506s, v(C@C) v(C@N); 1412s, 1366s, 1299s
m(NCS); 1217s, m(Ar–F); 1152s, m(N–N); 816s, m(CS); 1655s, 1590s
3218m,
m(NY); 2838m, v(CH); 1584mw, d(NH); 1561m, 1512s,
vas(COO); 1367ms, 1455s, vs(COO); 665mw, d(py); 427ms, v(Zn–
N); 372ms, v(Zn–S); 250ms, v(Zn–Npyr), 376ms, 346ms, 328ms,
. d (ppm): 8.49 (d, 1H,
v(Zn–O) cmꢀ1 1H NMR (DMSO-d6)
J = 4.3 Hz, C(1)H); 7.58 (t, 1H, J = 6 Hz, C(2)H); 7.86 (t, 1H,
J = 8.5 Hz, C(3)H); 8.11 (t, 1H, J = 7.8 Hz, C(4)H); 2.66 (s, 1H,
C(7)H); 4.11 (t, 4H, J = 3.7 Hz, C(9,11)H); 3.17 (t, 4H, J = 5 Hz,
C(10,12)H); 7.04–7.08 (m, 2H, C(14,18)H); 7.10–7.11 (m, 2H,
C(15,17)H); 1.79 (s, 3H, OAc); 13C NMR d (ppm): 150.76 C(1);
125.48 C(2); 141.06 C(3); 122.45 C(4); 155.27 C(5); 145.82 C(6);
14.07 C(7); 180.73 C(8); 50.13 C(9,11); 47.09 C(10,12); 148.80
C(13); 118.42 C(14); 116.44 C(15); 158.50 C(16); 116.09 C(17);
118.54 C(18); 23.96, 176.50 CH3COOꢀ; MS (ESI, m/z): 960 [3+H]+.
Scheme 1. The numbering scheme for HAcPipPheF, 1, showing the positively
charge N12 and the negatively charged S.

T.P. Stanojkovic et al. / Journal of Inorganic Biochemistry 104 (2010) 467–476
469
Elemental analysis are consistent with ZnC20H22N5SFJ2 found: C,
49.7; H, 4.9; N, 14.1; S, 6.5; Zn, 13.45. Calc.: C, 50.0; H, 4.6; N,
14.6; S, 6.7; Zn, 13.60. Suitable crystals for X-ray study were ob-
tained by crystallization from a fresh solution of CHCl3/THF (where
THF is tetrahydrofurane).
hydroxyethyl)1-piperazino]ethanosulfonosäure (Hepes) adjusted
to pH 7.2 by bicarbonate solution. RPMI-1640, FBS, Hepes, and
L
-glutamine were products of Sigma Chemical Co., St. Louis, MO.
The final concentrations of the compounds were 50, 10, 1, 0.1,
and 0.01 M.
l
2.3. X-ray crystallography
2.4.1.2. Tumor cell lines. Human cervix adenocarcinoma HeLa cells,
human chronic myelogenous leukaemia K562, human breast can-
cer MDA-MB-361 and MDA-MB-453 cells were cultured as a
monolayer, while were grown in a suspension in the complete
nutrient medium, at 37 °C in humidified air atmosphere with 5%
CO2. For the growth of MDA-MB-361 and MDA-MB-453 cells com-
plete medium was enriched with 1.11 g/L glucose. Neoplastic HeLa
cells (2000 cells per well), MDA-MB-361 cells (7000 cells per well),
MDA-MB-453 cells (3000 cells per well), were seeded into 96-well
microtiter plates. Twenty-four hours later, after the cell adherence,
five different, double diluted, concentrations of investigated com-
pounds were added to the wells, except for the control cells to
which a nutrient medium only was added. K562 cells (3000 cells
per well) were seeded, 2 h before addition of investigated com-
pounds to give the desired final concentrations. Nutrient medium
Crystal data and experimental details are listed in Table 1. A yel-
low prismatic crystal of 1 and yellow plate crystals of 2 and 3 were
mounted on a glass fiber and used for data collection. Crystal data
were collected at 100.0(1) K, using a Bruker X8 KappaAPEXII dif-
fractometer. Graphite monochromated Mo K(alpha) radiation
(lambda = 0.71073 Å) was used throughout. The data were pro-
cessed with APEX2 [14] and corrected for absorption using SADABS
(transmissions factors: 1.000–0.936, 1.000–0.766 and 1.000–
0.873) for 1, 2 and 3, respectively [15]. The structure was solved
by direct methods using the program SHELXS-97 [16] and refined
by full-matrix least-squares techniques against F2 using SHELXL-
97 [17]. Positional and anisotropic atomic displacement parame-
ters were refined for all non-hydrogen atoms. Hydrogen atoms
were located in difference maps and included as fixed contribu-
tions riding on attached atoms with isotropic thermal parameters
1.2 times those of their carrier atoms. Criteria of a satisfactory
complete analysis were the ratios of ‘‘rms” shift to standard devia-
tion less than 0.001 and no significant features in final difference
maps. Atomic scattering factors from ‘‘International Tables for
Crystallography” [18] and molecular graphics from PLATON [19].
was RPMI-1640, supplemented with L-glutamine (3 mM), strepto-
mycin (100 lg/mL), and penicillin (100 IU/mL), 10% heat inacti-
vated (56 °C) FBS and 25 mM Hepes, and the pH of the medium
was adjusted to 7.2 by bicarbonate solution. The cultures were
incubated for 72 h.
2.4.1.3. MTT test. Cell survival was determined by MTT test [20,21]
2.4. Biological experiments
72 h upon addition of the compounds. Briefly, 20 lL of MTT solu-
tion (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
2.4.1. Antiproliferative assay in vitro
mide, 5 mg/mL in phosphate buffered saline) was added to each
2.4.1.1. Compounds. Stock solutions of investigated compounds,
were prepared in DMSO at concentrations of 10 mM and after-
wards they were diluted with complete nutrient medium
well. Samples were incubated for further four h. Then, 100 lL of
10% SDS was added to extract the insoluble product formazan,
resulting from the conversion of the MTT dye by viable cells. The
number of viable cells in each well was proportional to the inten-
sity of the absorbance of light, which was then read in an ELISA
plate reader at 570 nm.
(RPMI-1640 without phenol red) supplemented with 3 mM
L-glu-
tamine, 100 g/mL streptomycin, 100 IU/mL penicillin, 10% heat
l
inactivated fetal bovine serum (FBS), and 25 mM: 2-[4-(2-
Table 1
Crystal data and structure refinement for 1–3.
1
2
3
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Space group
Unit cell dimensions
a (Å)
C18H20FN5S
357.45
100(2)
0.71073
Monoclinic, P 21/c
0.31 ꢁ 0.13 ꢁ 0.09
18.0664(10)
12.0592(6)
C36H38F2N10S2Zn
778.25
100(2)
0.71073
Monoclinic, P 21/c
0.35 ꢁ 0.12 ꢁ 0.06
9.3680(6)
33.020(2)
11.6627(7)
C40H44F2N10O4S2Zn2
961.71
100(2)
0.71073
Monoclinic, P 21/c
0.48 ꢁ 0.35 ꢁ 0.04
9.8053(5)
28.0511(12)
15.6225(9)
b (Å)
c (Å)
7.8641(5)
b (°)
95.90
1705.10(17)
4, 1.392
107.448(2)
3441.6(4)
4, 1.502
0.890
102.903(2)
4188.5(4)
4, 1.525
1.308
V (Å3)
Z, Dcalcd (Mg mꢀ3
)
)
l
(Mo Ka) (mmꢀ1
0.211
F(0 0 0)
752
1616
1984
h Range (°)
1.13–26.02
1.23–26.40
1.45–26.42
Reflections collected/unique
Absorption correct.
Range of h, k, l
Refinement method
Data/restraints/param.
Goodness-of-fit
33408/3347 [R(int) = 0.0468]
Empirical
ꢀ22 6 h 6 22, 0 6 k 6 14, 0 6 l 6 9
Full-matrix least-sq. on F2
3347/0/226
1.040
24055/7063 [R(int) = 0.0354]
Empirical
ꢀ11 6 h 6 11, 0 6 k 6 41,0 6 l 6 14
Full-matrix least-sq. on F2
7063/0/460
48308/8607 [R(int) = 0.0392]
Empirical
ꢀ11 6 h 6 12, 0 6 k 6 34,19 6 l 6 0
Full-matrix least-sq. on F2
8607/0/541
1.008
1.015
Final R indices
R1 = 0.0322
R1 = 0.0411
R1 = 0.0403
[I > 2sigma(I)]
wR2 = 0.0765
R1 = 0.0446
wR2 = 0.0833
ꢀ0.214 and 0.215
wR2 = 0.0882
R1 = 0.0629
wR2 = 0.0969
ꢀ0.479 and 1.181
wR2 = 0.0912
R1 = 0.0645
wR2 = 0.1017
ꢀ0. 446 and 0.745
R indices (all data)
Min. and Max. Resd.
Dens. (e/Å3)

470
T.P. Stanojkovic et al. / Journal of Inorganic Biochemistry 104 (2010) 467–476
2.4.2. Morphological analysis of HeLa cells death
difference, [mas(COO)–msym(COO)] between these frequencies for 3
In order to determine the mode of HeLa cell death induced by
the investigated compounds, morphological analysis by micro-
scopic examination of acridine orange and ethidium bromide
stained cells was performed. HeLa cells were seeded overnight on
coverslips (5 ꢁ 104 cells) in 2 mL of complete medium, and on next
day treated with investigated compounds for 24 h. Concentrations
applied corresponded to double IC50 concentrations of investigated
compounds. Afterwards, the cells were examined for morphologi-
cal features of apoptosis and necrosis by fluorescence microscopy
(287 and 135 cm–1) is close to that found for anisobidentate che-
late and bridging acetate mode. This is totally consistent with the
X-ray structure of 3. The Zn(II) donor atom stretching frequencies
are as follows: v(Zn–Nimine), 420–430; v(Zn-S), 380–370; v(Zn–
Npyr), 255–240 and Zn–O, 375–330 cm–1
.
3.2.3. NMR spectra
Peak assignments were based on 2D NMR data {proton–proton
correlated spectroscopy (1H–1H COSY), proton–carbon heteronu-
clear single-quantum coherence (1H–13C HSQC) and proton–car-
bon heteronuclear multiple-bond correlation (1H–13C HMBC)}. In
the 1H and 13C NMR spectra of the Zn(II) complexes 2 and 3 the
hydrogen and carbon atoms of the pyridyl ring were shifted upon
coordination, which indicated variations in the electron density of
the pyridyl ring. The C@S resonance of the thiosemicarbazone moi-
ety in 1 resonated at 184.37 ppm, in the complexes 2 and 3, upfield
shifts were observed, indicating increased electron density at this
using acridine orange and ethidium bromide stains (15
ture: 3 g/mL AO and 10 g/mL EB).
lL of mix-
l
l
2.4.3. Cell cycle determination
Aliquots of 5 ꢁ 105 control or cells were treated with investi-
gated compounds for 24 h (concentrations corresponded to
2 ꢁ IC50 values) were fixed in 70% ethanol on ice for at least 1 week
and centrifuged. The pellet was treated with RNase (100 lg/mL) at
37 °C temperature for 30 min and then incubated with propidium
iodide (PI) (40 g/mL) for at least 30 min. DNA content and cell cy-
site on complexation and due probably to back
lato sulphur [11].
p-bonding for thio-
l
cle distribution were analyzed using a Becton Dickinson FAC-Scan
flow cytometer. Flow cytometry analysis was performed using a
CellQuestR (Becton Dickinson, San Jose, CA, USA), on a minimum
of 10,000 cells per sample [22].
3.3. Crystal structures
Selected interatomic distances and bond angles are collected in
Table 2. Compounds 1–3 were recrystallized from CHCl3/hexane,
CHCl3/THF and CHCl3/hexane, to furnish yellow prisms suitable
for single-crystal X-ray analysis.
3. Results and discussion
3.1. Chemistry
Transamination of 4-methyl-4-phenyl-3-thiosemicarbazide in
the presence of 1-(4-fluorophenyl)-piperazine and 2-acetylpyri-
dine gives the ligand HAcPipPheF, 1. The Zn(II) complexes 2 and
3 were prepared by refluxing ZnCl2 or [Zn(CH3COO)2(H2O)2] with
the 1 in EtOH solution in a molar ratio metal (Zn(II)) to ligand of
1:2 and 1:1 respectively. The apparent pH value was adjusted to
8. The formula of compounds 1–3 was confirmed by elemental
analysis, spectroscopic studies and X-ray diffraction analysis.
3.3.1. Crystal structure of HAcPipPheF
Fig. 1a shows a perspective view of the HAcPipPheF molecule
with the numbering scheme. HAcPipPheF is the bifurcated E0
conformation similar to that found in 2-acetylpyridine 3-hexam-
ethyleneiminylthiosemicarbazone, HAchexim [25] and 2-pyridine-
formamide hexamethyleneiminylthiosemicarbazone, HAmhexim
[26]. The thiosemicarbazone moiety shows an E configuration
about the bonds C(16)–N(12), N(12)–N(13) and Z configuration
about the bond C(17)–N(13). The thiosemicarbazone moiety is al-
most planar except for the sulphur atom, that presents a deviation
from planarity, of the order of 0.253(1) Å. The bond distance C(17)–
S(1) is formally a single bond in 1 has a length of 1.712(2) Å,
Scheme 1. The bond distances and angles for 1 are similar to those
reported for the bifurcated HAchexim [25] and HAmhexim [26].
The bond distance C(17)–S(1) has a length of 1.70(1) Å in HAchex-
im [25] and 1.732(4) in HAmhexim [26] and is formally a single
bond, but in 2-hydroxy-4-methoxyacetophenone N4-dimethyl thi-
osemicarbazone is 1.65(2) Å and is formally a double bond [27].
The C(16)–N(12)–N(13) angle is 124.16(13)° for 1, 125(1)° for
HAchexim [25] and 122.8(3)° for HAmhexim [26]. The azomethine
C(16)–N(12) bond is a double bond, while the N(12)–N(13) and
both thioamide C(17)–N(13) and C(17)–N(14) bond distances exhi-
bit partial double-bond character. This is indicative of a greater
conjugation and an extensive delocalized electron density on the
thiosemicarbazone backbone. The dihedral angle between the
mean planes of the N4-1-(4-fluorophenyl) and the piperazinyl ring
is 32.44(7)°.
3.2. Spectroscopy
3.2.1. Mass spectra
The mass spectra of the soluble compounds 1–3 were recorded
in MeOH solution. The ligand shows a peak at m/z 358 amu due to
the parent ion fragment [HacPyPipF+H]+ and at 380and 326 amu,
due to the fragments [HacPyPipF+Na]+ and [HacPyPipFꢀS]+ respec-
tively. The monomer zinc(II) complex 2 shows peak at 779 amu
due to [2+H]+ and the dimer complex 3 shows peak at 957 amu
due to [3+H]+. Each spectrum is associated with daughter peaks
due to fragmentation.
3.2.2. Infrared spectroscopy
The significant IR bands of zinc(II) complexes are close in energy
to those found in other compounds with tridentate coordination
[23,24]. Coordination of the azomethine nitrogen to zinc(II) is sug-
gested by the shift of the
with the occurrence of the
the appearance of the band assignable to Zn–N. The thioamide
band, which contains considerable (CS) character is found at a
m
(C@N) band to lower frequencies along
m
(N–N) band to higher frequency and
The monomers of 1 form hydrogen-bonded dimer linked by two
weak C(12)–H(12)ꢂ ꢂ ꢂS(1) hydrogen bonds involving the carbon
C(12)–H(12) hydrogen atom and the sulphur S(1) and vice versa
of centrocymmetrically related pairs of molecules. The observed
hydrogen-bonding pattern is of the DA = DA type. Intra and inter-
molecular hydrogen bonds stabilize the structure, while the crystal
m
lower frequency, suggesting coordination of the metal through sul-
phur. The breathing motion of the pyridine ring is shifted to a high-
er frequency upon complexation and is consistent with pyridine
ring nitrogen coordination. Two bands are assigned to
(Zn–S) and (Zn–Npyr) for 2 indicating non-linear N–Zn–N, S–
Zn–S and Npyr–Zn–Npyr, moieties. The as(COO) band appears at
1654 and 1580 and
sym(COO) at 1367 and 1455 cmꢀ1 for 3. The
m(Zn–N),
m
m
packing is determined by
interaction is that between the pair of pyridyl rings at a centre-
to-centre separation of 3.9340(9) Å, Fig. 1b.
p–p interactions. The most significant p–
m
p
m

T.P. Stanojkovic et al. / Journal of Inorganic Biochemistry 104 (2010) 467–476
471
Table 2
Selected bond lengths (Å) and angles (°) for 1–3.
1
2
3
S(1)–C(17)
F(1)–C(23)
N(11)–C(11)
N(11)–C(15)
N(12)–C(16)
N(12)–N(13)
N(13)–C(17)
N(14)–C(17)
N(14)–C(27)
N(14)–C(18)
N(15)–C(20)
N(15)–C(19)
1.712(2)
1.363(2)
1.334(2)
1.352(2)
1.297(2)
1.355(2)
1.347(2)
1.364(2)
1.462(2)
1.463(3)
1.426(2)
1.462(2)
Zn(1)–N(12)
Zn(1)–N(32)
Zn(1)–N(31)
Zn(1)–N(11)
Zn(1)–S(2)
Zn(1)–S(1)
S(1)–C(17)
S(2)–C(37)
F(1)–C(23)
F(2)–C(43)
N(12)–C(16)
2.114(2)
2.138(2)
2.221(2)
2.252(2)
2.4271(7)
2.4545(8)
1.742(3)
1.732(3)
1.360(3)
1.368(3)
1.291(3)
1.294(3)
Zn(1)–O(21)
Zn(1)–O(11)
Zn(1)–N(12)
Zn(1)–N(11)
Zn(1)–S(1)
Zn(1)–Zn(2)
Zn(2)–O(12)
Zn(2)–O(21)
Zn(2)–N(32)
Zn(2)–N(31)
Zn(1)–O(22)
S(1)–C(17)
2.041(2)
2.048(2)
2.084(2)
2.133(2)
2.3566(8)
3.5529(5)
2.000(2)
2.052(2)
2.113(2)
2.126(2)
2.696(2)
1.742(3)
1.751(3)
99.39(8)
141.87(9)
117.50(9)
96.15(8)
88.72(9)
76.46(9)
103.65(6)
98.12(7)
81.50(6)
157.62(7)
100.32(8)
108.13(9)
150.42(8)
97.58(9)
92.78(8)
75.86(9)
110.39(6)
97.10(6)
80.71(6)
147.97(6)
N(15)–C(26)
1.469(2)
N(32)–C(36)
S(2)–C(37)
C(11)–N(11)–C(15)
C(16)–N(12)–N(13)
C(17)–N(13)–N(12)
C(17)–N(14)–C(27)
C(17)–N(14)–C(18)
C(27)–N(14)–C(18)
C(20)–N(15)–C(19)
C(20)–N(15)–C(26)
C(19)–N(15)–C(26)
N(11)–C(11)–C(12)
117.41(13)
124.16(13)
111.21(12)
121.61(12)
121.93(12)
112.67(11)
115.50(12)
114.03(11)
110.26(11)
123.84(14)
N(12)–Zn(1)–N(32)
N(12)–Zn(1)–N(31)
N(32)–Zn(1)–N(31)
N(12)–Zn(1)–N(11)
N(32)–Zn(1)–N(11)
N(31)–Zn(1)–N(11)
N(12)–Zn(1)–S(2)
N(32)–Zn(1)–S(2)
N(31)–Zn(1)–S(2)
N(11)–Zn(1)–S(2)
N(12)–Zn(1)–S(1)
N(32)–Zn(1)–S(1)
N(31)–Zn(1)–S(1)
N(11)–Zn(1)–S(1)
S(2)–Zn(1)–S(1)
159.46(8)
91.90(8)
74.26(8)
74.33(8)
90.05(8)
89.08(8)
112.98(6)
79.65(6)
153.90(6)
90.20(6)
79.90(6)
115.40(6)
93.52(6)
154.17(6)
98.35(3)
O(21)–Zn(1)–O(11)
O(21)–Zn(1)–N(12)
O(11)–Zn(1)–N(12)
O(21)–Zn(1)–N(11)
O(11)–Zn(1)–N(11)
N(12)–Zn(1)–N(11)
O(21)–Zn(1)–S(1)
O(11)–Zn(1)–S(1)
N(12)–Zn(1)–S(1)
N(11)–Zn(1)–S(1)
O(12)–Zn(2)–O(21)
O(12)–Zn(2)–N(32)
O(21)–Zn(2)–N(32)
O(12)–Zn(2)–N(31)
O(21)–Zn(2)–N(31)
N(32)–Zn(2)–N(31)
O(12)–Zn(2)–S(2)
O(21)–Zn(2)–S(2)
N(32)–Zn(2)–S(2)
N(31)–Zn(2)–S(2)
Zn(1)–O(21)–Zn(2)
120.46(10)
Fig. 1. (a) Labelled ORTEP diagram of 1 with 50% thermal probability ellipsoids and (b) packing diagram of 1 and along the c axis.
3.3.2. Crystal structure of [Zn(AcPipPheF)2]
perspective view of the centrosymmetric structure of
[Zn(AcPipPheF)2] is shown in Fig. 2. The two anionic AcPipPheF li-
gands act as tridentates which coordinate to zinc(II) through the
pyridyl nitrogen, N(11) and N(31), the azomethine nitrogen,
N(12) and N(32) and the thiolato sulphur atoms. The tridentate li-
A

472
T.P. Stanojkovic et al. / Journal of Inorganic Biochemistry 104 (2010) 467–476
Fig. 2. (a) Labelled ORTEP diagram of 2 with 40% thermal probability ellipsoids and (b) hydrogen-bonding pattern of 2.
gands have a ZEZ configuration based on the C(15)–C(16), N(13)–
C(17), N(14)–C(17) and C(35)–C(36), N(33)–C(37), N(34)–C(37)
bonds for the two ligands respectively. The zinc(II) metal ion is
coordinated in a distorted octahedral configuration in a trans-
N(12)N(32)-cis-N(11)N(31)-cis-S configuration. The trans angle
involving the two imine nitrogens, N(12)–Zn–N(32), is 159.46(8)°
is far from 180°. The N(11)–Zn(1)–N(31) angle is 89.08(8)°, while
the S(1)–Zn–S(2) angle is 98.35(3)° and demonstrate that the two
ligands exert significant steric effects on each other. The smallest
cis bond angles about the Zinc centre occur for donor atoms in
the same ligand {e.g. N(32)–Zn–N(31), 74.26(8)° and N(12)–Zn–
N(11) 74.33(8)°}. AcPipPheF ligands have essentially equivalent
Zn–S, Zn–N(imine) and Zn–N(pyridine) bond distances.
Coordination lengthens the thiosemicarbazone moiety’s C(17)–
S(1) and C(37)–S(2) bonds from 1.712(2) Å in HAcPipPheF to
1.742(3) and 1.732(3) Å respectively. The bond length S–C indi-
cates an increased single-bond character and both thioamide bond
distances indicate increased double-bond character and suggest a
charge delocalization in the thiosemicarbazone moiety. The li-
gands are not planar, as indicated by the dihedral angle between
the mean planes of the pyridyl ring and the chelate ring Zn–S–C–
N–N, with values of 12.40(11) and 6.64(10) for the two ligands
respectively. The two chelated rings are planar, as indicated by
the dihedral angle between the mean planes of the Zn–S–C–N–N
and the Zn–N–C–C–N, with values of 2.62(9)° and 1.37(3)° for
the two ligands respectively. The dihedral angle between the mean
planes of the N4-1-(4-fluorophenyl) and the piperazinyl ring is
67.34(14)° and 49.63(14)° for the two ligands respectively. C–
Hꢂ ꢂ ꢂS and C–Hꢂ ꢂ ꢂF2 intermolecular bond connect the monomers
of 2. Intra and intermolecular hydrogen bonds stabilize the struc-
ture, while the crystal packing is determined by
Table 3. A double interaction occurs between the pyridyl ring
and the N4-1-(4-fluorophenyl) ring (symmetry operation; 1 + x, y,
centre-to-centre separation of
p–p interactions,
p–p
1 + z; ꢀ1 + x, y, ꢀ1 + z) at
a
3.788(2) and 3.778(2) Å respectively.
3.3.3. Crystal structure of [Zn(AcPipPheF)(OAc)]2
The Zn(II) ions are linked by two acetato groups. One of them
bridges in the classical g1
:
g1
:
l2 fashion with Zn(2)–O(12) and
Zn(1)–O(11) distances of 2.000(2) and 2.048(2) respectively and
the other one bridges in the less common g2
2 fashion; this lat-
:
g1
:l
ter has an oxygen atom O(22) bound terminally to one Zn atom,
and the other oxygen atom O(21) bound in a l2 bridging manner
to both Zn atoms, forming a monoatomic bridge with Zn(1)–
O(21) and Zn(2)–O(21) distances of 2.041(2) and 2.052(2) Å
respectively. The Zn(2) atom is, however, subjected to a sixth
weaker interaction in the form of a carboxylato oxygen atom,
O(22), approaching the Zn(2) atom. The distance Zn(2)ꢂ ꢂ ꢂO(22)
2.696(2) Å is considered as a weak bond distance. One terminal
AcPipPheF monoanion at each metal ion complete a five and six –
coordination for Zn(2) and Zn(1) metal ions respectively, Fig. 3.
The zinc metal centres are connected by two inequivalent bridging
acetate ligands to give a Znꢂ ꢂ ꢂZn distance of 3.5529(5) Å. A similar
dimer structure was found for [Zn(Amhexim)(OAc))]2 where Amh-
exim represents the anion of 2-pyridineformamide3Hexamethyl-
eneiminylthiosemicarbazone [26].
The two anionic AcPipPheF ligands act as tridentates which
coordinate to zinc(II) through the pyridyl nitrogen, N(11) and
N(31), the azomethine nitrogen, N(12) and N(32) and the thiolato
sulphur atoms. The tridentate ligands have a ZEZ configuration

T.P. Stanojkovic et al. / Journal of Inorganic Biochemistry 104 (2010) 467–476
473
Table 3
Inter- and intra-molecular hydrogen bonds and
p
–
p
intermolecular interactions.
Cg–Cgb
1a
bc
CgI–Perpd
CgJ–Perpe
Cg(1)–˜ Cg(1)i
3.9340(9)
3.9341(9)
A
24.67
25.84
Dꢂ ꢂ ꢂA
3.6565(16)
2.8199(13)
2.6287(18)
3.5751(6)
ꢀ3.5407(6)
Hꢂ ꢂ ꢂA
2.84
2.26
2.22
ꢀ3.5406(6)
3.5751(6)
hD – Hꢂ ꢂ ꢂA
143
119
106
Cg(1)–˜ Cg(1)ii
D
H
C(12)
N(12)
N(12)
H(12)
H(12A)
H(12A)
S(1)iii
S(1)
N(11)
2a
Cg(10)–˜Cg(5)iv
Cg(5)–˜ Cg(10)v
D
3.788(2)
3.778(2)
A
28.93
19.44
Dꢂ ꢂ ꢂA
ꢀ3.5721(12)
3.3155(12)
Hꢂ ꢂ ꢂA
3.3155(12)
ꢀ3.5721(12)
H
hD–Hꢂ ꢂ ꢂA
C(21)vi
C(32)vii
C(38)vi
C(18)
C(47)
C(38)
H(21)
H(32)
S(2)
F(2)
F(2)
S(1)
3.595(3)
3.323(3)
3.264(4)
3.071(3)
3.032(3)
2.704(4)
2.752(4)
2.698(4)
2.85
2.46
2.38
2.53
2.55
2.26
2.37
2.25
138
153
149
114
115
107
104
109
H(38A)
H(18B)
H(47A)
H(38B)
H(48B)
H(27B)
S(2)
N(33)
N(33)
N(13)
C(48)
C(27)
3a
Cg(5)–˜ Cg(7)viii
Cg(7)–˜ Cg(5)vi
Cg(9)–˜ Cg(7)viii
D
4.0115(15)
4.0115(15)
3.744(2)
A
29.36
36.26
26.41
Dꢂ ꢂ ꢂA
3.340(4)
3.184(4)
3.725(3)
3.326(4)
3.087(4)
2.948(3)
2.768(4)
2.971(3)
2.825(4)
ꢀ3.2346(10)
3.4961(11)
ꢀ3.3241(11)
Hꢂ ꢂ ꢂA
2.53
2.43
2.82
2.56
2.41
2.46
2.36
2.50
2.40
3.4961(11)
ꢀ3.2347(10)
3.3534(11)
hD–Hꢂ ꢂ ꢂA
140
134
163
140
129
109
104
110
106
H
C(2)ix
H(2C)
H(13)
H(24)
H(32)
H(33)
H(18B)
H(28C)
H(38B)
H(48B)
F(2)
O(12)
S(1)
O(22)
O(22)
S (1)
N(13)
S(2)
C(13)vi
C(24)x
C(32)xi
C(33)viii
C(18)
C(28)
C(38)
C(48)
N(33)
D, Donor; A, Acceptor.
a
Where Cg(1) is referred to the centroid N11–C11–C12–C13–C14–C15 for 1; Cg(5) and Cg(10) are referred to the centroids N11–C11–C12–C13–C14–C15 and C40–C41–
C42–C43–C44–C45 respectively for 2; Cg(5), Cg(7) and Cg(9) are referred to the centroids Zn2N32C36C35N31, N11–C11–C12–C13–C14–C15 and N31–C31–C32–C33–C34–
C35 for 3.
b
Cg–Cg is the distance between ring centroids; symmetry transformations, (i)= x, 3/2 ꢀ y, ꢀ1/2 + z; (ii) x, 3/2 ꢀ y, 1/2 + z; (iii) ꢀx, 1 ꢀ y, 1 ꢀ z; (iv) 1 + x, y,1 + z; (v) ꢀ1 + x, y,
ꢀ1 + z; (vi) ꢀ1 + x, y, z; (vii) ꢀ2 + x, y, ꢀ1 + z; (viii) 1 + x, y, z; (ix) 1 ꢀ x, 1/2 + y, ꢀ5/2 ꢀ z; x, x,1/2 ꢀ y, 1/2 + z; (xi) ꢀx, ꢀy, ꢀ1 ꢀ z.
c
Where b is the angle Cg(I)–˜Cg(J) or Cg(I)–˜Me vector and normal to plane I (°).
CgI–Perp is the perpendicular distance of Cg(I) on ring J.
CgJ–Perp is the perpendicular distance of Cg(J) on ring I.
d
e
based on the C(15)–C(16), N(13)–C(17), N(14)–C(17) and C(35)–
C(36), N(33)–C(37), N(34)–C(37) bonds for the two ligands
respectively.
Hꢂ ꢂ ꢂS and C–Hꢂ ꢂ ꢂF(2) intermolecular hydrogen bonds connect the
monomers of 3. Also, the terminal oxygen O(22) forms two C–
Hꢂ ꢂ ꢂO(22) intermolecular hydrogen bonds. Intra and intermolecu-
lar hydrogen bonds stabilize the structure, while the crystal pack-
Analysis of the shape determining angles for Zn(2), using the
approach of Reedijk and co-workers [28], yields a
s
= (
s
a
ꢀ b)/60)
ing is determined by
p–p interaction is that between the pyridyl rings at a centre-to-
centre separation of 3.744(2) Å.
p–p interactions, Table 3. The most significant
{(150.42 ꢀ 147.97)/60 = 0.04) value of 0.04 for Zn(2), (
= 0.0 and
1.0 for SP and TBP geometries respectively). The metal coordination
geometry is therefore described as distorted square pyramidal
with the O(12) atom occupying the apical position. The donor
O(12) atom is chosen as apex by the simple criterion that it should
not be one of the oxygen atoms which define either of the two larg-
3.4. Biological evaluation
3.4.1. In vitro antiproliferative activity
est L–Zn–L angles,
a
and b and the atoms N(31), N(32), S(2) and
The results of cytotoxic activity in vitro are expressed as IC50 –
O(21) define the planar plane (maximum deviation 0.006(2) Å).
The Zn(2) atom is 0.5205(3) Å out of the basal plane. The Zn(1) me-
tal ion is coordinated in a distorted octahedral configuration. The
ligands are not planar, as indicated by the dihedral angle between
the mean planes of the pyridyl ring and the chelate ring Zn–S–C–
N–N, with values of 8.89(11)° and 14.08(11)° for the two ligands
respectively. The dihedral angle between the mean planes of the
six-membered ring Zn(1)–O(11)–C(2)–O(12)–Zn(2)–O(21) and
the five-membered ring Zn–N–C–C–N is 89.39(9)° and 80.95(9)°
for the two ligands respectively. The dihedral angle between the
mean planes of the N4-1-(4-fluorophenyl) and the piperazinyl ring
is 39.55(11)° and 33.30(14)° for the two ligands respectively. C–
the concentration of compound (in M) that inhibits a proliferation
rate of the tumor cells by 50% as compared to control untreated
cells. The compounds 1–3 were tested for their antiproliferative
activity in vitro against the cells of four human cancer cell lines:
HeLa (cervix adenocarcinoma cell line), K562 (chronic myeloge-
nous leukaemia), MDA-MB-361 and MDA-MB-453 (breast cancer
cell lines). The antiproliferative activity of compounds is presented
in Table 3 along with the activity of cis-platin and ZnCl2. Results
showed that the ligand as well as the complexes demonstrated
excellent antiproliferative activity, IC50 values range from 26 to
90 nM, against all cell lines tested, while for cis-platin the IC50 val-
ues range from 2 to 17 lM and for the zinc salt, ZnCl2, the IC50 val-

474
T.P. Stanojkovic et al. / Journal of Inorganic Biochemistry 104 (2010) 467–476
Fig. 3. (a) Labelled ORTEP diagram of 3 with 40% thermal probability ellipsoids and (b) packing diagram of 3 along the a axis.

T.P. Stanojkovic et al. / Journal of Inorganic Biochemistry 104 (2010) 467–476
475
Table 4
IC50 (M) values of the studied compounds.
Compounds
HeLa
MDA-MB-361
MDA-MB-453
K562
1
2
3
58 8 ꢁ 10ꢀ9
55 2 ꢁ 10ꢀ9
35 2 ꢁ 10ꢀ9
2.1 0.2 ꢁ 10ꢀ6
85.4 0.2 ꢁ 10ꢀ6
90 2 ꢁ 10ꢀ9
59 5 ꢁ 10ꢀ9
61 9 ꢁ 10ꢀ9
30 2 ꢁ 10ꢀ9
3.6 0.5 ꢁ 10ꢀ6
93.0 0.2 ꢁ 10ꢀ6
26 6 ꢁ 10ꢀ9
71 2 ꢁ 10ꢀ9
25.5 6 ꢁ 10ꢀ9
7.9 0.2 ꢁ 10ꢀ6
56 15 ꢁ 10ꢀ9
42 9 ꢁ 10ꢀ9
Cis-platin
ZnCl2
17.1 1.2 ꢁ 10ꢀ6
91.4 0.5 ꢁ 10ꢀ6
81.9 0.7 ꢁ 10ꢀ6
Concentrations of examined compounds that induced a 50% decrease in HeLa, MDA-MB-361, MDA-MB-453, and K562 cell survival (expressed as IC50 (M)) the compounds
were incubated with cells for 72 h. At the end of this incubation period, antiproliferative activity in vitro was determined by the MTT assay. Results are presented as the mean
value SD of three independent experiments.
ues range from 81 to 93
activity in all these four lines (see Table 4).
l
M. The ZnCl2 exhibits poor cytotoxic
tests. Interestingly enough, 1–3 were found to be more potent
cytotoxic agent than the prevalent benchmark metallodrug, cis-
platin, under the same experimental conditions measured by us.
The superior activity of 1–3 assumes significance in light of the fact
that cis-platin is undisputedly the most studied and widely used
metallopharmaceutical for cancer therapy known to date.
Platinum(II) complexes of N4-ethyl 2-formyl and 2-acetylpyri-
dine thiosemicarbazones showed cytotoxicity and were found to
be able to overcome the cis-platin resistance of A2780/Cp8 cells
[13]. Pt(II) or Pd(II) complexes with N4-ethyl 2-acetylpyridine thi-
osemicarbazone were tested in a panel of human tumor cell lines
of different origins (breast, colon, and ovary cancers), and cis-pla-
tin-refractory/resistant cell lines and were found to exhibit very
remarkable growth inhibitory activities with mean IC50 values of
0.9–0.5 nM and support the hypothesis that both [Pt(Ac4Et)2]
and [Pd(Ac4Et)2] complexes can be characterized by cellular phar-
macological properties distinctly different from those of cis-platin
[29]. The complexes [ZnCl2(Fo4Npypipe)] and [ZnCl2(Ac4Npypipe)]
where Fo4Npypipe and Ac4Npypipe are the monoion of 2-formyl
pyridine N(4)-1-(2-pyridyl)-piperazinyl thiosemicarbazone and 2-
acetyl pyridine N(4)-1-(2-pyridyl)-piperazinyl thiosemicarbazone
have been evaluated in vitro against MCF-7, T24, A-549 and L-
929 cell lines and it was found to exhibit remarkable antiprolifer-
3.4.2. Fluorescence microscopy
Compounds 1–3 at a concentration of 2 ꢁ IC50 nM, after a 24 h
continuous action, effectively induce cell apoptosis, rounding and
detachment in HeLa cells. In addition to their growth inhibition
activities, marked cytopathological effects, spherical morphology
and detachment of the cells, were observed on the HeLa cells trea-
ted with the compounds.
3.4.3. Cell cycle analysis
Flow cytometry was used to determine the effects of com-
pounds 1–3 on the cell cycle phase distribution of each of the tu-
mor cell lines. The effect of compounds upon the cell cycle was
assessed after incubating cells with the compounds at their
2 ꢁ IC50 nM. Incubation of the K562, HeLa and MDA-MB-453 cells
with 1–3 led to accumulation of more cells in S phase and a reduc-
tion of cell population in G2/M phase, Table 5. The S-phase cell cy-
cle arrest observed in the cells treated with the compounds
suggests that these new complexes inhibit DNA synthesis of the
malignant cells. It has been reported that thiosemicarbazones
ative activity with mean IC50 values of 0.2–20 lM [11].
The IC50 values for 1 against MDA-MB-361 and MDA-MB-453
cell lines are 90 and 59 nM respectively and against HeLa and
K562 cell lines are 59 and 26 nM respectively. Ligand 1 is 190
and 61 times more active than cis-platin against MDA-MB-361
and MDA-MB-453 and 36 and 303 times more active than cis-pla-
tin against HeLa and K562 cell lines. The IC50 values for 2 against
MDA-MB-361 and MDA-MB-453 cell lines are 56 and 61 nM
respectively and against HeLa and K562 cell lines are 55 and 71
nM respectively. The Zn(II) complex 2 is 305 and 59 times more ac-
tive than cis-platin against MDA-MB-361 and MDA-MB-453 and 38
and 111 times more active than cis-platin against and K562 cell
lines. In the case of HeLa and MDA-MB-453 cell lines the ligand
and complex 2, the IC50 values are in the same nM range 30–38
times for the former and 60 times for the latter cell line more cyto-
toxic compared to cis-platin. In this case the observed cytotoxicity
is probably due to the cytotoxicity of the ligand and the metal com-
plexes may be a vehicle for activation of the ligand as the cytotoxic
agent. Ligand 1 and complex 2 exhibited high activity as anticancer
agent against all four cancer cell lines, and 1 exhibits the highest
selectivity against K562.
Table 5
Effect of 1–3 on cell cycle progression.a
G0/G1
S
G2/M
Hela
Control
52.52
51.17
59.32
57.33
16.17
30.22
17.44
23.17
28.15
13.86
19.92
14.93
1
2
3
K-562
Control
49.81
46.6
48.19
46.36
21.45
32.86
35.18
31.98
19.68
16.31
12.1
1
2
3
17.09
The IC50 values for 3 against MDA-MB-361 and MDA-MB-453
cell lines are 42 and 30 nM respectively and against HeLa and
K562 cell lines are 35 and 26.5 nM respectively. The Zn(II) complex
3 is 406 and 120 times more active than cis-platin against MDA-
MB-361 and MDA-MB-453 and 60 and 298 times more active than
cis-platin against HeLa and K562 cell lines. The most outstanding
results are obtained from the activity of compound 3, which is
60–405 times better tha cis-platin. According to these results, 3
is the most active compound of this study. It is noteworthy the
high selectivity against MDA-MB-453 and K562 cancer cell lines.
The mentioned evident differences in the antiproliferative ac-
tion of the ligand and its zinc(II) complexes indicate that the zin-
c(II) complexes really exist under the condition of the biological
MDA-MB-361
Control
1
2
3
50.78
70.14
76.28
72.2
17.31
9.56
6.42
6.5
29.44
18.99
16.13
20.06
MDA-MB-453
Control
1
2
3
67.11
55.98
67
14.27
22.29
15.03
24.07
14.93
14.83
12.24
8.21
60.83
a
Effect of 1–3 on cell cycle phase distribution. HeLa, K562, MDA-MB-361, and
MDA-MB-453 cell lines were exposed to compounds (2 ꢁ IC50 nM) for 24 h and
then collected for analysis of cell cycle phase distribution using flow cytometry.
Percentage of cells under different stages of cell cycles (G0–G1, S, G2–M) is shown.

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Supplementary data associated with this article can be found, in
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