Chemical and biological profiles of novel copper(II) complexes containing S-donor ligands for the treatment of cancer

Article abstract of DOI:10.1021/ic800404e

In the last years, we have synthesized some new platinum(II), palladium(II), gold(I/III) complexes with dithiocarbamato derivatives as potential anticancer drugs, to obtain compounds with superior chemotherapeutic index in terms of increased bioavailability, higher cytotoxicity, and lower side effects than cisplatin. On the basis of the obtained encouraging results, we have been studying the interaction of CuCl₂ with methyl-/ethyl-/tert- butylsarcosine-dithiocarbamato moieties in a 1:2 molar ratio; we also synthesized and studied the N,N-dimethyl- and pyrrolidine-dithiocarbamato copper complexes for comparison purposes. The reported compounds have been successfully isolated, purified, and fully characterized by means of several spectroscopic techniques. Moreover, the electrochemical properties of the designed compounds have been studied through cyclic voltammetry. In addition, the behavior in solution was followed by means of UV - vis technique to check the stability with time in physiological conditions. To evaluate their in vitro cytotoxic properties, preliminary biological assays (MTT test) have been carried out on a panel of human tumor cell lines. The results show that cytotoxicity levels of all of the tested complexes are comparable or even greater than that of the reference drug (cisplatin).

Full text of DOI:10.1021/ic800404e

Inorg. Chem. 2008, 47, 6336-6343
Chemical and Biological Profiles of Novel Copper(II) Complexes
Containing S-Donor Ligands for the Treatment of Cancer
†
Lorena Giovagnini, Sergio Sitran, Monica Montopoli, Laura Caparrotta, Maddalena Corsini,
‡
§
§
|
|
Claudia Rosani, Piero Zanello, Q. Ping Dou, and Dolores Fregona*
|
⊥
,†
Department of Chemical Sciences, UniVersity of PadoVa, Via Marzolo 1, 35131 PadoVa, Italy,
Institute of Inorganic and Surfaces Chemistry, CNR Research Area, Corso Stati Uniti 4,
3
5127 PadoVa, Italy, Department of Pharmacology and Anaesthesiology, UniVersity of PadoVa,
Via L.go Meneghetti 2, 35131, PadoVa, Italy, Department of Chemistry, UniVersity of Siena, Via A.
Moro, 53100 Siena, Italy, and The PreVention Program, Barbara Ann Karmanos Cancer Institute
and Department of Pathology, School of Medicine, Wayne State UniVersity,
Detroit, Michigan 48202
Received March 5, 2008
In the last years, we have synthesized some new platinum(II), palladium(II), gold(I/III) complexes with dithiocarbamato
derivatives as potential anticancer drugs, to obtain compounds with superior chemotherapeutic index in terms of
increased bioavailability, higher cytotoxicity, and lower side effects than cisplatin. On the basis of the obtained
2
encouraging results, we have been studying the interaction of CuCl with methyl-/ethyl-/tert-butylsarcosine-
dithiocarbamato moieties in a 1:2 molar ratio; we also synthesized and studied the N,N-dimethyl- and pyrrolidine-
dithiocarbamato copper complexes for comparison purposes. The reported compounds have been successfully
isolated, purified, and fully characterized by means of several spectroscopic techniques. Moreover, the electrochemical
properties of the designed compounds have been studied through cyclic voltammetry. In addition, the behavior in
solution was followed by means of UV-vis technique to check the stability with time in physiological conditions.
To evaluate their in vitro cytotoxic properties, preliminary biological assays (MTT test) have been carried out on a
panel of human tumor cell lines. The results show that cytotoxicity levels of all of the tested complexes are comparable
or even greater than that of the reference drug (cisplatin).
1
,2
Introduction
and overgrowth of a drug-resistant tumor-cell population.
In 1971, J. Folkman hypothesized that tumors are angiogen-
esis-dependent and that antiangiogenic therapy might rep-
resent a good alternative for the treatment of solid tumors.
Currently, a large variety of chemotherapeutic drugs are
being used to treat cancer. Unfortunately, many compounds
demonstrate restricted efficacy due to problems of delivery
and penetration and a modest degree of selectivity for the
tumor cells, causing severe damage to healthy tissues.
However, the activity of these compounds is mainly limited
by the development of drug resistance. Tumor cells are a
rapidly changing target because of their genetic instability,
heterogeneity, and high rate of mutation, leading to selection
3
A promising way of cancer research is the study of a group
of compounds called angiogenesis inhibitors. These are drugs
that block the development of new blood vessels, which
supply tissues with oxygen and nutrients necessary for
4
,5
survival and growth. In cancerous tissues, tumors cannot
grow or metastasize without the development of new blood
vessels. Solid tumors cannot grow beyond the size of 1 to 2
3
mm without inducing the formation of new blood vessels
*
To whom correspondence should be addresses. Tel.: +39-049-8275159.
6
Fax: +39-02-700500560. E-mail: dolores.fregona@unipd.it.
to supply the nutritional and other needs of the tumor.
†
Department of Chemical Sciences, University of Padova.
Institute of Inorganic and Surfaces Chemistry, CNR Research Area.
Department of Pharmacology and Anaesthesiology, University of
‡
§
(1) Guo Hazlehurst, L. A.; Landowski, T. H.; Dalton, W. S. Oncogene
2003, 22, 7396–7402.
Padova.
|
Department of Chemistry, University of Siena.
Barbara Ann Karmanos Cancer Institute and Department of Pathology,
(2) Smythe, L. A. M; Stinson, S. F.; Mullendore, L. A.; Monks, A.;
Scudiero, D. A. Cancer Res. 1992, 52, 3029–3034.
(3) Folkman, J. N. Engl. J. Med. 1971, 285, 1182–1186.
⊥
School of Medicine, Wayne State University.
6336 Inorganic Chemistry, Vol. 47, No. 14, 2008
10.1021/ic800404e CCC: $40.75  2008 American Chemical Society
Published on Web 06/21/2008

Copper(II) Complexes Containing S-Donor Ligands
Endothelial cells are the source of new blood vessels and
have a remarkable ability to divide and migrate. In an attempt
to isolate a peptide, endothelial stimulating growth factor,
toward a number of tumor cell lines sensible and resistant
to cisplatin, which is a widely used drug in chemotherapy
toward different types of tumors.
2
1–28
7
McAuslan and Reilly noted a high concentration of copper
In addition, these complexes were proved to induce very
low toxicity levels, in particular regarding its nephrotoxic
salts. They postulated that copper was the active principle
in angiogenesis. Copper, but not other trace metals, stimu-
lated the directional migration of endothelial cells and, more
recently, copper was found to directly stimulates the in vitro
2
9
effects. Considering the potential advantages in terms of
noticeable in vitro and in vivo antitumor activity, lack of
cross-resistance with cisplatin, and reduced adverse side
effects, further studies on biochemical features of the
dithiocarbamato complexes are warranted.
8
proliferation of endothelial cells. Copper metabolism is
profoundly altered in neoplastic development in human
9,10
cancer and in tumor-bearing animals. Serum copper levels
correlate with tumor incidence, tumor weight, malignant
progression, and recurrence in a variety of human cancers:
Hodgkin’s lymphoma, sarcoma, leukemia, and cancer of the
Dithiocarbamates are a class of metal-chelating compounds
that have previously been used in the treatment of bacterial
30,31
and fungal infections, and in the treatment of AIDS.
For
example, pyrrolidine dithiocarbamate (PyDT) is a synthetic
1
1–14
cervix, breast, liver, and lung
mors.
as well as brain tu-
antioxidant and inhibitor of NF-κB, a transcription factor
1
5,16
Consistently, the high serum and tissue levels of
32,33
capable of binding copper.
PyDT and other dithiocar-
copper, found in many types of human cancers, support the
idea that copper could be used as a potential tumor-specific
target.
bamates have been found to induce apoptosis in conjunction
34
with copper in different types of cancer cells. Previously,
a synthetic PyDT containing copper was found to be a potent
Recent studies show that some organic prodrugs, used to
treat Wilson’s disease (characterized by copper accumulation
in different organs), present also an antiangiogenic effect
35
proteasome inhibitor and apoptosis inducer. Moreover,
PyDT is capable of binding copper spontaneously, forming
new complexes that have proteasome-inhibitory and apo-
ptosis-inducing activities to tumor but not to normal/
nontransformed breast cells. It was also found that prema-
lignant or cancer breast cells cultured to contain elevated
1
7
toward some murine tumors. The activity of these drugs
is related not only to their ability to remove copper but also
to the cytotoxic properties of the complexes formed between
the organic prodrugs and the copper ion. In fact, we have
recently demonstrated a correlation between the antiangio-
genic and the antitumor properties of some copper protea-
3
6
copper are sensitive to treatment with PyDT.
Most recently, we reported that a gold(III) dimethyldithio-
carbamato complex could inhibit the activity of a purified
1
8,19
some inhibitors;
moreover, the inhibition of the protea-
2
0S and 26S proteasome in human breast cancer cell cultures
somal activity can be achieved by targeting tumor cellular
copper with the nontoxic compound DSF (disulfiram, a
member of the dithiocarbamate family, capable of binding
copper), resulting in selective apoptosis induction of tumor
3
7
and xenografts.
(
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Inorganic Chemistry, Vol. 47, No. 14, 2008 6337

Giovagnini et al.
These findings altogether prompted us to study, as che-
motherapeutics, new copper(II) dithiocarbamato derivatives
of the esters of sarcosine; we have also synthesized, following
Scheme 1. Reaction Leading to the Synthesis of methyl/ethyl/
tert-butyl-Sarcosinedithiocarbamic Acid
3
8,39
a different reaction with respect to literature data,
and
studied the N,N-dimethyl-/pyrrolidine dithiocarbamate (DMDT/
PyDT)) complexes for comparison purposes.
4
washed with water and dried in a desiccator with P O10, the final
yield being 90-95%.
bis(dimethylcarbamodithioato-kS,kS′)-copper(II), [Cu(DM-
DT) ]. Anal. Calcd for C (M.W. ) 303.98 g/mol), %:
2
6 2 4
H12CuN S
Experimental Section
C, 23.24; H, 3.90; N, 8.89; S, 40.75. Found, %: C, 23.71; H, 3.98;
Chemicals. N,N-dimethyl-/pyrrolidine dithiocarbamate, methyl/
ethyl/tert-butylsarcosine hydrochloride (Fluka), carbon disulfide
N, 9.21; S, 41.12. FTIR (KBr, nujol, ν˜ max): ν(N-CSS) ) 1524;
a,s(SCS)) 976, 563; ν(SCuS) ) 352 cm- . H NMR (DMSO-d
1
1
,
ν
6
(
Aldrich), copper(II) chloride (J. T. Baker-Chemicals) were used
δ): 3.52 (12H, s, br, CH
bis(1-pyrrolidinecarbamodithioato-kS,kS′)-copper(II), [Cu-
(PyDT) ]. Anal. Calcd for C10 (M.W. ) 356.04 g/mol),
%: C, 33.70; H, 4.53; N, 7.87; S, 36.02. Found, %: C, 33.66; H,
4.72; N, 8.10; S, 36.22. FTIR (KBr, nujol, ν˜ max): ν(N-CSS) )
3
N).
as supplied. All other reagents and solvents were of high purity
and were used as purchased without any further purification.
Instrumentation. Electronic spectra were recorded at 298 K in
the range of 190-750 nm with a PerkinElmer Lambda 15 double-
beam spectrophotometer, using fresh 100 µM solutions of the
samples; in both DMSO and PBS/DMSO (in the latter case, the
complexes were previously dissolved in DMSO, to a final DMSO
concentration of 0.5% (v/v)). FTIR spectra were recorded at room
temperature in nujol between two polyethylene tablets on a Nicolet
2
2 4
H16CuN S
1500; νa,s(SCS)) 948, 580; ν(SCuS) ) 324 cm-
1
.
1
H NMR
-N).
, (CH C. The
dithiocarbamato ligands were obtained by reacting in water at
0 °C R-sarcosinehydrochloride (RSHCl (RO(O)CCH N-
(CH )H ] Cl ), CS , and NaOH in equimolar ratio. After 1 h, the
(DMSO-d
6
, δ): 2.01 (4H, m, CH
2
-); 3.85 (4H, m, CH
3 3
, CH CH )
2
Synthesis of [Cu(RSDT)
2
], R ) CH
3
3
2
)
2
Magna 740 FTIR spectrophotometer for the range 600-50 cm-
and in solid KBr on a Nicolet-NEXUS 670 FTIR spectrophotometer
for the range 4000-400 cm- . H NMR spectra were recorded at
1
,
3
2
+
-
2
pH value dropped from 10-11 to 6-7, according to the reaction
shown in Scheme 1.
The solution thus obtained was slowly added under stirring to
1 1
2
98 K in DMSO-d on a Bruker Avance DRX400 spectrometer
6
equipped with a Silicon Graphics workstation operating in Fourier
transform, using tetramethylsilane (TMS, δ ) 0.00) 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 mea-
surements were carried out in the range 25-1300 °C in alumina
2
an aqueous cold (0 °C) solution (2 mL) of CuCl in a 1:4 metal-
to-ligand molar ratio, leading to the immediate precipitation of a
brown solid that was filtered off, washed with water, and dried in
a desiccator with P
bis[methyl N-(dithiocarboxy-kS,kS′)-N-methylglycinato]-cop-
per(II), [Cu(MSDT) ]. Anal. Calcd for C10 (M.W.
4
O10, the final yield being 82-85%.
2
2 4 4
H16CuN O S
3
-1
) 420.18 g/mol), %: C, 28.59; H, 3.84; N, 6.67; S, 30.53. Found,
%: C, 28.42; H, 3.15; N, 6.46; S, 30.37. Mp ) 199,0 °C (dec.).
crucibles under air (flux rate 30 cm ·min ) and at a heating rate
-
1
of 10 °C·min , using alumina as reference. Elemental analyses
were performed in duplicate with a PerkinElmer 2400 CHN
microanalyzer; S, was determined by Sch o¨ ninger method and with
a Carlo Erba 1108 CHNS-O microanalyser. The apparatus for
electrochemistry and spectroelectrochemistry has been described
FTIR (KBr, nujol, ν˜ max): ν(N-CSS) ) 1506; ν(CdO) ) 1752;
ν(C-OMe) ) 1206; νa,s(SCS) ) 970, 570; νa,s(SCuS) ) 366 cm-
1
.
1
H NMR (DMSO-d
N); not detected (4H, CH
bis[ethyl N-(dithiocarboxy-kS,kS′)-N-methylglycinato]-cop-
per(II), [Cu(ESDT) ]. Anal. Calcd for C12 (M.W.
6
, δ): 3.81 (6H, s,br, CH
3
O); not detected (6H,
CH
3
2
N).
4
0
elsewhere. Electrochemical experiments were performed either
in deaerated CH Cl solution containing [NBu ][PF ] as supporting
electrolyte (0.2 mol dm ) or in deaerated H O-DMSO solution
1:1) using NaCl as supporting electrolyte (0.1 mol dm-3). All
potential values are referred to the saturated calomel electrode
SCE). In CH Cl solution, the one-electron oxidation of ferrocene
occurs at +0.41 V, whereas in H O-DMSO solution it occurs at
0.12 V. IC50 values were estimated by nonlinear regression
2
2 4 4
H20CuN O S
2
2
4
6
-3
) 448.11 g/mol), %: C, 32.16; H, 4.50; N, 6.25; S, 28.62. Found,
%: C, 31.93; H, 4.47; N, 6.15; S, 28.05. Mp ) 209,0 °C (dec.).
FTIR (KBr, nujol, ν˜ max): ν(N-CSS) ) 1506; ν(CdO) ) 1733;
2
(
ν(C-OEt) ) 1206; νa,s(SCS)) 970, 572; νa,s(SCuS) ) 361 cm-
1
.
(
2
2
1
H NMR (DMSO-d
CH ); not detected (6H, CH
bis[tert-butyl N-(dithiocarboxy-kS,kS′)-N-methylglycinato]-
copper(II), [Cu(TSDT) ]. Anal. Calcd for C16 (M.W.
504.22 g/mol), %: C, 38.11; H, 5.59; N, 5.55; S, 25.44. Found,
: C, 37.99; H, 5.51; N, 5.12; S, 25.32. Mp ) 208,0 °C (dec.).
6
, δ): 1.22 (6H, br, CH
3
CH
2
); 4.27 (4H, br,
2
CH
3
2
3
N), not detected (4H, CH
2
N).
+
analysis using GraphPad Prism software (San Diego, Ca. USA).
Data are the means (95% confidence intervals (CI) of quadruplicate
determinations from three independent experiments; the absorbance
at 570 nm was determined using a Victor2, multilabel counter
2
2 4 4
H28CuN O S
)
%
FTIR (KBr, nujol, ν˜ max): ν(N-CSS) ) 1505; ν(CdO) ) 1737;
(
Wallac Instruments, UK).
-1
ν(C-Ot-bu) ) 1207; νa,s(SCS) ) 967, 566; νa,s(SCuS) ) 354 cm .
2 2
Synthesis of [Cu(DMDT) ] and [Cu(PyDT) ]. The synthesis
41
1
6
H NMR (DMSO-d , δ): 1.46 (18H, br, (CH
3 3
) C); not detected (6H,
of these complexes was made following the literature. The powder
of CuCl was added to a water solution of DMDT sodium salt
(CH NCS ]Na or PyDT ammonium salt [PyNCS ]NH in molar
CH
3
N), not detected (4H, CH
2
N).
2
Cell Lines and Culture Conditions. Human ovarian carcinoma
[
3
)
2
2
2
4
(
2008 wild type and C13 cisplatin resistant subclone) and cervix
ratio 1:2 metal-to-ligand under vigorous stirring, leading to the
immediate precipitation of a brown solid that was filtered off and
squamous carcinoma (A431 wild type and A431Pt cisplatin resistant
subclone) cell lines were grown incubated with RPMI 1640
medium, 10% fetal bovine serum, 2% glutamine, and 1% pen-strep
in humidified condition at 5% CO and 37 °C. All cell lines have
2
a duplication time of about 24 h. Cells were collected every 2 days
(
38) Borchert, A.; Wiedner, W. Arch. Exp. Veterinaermed. 1958, 12, 242–
2
55.
(
(
39) Janssen, M. J. Recl. TraV. Chim. Pays-Bas 1956, 75, 1411–1422.
40) Fabrizi de Biani, F.; Corsini, M.; Zanello, P.; Yao, H.; Bluhm, M. E.;
Grimes, R. N. J. Am. Chem. Soc. 2004, 136, 11360–11369.
with minimum amount of 0.05% trypsin/0.02% EDTA and seeded
6
1
× 10 in 100 mm dish. All reagents were from Cambrex (NY,
(
41) Shinobu, L. A.; Jones, S. G.; Jones, M. M. Acta Pharmacol. Toxicol.
1
984, 54, 189–194.
USA).
6338 Inorganic Chemistry, Vol. 47, No. 14, 2008

Copper(II) Complexes Containing S-Donor Ligands
Scheme 2. Chemical Drawing of the Investigated Copper(II)
Complexes: [Cu(RSDT)2] (a), [Cu(PyDT)2] (b), [Cu(DMDT)2] (c)
increase of the C-N double-bond character. On the contrary,
for the complexes obtained through the template reaction,
this behavior cannot be observed as we were not able to
isolate the free R-sarcosinedithiocarbamato precursor; any-
way, the values recorded for ν(C-N) modes are in agreement
with the ones observed for DMDT or PyDT complexes and
with those reported in the literature for analogous metal
4
4–46
derivatives of R-amino acid dithiocarbamates.
The
-
1
presence of only one band in the region 1050-950 cm ,
assigned to the ν(S-C-S) asymmetric mode, is assumed to
indicate a symmetrical bonding of the dithiocarbamato
4
7
-1
moiety, acting in a bidentate mode. The 420-240 cm
4
8
region is associated with ν(M-S) vibrations. New bands,
absent in the starting material, are observed in the 370-320
-
1
cm region, and they can be attributed to the metal-sulfur
In Vitro Cytotoxicity Studies. For the biological tests, the
compounds were dissolved in DMSO, aliquoted, and stored at -
49
stretching mode.
Thermal Analysis. The thermal behavior of the synthe-
sized compounds has been studied to confirm the proposed
stoichiometry. The results have been summarized in Table
8
0 °C. Compounds and cisplatin (Pharmacia & Upjohn, Milano,
Italy) were dissolved in IMDM (Biochrome KG, Berlin, Germany)
and filter sterilized (0.2 µM) immediately before use. The final
DMSO concentration (0.1%) had no effect on cell killing. Cyto-
toxicity was measured by inhibition of cancer cell viability,
determined using the 3-(4,5-dimethyl-thizol-2-yl)-2,5-diphenyltet-
1
; a good correlation exists between calculated and found
values. The thermal behavior of the complexes was recorded
up to 1200 °C in air flux. All of the copper complexes
undergo the same degradation pathway, leading to the
corresponding metal-oxide (CuO) as the final product.
Moreover, [Cu(DMDT) ] and [Cu(PyDT) ] decompose gradu-
4
2
razolium bromide (MTT) assay. Cells were plated in 200 µl of
medium in 96 well microtiter plates. Following overnight incuba-
tion, cells were exposed for 24 h to the different sulfured copper
complexes. At the end of incubation, cells were washed and fresh
medium was replaced. After 48 h, to each well was added 20 µl of
2
2
ally to CuO in the 180-1100 °C interval without melting.
Electrochemistry and Spectroelectrochemistry. The
inherent electrochemical properties of these copper(II) dithio-
carbamato derivatives have been preliminary studied through
cyclic voltammetry soon after dissolution. Dithiocarbamates
are redox-active ligands, the electron transfer activity of
which is influenced by the electrode material as well as by
the solvent. In fact, at platinum or glassy carbon electrodes
they undergo oxidation to radical species, which rapidly
dimerize in dimethylsulfoxide (DMSO) solution, whereas no
5
mg/ml MTT, and cells were incubated for 4 h at 37 °C. The cells
were then lysed by adding 200 µl of acid isopropanol, and the
absorbance at 570 nm was determined. Triplicate determinations
and at least three separate experiments have been performed. IC50
defined as the concentration causing 50% reduction of overall cell
viability as compared to untreated cells, was extrapolated from
dose-response curves of each copper complex.
,
Results and Discussion
5
0
2 2
process can be detected in CH Cl solution. Dithiocarba-
Synthesis of the Complexes. By the reaction of the
mato ligands have stabilizing ability toward copper ions, and
their copper(II) complexes undergo reversibly oxidation and
reduction processes to their monocations and monoanions,
DMDT sodium salt [(CH
salt [PyNCS ]NH with CuCl
ratio, we have obtained the [Cu(DMDT)
3
)
2
NCS
in a 1:2 metal to ligand molar
] and [Cu(PyDT)
2
]Na or PyDT ammonium
2
4
2
2
2
]
5
0
respectively. Some characterization studies on dithiocar-
bamates of copper(I), copper(II), and copper(III) have been
complexes in which the dithiocarbamato ligands coordinate
the metal center through the sulfured atoms. The sulfured
ligands derived from the methyl-, ethyl-, and tert-butyl- esters
5
1,52
reported in the past.
In CH Cl solution, all of the copper(II) complexes exhibit
2
2
of sarcosine were obtained by reacting RSHCl (R)CH
CH -CH , C(CH ), CS , and NaOH in equimolar ratio;
the copper(II) complexes [Cu(RSDT) ] were obtained by
adding CuCl in 1:4 metal-to-ligand molar ratio (Scheme 2).
3
,
both oxidation and reduction processes with a certain
chemical reversibility in the cyclic voltammetric time scale.
3
2
3
)
3
2
2
2
(
44) Criado, J. J.; Carrasco, A.; Macias, B.; Salas, J. M.; Medarde, M.;
FTIR spectroscopy. In the FTIR spectra of all of the
synthesized complexes, the ν(C-N) band is observable in
Castillo, M. Inorg. Chim. Acta 1986, 124, 37–42.
(45) Criado, J. J.; Fernandez, I.; Macias, B.; Salas, J. M.; Medarde, M.
-1
Inorg. Chim. Acta 1990, 174, 67–75.
the range 1493-1539 cm ; this band defines a C-N bond
(
46) Criado, J. J.; Lopez-Arias, J. A.; Macias, B.; Fernandez-Lago, L. R.;
-1
order intermediate between a single bond (1350-1250 cm )
Salas, J. M. Inorg. Chim. Acta 1992, 193, 229–235.
-
1
43
(47) Kellner, R.; Nikolov, G. S.; Trendafilova, N. Inorg. Chim. Acta 1984,
and a double bond (1690-1640 cm ); in fact, moving
8
4, 233–239.
-
1
from DMDTNa (ν(C-N) ) 1486 cm ) or PyDTNH
4
(
(
(
48) Brown, D. A.; Glass, W. K.; Burke, M. A. Spectrochim. Acta 1976,
-1
3
2A, 137–143.
(
v(C-N) ) 1410 cm ) to its metal derivatives, the ν(C-N)
49) Desseyn, H. O.; Fabretti, A. C.; Forghieri, F.; Preti, C. Spectrochim.
Acta, Part A 1985, 42, 1105–1108.
vibrational mode is shifted to higher energies, showing an
50) Bond, A. M.; Martin, R. L. Coord. Chem. ReV. 1984, 54, 23–98.
(
(
42) Hansen, M. B.; Nielsen, S. E.; Berg, K. J. Immunol. Methods. 1989,
19 (2), 203–210.
43) Herlinger, A. W.; Wenhlod, S. N.; Long, T. V. J. Am. Chem. Soc.
970, 92, 6474–6481.
(51) Hendrikson, A. R.; Martin, R. L.; Rohde, N. M. Inorg. Chem. 1976,
1
15, 2115–2119.
(52) Hogart, G.; Pateman, A.; Redmond, S. P. Inorg. Chim. Acta 2000,
306, 232–236.
1
Inorganic Chemistry, Vol. 47, No. 14, 2008 6339

Giovagnini et al.
Table 1. Thermogravimetric (TG) and Differential Scanning Calorimetric (DSC) Data in Air Flux
DSC
decomposition
interval [°C]
TG weight loss [%]
(calculated) to CuO
a
compound
peak temperature [°C] (process )
[Cu(DMDT)2]
[Cu(MSDT)2]
[Cu(ESDT)2]
200-1100
125-1200
170-1200
(-73.83)-74.71
(-81.06)-80.84
(-82.24)-82.71
314(exo)/361(exo)/1044(endo)
199(endo/melting)/294(exo)/502(exo)/1041(endo)/1127(exo)
178(endo/melting)/209(endo)/301(exo)/468(exo)/518(endo)/1040(endo)/
1
132(endo)
[
[
Cu(TSDT)2]
Cu(PyDT)2]
100-1100
180-1100
(-84.22)-83.91
(-77.66)-78.83
208(endo/melting)/303(exo)/384(exo)/1043(endo)
288(exo)/338(exo)/396(exo)/1025(endo)/1033(endo)
a
endo/exo/melting ) endothermic/exothermic/melting process.
Table 2. Formal Electrode Potentials (V vs SCE) and peak-to-peak
separation (mV) for the redox changes exhibited by the
Cu(II)-dithiocarbamate complexes in different solutions
E°′ [Cu(III)/
Cu(II)]
E°′ [Cu(II)/
Cu(I)]
a
a
compound
∆Ep
∆Ep
solvent
[
[
[
[
[
Cu(DMDT)2]
+0.41
74
79
96
84
82
75
69
-0.57
-0.29
-0.46
69 CH2Cl2
88 H2O-DMSO (1:1)
140 CH2Cl2
H2O-DMSO (1:1)
180 CH2Cl2
90 H2O-DMSO (1:1)
134 CH2Cl2
+
0.47
Cu(MSDT)2]
Cu(ESDT)2]
Cu(TSDT)2]
Cu(PyDT)2]
a
+0.55
0.34
+0.55
0.52
+0.52
0.50
+0.45
0.54
b
+
-0.35
-0.46
-0.20
-0.48
-0.50
-0.52
+
+
H2O-DMSO (1:1)
88 CH2Cl2
74
108
b
+
-0.23
H2O-DMSO (1:1)
Measured at 0.2 Vs-1
. From Osteryoung square wave voltammetry.
b
Figure 1. Cyclic voltammetric response recorded at a platinum electrode
-
3
-3
in a CH2Cl2 solution of [Cu(ESDT)2] (0.4 ·10 mol dm ); [NBu4][PF6]
-
3
-1
(
0.2 mol dm ) as supporting electrolyte; scan rate 0.2 V s .
A representative example is shown in Figure 1, which refers
to [Cu(ESDT) ]. Controlled potential coulometry in cor-
respondence of the anodic process (E ) +0.8 V) consumed
2
w
one electron per molecule, and cyclic voltammetric tests on
the resulting solution gave rise to profiles quite similar to
the original ones, the chemical reversibility of the electron
removal, not only in the short times of cyclic voltammetry
(ipc/ipa constantly equal to the unity in the scan rate range
1
-
from 0.02 to 2.0 Vs ) but also in the longer times of
macroelectrolysis.
The reduction pattern appears more complicated: the
current ratio ipa/ipc decreases with the increase of the scan
rate, and the peak-to-peak separation notably swerves from
the 60 mV expected for an electrochemically reversible
Figure 2. UV-vis spectral changes recorded in a OTTLE cell in CH2Cl2
-3
-3
solution of [Cu(ESDT)2] (0.4 × 10 mol dm ): (a) upon stepwise
oxidation (Ew ) +0.7 V, vs pseudoreference silver electrode); (b) upon
stepwise reduction (Ew ) -0.7 V).
process. An E
r r
C mechanism, in which the quasireversible
copper(II)/copper(I) reduction is coupled to a slow, reversible
5
3
chemical side-reaction, could account for this trend.
Controlled potential coulometry (E ) -0.8 V) consumed
w
one-electron addition. The original neutral species show in
approximately 0.6 electrons per molecule, leading to a cyclic
voltammetric profile in which only marginal residues of the
monoanion could be identified.
2 2
CH Cl two main bands at λmax ) 284 and 428 nm. In
agreement with the color changes observed in the exhaustive
oxidation (from yellow-brown to yellow), upon stepwise
oxidation, the UV band tends to disappear, whereas the
visible band progressively increases with a blue shift of 15
nm.
Upon reduction, the visible band tends to disappear and,
as a possible consequence of the mentioned slow chemical
side-reaction, a new band at 350 nm tends to appear (in
accordance with the pale-yellow color observed upon
exhaustive reduction).
The formal electrode potentials of the copper(III)/cop-
per(II)/copper(I) sequence for all of the complexes in CH
solution are collected in Table 2 together with those recorded
in H O-DMSO (1:1) solution, the electrochemical behavior
2 2
Cl
2
of which will be discussed below.
As proved by the substantially constant HOMO-LUMO
gap (∼1 eV), the location of the redox potentials is governed
by the inductive effects of the dithiocarbamato substitu-
5
0,51
ents.
Figure 2 illustrates the UV-vis spectral changes of
Cu(ESDT) ] recorded upon either one-electron removal or
(
53) Zanello, P. Inorganic Electrochemistry. Theory, Practice and Ap-
plication; RSC: UK, 2003.
[
2
6340 Inorganic Chemistry, Vol. 47, No. 14, 2008

Copper(II) Complexes Containing S-Donor Ligands
Table 3. UV-vis Spectral Parameters Recorded upon Reduction and
Oxidation of the Copper(II)-Dithiocarbamate Complexes in CH2Cl2
Solution
compound
λmax/nm
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
Cu(DMDT)2]
Cu(DMDT)2]
Cu(DMDT)2]
Cu(MSDT)2]
281
428
413
+
-
281
285
426
414
+
Cu(MSDT)2]
Cu(MSDT)2]
Cu(ESDT)2]
-
285
284
428
413
+
Cu(ESDT)2]
Cu(ESDT)2]
Cu(TSDT)2]
Cu(TSDT)2]
Cu(TSDT)2]
Cu(PyDT)2]
-
289
285
426
417
+
-
285
280
427
412
+
-
Cu(PyDT)2]
Cu(PyDT)2]
280
Figure 3. UV-vis spectral with time of [Cu(TSDT)2] complex in DMSO
-
5
As shown, the appearance of isosbestic points in both
processes confirms the relative stability of both [Cu(ES-
DT) ] and [Cu(ESDT) ] ions.
2 2
A similar spectroelectrochemical behavior is shown by the
other complexes, and the spectral characteristics of the whole
solution, 2.46 × 10 M.
located in the d orbitals of the metal center, whereas bands
+
-
3
, 4, and 5 are typical of dithiocarbamato moieties. The two
strong bands at around 287 and 270 nm (bands 4 and 5
respectively) can be attributed to intramolecular intraligand
series in CH
The redox behavior of the complexes has been examined
also in H O/DMSO (1:1) solution. The complexes tend to
2 2
Cl solution are reported in Table 3.
π* r π transitions located in the S-C-S ed N-C-S
49,54–56
moieties respectively.
Band 3, at about 360 nm, is
2
ascribed to π* r n transition in sulfur atoms; moreover,
band 1 is related to a dxy f dz2 transition, and the broad
precipitate, thus hampering detailed electrochemical studies.
The most stable, and relatively most soluble, derivative
band 2 could be an overlapping of dxy f dxz and dxy f dyz
resulted to be [Cu(ESDT)
voltammetric profile).
2
] (Supporting Information for the
5
7
transitions, and the presence of an asymmetry toward low
energies is probably caused by the presence of the dxy
f
The copper(III)/copper(II)/copper(I) sequence is observed
also in this medium, and both processes are complicated by
concomitant chemical reactions, probably because the sta-
bilizing planar and tetrahedral geometries of the electrogen-
erated copper(III) and copper(I) species are here distorted
by the coordination of DMSO or water molecules.
58,59
2
d
x2-y transition.
On the other hand, when dissolved in DMSO/PBS, the
samples give rise to a cloudy solution, but the spectra are
very similar to those in DMSO, and the λ of the main
absorptions remain unchanged with time (Supporting Infor-
mation). The only difference is in the relative intensity of
the absorptions: the overall effect is an approximately
constant intensity for band 1 accompanied by a regular drop
of the other absorptions that also present in a few cases a
small red shift, probably caused by a major polarity of the
aqueous solvent compared with the DMSO (Figure 4, also
Solution Properties Analyzed by UV-vis Absorption
Spectroscopy. The evolution with time of all of the
complexes was followed by UV-vis spectroscopy in dif-
ferent media for 24 h (Supporting Information). The com-
pounds were initially dissolved in DMSO, giving a final
-5
concentration of 10 M.
60
Supporting Information). The drawdown of the baseline
For the UV-vis studies at pH 7.4, we have dissolved the
with time, is accompanied by the formation of an insoluble
residue and by a dramatic lowering the bands 4 and 5
intensity when if compared with the spectra in DMSO (ε5/
ε2(DMSO) ) 2.63/3.03; ε5/ε2(DMSO/PBS) ) 1.23/1.84).
To clarify this behavior, we performed IR spectra (Supporting
Information) of the solid species formed in PBS solution:
-
3
compounds in DMSO (10 M), and then we have diluted
the DMSO solution in phosphate buffer saline (PBS)
concentration of 100 µM, consistent with the maximum
concentration of the complexes used for the biological tests.
All the complexes behave similarly and are quite inert in
DMSO at 298 K; in fact, the slow spectral changes with
time, that is, a decrease in absorbance values, are caused by
a slow formation of a brown solid derived from a diminished
solubility of the complexes, and the nature of the solid
obtained has been confirmed by IR spectra (data not shown).
(54) Terent’ev, A. P.; Vozzhennikov, V. M.; Kolnikov, O. V.; Zvonkova,
Z. V.; Rukhadze, E. G.; Glushkova, V. P.; Berezkin, V. V. Dokl. Akad.
Nauk SSSR 1965, 160, 405–408.
(
55) Janssen, M. J. Recl. TraV. Chim. Pays-Bas 1960, 79, 454–463.
(56) Kurashvili, L. M.; Zavorokhina, N. A. Zh. Prikl. Spektrosk. 1974, 21,
6
76–679.
2
Figure 3 illustrates the [Cu(TSDT) ] complex spectrum with
(
(
(
57) Choi, S. N.; Menzel, E. R.; Wasson, J. R. J. Inorg. Nucl. Chem. 1977,
time as an example. Each complex is characterized by five
absorption bands as shown in Table 4 (Supporting Informa-
tion); for some complexes, bands 1 and 3 are only detectable
39, 417–422.
58) Takami, F.; Wakahara, S.; Maeda, T. Tetrahedron Lett. 1971, 28,
2
645–2648.
59) J o¨ rgensen, C. K. J. Inorg. Nucl. Chem. 1962, 24, 1571–1558.
-3
-4
as shoulders, only on concentrated (10 and 10 M) mother
solutions (Supporting Information). For all the studied
complexes, bands 1 and 2 are related to electronic transitions
(60) Skoog, D. A.; Leary, J. L. Applicazioni Della Spettroscopia di
Assorbimento Molecolare Nell’ultraVioletto e nel Visibile. In Chimica
Analitica Strumentale, 4th ed.; EdiSES, s.r.l., Eds., Napoli, 1995; pp
220-254..
Inorganic Chemistry, Vol. 47, No. 14, 2008 6341

Giovagnini et al.
-
5
Table 4. UV-vis Copper(II) Complex Absorptions in the 190-750 nm Range in DMSO Solvent (10 M)
-
1
-1
λmax/nm (ꢀ/cm
M )
compound
band 1
band 2
band 3
band 4
band 5
[Cu(DMDT)2]
[Cu(MSDT)2]
[Cu(ESDT)2]
[Cu(TSDT)2]
[Cu(PyDT)2]
436.4 (10 188)
438.8 (9038)
493.0 (9859)
438.4 (7528)
437.0 (11 750)
286.4 (16 886)
288.2 (15 657)
287.2 (16 766)
288.2 (13 166)
286.8 (18 189)
269.2 (28 251)
270.6 (27 368)
270.4 (29 125)
270.6 (22 830)
269.8 (30 948)
634.0 (32)
639.8 (37)
367.0 (636)
367.0 (315)
358.0 (475)
629 (178)
the only difference is a shoulder at about 1080 cm- for
1
equally active in wild type and cisplatin resistant cells,
indicating lack of cross-resistance with cisplatin. [Cu(ES-
DT) ] was the most active compound with IC < 0.5 µM
[
Cu(TSDT)
nated DMSO molecule. On the contrary, for the solid
obtained from [Cu(MSDT) ] complex, a new double band
assignable to the ν(C-N), appears at 1460-1470 cm at
2
]; this band might be assigned to an S-coordi-
6
1
2
50
2
in all cell lines also in comparison with cisplatin. Further-
more, [Cu(ESDT) ], [Cu(MSDT) ], and [Cu(DMDT) ] were
all more active than cisplatin in ovarian carcinoma cells
-
1
2
2
2
-1
lower energies compared to the starting species (1508 cm ).
This behavior can be explained, considering that ν(C-N)
shifts toward higher energies on passing from the free ligand
to the complexes, showing an increased double-bond char-
acter upon coordination. In this case, the appearance of a
new absorption at lower energies could be caused by a partial
breakage of the metal-dithiocarbamate coordination assisted
by a DMSO molecule. Altogether, the IR spectra do not
show big differences, suggesting that only a few changes
on the coordination sphere after solubilization in DMSO/
PBS occur.
Biological Assay. Human ovarian and cervix carcinoma
cell lines (2008 and A431) and the cisplatin resistant
subclones (C13 and A431Pt) were exposed for 24 h to
increasing concentrations (0.01-100 µM) of the different
copper-dithiocarbamato complexes or to cisplatin, as a
reference antineoplastic drug. Cytotoxicity was analyzed by
measuringcellviabilitybyMTTassay(Figure5).Dose-response
curves clearly show a dose-related inhibition of cell viability
caused by all the copper compounds, both in wild-type and
in cisplatin-resistant cells. Some of these compounds were
even more potent than cisplatin, mainly in cisplatin-resistant
subclones. IC50 values calculated from dose-response curves
are reported in Table 5. As expected, cisplatin was more
potent in wild-type cancer cells (0.19 µM and 0.78 µM in
A431 and 2008 cells) than in resistant subclones (3.5 µM
and 5.4 µM in A431Pt and C13). By contrast, especially
(2008-C13) and also in A431Pt cells.
The consistent evidence in the literature correlating copper,
but not other metal, with the biochemistry of the tumor and
the micromolar IC50 concentration in the present cytotoxicity
studies suggest that the molecular mechanism of copper
might target mainly specific proteins of relevance in regulat-
ing the balance of proliferation/apoptosis in cancer cells. Data
are encouraging to further investigate the potential copper
pharmacological activity in in vitro biological models.
Final Remarks. Our previous findings stimulated us to
study, as chemotherapeutics, new copper(II) dithiocarbamato
complexes derived from the esters of sarcosine. The conclu-
sions reached upon application of the spectroscopic tech-
niques suggest that coordination in all the copper(II)
derivatives takes place in a chelate manner, where the copper
atom is coordinated to the sulfur-donating atoms. The
presence of a paramagnetic copper(II) metal center in high-
4
3
6
2
9
spin d electronic configuration was confirmed by the
particularly severe shifts and broadening of the NMR signals
of the coordinated ligands. In the Supporting Information,
1
we reported the H NMR of the [Cu(ESDT)
2
] complex,
) signals are not recorded,
-CH - signals are in the
where the N(CH
3
) and N(CH
- and CH
2
whereas CH -CH
3
2
3
2
expected region of the NMR spectrum, although they have
lost their multiplicity.
[
2 2 2
Cu(ESDT) ], [Cu(MSDT) ], [Cu(DMDT) ] were almost
Figure 5. Cytotoxicity of sulfur-copper complexes in A431 and 2008
and in cisplatin resistant subclones (A431Pt and C13). Cell lines were treated
-8
-4
for 24 h with increasing concentration (10 to 10 M) of copper complexes
or cisplatin. The percent of live cells was quantified by the MTT assay as
described in the Experimental Section. Cell viability has been expressed as
a percent of absorbance of untreated cells.
Figure 4. UV-vis spectral with time of [Cu(TSDT)2] in DMSO/PBS
solution, 101µM.
6342 Inorganic Chemistry, Vol. 47, No. 14, 2008

Copper(II) Complexes Containing S-Donor Ligands
a
Table 5. IC50 Values of Cytotoxicity by Different Sulfured Ccopper Complexes
compound
A431 mean IC50 (µM) 95% CI A431Pt mean IC50 (µM) 95% CI 2008 mean IC50 (µM) 95% CI C13 mean IC50 (µM) 95% CI
Cisplatin
0.19
3.54
0.78
5.45
0
0.72
.06 to 0.54
0.74 to 17.04
0.32
0.26 to 0.60
0.24
3.35 to 8.85
0.36
[
[
[
[
[
Cu(DMDT)2]
Cu(MSDT)2]
Cu(ESDT)2]
Cu(TSDT)2]
Cu(PyDT)2]
a
0
0.29
0
0.01
0
0.59
0
2.80
.05 to 0.60
0.01 to 12.57
0.32
0.10 to 0.98
0.49
0.79 to 3.01
2.60
0.45 to 15.27
2.80
0.12 to 0.49
0.63
0.22 to 1.80
0.26
0.09 to 0.76
1.03
0.34 to 3.16
0.99
0.07 to 1.84
0.23
0.06 to 0.92
0.16
0.03 to 0.87
1.56
0.04 to 5.50
0.63
.10 to 0.80
.0006 to 0.03
.14 to 2.45
0
.77 to 10.41
1.63 to 4.79
0.15 to 6.68
0.09 to 4.17
The data are expressed as µM concentration (95% confidence Intervals (CI). Each value represents the average of three to four sets of independent
experiments.
Essential requirements for the pharmacological evaluation
of new metal complexes as cytotoxic agents are an ap-
preciable solubility in water and a sufficient chemical stability
under physiologically relevant conditions. Unfortunately, the
reported copper(II) complexes are not soluble in water; thus,
before any further biological investigation, they had to be
previously dissolved in DMSO and then added to PBS
solution or to the growth medium containing cells. For this
reason, we studied in different media their redox behavior
through cyclic voltammetry, and the evolution with time of
all the complexes was followed by UV-vis spectroscopy
for 24 h. When dissolved in DMSO/PBS, the compounds
react slowly, with a partial breakage of the metal-ligand
coordination supported by a solvent molecule, suggesting
that only a few changes on the coordination sphere after
solubilization in PBS occur.
out the occurrence of cross-resistance phenomena and
supporting the initial hypothesis of a different antitumor
activity mechanism of action compared with platinum(II)
compounds.
In conclusion, the chemical characterization carried out
in different media allowed us to establish that the five
investigated copper(II) dithiocarbamato derivatives are suf-
ficiently stable within a physiological-like environment, thus
representing the essential prerequisite for any further phar-
macological evaluation.
The encouraging chemical and biological properties of the
copper(II) dithiocarbamato complexes warrant further studies
to assess their pharmacological properties in vivo and to
elucidate the actual mechanism of their biological activity.
Acknowledgment. The authors gratefully acknowledge
Mrs. Anna Moresco of the Institute of Inorganic and Surfaces
Chemistry, CNR Research Area, Padua, Italy for technical
support, and the Department of Chemical Sciences, Univer-
sity of Padova for financial support (postdoctoral fellowship
for L.G.).
The citotoxic properties of the complexes were then
studied on human ovarian and cervix carcinoma cell lines
(
(
A431 and 2008) and the cisplatin resistant subclones
A431Pt and C13). All the copper dithiocarbamato deriva-
tives here reported show generally a good activity toward
all the cancer cells. Nevertheless, among the compounds,
the copper(II)/ESDT complex appeared to be the most stable
and relatively most soluble. This finding well correlates with
Supporting Information Available: 1H NMR spectrum of
2 6
[Cu(ESDT) ] complex in DMSO-d . Cyclic and Osteryoung square
wave voltammetric responses; UV-vis spectral data with time of
copper(II) dithiocarbamate complexes in DMSO solution; UV-vis
2
the results of the biological assays; in fact [Cu(ESDT) ] was
2
spectra with time of [Cu(TSDT) ] in DMSO solution at 298 K,
proved to be much more cytotoxic in vitro, with IC50 < 0.5
µM, than cisplatin even toward human tumor cell lines
resistant to cisplatin itself with activity levels comparable
to those on the corresponding wild-type cancer cells, ruling
.03 × 10-3M mother solutions; UV-vis spectral data with time
1
of copper(II) dithiocarbamate complexes in DMSO/PBS solution;
UV-vis spectra with time of [Cu(PyDT) ] in DMSO/PBS solution,
2
99 µM; FTIR spectra details of the [Cu(TSDT) ] (a) and [Cu(MS-
2
2
DT) ] (b) original complex and the brown precipitate from PBS
solution. This material is available free of charge via the Internet
at http://pubs.acs.org.
(
61) Enanas, I. P.; Spencer, A.; Wilkinson, G. J. Chem. Soc., Dalton Trans.
1
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(
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