Synthesis, characterization, and biological activity of a new palladium(II) complex with deoxyalliin

Article abstract of DOI:10.1139/v05-003

Synthesis, characterization, and biological activity of a new water-soluble Pd(II)-deoxyalliin (S-allyl-L-cysteine) complex are described in this article. Elemental and thermal analysis for the complex are consistent with the formula [Pd(C₆H₁₀NO₂S)₂]. ¹³C NMR, ¹H NMR, and IR spectroscopy show coordination of the ligand to Pd(II) through S and N atoms in a square planar geometry. Final residue of the thermal treatment was identified as a mixture of PdO and metallic Pd. Antiproliferative assays using aqueous solutions of the complex against HeLa and TM5 tumor cells showed a pronounced activity of the complex even at low concentrations. After incubation for 24 h, the complex induced cytotoxic effect over HeLa cells when used at concentrations higher than 0.40 mmol/L. At lower concentrations, the complex was nontoxic, indicating its action is probably due to cell cycle arrest, rather than cell death. In agreement with these results, the flow cytometric analysis indicated that after incubation for 24 h at low concentrations of the complex cells are arrested in G0/G1.

Full text of DOI:10.1139/v05-003

104
Synthesis, characterization, and biological activity
of a new palladium(II) complex with deoxyalliin
Pedro P. Corbi, Antonio C. Massabni, Andréia G. Moreira, Francisco J. Medrano,
Miriam G. Jasiulionis, and Claudio M. Costa-Neto
Abstract: Synthesis, characterization, and biological activity of a new water-soluble Pd(II)–deoxyalliin (S-allyl-L-
cysteine) complex are described in this article. Elemental and thermal analysis for the complex are consistent with the
1
formula [Pd(C6H10NO2S)2]. 13C NMR, H NMR, and IR spectroscopy show coordination of the ligand to Pd(II)
through S and N atoms in a square planar geometry. Final residue of the thermal treatment was identified as a mixture
of PdO and metallic Pd. Antiproliferative assays using aqueous solutions of the complex against HeLa and TM5 tumor
cells showed a pronounced activity of the complex even at low concentrations. After incubation for 24 h, the complex
induced cytotoxic effect over HeLa cells when used at concentrations higher than 0.40 mmol/L. At lower concentra-
tions, the complex was nontoxic, indicating its action is probably due to cell cycle arrest, rather than cell death. In
agreement with these results, the flow cytometric analysis indicated that after incubation for 24 h at low concentrations
of the complex cells are arrested in G0/G1.
Key words: palladium(II), deoxyalliin, S-allyl-L-cysteine, tumor cells, cancer.
Résumé : On décrit la synthèse, la caractérisation et l’activité biologique d’un nouveau complexe, soluble dans l’eau,
de Pd(II)–désoxyalliine (S-allyl-L-cystéine). Les analyses élémentaire et thermique du complexe sont en accord avec la
1
formule [Pd(C6H10NO2S)2]. Les spectres de RMN du 13C et du H ainsi que la spectroscopie infrarouge permettent de
montrer que la coordination du ligand au Pd(II) se fait par les atomes de soufre et d’azote dans une géométrie plan
carré. Le résidu final du traitement thermique a été identifié comme un mélange de PdO et de Pd métallique. Des es-
sais antiproliférations à l’aide de solutions aqueuses du complexe ont permis de démontrer l’activité prononcée du
complexe contre les cellules cancéreuses HeLa et TM5, même à faibles concentrations. Après une incubation de 24 h,
le complexe utilisé à des concentrations supérieures à 0,40 mmol/L induit un effet cytotoxique vis-à-vis des cellules
HeLa. À des concentrations plus faibles, le complexe n’est pas toxique, ce qui indique que son action est probablement
due à un arrêt du cycle cellulaire plutôt qu’à une mort de la cellule. En accord avec ces résultats, l’analyse de
l’écoulement cytométrique indique que, après une incubation de 24 h à de faibles concentrations de complexe, les cel-
lules de G0/G1 s’arrêtent.
Mots clés : palladium(II), désoxyalliine, S-allyl-L-cystéine, cellules cancéreuses, cancer.
[Traduit par la Rédaction] Corbi et al. 109
Introduction
sity in 1965 (4), but only in 1978 was this compound
approved worldwide for cancer treatment (5). Toxic side ef-
fects of cisplatin, mainly nephrotoxicity, neurotoxicity (2),
and ototoxicity (6), have limited its use and led to the devel-
opment of second generation drugs. Interest in developing
new complexes with lower side effects than cisplatin, but at
the same time with high activity against tumors, has stimu-
lated syntheses of many new complexes of platinum(II) and
The most largely used metal-based drug is cisplatin (cis-
diammindichloroplatinum(II)), which has been used for the
treatment of several human cancers, particularly testicular,
ovarian, bladder, head, and neck (1, 2). For a review see
ref. 3. The anticancer properties of cisplatin were first ob-
served by Barnet Rosenberg at the Michigan State Univer-
Received 12 July 2004. Published on the NRC Research Press Web site at http://canjchem.nrc.ca on 12 February 2005.
P.P. Corbi1 and A.C. Massabni. Departamento de Química Geral e Inorgânica, Instituto de Química – UNESP, CP 355, CEP
14801–970, Araraquara, SP, Brazil.
A.G. Moreira. Departamento de Química Geral e Inorgânica, Instituto de Química – UNESP, CP 355, CEP 14801-970, Araraquara,
SP, Brazil and Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto – USP, 14049-900, Ribeirão
Preto, SP, Brazil.
F.J. Medrano. Laboratório Nacional de Luz Síncrotron – LNLS, CP 6192, CEP 13084-971, Campinas, SP, Brazil.
M.G. Jasiulionis. Departamento de Micro-Imuno-Parasitologia, Escola Paulista de Medicina – UNIFESP, 04023-062, São Paulo,
Brazil.
C.M. Costa-Neto. Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto – USP, 14049-900,
Ribeirão Preto, SP, Brazil.
1Corresponding author (e-mail: pedrocorbi@yahoo.com).
Can. J. Chem. 83: 104–109 (2005)
doi: 10.1139/V05-003
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Corbi et al.
105
also of palladium(II) (7, 8). Pd(II) complexes containing S
and N donor ligands have been prepared and biological
assays showed their activities against several tumor lines
(HL-60, HeLa, 3T3, JURKAT, Pam212, and Pam-ras) (7).
Deoxyalliin (S-allyl-L-cysteine, C6H11NO2S), a sulfur-
containing amino acid derived from cysteine, is a product of
vegetal origin present in onion and garlic bulbs (9, 10).
More recent studies have shown that deoxyalliin may be
considered a biological antagonist of nitrosomorpholine, the
substance responsible for the development of hepatic cancer
in humans (11). It also exhibits the capacity to inhibit prolif-
eration of malignant cells from the human nervous system
and thorax (12, 13).
Preparation of the Pd(II) complex
The potassium salt solution of deoxyalliin was prepared
by adding 1.12 × 10–3 mol of potassium hydroxide to a
methanolic solution containing 1.12 × 10–3 mol of deoxy-
alliin (molar proportion of 1:1). This reaction was carried
out at room temperature with stirring for 1 h.
The Pd(II) complex was synthesized by adding 5.6 ×
10–4 mol of a freshly prepared methanolic solution of
Li2PdCl4 to the solution of the potassium salt of deoxyalliin
previously prepared, containing 1.12 × 10–3 mol of the
ligand (molar proportion Pd(II):deoxyalliin of 1:2). This re-
action was carried out at room temperature with stirring. A
pale yellowish solid of the complex was precipitated slowly.
After 2 h of constant stirring, the complex was filtered,
washed with cooled methanol, and dried in a desiccator un-
der P4O10. Final yield of the synthesis was about 70%. Anal.
calcd. for [Pd(C6H10NO2S)2] (%): C 33.8, H 4.72, N 6.56, S
15.0, Pd 24.9; found: C 33.4, H 4.60, N 6.40, S 14.7, Pd
23.7. No single crystals of the complex were obtained for X-
ray structure determination. The lithium tetrachloropalladate
solution was prepared by the reaction of PdCl2 and LiCl in
methanol under stirring and reflux for 30 min.
Complexes containing Pt(II) or Pd(II) with mixed ligands
such as amino acids and 1,10-phenanthroline displayed
cytotoxic activities in vitro against Molt-4, a human leukae-
mia cell line (1). A study of S-alkyl and S-alkylaryl cysteine
1
complexes with platinum(II) characterized by 13C NMR, H
NMR, and IR absorption spectroscopy has also been re-
ported (14). Biological tests were not performed for the lat-
ter complexes. The present article describes the synthesis
and characterization of a new palladium(II) complex with
deoxyalliin and its biological action against proliferation of
tumor cells.2
Cell culture
HeLa (human cervix cancer, ATCC CCL-2) and TM5
(murine melanoma, (15)) cell lines were cultured at 37 °C in
a humidified atmosphere containing 5% CO2, using, respec-
tively, DMEM supplemented with 10% of Fetal Calf Serum
(FCS) and RPMI at pH 6.9 with 5% FCS. Penicillin (100
U/mL) and streptomycin (100 µg/mL) were used as antibiot-
ics.
Experimental
Materials
L-Deoxyalliin of analytical purity was purchased from
LKT Laboratories. Palladium(II) chloride and lithium chlo-
ride of analytical purity were purchased respectively from
Acros and Mallinckrodt Laboratories. Elemental analyses for
carbon, hydrogen, nitrogen, and sulfur were performed with
a CHNS-O EA1110 analyzer (CE Instruments); cystine was
used as a reference substance. IR spectra were recorded on a
FT-IR spectrophotometer spectrum 2000 (PerkinElmer) with
Determination of the cell number and cell viability
A stock sample solution was prepared by dissolving the
Pd(II) complex in a phosphate-buffered saline solution
(PBS). PBS was used as the vehicle for the Pd(II) complex
in all biological experimental procedures. Final different
concentrations of the complex were achieved by dilution of
the stock solution directly into the cell’s medium. Cells were
plated (25 × 104 cells/60 mm dish) 24 h prior to the begin-
ning of the experiment. 48 h after addition of the complex or
the vehicle, cells were detached and counted in individual
samples using an impedance-based automated counter
(CELM, Barueri, Brazil). To evaluate the immediate
cytotoxic action of the Pd(II) complex, the MTT method
(16) was applied to the cells after incubation for 24 h.
Briefly, 24 h after plating 2.5 × 104 cells/well in a 24-well
plate, the complex or the vehicle was added and the cells in-
cubated for 24 h in culture conditions. After that, 100 µL of
a MTT solution (5 mg/mL in PBS) were added and the cells
were incubated for another period of 3 h. After washing with
PBS, 200 µL of isopropanol were added, and cell viability
was determined by absorbance measurements at 570 nm.
1
samples prepared as KBr or CsI pellets. 13C NMR and H
NMR were recorded on a Varian 500 MHz spectrometer;
samples were analyzed in deuterium oxide solutions. Ther-
mal analyses were performed on a Thermoanalyzer TG/DTA
simultaneous SDT 2960 (TA Instruments) in the following
conditions: synthetic air, 100 cm3/min and heating rate of
10 °C/min, from 40 to 1100 °C. Palladium content was
determinated by analyzing the residue of the thermal treat-
ment at 900 °C. Powder X-ray analysis was performed on a
D 5000 Siemens diffractometer using CuKα1 radiation (λ =
1.5406 Å) with a graphite diffracted beam monochromator.
The sample was scanned over the 2θ range from 4° to 70° in
0.05° steps. The counting time was 1.0 s/step. Cisplatin was
purchased from Acros and MTT from Sigma. Media
(DMEM and RPMI) and antibiotics for cell culture were
purchased from Life Technologies, Inc. (Gaithersburg,
Maryland) and FCS from Cultilab (Campinas, Brazil).
Flasks and 24-well plates for cell culture were purchased
from Costar (Corning Inc., Corning, N.Y.).
Cell cycle analysis by flow cytometry
Flow cytometric analysis of the Pd(II)-complex-treated
TM5 cells was performed using a FACScan (Becton
Dickinson). For synchronization, the cells were submitted to
2 Abbreviations: phosphate-buffered saline (PBS), Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute medium
(RPMI), fetal calf serum (FCS), American type cell collection (ATCC).
© 2005 NRC Canada

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Can. J. Chem. Vol. 83, 2005
Fig. 1. IR absorption spectra for: (a) deoxyalliin, (b) potassium
S-allyl-L-cysteinate, and (c) the Pd(II) complex.
a serum depletion for 24 h to induce M block, followed by
addition of fresh serum to stimulate growth in the presence
(0.20 mmol/L) or absence of the Pd(II) complex. After 24 h,
cells were washed, fixed with ice-cold ethanol (70% final
concentration), resuspended in 0.5 mL of a solution of
propidium iodide (20 µg/mL) and 200 µg/mL of Dnase-free
Rnase in PBS, and incubated for 30 min at 37 °C. For flow
cytometric analysis, at least 10 000 cells were evaluated us-
ing a FACSCalibur™. Cell cycle distribution and pre-G1
fraction were determined and quantified using the
CellQUEST™ program.
Effect of the complex on melanoma growth in C57BL/6
mice
Groups of six C57BL/6 mice weighing between 25 and
30 g were selected. For tumor induction, melanoma TM5
cells (2 × 105) were injected subcutaneously into the dorsal-
side of each mouse. In the subsequent 5 days, the complex
(24 mg/kg and 72 mg/kg) or the vehicle (PBS) was adminis-
trated subcutaneously into the dorsal-side of each animal at
a distance (1 cm) from the site of the cells injection. Detec-
tion of tumors were followed for 30 days, after which ani-
mals were sacrificed. C57BL/6 mice were kept at Centro de
Desenvolvimento de Modelos Experimentais para Medicina
e Biologia (CEDEME, UNIFESP, SP, Brasil) under the In-
ternational Guiding Principles for Biomedical Research In-
volving Animals (CIOMS), Genebra (1985), and the Guide
to the Care and Use of Experimental Animals, Canadian
Council on Animal Care (www.ccac.ca).
Fig. 2. The schematic structure of deoxyalliin.
Results and discussion
IR spectroscopy
The IR spectra of deoxyalliin, potassium S-allyl-L-
cysteinate, and [Pd(C6H10NO2S)2] complex are shown in
Fig. 1.
Two very well resolved bands in the range 3400–
3000 cm–1 are an indication of coordination of the amino
group to Pd(II) (17). The IR spectrum of potassium S-allyl-
L-cysteinate shows a broad band at 3350–2575 cm–1, which
corresponds to the NH2 vibrational frequencies, while in the
spectrum of deoxyalliin, the frequencies related to the amino
group are observed in the range 3160–2675 cm–1 (broad
band). Enlargement and shifting of the band show that the
two hydrogen atoms of the amino group are involved in the
formation of hydrogen bonds, which occur with cysteine and
its derivatives, as deoxyalliin (17). The amino group involve-
ment in the ligand coordination is attested by the presence of
two very well resolved bands at 3220 and 3079 cm–1 for the
Pd(II) complex (17). Moreover, the absence of the band at
2050–2150 cm–1 in the complex spectrum, in comparison to
the free ligand and its potassium salt, confirms coordination
through the amino group. The band at 2050–2150 cm–1 is as-
signed to a noncoordinated NH2 group, as it occurs for the
free ligand (18).
band attributed to the free ionized carboxylate group appears
at 1620 cm–1 (14).
The IR spectra of the complexes were also measured in
the region 700–150 cm–1 to identify frequencies related to
M—S and M—N bonds. The IR spectrum shows frequencies
at 342 cm–1 and at 554 cm–1, which could be assigned to
ν(M-S) and ν(M-N), respectively. These attributions are in
agreement with the literature values, being similar to those
found for an ethyl cysteinate complex of palladium(II) (19)
and for other metal complexes with amino acids (14). Con-
sidering the presence of two well-defined bands for ν(M-N)
and ν(M-S), a trans configuration could be proposed for the
Pd(II) complex, as it occurs for complexes of the type [Pt(L-
H+)2], where L is an S-alkyl-L-cysteine derivative (14).
1H and 13C NMR spectrometry
1H NMR and 13C NMR spectra of the Pd(II) complex are
useful for assigning ligand to metal bonding sites. The NMR
spectra of the complex were analyzed in comparison to the
spectra of the free ligand. The ligand structure is shown in
Fig. 2.
The IR spectrum of [Pd(C6H10NO2S)2] also shows a
strong absorption band at 1617 cm–1, which can be assigned
to a free ionized carboxylate group. In the case of Pt(II)
complexes with S-alkyl cysteine derivatives, in which the
carboxylate group was not coordinated to the metal ion, the
The 1H NMR spectrum of the Pd(II) complex with
deoxyalliin is consistent with coordination of the ligand to
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Corbi et al.
107
Fig. 3. 13C NMR for: (a) deoxyalliin and (b) the Pd(II) complex.
the metal through the S and N atoms. Protons near the coor-
dination sites are shifted downfield by 0.2–0.7 ppm. It was
also observed that the chemical shifts for H(4a, 4b) (see
Fig. 2) varies from 2.9 ppm for the ligand spectrum to
3.6 ppm for the complex. It was also observed that H(2a)
varies from 3.8 ppm for the ligand to 4.0 ppm for the com-
plex. These results are in agreement with the literature for
Pd(II) and Pt(II) complexes involving amino acid derivatives
showing S,N-coordination sites to the metal ions (14).
the chemical shift of the COOH group. Pronounced changes
are observed for the chemical shifts of C2, C3, and C4 in the
spectrum of the Pd(II) complex when compared to the free
ligand (see Figs. 2, 3a, and 3b). These changes are other in-
dications for coordination of deoxyalliin to Pd(II) through
the N and S atoms of the amino acid structure.
Thermal analysis
TG and DTA curves for the Pd(II) complex are shown in
Fig. 4. According to the thermogravimetric data, the compo-
sition of the complex formulated as [Pd(C6H10NO2S)2] is
confirmed. Oxidation of the coordinated ligand starts at tem-
peratures near 200 °C. The residue formed after the thermal
treatment of the Pd(II) complex at 900 °C was identified by
powder X-ray diffractometry as a mixture of PdO (20) and
metallic Pd (21).
13C NMR data are in agreement with S and N coordina-
1
tion proposed by analyzing the H NMR and IR spectra. 13C
NMR chemical shifts for deoxyalliin and the Pd(II) complex
are shown in Fig. 3.
According to the 13C NMR data, the chemical shift at
173 ppm in the spectrum of the ligand is assigned to the car-
bon atom of the COOH group (C1 in Figs. 2, 3a, and 3b). In
the spectrum of the complex, no changes are observed for
DTA of the Pd(II) complex shows a strong exothermic
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Can. J. Chem. Vol. 83, 2005
Fig. 4. TG (solid) and DTA (dot) curves for [Pd(C6H10NO2S)2].
Fig. 5. Proliferation profiles for HeLa and TM5 cells after treat-
ment with the Pd(II) complex in different concentrations for
48 h. The data represent an average of three to six independent
experiments done in duplicate.
peak with a maximum at 290 °C and a weak peak at 257 °C
(see Fig. 4). These effects are assigned to ligand oxidation
of the complex in two steps.
Fig. 6. Profiles for HeLa proliferation after treatment with the
Pd(II) complex in different concentrations. The data represent an
average of three independent experiments done in duplicate.
Powder X-ray diffractometry
Powder X-ray diffraction data for [Pd(C6H10NO2S)2] have
been successfully indexed in supposition of an orthorhombic
system with the following lattice cell parameters: a =
10.740 Å, b = 19.999 Å, and c = 5.2470 Å. A complete dis-
cussion of the powder X-ray indexation of this new complex
has already been described (22).
Antiproliferative and cell viability analyses
The profile of the cell proliferation assays using HeLa and
TM5 cells (Fig. 5) shows a blockage action of the Pd(II)
complex starting at low concentrations such as
0.050 mmol/L, especially for HeLa cells. At 0.40 mmol/L,
HeLa proliferation was totally eliminated and TM5 cell pro-
liferation was only about 25% when compared to the con-
trol. Similar results were obtained when cisplatin was used:
~70% inhibition at 0.40 mmol/L (data not shown here).
The Pd(II) complex was also analyzed for HeLa cells after
a longer exposure period of time (Fig. 6). The results
showed that after 7 days of exposure to the Pd(II) complex
(concentrations of 0.10 and 0.20 mmol/L), cell proliferation
was about 75% inhibited. When the concentration was in-
creased to 0.40 or 0.80 mmol/L, cell proliferation was com-
pletely inhibited.
The evaluation of the immediate cytotoxic action of the
Pd(II) complex was analyzed after an incubation time of
24 h. After this period of incubation, a relevant effect was
found only at high concentrations of the complex (0.40 and
0.80 mmol/L) suggesting that the antiproliferative action of
the Pd(II) complex observed at lower concentrations is prob-
ably due to cell cycle arrest rather than cell death. In the
case of cell death, cell viability would appear reduced at
lower concentrations of the complex when compared to the
control.
the other hand, cells were mainly arrested in G0/G1, when
compared to the vehicle treated group. These results corrob-
orate the data discussed here for the cytotoxic action of the
compound after an incubation time of 24 h.
In vivo assays
The results obtained for the melanoma growth assay using
C57/BL6 mice were considered nonsignificant due to the
short delay (~48 h) observed for the appearance of tumors in
the group treated with the complex (72 mg/kg body weight)
when compared to the control group. No side effects, e.g.,
weight loss, were detected. This modest result warrants fur-
ther in vivo investigation and optimization of dosage of the
complex.
Flow cytometry analysis
As can be observed in Fig. 7, TM5 melanoma cells treated
during 24 h with the Pd(II) complex at the concentration of
0.20 mmol/L did not present an apoptosis pattern, as seen by
the low level of hypodiploid cells, which are detected when
DNA fragmentation and (or) loss of DNA is occurring. On
Final conclusions
Based on the chemical and spectroscopic results, a sche-
matic structure for the Pd(II) complex with deoxyalliin is
proposed in Fig. 8.
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Corbi et al.
109
Fig. 7. Flow cytometric analysis of TM5 cells subjected to
0.20 mmol/L of Pd(II) complex or the vehicle (PBS) for 24 h.
Hypodiploid cells correspond to cells that are undergoing
apoptosis. The data represent an average of two independent ex-
periments.
tion is similar to other palladium complexes and also to
cisplatin when tested against HeLa cells (8, 23–25).
Acknowledgments
This study was supported by grants from FAPESP (São
Paulo State Research Foundation, Proc. 03/02616-7) and
CNPq (Brazilian National Research Council). The authors
are grateful to Dr. Nivaldo Boralle for the NMR spectra and
also to Dra. Marisa S. Crespi for thermal analyses.
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Biochem. 83, 31 (2001).
This work describes a new palladium(II) complex with in
vitro biological activity in the µmol/L concentration. Its ac-
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