Zinc(II) complexes with dithiocarbamato derivatives: Structural characterisation and biological assays on cancerous cell lines

Article abstract of DOI:10.1016/j.jinorgbio.2012.09.004

Zinc is one of the most important trace elements in the body and it is essential as a cofactor for the structure and function of a number of cellular molecules including enzymes, transcription factors, cellular signalling proteins and DNA repair enzymes. On the other hand, recent studies have shown that zinc could play a role both in the development of various cancers and in the induction of apoptosis in some cell types, however, no established common relationships of zinc with cancer development and progression have been identified. To date, in our research group different metal-dithiocarbamato complexes have been designed that were expected to resemble the main features of cisplatin together with higher activity, improved selectivity and bioavailability, and lower side-effects. On the basis of the obtained encouraging achievements with other metals (such as gold and copper) we have decided to enlarge the studies to the complexes of zinc(II) using the same ligands. Hereby, we report the results on the synthesis and characterisation of ZnL₂ complexes with five different dithiocarbamato derivatives, such as dimethyl-(DMDT), pyrrolidine-(PyDT), methyl-(MSDT), ethyl-(ESDT) and tert-butyl-(TSDT) sarcosinedithiocarbamate. All the obtained compounds have fully been characterised by means of several spectroscopic techniques. In addition, the crystal structure of [Zn(MSDT)₂]₂ dinuclear complex is also reported. In order to evaluate the in vitro cytotoxic properties, some biological assays have been carried out on a panel of human tumour cell lines sensible and resistant to cisplatin. Some of the tested compounds show cytotoxicity levels comparable or even greater than the reference drug (cisplatin).

Full text of DOI:10.1016/j.jinorgbio.2012.09.004

Journal of Inorganic Biochemistry 117 (2012) 131–139
Contents lists available at SciVerse ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Zinc(II) complexes with dithiocarbamato derivatives: Structural characterisation and
biological assays on cancerous cell lines
Eszter Márta Nagy a, Sergio Sitran b, Monica Montopoli c, Monica Favaro b, Luciano Marchiò d,
Laura Caparrotta c, Dolores Fregona a,
⁎
a
Department of Chemical Sciences, University of Padova, Via Marzolo 1, 35131 Padova, Italy
Institute of Inorganic and Surfaces Chemistry, C.N.R. Research Area, Corso Stati Uniti 4, 35127 Padova, Italy
Department of Pharmacology and Anaesthesiology, University of Padova, L.go Meneghetti 2, 35131, Padova, Italy
Department of General and Inorganic Chemistry, Analytical Chemistry, Physical Chemistry, University of Parma, V.le G.P. Usberti 17/A, 43100 Parma, Italy
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 May 2012
Received in revised form 2 September 2012
Accepted 3 September 2012
Available online 13 September 2012
Zinc is one of the most important trace elements in the body and it is essential as a cofactor for the structure
and function of a number of cellular molecules including enzymes, transcription factors, cellular signalling
proteins and DNA repair enzymes. On the other hand, recent studies have shown that zinc could play a
role both in the development of various cancers and in the induction of apoptosis in some cell types, howev-
er, no established common relationships of zinc with cancer development and progression have been iden-
tified. To date, in our research group different metal-dithiocarbamato complexes have been designed that
were expected to resemble the main features of cisplatin together with higher activity, improved selectivity
and bioavailability, and lower side-effects. On the basis of the obtained encouraging achievements with other
metals (such as gold and copper) we have decided to enlarge the studies to the complexes of zinc(II) using
the same ligands. Hereby, we report the results on the synthesis and characterisation of ZnL2 complexes
with five different dithiocarbamato derivatives, such as dimethyl-(DMDT), pyrrolidine-(PyDT), methyl-
(MSDT), ethyl-(ESDT) and tert-butyl-(TSDT) sarcosinedithiocarbamate. All the obtained compounds have
fully been characterised by means of several spectroscopic techniques. In addition, the crystal structure of
[Zn(MSDT)2]2 dinuclear complex is also reported. In order to evaluate the in vitro cytotoxic properties,
some biological assays have been carried out on a panel of human tumour cell lines sensible and resistant
to cisplatin. Some of the tested compounds show cytotoxicity levels comparable or even greater than the ref-
erence drug (cisplatin).
Keywords:
Zinc(II) complexes
Dithiocarbamate
Cancer
Cell cycle
Metallopharmaceuticals
© 2012 Elsevier Inc. All rights reserved.
1. Introduction
efficacy due to problems of delivery and penetration, and low selectivity
for the tumour cells causing severe damage to healthy tissues [7,8].
The clinical success of cisplatin and related platinum-based drugs
as anticancer agents [1] encouraged the researchers to discover more
selective and less toxic metallopharmaceuticals [2]. The studies are
based on the observations that using cisplatin in curative therapy is
associated with some serious clinical problems, such as severe normal
tissue toxicity and resistance to the treatment [3,4]. Tumour cells are
a rapidly changing target because of their genetic instability, hetero-
geneity and high rate of mutation, leading to selection and over-
growth of a drug-resistance tumour cell population [5,6]. In fact, the
activity of the anticancer agents is mainly limited by the development
of drug resistance. Furthermore, the therapeutic application of metal
complexes is still an unexplored area of research. To date, many
metal-based chemotherapeutic agents have been developed to treat
or cure a variety of cancers but most of them demonstrate restricted
Zinc is the second most prominent trace metal in the human body
after iron [9]. It is essential to the growth and development of microor-
ganisms, plants and animals, and plays an important role in various bi-
ological systems: it is involved in homeostasis, in immune responses, in
oxidative stress, in apoptosis and in ageing. As an indispensable cofac-
tor, zinc is required for numerous cellular processes and may be a
major regulatory ion in the metabolism of cells. Zinc regulates both en-
zymatic activity and the stability of the proteins as an activator or as an
inhibitor ion [10,11]. In addition, it is essential for neurogenesis,
synaptogenesis, neuronal growth, and neurotransmission [12]. Zinc is
not stored in the body and excess intakes result in reduced absorption
and increased excretion. In addition, it is neither cytotoxic nor system-
atically toxic, not carcinogenic, mutagenic or teratogenic. Nonetheless,
accumulation of zinc in cells has wide-ranging effects from stimulation
to inhibition of cell growth, depending upon its intracellular level and
the cell type [13,14]. In fact, evidence exists that zinc is capable of induc-
ing apoptosis at appropriate concentrations, and that even high
⁎
Corresponding author. Tel.: +39 049 8275159; fax: +39 02 700500560.
E-mail address: dolores.fregona@unipd.it (D. Fregona).
0162-0134/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jinorgbio.2012.09.004

132
E.M. Nagy et al. / Journal of Inorganic Biochemistry 117 (2012) 131–139
concentrations can produce irreversible changes in exposed cells lead-
ing to toxic cell death. Apoptosis or ‘programmed cell death’ is a generally
regulated mechanism of cell turnover that occurs during embryonic de-
velopment, normal cellular homeostasis and spontaneous or drug-
induced tumour cell death [15]. Experimental studies have shown a sig-
nificant efficacy of zinc in the prevention of colon and thyroid cancer
through induction of cell cycle arrest and apoptosis [16,17]. Moreover, a
number of Zn(II) complexes have been utilised as insulinomimetic and
anti-diabetic [12,18,19], antibacterial or antimicrobial agents [20–24].
Despite the variety of physiological roles of the zinc(II) ions and the
wide repertoire of its complexes utilised in biological field, only a few
data on the cytotoxicity of zinc-based compounds against human can-
cer cell lines can be found [17,25–28].
In the last years, we have designed and synthesised new platinum(II),
palladium(II), gold(I/III), ruthenium(III) and copper(II) complexes with
dithiocarbamato derivatives as potential anticancer drugs, to obtain com-
pounds with superior chemotherapeutic index in terms of increased bio-
availability, higher cytotoxicity and lower side effects than cisplatin
[29–40]. Some complexes have shown very low toxicity levels, in partic-
ular regarding their nephrotoxicity [41]. Dithiocarbamato ligands are a
class of metal chelating compounds that have previously been used
for bacterial and fungal infections and in the treatment of AIDS
[42,43]. In addition, dithiocarbamates are known to exert pro-oxidant
and anti-oxidant effects both in vitro and in vivo [44,45]. For example,
pyrrolidine dithiocarbamate (PyDT) potently inhibits the activation of
NF-κB, a transcription factor that plays a pivotal role in the expression
of a wide range of genes involved in chronic inflammatory diseases.
However, the real molecular mechanisms through which PyDT could
elicit antioxidant effects in a cellular setting, remain unclear [46,47].
Furthermore, dithiocarbamates in conjunction with Cu(II) have been
found to induce apoptosis in different types of cancer cells, acting as po-
tent proteasome inhibitor and apoptosis inducer [48–50]. Most recently,
we have reported on new Cu(II) complexes with methyl-, ethyl and
tert-buthylsarcosinedithiocarbamato ligands that have generally shown
a good activity on a panel of human tumor cell lines [38]. Additionally,
the studied complexes have been found active toward cisplatin resistant
cell lines too, ruling out the occurrence of cross-resistance phenomena.
Moreover, these complexes could inhibit the activity of a purified 20S
and 26S proteasome in human breast cancer cell cultures and xenografts
as it has been found with gold(III) dimethyldithiocarbamato complex
[51].
Taking advantage of both the bioavailability and versatile coordi-
nation ability of the zinc(II) ion, which does not change its oxidation
state as opposed to copper(II), prompted us to explore the role of this
metal in the growth and survival of cancer cells designing new Zn(II)
dithiocarbamate complexes as potential anticancer agents with low
toxicity in vivo [41] and most likely with new cellular targets and
mode of action, in comparison to the traditional metal-based drugs.
Hereby we report on the synthesis of new Zn(II) dithiocarbamato
complexes derived from the esters of methyl-, ethyl and tert-
buthylsarcosine. The corresponding Zn(II) complexes with dimethyl-
dithiocarbamato (DMDT) and PyDT have also been synthetised for com-
parison. All complexes have been characterised by elemental analysis,
1H NMR, 13C NMR and FT-IR spectroscopies, UV–visible spectrophotome-
try and thermogravimetric analyses. In addition, single crystals of
[Zn(MSDT)2]2 dinuclear complex have been obtained and the X-ray struc-
ture has been determined. Moreover, cytotoxic activity assays have been
carried out with all the complexes on different human tumour cell lines.
hydrochloride from Fluka; carbon disulphide and (methyl sulfoxide)-d6
from Aldrich. All other reagents and solvents (Aldrich and Fluka) were
of high purity and used as purchased without any further purification.
2.2. Instrumentation
Electronic absorption spectra were recorded at 298 K in dimethyl-
sulfoxide over the range 250–700 nm on a Perkin-Elmer Lambda 15 dou-
ble beam spectrophotometer. The path length was 1 cm (quartz cell), the
metal complex concentrations were in the range 1.5–2.5·10−5 M.
FT-IR spectra were recorded at room temperature in nujol between
two polyethylene tablets on a Nicolet Nexus 870 FT-IR spectrophotom-
eter in the range 600–50 cm−1, and in solid KBr on a Nicolet-Nexus
5SXC FT-IR spectrophotometer in the range 4000–400 cm−1
.
1H NMR spectra were recorded at 298 K in (dimethyl-sulfoxide)-d6
on a Bruker Avance DRX300 spectrophotometer equipped with a
Silicon Graphics workstation operating in Fourier transform, using
tetramethylsilane (TMS, δ=0.00 ppm) as external standard.
The thermogravimetric (TG) and differential scanning calorimetry
(DSC) curves were recorded with a TA Instruments thermobalance
equipped with a DSC 2929 calorimeter; the measurements were carried
out in the range 25–1300 °C in alumina crucibles under air (flux rate
30 cm3 min−1) and at a heating rate of 10 °C min−1, using alumina
as reference.
Elemental analyses were carried out on
microanalyser.
a Leco CHNS-932
Single crystal data were collected with a Bruker Smart APEXII area
detector diffractometer, Mo Kα: λ=0.71073 Å. A summary of data
collection and structure refinement for [Zn(MSDT)2]2 is reported in
Table 1. The unit cell parameters were obtained using 60 ω-frames
of 0.5° width and scanned from three different zones of reciprocal lat-
tice. The intensity data were integrated from several series of expo-
sures frames (0.3° width) covering the sphere of reciprocal space
[52]. An absorption correction was applied using the program
SADABS [53] with minimum and maximum transmission factors of
0.641 and1.000, respectively. The structure was solved by direct
methods (and SIR2004 [54]) and refined on F2 with full-matrix least
squares (SHELXL-97 [55]), using the Wingx software package [56].
Non hydrogen atoms were refined anisotropically for all compounds
and the hydrogen atoms were placed at their calculated positions.
Graphical material was prepared with the ORTEP3 for Windows
[57]. CCDC 832168 contains the supplementary crystallographic
data for this paper.
Table 1
Summary of the structure determination for [Zn(MSDT)2]2.
Chemical formula
Formula weight
Colour, habit
T [K]
C10H16N2O4S4Zn
421.86
Colourless, block
293(2)
λ [Å]
0.71073
Crystal system
Space group
a [Å]
b [Å]
c [Å]
Monoclinic
P21/c
10.627(1)
11.659(1)
14.838(2)
106.774(1)
1760.2(3)
4
β [deg]
V [Å3]
Z
ρ (calc) [Mg/m3]
1.592
1.882
μ [mm−1
]
2. Experimental
θ range [deg]
No. of rflcn/unique
GooF
R1
2.00 to 30.61
28105/5417
1.006
2.1. Chemicals
0.0321
wR2
0.0797
Zn(II) chloride was purchased from J.T. Baker-Chemicals;
sodium N,N-dimethyldithiocarbamate hydrate, ammonium
pyrrolidinedithiocarbamate and methyl-/ethyl-/tert-butylsarcosine
R1=Σ||Fo|–|Fc||/Σ|Fo|, wR2=[Σ[w(Fo2–Fc2)2]/Σ[w(Fo2)2]]½, w=1/[σ2(Fo2)+(aP)
2+bP], where P=[max(Fo2,0)+2Fc2]/3.

E.M. Nagy et al. / Journal of Inorganic Biochemistry 117 (2012) 131–139
133
S
S
S
S
H3C
H3C
S
S
S
S
CH3
CH3
N
C
Zn
C
N
N
C
Zn
C
N
[Zn(DMDT) 2]
[Zn(PyDT)2]
O
CH2 C
CH3
H3C
H2C
S
S
S
O
O
R
N
C
Zn
Zn
C
N
N
S
R
O
C
O
R = Et[Zn(ESDT)2]
tBu [Zn(TSDT)2]
O
CH2 C
CH3
H3C
H2C
S
S
Me
N
C
C
S
Me
O
C
O
S
S
O
H3C
H2C
S
S
CH3
N
C
Zn
C
N
CH2 C
O Me
S
Me O
C
O
[Zn(MSDT)2]2
Fig. 1. Chemical drawing of the investigated zinc(II) dithiocarbamato complexes.
Observation of samples and identification of elements have been
performed by SEM Philips Model XL 40 LaB6 instrument. The semi-
quantitative elemental analyses were obtained using an EDAX DX
Prime X-ray energy dispersive spectrometer equipped with a thin be-
ryllium window, using an accelerating voltage of 25 keV. Analyses
were performed in high vacuum condition and the samples were car-
bon coated before SEM–EDS investigation.
CS2 and NaOH in equimolar ratio. After 1 h pH value turned from
10–11 to 6–7, according to the reaction pathway previously reported
[38]. Thus the above prepared solution was slowly added to an aqueous
solution of ZnCl2 in 1:2 metal-to-ligand molar ratio under vigorous stir-
ring, leading to the immediate precipitation of a white solid. The solid
was filtered off, washed with water and dried in vacuum over P4O10
,
giving a final yield of 77–85%.
2.3. Synthesis of the metal complexes
2.3.2.1. Bis(μ-methyl-N-(dithiocarboxy-kS,kS’)-N-methylglycinato)-
[bis(methyl-N-(dithiocarboxy-kS,kS’)-N-methylglycinato)zinc(II)]
[Zn(MSDT)2]2. (Found: C, 28.51; H, 3.85; N, 6.48; S, 30.50. Calc. for
2.3.1. [Zn(DMDT)2] and [Zn(PyDT)2]
Solid ZnCl2 was added to an aqueous solution of sodium N,
C
10H16ZnN2O4S4 (M.W. =421.38 g/mol): C, 28.47; H, 3.79; N, 6.64;
S, 30.37%) FT-IR (KBr or nujol, νmax/cm−1): ν(C_O)=1747, 1739;
ν(N\CSS)=1494; ν(C\OMe)=1217, 1215; νa,s(SCS)=968, 611;
N-dimethyldithiocarbamate
hydrate
or
ammonium
pyrrolidinedithiocarbamate in molar ratio metal-to-ligand 1:2,
under vigorous stirring leading to the immediate precipitation of a
white solid. The precipitate was filtered off, washed with water and
n-pentane and dried in vacuum over P4O10, giving a final yield of
75–80%.
ν
a,s(ZnS)=381, 369. 1H NMR: δH (DMSO-d6, ppm): 3.38 (6H, s,
CH3\O); 3.67 (6H, s, CH3N); 4.70 (4H, s, CH2\N). 13C NMR: δC
(DMSO-d6, ppm): 44.18 (CH3\N); 52.32 (CH3\O); 57.56
(CH2\N); 168.36 (COO); 207.39 (CS). EDS calculated element
ratio Zn:S=0.26.
2.3.1.1. [Bis(dimethylcarbamodithioato-kS,kS′)zinc(II)] [Zn(DMDT)2].
(Found: C, 23.72; H, 4.04; N, 9.12; S, 41.88. Calc. for C6H12ZnN2S4
(M.W.=305.78 g/mol): C, 23.57; H, 3.96; N, 9.16; S, 41.94%) FT-IR
(KBr or nujol, νmax/cm−1): ν(N-CSS)=1525 br; νa,s(SCS)=974,
565; ν(ZnS)=381. 1H NMR: δH (DMSO-d6, ppm): 3.38 (12H, s,
CH3N). 13C NMR: δC (DMSO-d6, ppm): 44.64 (CH3\N); 203.72 (CS).
EDS calculated element ratio Zn:S=0.25.
2.3.2.2. [Bis(ethyl-N-(dithiocarboxy-kS,kS’)-N-methylglycinato)zinc(II)]
[Zn(ESDT)2]. (Found: C, 32.06; H, 4.52; N, 6.18; S, 28.70. Calc. for
C
12H20ZnN2O4S4 (M.W. =449.38 g/mol): C, 32.04; H, 4.45; N,
6.23; S, 28.48%) M.p. =219.3 °C (dec.). FT-IR (KBr or nujol,
ν
ν
max/cm−1): ν(C_O)=1737; ν(N\CSS)=1498; ν(C-OEt)=1206;
a,s(SCS)=971, 576; νa,s(ZnS)=382. 1H NMR: δH (DMSO-d6, ppm):
1.99 (6H, t, CH3\CH2); 4.13 (4H, q, CH3\CH2); 3.38 (6H, s, CH3N),
4.68 (4H, s, CH2\N). 13C NMR: δC (DMSO-d6, ppm): 14.10
(CH3\CH2); 43.99 (CH3\N); 57.48 (CH2\N); 60.93 (CH3\CH2);
167.61 (COO); 207.14 (CS). EDS calculated element ratio Zn:S=0.24.
2.3.1.2. [Bis(1-pyrrolidinecarbamodithioato-kS,kS′)zinc(II)] [Zn(PyDT)2].
(Found: C, 33.52; H, 4.52; N, 7.76; S, 35.70. Calc. for C10H16ZnN2S4
(M.W.=357.90 g/mol): C, 33.56; H, 4.50; N, 7.83; S, 35.83%) FT-IR
(KBr or nujol, νmax/cm−1): ν(N\CSS)=1482, 1439; νa,s(SCS)=945,
582; ν(ZnS)=344. 1H NMR: δH (DMSO-d6, ppm): 1.97 (8H, m,
CH2\CH2); 3.59 (8H, m, CH2\N). 13C NMR: δC (DMSO-d6, ppm):
26.13 (CH2\CH2); 54.05 (CH2\N); 199.21 (CS). EDS calculated ele-
ment ratio Zn:S=0.27.
2.3.2.3. [Bis(ter-butyl-N-(dithiocarboxy-kS,kS’)-N-methylglycinato)
zinc(II)] [Zn(TSDT)2]. (Found: C, 38.12; H, 5.60; N, 5.48; S,
25.19. Calc. for
C16H28ZnN2O4S4 (M.W. =505.38 g/mol): C,
37.99; H, 5.54; N, 5.54; S, 25.33%) M.p.=196.3 °C (dec.). FT-IR (KBr
or nujol,
ν
max/cm−1): ν(C_O)=1741, 1730; ν(N\CSS)=1501;
2.3.2. [Zn(RSDT)2]
ν(C-OtBu)=1154; νa,s(SCS)=970, 563; νa,s(ZnS)=381. 1H NMR: δH
(DMSO-d6, ppm): 1.43 (18H, s, (CH3)3C); 3.38 (6H, s, CH3\N), 4.58
(4H, s, CH2\N). 13C NMR: δC (DMSO-d6, ppm): 27.75 (CH3\C); 44.03
The dithiocarbamato ligands were prepared in water reacting the
R-sarcosinehydrochloride (RSHCl=(RO(O)CCH2N(CH3)H2]+Cl–) with

134
E.M. Nagy et al. / Journal of Inorganic Biochemistry 117 (2012) 131–139
the cells were collected with trypsin and fixed on ice with cold 70%
ethanol for 30 min, washed and resuspended in PBS solution
containing 0.05 mg/ml propidium iodide (Molecular Probes,
Invitrogen, UK) and 0.2 μg/ml RNAase–DNAase free (Sigma, St.
Louis, MO, USA) and incubated for 30 min at room temperature in
the dark. About 3·104 cells/sample were analysed on Epics XL-flow
cytometre (Beckman Coulter, USA), with an argon laser λexc 488 nm
and a photomultiplier PMT2 at λem 575 20 nm. The percentages of
cells in the different phases of cell cycle were calculated with the
Multicycle software provided by the manufacturer, considering dip-
loid cycle and correcting for cell clusters.
(CH3\N); 58.15 (CH2\N); 81.50 (C\CH3); 168.17 (COO); 211.41 (CS).
EDS calculated element ratio Zn:S=0.27.
2.4. Biological assay
2.4.1. Cell culture
The 2008 cell line derived from human ovarian carcinoma and the
A431 cells from human cervix squamous carcinoma. Their parental
cisplatin-resistant variants (C13 and A431/Pt respectively) were gen-
erated by in vitro exposure to increasing concentrations of cisplatin
and selection until persistent 9 to 12-fold resistance to cisplatin
[58,59]. The cells were routinely grown in humidified condition at 5%
CO2 and 37 °C, incubated with RPMI 1640 medium, 10% FBS, 2% gluta-
mine and 1% pen-strep. The cells were collected every 2 days with min-
imum amount of 0.05% trypsin–0.02% EDTA and seeded 1·106 in
100 mm dish with RPMI 1640 medium, 10% FBS, 2% glutamine and 1%
pen-strep. All reagents were from Lonza (Basel, Switzerland).
2.4.4. Statistical analysis
Analysis of data has been performed by using GraphPad Prism
software (San Diego, CA). Cytotoxicity data are the means of quadru-
plicate determinations from at least three independent experiments
with 95% confidence interval. Cell cycle data are expressed as per-
centage of cell population in each cell phase and are the means
SEM from at least three independent experiments. Comparison of dif-
ferences among groups was carried out using a two-tailed Student's t
test. Significance was considered at pb0.05.
2.4.2. Cytotoxicity
Cell viability was determined by the 3-(4,5-dimethyl-thizol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As the tested
compounds are poorly soluble in water, stock solutions of the Zn(II)
complexes and cisplatin were made in dimethyl sulfoxide and then
diluted with complete medium giving a final concentration of 0.4%
(v/v) of dimethyl sulfoxide. At this concentration dimethyl sulfoxide
has no discernible effect on cell killing. All the tested complexes
have been proved, by 1H NMR studies, to be stable in dimethyl sulfox-
ide over 72 h. Cells were seeded in 96-well microtitre plates and fol-
lowing overnight incubation, they were exposed for 24 h to a range of
different concentrations of the testing compounds, then the medium
was changed and the cells were incubated for 48 h further. At the end
of the incubation the cells were added with a solution of 20 μl/well
MTT (5 mg/ml medium) (Sigma, St. Louis, MO, USA), incubated for
4 h at 37 °C and then lysed by the addition of 200 μl of isopropanol
acid. The absorbance at 570 nm was determined at Victor3, multilabel
counter, Wallac Instruments. The reported values are the average of
triplicate determinations on at least three separate experiments. IC50
for each compound tested was calculated from the dose–response cyto-
toxic curves as the concentration causing the 50% reduction of cell num-
ber in comparison to untreated cells.
3. Results and discussions
3.1. Synthesis of the metal complexes
For the synthesis of [Zn(DMDT)2] and [Zn(PyDT)2] the commer-
cially available N,N-dimethyldithiocarbamate sodium salt and
pyrrolidinedithiocarbamate ammonium salt were reacted directly
with ZnCl2 in aqueous solution using 1:2 metal to ligand molar
ratio. The immediate formation of the complexes as white precipi-
tate was observed.
Since all the attempts to isolate the sarcosinedithiocarbamato de-
rivatives without causing their degradation during the separation/
purification process were proved unsuccessful, the corresponding
[Zn(RSDT)2] complexes were obtained by a one-pot reaction. Firstly
the methyl-, ethyl- and tert-butylsarcosine hydrochloride was reacted
with CS2 and NaOH in equimolar ratio at 0 °C in an aqueous solution,
followed by ZnCl2 in 1:2 metal to ligand molar ratio yielding the com-
plexes as white precipitate. Elemental analysis confirmed the Zn:L 1:2
stoichiometry for all the five derivatives. The proposed chemical
structures of each zinc(II) complex are shown in Fig. 1. Moreover,
the formation of a dinuclear species was found in the case of the
methylsarcosinedithiocarbamate derivative (MSDT) that was con-
firmed by both the FT-IR and Thermal analyses (see Sections 3.3
2.4.3. Cell cycle and apoptosis
The cells were seeded at a density of 1.5·105 cells/6 wells and
after 24 h of incubation they were exposed to testing compounds
according to the experimental protocol. At the end of the treatment
Fig. 2. Ortep drawing of [Zn(MSDT)2]2 at the 30% thermal ellipsoids probability level. Symmetry code=−x; −y; −z.

E.M. Nagy et al. / Journal of Inorganic Biochemistry 117 (2012) 131–139
135
Table 2
Selected bond lengths (Å) and angles (°) for [Zn(MSDT)2]2.
bond (1350–1250 cm−1) and a double bond (1690–1640 cm−1
)
[74]. In fact, passing from DMDTNa (νN\CSS=1486 cm−1) or
PyDTNH4 (νN\CSS=1410 cm−1
) to its metal derivatives, the
Zn\S(11)
Zn\S(12)
Zn\S(21)
Zn\S(22)′
S(22)′\Zn\S(21)
S(22)′\Zn\S(12)
S(12)\Zn\S(11)
2.4616(6)
2.3590(5)
2.3329(6)
2.3117(6)
129.46(2)
108.91(2)
106.96(2)
C(11)\S(11)
C(11)\S(21)
C(12)\S(12)
C(12)\S(22)
S(21)\Zn\S(12)
S(22)’\Zn\S(11)
S(21)\Zn\S(11)
1.711(2)
1.733(2)
1.737(2)
1.715(2)
117.94(2)
108.24(2)
75.99(2)
ν(N\CSS) vibrational mode is shifted to higher energies, showing
an increase of the N\C double bond character. For the ligands
obtained through the template reaction this correlation cannot be
done since the isolation of the free R-sarcosinedithiocarbammic
acid or salt precursor is not possible as they are instable in solid
phase. Anyway, for all the zinc(II) complexes the values recorded
for ν(N\CSS) modes are in agreement with the results reported
in the literature for analogous metal derivatives of α-amino acid
dithiocarbamates [25–27]. Furthermore, the presence of only one
band in the region 1050–950 cm−1, assigned to ν(S\C\S) asym-
metric mode, is assumed to indicate a symmetrical bonding of the
dithiocarbamato moiety, acting in a bidentate mode [28]. In addition,
the 420–240 cm−1 region is associated with ν(M\S) vibrations [51].
In fact, new bands in the spectra of the metal complexes are observed
in the 350–380 cm−1 region, absent in the case of free ligands, that
can be attributed to the zinc–sulphur stretching [75]. Moreover, two
bands for ν(Zn\S) vibrations at 381 and 369 cm−1 are observed for
the [Zn2(MSDT)4] dinuclear complex, confirming the presence of two
different metal–ligand coordination modes (asymmetric coordination
of the ligand) in solid phase, as it has been proved by the crystal struc-
ture of the complex.
Symmetry code=−x; −y; −z.
and 3.4). As well, the dimeric structure of the Zn–MSDT complex has
also been verified by X-ray characterisation, corresponding to
[Zn(MSDT)2]2 (i.e. [Zn2(MSDT)4]).
3.2. Structural characterisation of [Zn(MSDT)2]2
Crystallographic data are given in Table 1, while molecular struc-
ture and selected bond distances are shown in Fig. 2 and Table 2, re-
spectively. The complex crystallises as a dimer in the monoclinic
space group P21/c. The asymmetric unit is represented by
a
Zn(MSDT)2 fragment, and the presence of a center of symmetry gen-
erates a dinuclear structure. Each metal is coordinated by four sul-
phur atoms deriving by three different ligands. One ligand acts as S,
S chelate with the S(11) and S(21) atoms, whereas the second ligand
bridges on two metal centres with the S(12) and S(22) atoms. The
small bite angle (~76°) of the chelate ligand is mainly responsible of
the distortion from the ideal tetrahedral geometry. This is also
reflected in the asymmetric coordination of the chelate ligand, which
is evidenced in the elongation of the Zn–S(11) distance (2.4616(6) Ǻ)
with respect to the Zn\S(12) one (2.3590(5) Ǻ). On the opposite, the
Zn\S distances that pertains to the bridging dithiocarbamate ligand
are in the narrow range of 2.3117(6)–2.329(6) Ǻ. The peripheral ester
moieties are arranged perpendicularly to the dithiocarbamato donor
moiety.
3.4. Thermal analysis
The thermal behavior of the synthesised compounds have been
studied by thermogravimetry (TG) and differential scanning calorim-
etry (DSC) techniques in a dynamic atmosphere of air, in order to es-
tablish the different decomposition processes and to confirm the
proposed stoichiometry. The experimental data agree to a good ex-
tent with the ones obtained by the other spectroscopic techniques,
and the results of such analysis, summarised in Table 3, indicate a
good correlation between calculated and found weight loss values
for all the investigated compounds.
As concerns the free dithiocarbamato ligands, the decomposition
process of DMDTNa∙H2O takes place in two well-defined steps. The
first weight loss at around 100 °C coincides with the endothermic
DSC peak and corresponds to the loss of the related crystallisation
water molecules. A second weight loss is recorded at higher temper-
atures (>200 °C) when the pyrolysis process leads to the total con-
version to sodium sulphate (data not shown).
The thermal behaviour of the metal complexes was recorded up to
1200 °C in air flux. Each of the zinc complexes undergoes a different
degradation pathway, leading to the corresponding metal-oxide
(ZnO) as final product for [Zn(PyDT)2], [Zn(ESDT)2] and [Zn(TSDT)2],
while to the metal (Zn) for [Zn(DMDT)2] and [Zn2(MSDT)4]. EDS
analysis after thermal degradation was performed to identify the na-
ture of the residue (Fig. S1 for the [Zn(DMDT)2] before and after
In the literature a number of dinuclear Zn(II) dithiocarbamato com-
plexes that exhibit the same type of Zn\S core of [Zn(MSDT)2]2 are
reported, and they differ mainly by the arrangements of functional
groups that decorate the peripheral dithiocarbamato ligand [60–73].
3.3. FT-IR spectroscopy
In the FT-IR spectra of all the synthesised complexes the
ν(N\CSS) band is observable in the region 1539–1493 cm−1. This
band defines a N\C bond order intermediate between a single
Table 3
Thermogravimetric (TG) and differential scanning calorimetric (DSC) data in air flux.
Decomposition TG/weight DSC/peak temperature [°C]
interval [°C]
loss [%]
(calc.)
(process)a
[Zn(DMDT)2] 250–800
−78.52
−77.69
(−77.26
to ZnO)
−83.64
(−84.50
to Zn)
−81.63
(−81.91
to ZnO)
−84.10
(−83.92
to ZnO)
251(endo-fus)/323(endo)/642(exo)
146(endo)/267(endo)/308(endo)
331(exo)/504(exo)/661(exo)
Table 4
[Zn(PyDT)2]
[Zn2(MSDT)4]
[Zn(ESDT)2]
[Zn(TSDT)2]
127–700
75–1100
170–1200
100–1200
UV–visible spectral data for the free DMDT and PyDT ligands, and for the Zn(II)
dithiocarbamato complexes in dimethyl sulfoxide.
130(endo)/212(endo)/563(exo)/
761(exo)1037(exo)
λmax in nm (ε, M−1 cm−1
)
DMDT
PyDT
262.0 (11,840)
263.0 (20,420)
260.7 (38,850)
261.2 (49,740)
261.0 (44,120)
261.2 (33,970)
261.2 (21,780)
299.0 (13,220)
297.0 (21,380)
277.0 (30,500)
275.2 (36,650)
281.0 (32,360)
280.6 (24,280)
281.2 (15,450)
115(endo)/219(endo)/320(exo)/
687(exo)/1050(exo)
[Zn(DMDT)2]
[Zn(PyDT)2]
[Zn2(MSDT)4]
[Zn(ESDT)2]
[Zn(TSDT)2]
~295 sh (~10,000)
~295 sh (~11,000)
~300 sh (~19,000)
~300 sh (~14,000)
~300 sh (~10,000)
136(endo)/196(endo)/525(exo)/
658(exo)/1050(exo)
a
Endo/exo/fus=endothermic/exothermic/fusion process.
sh — shoulder.

136
E.M. Nagy et al. / Journal of Inorganic Biochemistry 117 (2012) 131–139
[Zn(DMDT)2]
[Zn(ESDT)2]
[Zn2(MSDT)4]
[Zn(PyDT)2]
[Zn(TSDT)2]
CisPt
C13
2008
100
75
50
25
0
100
75
50
25
0
-9
-8
-7
-6
-5
-4
-3
-9
-8
-7
-6
-5
-4
-3
[LogM]
[LogM]
A431
A431 Pt
100
75
50
25
0
100
75
50
25
0
-9
-8
-7
-6
-5
-4
-3
-9
-8
-7
-6
-5
-4
-3
[LogM]
[LogM]
Fig. 3. Cytotoxicity of zinc(II) dithiocarbamato complexes in 2008 and A431 cell lines and in cisplatin-resistant variants (C13 and A431Pt). Cells were exposed to the complexes for
24 h and cell viability was measured by MTT assay. Results are expressed as a percent of absorbance unit of untreated cells. Mean SEM is of at least three independent
experiments.
thermal degradation). For detailed description of the thermograms of
each zinc(II) complex see Supplementary Materials.
Moreover, the correlation between absorbance and concentration on
the UV–visible spectra of the zinc(II) complexes has been studied. The re-
sults of the [Zn(DMDT)2] and [Zn(MSDT)2]2 complexes are shown in the
Supplementary material as example (Figs. S4 and S5). While a linear re-
lation between absorbance and concentration is observed with all the
mononuclear complexes, deviation from Lambert–Beer law occurs
for [Zn(MSDT)2]2. This condition confirms the presence of dimeric
[Zn(MSDT)2]2 species in solution.
3.5. Electronic absorption spectroscopy
The UV–visible spectra of the Zn(II) dithiocarbamato complexes in
dimethyl-sulfoxide are shown in electronic supplementary informa-
tion (Fig. S3). The electronic spectroscopic data of the DMDT and
PyDT ligands and the studied metal complexes are summarised in
Table 4.
3.6. Biological assay
Two intense absorption bands are present on the spectra of the
free ligands at around 260 nm and 300 nm (Table 4 for DMDT and
PyDT, while RSDT ligands were prepared in situ and were not isolated
as pure compounds). These bands with very large molar extinction
Human ovarian and cervix carcinoma cell lines (2008 and A431) and
their cisplatin-resistant subclones (C13 and A431Pt) were exposed for
24 h to zinc(II) dithiocarbamato complexes or to cisplatin, as reference
drug, all at 0.01−100 μM concentration range. Cytotoxicity was mea-
coefficients (ε~105 M−1 cm−1
) correspond to the intraligand
π ←π transition located on the N\C\S and the S\C\S moieties of
the dithiocarbamato ligands, respectively [76].
sured by MTT assay (Fig. 3) and the results clearly show
a
⁎
concentration-dependent inhibition of cancer cell viability by all the
zinc compounds. IC50 values calculated from the concentration–response
curves are reported in Table 5.
For all the studied zinc(II) complexes the absorption spectra show a
similar pattern with three strong absorption bands in the UV region
(250–320 nm, Table 4) that are probably assigned to the above-
mentioned intraligand transitions [36,77,78]. In addition, Zn(II) ion has
3d10 electron configuration therefore no d–d or LMCT bands are feasible
due to the completely filled d orbitals [79]. The band at ~260 nm appears
almost at the same position for all complexes, while the band at ~280 nm
and at ~300 nm (as a shoulder) are slightly shifted to lower energies
(Δ~5 nm) in the case of the sarcosinedithiocarbamato derivatives
(RSDT), respect to the complexes with DMDT and PyDT. Indeed, the for-
mer bands are usually dependent on the nature of the alkyl moieties
bound to the nitrogen atom [80]. Besides, the intraligand π*←n transi-
tion of both the in-plane non-bonding sulphur orbital and the carbonyl
group of the sarcosinedithiocarbamato moiety should be recorded [76].
However these are low intensity bands and are not always detected due
to the overlap of the more intense bands.
As expected, cisplatin is more potent in wild-type cancer cells
(0.19 μM and 0.78 μM in A431 and 2008 cells, respectively) than in re-
sistant subclones (3.5 μM and 5.4 μM in A431Pt and C13). However,
some of the zinc complexes are even more potent than cisplatin, such
as [Zn(DMDT)2], that is the most active compound (IC50 b0.9 μM in all
cell lines). Furthermore, [Zn(DMDT)2], [Zn2(MSDT)4] and [Zn(PyDT)2]
show similar potency in wild-type 2008 and cisplatin-resistant C13
cells, indicating the lack of cross-resistance with cisplatin. Moreover,
[Zn(DMDT)2], [Zn(ESDT)2] and [Zn(TSDT)2] are all more active than cis-
platin in cisplatin-resistant A431Pt cells.
The effect of zinc complexes and cisplatin has also been investigat-
ed on cell cycle of 2008–C13 and A431–A431Pt cells. Exemplificative
cytograms for cisplatin and [Zn(DMDT)2] are shown in Fig. 4. It can
be observed that cisplatin and [Zn(DMDT)2] affect in a different way
on the cell distribution in the cycle phase, without suggesting a

E.M. Nagy et al. / Journal of Inorganic Biochemistry 117 (2012) 131–139
137
Table 5
pronounced in wild type (2008 and A431) than in cisplatin resistant
(C13 and A431Pt) cancer cells. The zinc complexes in 2008 and C13
cells (Fig. S7) caused significant apoptosis (sub G0/G1) but unlike cis-
platin, they induced mainly decreased G2/M phase; in A431 and
A431Pt cells (Fig. S8) similarly decreased G2/M, also altering S phase.
IC50 values of Zn(II) dithiocarbamato complexes and cisplatin (CDDP) in 2008, C13,
A431 and A431Pt cell lines. Each value expressed as micromolar concentration repre-
sents the average of at least three sets of independent experiments.
Mean IC50 (μM) 95% CI
2008
C13
A431
A431Pt
4. Conclusions
CDDP
0.19
3.54
0.78
5.45
0.06 to 0.54
0.74 to 17.04
0.26 to 0.60
3.35 to 8.85
Cisplatin was approved by the U.S. Food and Drug Administration
in 1979 as the first metal-based compound to block DNA replication
and cell division. Despite its high neuro- and nephrotoxicity and ac-
quired drug resistance as major drawbacks, cisplatin and its ana-
logues are currently amongst the most effective chemotherapeutic
agents in clinical use. The enormous success of cisplatin has raised
great interest in the study of platinum and non-platinum metal com-
plexes to be used as antitumour agents. Recently, we have reported
on a new class of metal complexes of dithiocarbamato ligands
(containing gold, platinum, palladium, ruthenium and copper) with
greater cytotoxic activity than cisplatin; moreover, in most cases
they showed no cross-resistance with cisplatin and very low in vitro
and in vivo nephrotoxicity levels in comparison to the reference
drug [29–40].
Based on these promising results and by the bioavailability of
the zinc(II) ion, we planned to design and study new zinc(II)
dithiocarbamato complexes as potential chemotherapeutics. In
this paper we have shown the synthesis and the detailed character-
isation of five bis(dithiocarbamato)zinc(II) complexes. By different
spectroscopic techniques we have established the (S,S) chelating na-
ture of the ligands. In addition, the X-ray structure of the
metylsarcosineditiocarbamato complex was also determined and it
revealed the dimeric structure of the complex, in which one ligand
[Zn(DMDT)2]
[Zn(PyDT)2]
[Zn2(MSDT)4]
[Zn(ESDT)2]
[Zn(TSDT)2]
0.22
0.08 to 0.57
0.17
0.04 to 0.65
1.72
0.12 to 14.68
0.17
0.04 to 0.75
1.03
0.44 to 2.41
0.87
0.37 to 2.05
0.82
0.35 to 1.89
2.00
0.75 to 5.70
5.70
4.20 to 7.83
4.31
1.15 to 16.00
0.62
0.14 to 2.65
2.10
0.59 to 7.55
9.81
7.13 to 13.54
12.34
4.12 to 36.95
1.90
0.63 to 5.69
0.10
0.02 to 0.52
6.20
0.41 to 5.14
23.00
9.18 to 37.00
0.29
0.05 to 1.59
0.54
0.14 to 2.08
CI — confidence intervals.
different mechanism of action. Furthermore, it clearly appears that
[Zn(DMDT)2] is equipotent in wild type and in cisplatin-resistant can-
cer cells, compared to the reference drug.
Detailed results of the cytofluorimetric assay, describing the effects
of Zn(II) complexes and cisplatin on the percentage of cell in the differ-
ent cycle phases (G0/G1, S, G2/M) or apoptosis (sub G0/G1) are shown in
Supplementary material (Figs. S6–S8). Cisplatin (Fig. S6) caused a sig-
nificant concentration-dependent reduction of G0/G1 as well an in-
crease of S and G2/M phases and of apoptosis (sub G0/G1), more
2008
C13
A431
A431Pt
(1)
(2)
(3)
Fig. 4. Cell cycle cytograms of 2008–C13 and A431–A431Pt after 24 h exposure to control (1), cisplatin (2) and [Zn(DMDT)2] (3) (c=1 μM). All pictures are typical of three inde-
pendent experiments each performed under identical conditions. The four labels at the bottom read “FL3 LIN” and the axis tick labels for all 12 panels are 0 to 1023 (X-axis) and 280
(Y-axis).

138
E.M. Nagy et al. / Journal of Inorganic Biochemistry 117 (2012) 131–139
acts as (S,S) chelate, while the second ligand bridges on two metal
centres resulting in distorted tetrahedral coordination to the metal.
Moreover, we demonstrate that zinc(II) dithiocarbamato com-
plexes cause significant reduction on the cell viability in ovarian
(2008) and cervix (A431) human carcinoma cells and in their
cisplatin-resistant variant (C13 and A431Pt respectively). Comparing
the IC50 values of previously studied copper complexes in the same
cancer cell lines [38] with the corresponding zinc derivatives, howev-
er, it is possible to deduce that in general zinc compounds are less po-
tent than copper ones on all the panel of cancer cells. These results
highlight that for the antiproliferative effect the metal centre plays
an important role, although the dithiocarbamato ligands are required
as both stabilizing moieties due to the so-called “chelate effect” and
intrinsic chemoprotectants (likewise sodium diethyldithiocarbamate
used for cisplatin therapy [81]). In the last case the interaction of the
metal centre with biologically-relevant macromolecules is potentially
avoided and, consequently, side-effects are reduced. Nonetheless, free
PyDT has been shown to induce pro-apoptotic and antiproliferative ef-
fects too in some cancer cell lines, such as T-cell leukaemia, lymphoma,
myleogenous leukaemia and renal cell carcinoma [82–85]. On the other
hand, dithiocarbamates themselves (i.e. not complexed) are well
known for their strong affinity to free or protein-bound metals remov-
ing them from organism, altering the metal distribution and affecting
the metal-enzyme activity, so resulting highly toxic (neuro- and hepa-
totoxicity) [86–89]. However, upon coordination to the metal ion, the
resulting complexes are rather stable and possible decomposition
with subsequent loss of the dithiocarbamato ligand is unlikely to
occur. Recently we confirmed this expectation by the in vitro and in
vivo toxicological studies carried out with some Pt(II) and Au(III)
dithiocarbamato derivatives with high anticancer activity and reduced
or no systemic and renal toxicity [32,41,90]. Concerning the Zn(II) com-
plexes reported in this work, further studies are necessary to elucidate
the behaviour of the compounds in the presence of biologically-
relevant macromolecules, as well as to comprehend their mechanism
of action.
strategy looking for differently acting metal antineoplastic drugs with se-
lective cytotoxicity on cancer cell with respect to normal ones.
Abbreviations
DMDT
PyDT
MSDT
ESDT
TSDT
TG
N,N-dimethyldithiocarbamate
pyrrolidinedithiocarbamate
methylsarcosinedithiocarbamate
ethylsarcosinedithiocarbamate
tert-butylsarcosinedithiocarbamate
termogravimetry
DSC
SEM
differential scanning calorimetry
scanning electron microscope
SEM–EDS Scanning Electron Microscope equipped with Energy Dis-
persive Spectrometer
MTT
3-(4,5-dimethyl-thizol-2-yl)-2,5-diphenyltetrazolium
bromide
FBS
fetal bovine serum
PBS
LMCT
phosphate buffered saline
ligand-to-metal charge transfer
Acknowledgements
The authors gratefully acknowledge Dr Loris Calore for the techni-
cal support, Consorzio Interuniversitario di Ricerca in Chimica dei
Metalli nei Sistemi Biologici (CIRCMSB) for the financial support and
the members of Centro Interdipartimentale di Ricerca “Ruolo degli
Ioni Metallici nelle Scienze Biomediche” (Biométion) for the stimulat-
ing discussions.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://
dx.doi.org/10.1016/j.jinorgbio.2012.09.004.
Furthermore, we have analysed the activity of the zinc complexes
on the cell cycle. The results show that the cytotoxicity of the com-
plexes is associated with cell cycle arrest and significant apoptosis
in ovarian and cervix cancer cells, with an evident activity at micro-
molar concentration in both sensitive and cisplatin-resistant cells.
Recently, we have published the cytotoxicity of [Zn(PyDT)2] and
[Cu(PyDT)2] in a panel of breast and prostate cancer cells were associat-
ed with inhibition of proteosomal chymotrypsyn-like activity [50]. In
fact, previous studies have reported the involvement of the ubiquitin–
proteasome pathway in a number of critical cell processes, including an-
tigen processing, selective elimination of misfolded proteins and onco-
genesis [91]. Ubiquitin–proteasome system is also critical for cell
cycling and apoptosis, because it regulates the synchronised degrada-
tion of short-lived proteins, such as cyclins, cyclin-dependent kinases
(cdk), cdk-inhibitors and anti-apoptotic Bcl-2 family, IAPs and caspases
[92]. Furthermore, the deregulation of proteasome catalytic process
may contribute to tumour drug resistance and altered immune surveil-
lance. Despite proteasome plays a relevant role in normal cell homeo-
stasis, it has been selected as a molecular target to force the cancer
cell cycle arrest [93] and apoptosis [94]. Moreover, Geraki et al. shown
that zinc is also required for the catalytic activity of the human
deubiquitinating isopeptidase T [95].
In conclusion the zinc(II) dithiocarbamato complexes affect cell cycle
progression differently as compared to cisplatin, indicating a different
mechanism of action; both cisplatin and [Zn(DMDT)2] are more active
in ovarian cancer than in cervix cancer cells, however, the complexes
are almost equipotent in wild type and in cisplatin-resistant cancer
cells. Even if it remains to be established how the cancer cell phenotype
might influence the potency of zinc-complexes, the versatile coordination
of zinc, that being essential metal, however, differently from copper does
not change its oxidation state in biological environment, might be a novel
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