On the cytotoxic activity of Pd(II) complexes of N,N-disubstituted- N′-acyl thioureas

Article abstract of DOI:10.1016/j.jinorgbio.2014.01.022

The rational design of anticancer drugs is one of the most promising strategies for increasing their cytotoxicity and for minimizing their toxicity. Manipulation of the structure of ligands or of complexes represents a strategy for which is possible to mo

Full text of DOI:10.1016/j.jinorgbio.2014.01.022

On the cytotoxic activity of Pd(II) complexes of N, N-disubstituted -N-acyl
thioureas
Ana M. Plut´ın, Rau´l Mocelo, Anislay Alvarez, Rau´l Ramos, Eduardo E.
Castellano, Marcia R. Cominetti, Angelica E. Graminha, Antonio G. Ferreira,
Alzir A. Batista
PII:
DOI:
Reference:
S0162-0134(14)00039-7
doi: 10.1016/j.jinorgbio.2014.01.022
JIB 9466
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
16 July 2013
29 January 2014
30 January 2014
Please cite this article as: Ana M. Plut´ın, Rau´l Mocelo, Anislay Alvarez, Rau´l
Ramos, Eduardo E. Castellano, Marcia R. Cominetti, Angelica E. Graminha, Anto-
nio G. Ferreira, Alzir A. Batista, On the cytotoxic activity of Pd(II) complexes of
N, N-disubstituted -N-acyl thioureas, Journal of Inorganic Biochemistry (2014), doi:
10.1016/j.jinorgbio.2014.01.022
This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.

ACCEPTED MANUSCRIPT
On the cytotoxic activity of Pd(II) complexes of N, N-
disubstituted -N´-acyl thioureas
Ana M. Plutín*a, Raúl Moceloa, Anislay Alvareza, Raúl Ramosa, Eduardo E.
Castellanob, Marcia R. Cominettic, Angelica E. Graminhad, Antonio G. Ferreirad, Alzir
A. Batista*d
aLaboratorio de Síntesis Orgánica, Facultad de Química, Universidad de La Habana, La
Habana, Cuba
bInstituto de Química de São Carlos, Universidade de São Paulo, São Carlos - SP, Brazil
cDepartamento de Gerontologia,Universidade Federal de São Carlos-São Carlos – SP,
Brazil
dDepartamento de Química,Universidade Federal de São Carlos-São Carlos – SP, Brazil
*Corresponding authors:
Alzir A. Batista, daab@ufscar.br; AnaMaría Plutín, anap@fq.uh.cu
Keywords: Acyl thiourea derivatives; Triphenylphosphine; Pd (II) complexes; X-Ray
structures; cytotoxicity
1

ACCEPTED MANUSCRIPT
Abstract
The rational design of anticancer drugs is one of the most promising strategies for
increasing their cytotoxicity and for minimizing their toxicity. Manipulation of the
structure of ligands or of complexes represents a strategy for which is possible to
modify the potential mechanism of their action against the cancer cells. Here we
present the cytotoxicity of some new palladium complexes and our intention is to show
the importance of non-coordinated atoms of the ligands in the cytotoxicity of the
complexes. New complexes of palladium (II), with general formulae [Pd(PPh3)2(L)]PF6
or [PdCl(PPh3)(L)], where L = N,N-disubstituted-N´-acyl thioureas, were synthesized
and characterized by elemental analysis, molar conductivity, melting points, IR,
NMR(1H, 13C and 31P{1H}) spectroscopy. The spectroscopic data are consistent with the
complexes containing an O, S chelated ligand. The structures of complexes with N,N-
dimethyl-N`-benzoylthiourea,
N,N-diphenyl-N`-benzoylthiourea,
N,N-diethyl-N´-
furoylthiourea, and N,N-diphenyl-N`-furoylthiourea were determined by X-ray
crystallography, confirming the coordination of the ligands with the metal through
sulfur and oxygen atoms, forming distorted square-planar structures. The N,N-
disubstituted-N´-acyl thioureas and their complexes were screened with respect to their
antitumor cytotoxicity against DU-145 (human prostate cancer cells), MDA-MB-231
(human breast cancer cells) and their toxicity against the L929 cell line (health cell line
from mouse).
2

ACCEPTED MANUSCRIPT
1. Introduction
The success of cisplatin in cancer therapies has triggered great interest to search
for more effective antitumor agents, and as a consequence many other transition metal
complexes have been obtained and tested against tumor cells. The number of metal
compounds in current clinical use for this purpose is very limited and concerns only the
platinum complexes [1]. However, it is important to search for other therapeutic agents
different from those of platinum, which are in clinical use, in order to overcome the
known drawbacks caused by them. Thus, a number of drug-design strategies have been
employed, mainly in the past twenty years, and literature reports palladium(II) and
platinum(II) complexes of phenylacetaldehyde thiosemicarbazone that are capable to, in
vitro, bind to DNA, and have enhanced capacity to form inter-strand cross links by
comparison with cisplatin [2-3].
The acyl thiourea ligand systems are very versatile and small structural changes
can be readily made that lead to very different chemical and physical properties.
Chelating thiourea ligands containing N, S and O donor atoms show broad biological
activity and the existence of metal ions bonded to biologically active compounds may
enhance their activities, and it was shown that the mode of binding of platinum to DNA
is affected by the substituent nature on the acyl thiourea [4]. Acyl thiourea derivatives
have been increasingly important with a wide diversity of applications in heterocyclic
chemistry, metal complexes, molecular electronics and exhibit an array of biological
activities [5-9]. Some of them are employed as fungicidal, antiviral, antimicrobial [10],
parasiticidal [11], antitumoral [12] and pesticidal agent [13–15]. Indeed cytotoxicity
studies using HeLa cancer cell lines have demonstrated that some of these platinum acyl
thioureas show cytotoxic behavior, with the antiproliferative effects being dependent on
3

ACCEPTED MANUSCRIPT
the nature/type of the substituent in the acyl thiourea ligand [16-18]. Having this in
mind our research group has been interested in developing new thiosemicarbazones or
similar ligands, including polydentate ones such as acyl thioureas, containing N, S and
O atoms with the capacity to form transition metal complexes. It turns out that these
ligands are able to bind a wide variety of metal ions in different coordination modes,
forming stable complexes. In most of the ones containing acyl thioureas, which were
structurally characterized, they act as bidentate O,S-monoanionic ligands [19-22].
Also, it is well known that palladium(II) complexes are potential candidates to be used
as metal based therapeutic agents, since this metal has a very similar chemistry to that of
platinum(II), some of whose compounds are well known as anticancer agents [23].
Here we report the synthesis, crystal structures and citotoxicity studies of new
palladium(II) complexes containing PPh3 and N, N-disubstituted-N´-acyl thioureas as
ligands. The N, N-disubstituted-N´-acyl thioureas used as ligands in this work were
synthesized by the procedure previously reported in literature [24]. Scheme 1 shows the
pathway for the synthesis of the Pd(II) complexes, which were obtained by reacting
methanolic
solutions
of
acyl
thioureas
with
the
precursor,
the
dichloro(bistriphenylphosphine)palladium(II).
The coordination between acyl thiourea derivatives and the precursor was
proceeded by an exchange reaction, which involved deprotonation of the acyl thioureido
group of the ligands during the complexes formation [25].
Insert Scheme 1
4

ACCEPTED MANUSCRIPT
2. Experimental
2.1. Material and measurements
Bis diphenylphosphine palladium(II) chloride was obtained from Strem. All
reagents were purchased with reagent grade and used without further purification.
Solvents were dried and used freshly distilled, unless otherwise specifically indicated.
Thin layer chromatography (TLC) was performed on 0.25 mm silica gel pre-
coated plastic sheets (40/80 mm) (Polygram_ SIL G/UV254, Macherey & Nagel,
Düren, Germany) using benzene/methanol (9/1) as eluent.
The IR spectra were recorded on a FTIR Bomem-Michelson 102 spectrometer in
the 4000–200 cm−1 region using CsI pellets. Conductivity values were obtained using
1.0 mM solutions of complexes in CH2Cl2, using a Meter Lab CDM2300 instrument.
The molar conductance measurements (Λ) were carried out in dichloromethane at 25oC,
1
31
13
using concentrations of 1.0 x 10-3 M for the complexes. H, P and C NMR were
recorded on a Bruker DRX 400 MHz, internally referenced to TMS, chemical shift (δ),
multiplicity (m), spin-spin coupling constant (J), integral (I). CDCl3 was used as
solvent, unless mentioned. The 31P shifts are reported in relation to H3PO4, 85%. Partial
elemental analyses were carried out on the Department of Chemistry of the Federal
University of São Carlos – UFSCar, in an instrument of CHNS staff EA 1108 of the
FISONS.
2.2. Synthesis of N,N-dialkyl-N´-acyl thioureas
Detailed descriptions of syntheses and characterization of ligands have been
reported elsewhere [24]. A solution of an appropriately acyl chloride (30 mmol) in
acetone (50 mL) was added drop wise to a suspension of KSCN (0.01 mol) in acetone
(30 mL). The mixture was stirred until a precipitate appeared (ammonium chloride),
5

ACCEPTED MANUSCRIPT
indicating the formation of the respective organic isothiocyanate. The corresponding
amine (40 mmol), dissolved in acetone was added, slowly and with constant stirring to
the resulting solution. The solution was cooled in an ice-water bath and the stirring was
continued at room temperature during 2–9 hours, until the reaction was completed (the
reaction progress was monitored by TLC). The reaction mixture was then poured into
600 mL of cold water. The solid N,N-disubstituted-N´-acyl thioureas were collected by
filtration and finally purified by recrystallization from ethanol. The identity of the
1
13
products was confirmed by comparing their H and C NMR data with those reported
in the literature [24]. The structures of the ligands are shown in Figure 1.
Insert Figure 1
2.3. Synthesis of the complexes:
The complexes were obtained from direct reactions of the precursor,
[PdCl2(PPh3)2], with the N,N-disubstituted-N´-acyl thioureas, in methanol solutions. The
complexes were separated from the reaction mixtures as yellow crystalline solids.
Filtration and further washing with hot water and hot hexane were enough to afford
pure and stable compounds, in about 80% yields. Thus, the general procedure for the
synthesis of the complexes is described: A solution of [PdCl2(PPh3)2] (1.49 mg; 0.002
mmol) in 5 mL of methanol, was added drop wise to a solution of corresponding N,N-
dialkyl-N´-acyl thiourea (0.002 mmol), dissolved in 30 mL of the same solvent, and
0.368 g (0.002 mmol) of KPF6. The reaction was heated under magnetic stirring at
80oC, for 2 hours. The reaction mixture was left in the refrigerator overnight. The
yellow solids obtained were filtered off and washed, successively, with hot water and
hot hexane (3 x 20 mL). The obtained compounds are stable in the air.
6

ACCEPTED MANUSCRIPT
1
The H and 13C NMR data, the elemental analyses (calculated values in
parenthesis), melting point temperature and molar conductivity (ΛM, 1.0 x 10-3 M in
CH2Cl2) for the complexes (1-7) are listed below and the other data used for the
characterization of the complexes will be find in the text:
(1) [PdCl(PPh3)2(N,N-dimethyl-N´-benzoylthioureato-k2O,S)]
1
NMR H, ppm:. 8.07-7.95 (m, 5H, Ph), 7.78-7.22 (m, 5H), 7.21-7.13 (m, 10H), 3.78
13
(3H, s, CH3), and .3.76 (3H, s, CH3). NMR C; ppm: 162. 68 (C=S); 161.84 (C=O);
133.87, 133.77, 133.40, 131.87, 131.76 - 116.34 (C-Ph), 43.85 (CH3), 43.61 (CH3);
31P{1H}, 23.39 (s). IR: ν(C=O),1539; ν(C=S), 799; ν(C=N), 1573 cm-1.
Anal. (%): Found (Calc.): C, 54.92 (55.00) ; H, 4.18 (4.29); N 4.58 (4.58); S, 5.13
(5.24); MP, 226-228 oC; ΛM = 1.4 Ω-1 cm2 mol-1).
(2) cis-[Pd(PPh3)2(N,N-diethyl-N´-benzoylthioureato-k2O,S)]PF6
1
NMR H; ppm: 8.18-8.07 (m, 1H), 7.96-7.86 (m, 5H), 7.69-7.10 (m, 5H), 7.05-6.92
(m, 10H), 3.84 (d, J = 7.07 Hz, 2H, CH2), 3.46 (d, J = 7.07 Hz, 2H, CH2)1.64 (t, J =
7.02, 7.02 Hz, 3H, CH3), 1.30 (t, J = 7.02, 7.02 Hz, 3H, CH3); NMR 13C; ppm: 170.30
(C=S); 169.86 (C=O); 136.22, 136.18, 135.89, 134.48, 133.82 - 126.01 (C-Ph), 47.46
(CH2), 46.63 (CH2), 13.13 (CH3), 12.20 (CH3); 31P{1H}, 26.68 ; 38.36 (d), 2JP-P = 32.40 Hz.
IR: ν(C=O),1528; ν(C=S), 787; ν(C=N), 1576 cm-1.
Anal. (%): Found (Calc.): C, 54.87 (55.01) ; H, 4.35 (4.49); N, 2.58 (2.77); S, 3.25
(3.17); MP, 190-193 oC; ΛM = 50.4 Ω-1 cm2 mol-1);
(3) cis-[Pd(PPh3)2(N,N-diphenyl-N´-benzoylthioureato-k2O,S)]PF6
1
NMR H, ppm: 7.43-7.18 (m, 1H), 7.03 (s, 1H), 6.13 (d, J = 1.60 Hz, 1H), 6.13-6.01
(m, 1H), NMR 13C, ppm: 174.07. (C=S); 162.92 (C=O); 150.00, 149.93, 146.60,
7

ACCEPTED MANUSCRIPT
2
144.00, 143.57, 134.53 – 112. 11 (C-Ph). 31P{1H}, 27.63 ; 37.71 (d) , JP-P = 29.16 Hz.
IR: ν(C=O), 1517; ν(C=S), 784; ν(C=N), 1586 cm-1.
Anal. (%): Found (Calc.): C, 60.59 (60.74) ; H, 4.19 (4.10); N, 2.45 (2.53); S, 2.83
(2.90); MP, 220-221 oC; ΛM = 52.7 Ω-1 cm2 mol-1).
(4) cis-[Pd(PPh3)2(N, N-dibencyl-N´-benzoylthioureato-k2O,S)]PF6
1
NMR H, ppm: 8.43-8.32 (m, 1H), 7.80-7.40 (m, 1H), 7.10-6.97 (m, 1H), 6.76-6.02
13
(m,1H), 5.29 (s, 2H, CH2), 5.06 (s, 2H, CH2). NMR C; ppm: 172.54 (C=S); 170.99
(C=O); 149.96, 149.88, 146.94, 134.64, 134.41 - 112.06 (C-Ph), 53.93 (CH2), 51.25
31
2
(CH2). P{1H}, 27.37 ; 37.95 (d) , JP-P = 29.97 Hz. IR: ν(C=O),1518; ν(C=S), 783;
ν(C=N), 1576 cm-1.
Anal. (%): Found (Calc.): C, 61.48 (61.35) ; H, 4.30 (4.35); N, 2.45 (2.47); S, 2.90
(2.82); MP, 160-162 oC; ΛM = 48.6 Ω-1 cm2 mol-1).
(5) cis-[Pd(PPh3)2(N, N-diethyl-N´-furoylthioureato-k2O,S)]PF6
NMR 1H, ppm: 7.78-7.50 (m, 1H), 7.46 – 6.80 (3H, m, furoyl), 3.12 (2H, c, CH2), 3.09
(2H, c, CH2), 1.31 (3H, t, CH3), 1.06 (3H, t, CH3). NMR 13C, ppm: 175.92. (C=S),
169.53 (C=O), 161.60, 155.11, 154.29, 153.77-150.16 (C-Ph), 148.02 - 111.99 (C-Ph
and furan ring ), 46.75 (CH2), 46.47 (CH2), 13.18 (CH3) 12.07 (CH3). 31P{1H}, 26.63 ;
38.52 (d) , 2JP-P = 32.40 Hz. IR: ν(C=O),1511; ν(C=S), 756; ν(C=N), 1581 cm-1.
Anal. (%): Found (Calc.): C, 55.09 (55.18) ; H, 4.30 (4.33); N, 2.95 (2.80); S, 3.15
(3.20); MP, 226-228 oC; ΛM = 59.6 Ω-1 cm2 mol-1).
(6) cis-[Pd(PPh3)2(N,N-diphenyl-N´-furoylthioureato-k2O,S)]PF6
1
13
NMR H, ppm: 7.42-7.03 (m, 1H, Ph), 6.13-6.01 (m, 1H, the furan ring), NMR C
ppm: 174.08 (C=S); 162.93 (C=O); 150.01, 149.94, 146.60, 144.03-129.78 and
8

ACCEPTED MANUSCRIPT
31
2
128.98- 112.10 (C-Ph and furoyl ring ). P{1H}, 26.78 ; 38.07 (d) , JP-P = 30.78 Hz.
IR: ν(C=O),1523; ν(C=S), 769; ν(C=N), 1574 cm-1.
Anal. (%): Found (Calc.): C, 58.95 (59.11) ; H, 4.00 (3.95); N 2.50 (2.55); S, 2.88
(2.92); MP, 300-302 oC; ΛM = 52.8 Ω-1 cm2 mol-1).
(7) cis-[Pd(PPh3)2(N,N-dibenzyl-N´-furoylthioureato-k2O,S)]PF6
1
NMR H; ppm: 7.92 – 6.94 (44H, m, C-Ph and furan ring), 5.29 (2H, s, CH2), 5.10
13
(2H, s, CH2). NMR C; ppm: 173.09. (C=S), 170.99 (C=O), 135.72, 135.29, 135.22,
134.67-129.01 and 128.96- 125.79 (C-Ph and furoyl ring ). 31P{1H}, 27.41 ; 37.96 (d) ,
2JP-P = 30.78 Hz. IR: ν(C=O),1534; ν(C=S), 752; ν(C=N), 1581 cm-1.
Anal. (%): Found (Calc.): C, 59.77 (59.77) ; H, 4.35 (4.21); N, 2.40 (2.49); S, 2.73
(2.85); MP, 200-202 oC; ΛM = 48.6 Ω-1 cm2 mol-1).
2.4 Crystal structure determination
Single crystals suitable for X-ray diffraction were obtained by slow evaporation of
CHCl3:n-hexane (3:1) solutions of the complexes 1, 3, 4, 5 and 6. Diffraction data were
collected on an Enraf-Nonius Kappa-CCD diffractometer with graphite-monochromated
Mo Ka radiation (λ = 0.71073 Å). The final unit cell parameters were based on all
reflections. Data collections were performed using the COLLECT program [26];
integration and scaling of the reflections were performed with the HKL Denzo-
Scalepack system of programs [27]. Absorption corrections were carried out using the
Gaussian method [28]. The structures were solved by direct methods with SHELXS-97
[29]. The models were refined by full-matrix least-squares on F2 by means of SHELXL-
97 [30]. The projection views of the structures were prepared using ORTEP-3 for
Windows [31]. Hydrogen atoms were stereochemically positioned and refined with the
riding model. Data collections and experimental details are summarized in Table 1.
9

ACCEPTED MANUSCRIPT
Relevant interatomic bond lengths and angles are listed in Table 2. Also included are
the CCDC deposit numbers for supplementary crystallographic data.
Insert Table 1
2.5 Cell culture assay
In vitro cytotoxicity assays on cultured human tumor cell lines still represent the
standard method for the initial screening of antitumoral agents. Thus, as a first step in
assessing their pharmacological properties, the new palladium(II) complexes were
assayed against human breast tumor cell lines MDA-MB-231 (ATCC: HTB-26), DU-
145 (ATCC: HTB-81) and against the L929 cell line (ATCC: CCL 1). The cells MDA-
MB-231 and L929 were routinely maintained in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% fetal bovine serum (FBS) for cell DU-145 were
maintained in RPMI-1640 supplemented with 10% FBS, at 37 ºC in a humidified 5%
CO2 atmosphere. After reaching confluence, the cells were detached by trypsinization
and counted. For the cytotoxicity assay, 1.0 x 104 cells.well-1 were seeded in 200 µL of
complete medium in 96-well assay microplates. The plates were incubated at 37 ºC in
5% CO2 for 24 h to allow cell adhesion. All tested compounds were dissolved in sterile
DMSO (stock solution with maximum concentration of 20 mmol L-1) and diluted to 20;
10; 5.0; 2,5; 0.62; 0.15; 0.039 mmol L-1. From each of these dilute samples 2 µL
aliquots were added to 200 µL medium giving a final concentration of DMSO of
approximately 1% and a final concentration of the complex diluted about 100 times.
Cells were exposed to the compounds during a 48 h-period. Cell respiration, as an
indicator of cell viability, was determined by the mitochondrial-dependent reduction of
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to formazan [32].
MTT solution (0.5 mg/mL) was added to the cell cultures and incubated for 3 hours,
10

ACCEPTED MANUSCRIPT
after which 100 µL of isopropanol was added to dissolve the precipitated formazan
crystals. The conversion of MTT to formazan by metabolically viable cells was
monitored in an automated microplate reader at 540 nm. The percent cell viability was
calculated by dividing the average absorbance of the cells treated with the test
compounds by that of the control; % cell viability was plotted against drug
concentration (logarithmic scale) to determine the IC50 (drug concentration at which
50% of the cells are viable in relation to the control), with the error estimated from the
average of 3 trials.
3. Results and discussion
All the synthesized palladium(II) complexes are of yellow color, and their
elemental analyses, melting point temperatures, and molar conductivities were listed in
the experimental section. These data suggest the formation of [Pd(PPh3)(Cl)(L)] for
complex (1) and [Pd(PPh3)2(L)]PF6 for complexes (2-7), in which L represents the
anionic ligand formed upon deprotonation, when the N, N-disubstituted-N´-acyl
thioureas coordinate to the metal center.
The most useful infrared spectral bands for determining the ligands mode of
coordination are given in the experimental section. The IR spectra of the ligands reveal
broad, strong absorption bands in the range of 3053 – 3267 cm-1, due to NH stretching
vibrations. As expected, these N–H bands present in the free ligands are absent in the IR
spectra of the complexes. On the other hand, the ν(C=N) band, present in the complexes
at 1573-1586 cm-1, is absent in the free ligands, suggesting the formation of the
heterocyclics, in which the nitrogen changes from sp3 to sp2 hybridization when
coordinating to the metal, as shown in Scheme 1. The coordination of the metal to the
carbonyl group decreases the C=O stretching vibration frequency by ca. 180 cm-1, when
11

ACCEPTED MANUSCRIPT
compared with the free ligand, in agreement with the literature [33]. Thus, considering
that the free ligands have their ν(C=O) bands at about 1680 cm-1, it is reasonable to
assign the bands in the range 1511-1539 cm-1 to the coordinated C=O group. The
absorptions at 815-878 cm-1 in the spectra of free bases, N, N-disubstituted-N´-acyl
thioureas, attributed to the ν(C=S) stretching vibrations, shift to the 752-799 cm-1 range
in the complexes spectra. This substantial change suggests deprotonation of the ligands,
indicating coordination through the sulfur atom with a formally C-S single bond [34-
37]. The absorptions at about 470 cm-1 in the IR spectra of the complexes can be
assigned to the M-O vibration mode [38-39] and the assignment of the Pd-S stretching
vibration bands at about 330 cm-1 are in accordance to the reported by Orysyk et al for
palladium(II) complexes with 1-allyl-3-(2-pyridyl)thioureas [40]. Thus, from the IR
data it is possible to suggest that the N, N-disubstituted-N´-acyl thioureas are attached
to the metal through the oxygen and sulfur chelating system. In the case of complex (1)
a chloride ion occupies one coordination position and the fourth coordination site of the
metal is occupied by a triphenylphosphine, while for the other complexes (2-7) there are
two coordinated triphenylphosphines. The molar conductivity of complex (1) shows that
it is neutral, while complexes (2-7) are anionic species [41].
The molecular structures of the complexes were also investigated by NMR (1H,
13C and 31P{1H}) spectroscopy and a comparative analysis on the basis of the
spectroscopic data corresponding to both, free and coordinated ligands with the metallic
ion was carried out.
The 1H NMR data for the complexes (1-7) are given in the experimental section.
The 1H NMR integrations and signal multiplicities are consistent with the proposed and
with the X-ray structures. The 1H NMR spectra of the free ligands show basically three
sets of well separated signals corresponding to their R1, R2 substituent and to the NH
12

ACCEPTED MANUSCRIPT
proton. The signals of the NH protons appear as broad singlets in the region between
8.35 – 8.80 ppm [24]. The absence of the N-H proton after the coordination of the
ligands to the metal is associated with an increase in electron density at the C-N bond
upon complexation [42-43]. A slight downfield shift was also observed in the aromatic
protons and in the protons of the nitrogen substituents in the coordinated ligands, when
compared to the chemical shift of the free ligands. The aromatic protons appear as a
13
complex pattern in the region δ 8.82–6.50 ppm. In the C NMR spectra the chemical
shifts of the C=S carbon atoms of the ligands moved up field after their coordination to
the metal for all complexes studied, but in the case of the C=O group, this tendency was
not observed for all complexes. The explanation for this is the fact that with the
deprotonation of the secondary amide and with the formation of a “N=C-S-Ru” species,
the sulfur atoms, and consequently their neighbor carbon atoms, became more shielded
in all complexes, something that does not happen with the carbon from the carbonyl
group, in the same extension. The 31P{1H} NMR spectrum of the precursor
[PdCl2(PPh3)2] in CH2Cl2 solution shows a singlet peak for phosphorous atoms at 23.83
ppm. In the spectra of complex (1) the singlet peak is shifted to 23.39 ppm, in
conformity with the presence of only one phosphorus species for the complex, in
solution. For the other complexes two doublets arise, at about 27 and 38 ppm, indicating
the presence of two magnetically different phosphorus atoms coordinated to the
palladium(II) ion. Thus, considering that for complex (1) the phosphorus atoms is trans
to the oxygen atom from the acyl thiourea ligand and that its chemical shifts is 23.39
ppm, it is reasonable to assign the 31P{1H} chemical shifts of the other complexes (2-7)
at about 27 ppm also to the phosphorus trans to the oxygen atoms and the chemical
shifts at about 38 ppm to the phosphorus trans to the sulfur atoms from the ligands.
-
Also in the region of -138-158 ppm a multiple signal of the PF6 function as outer–
13

ACCEPTED MANUSCRIPT
sphere anions for the complexes obtained is observed, except for complex (1),
indicating that this complex is neutral, in accordance to the molar conductivity
measurements for these complexes (see experimental section). These data are consistent
with those obtained for some palladium(II) thiosemicarbazones complexes [35].
The structures of complexes (1), (3), (4), (5) and (6) were determined by X-ray
diffraction analysis. Their ORTEP views are in Figure 2 and selected bond lengths (Å)
and angles (o) for the complexes are listed in Table 2. In the structures of the complexes
the N,N-disubstituted-N´-acyl thioureas adopts a cis conformation, bounded to the
central ion Pd(II) by the oxygen and sulfur atoms, and with two PPh3 ligands, which are
also in the cis fashion, except for the complex (1), which has just one
triphenylphosphine in its structure. In all complexes the Pd(II) ion is nearly planar,
fourfold environment. The thione C=S bond in the coordinated anionic ligands becomes
formally a single bond making it longer (average = 1.7418 Ả) than the C=S bond of the
neutral moieties [24,25], and the N(2)-C bond (average 1.3252 Ả) (Figure 2, Table 2),
which becomes a double bond in the anionic moieties, is typical for C=N distances [44].
The N(1)-C distance, 1.341 Ả (average), is anionic and in the amine form. Thus the
metal is coordinated to the negatively charged organic molecules, which act as bidentate
ligands, through oxygen (average distance Pd-O = 2.059 Ả) and sulfur (average distance
Pd-S = 2.2924 Ả) atoms. The Pd-S average distance is close to the ones found for other
palladium(II) thiosemicarbazones complexes [36]. The remaining binding sites are
occupied by triphenylphosphines (average distance Pd-P1 = 2.2577 Ả and Pd-P2 =
2.3455 Ả), or by the chloride in the case of complex (1), where the distance Pd-Cl is
2.3269(9) Ả, which is close to those reported in the literature [36]. The distances for the
C–S, C-N and C-O bonds in the chelate rings, listed in Table 2, are characteristic of
single and double bond lengths, respectively [44]. The C–O bond distances are slightly
14

ACCEPTED MANUSCRIPT
sensitive to the coordination of the ligand to the metal. In the case of the bis-
triphenylphosphine-N, N-dietyl-N'-furoilthioureato-k2O,S) the C-O distance for the free
ligand is 1.226(3) Å (average for two independent molecules per asymmetric unit), and,
1.2605(4) Å, after its coordination to the metal [25].
Insert Figure 2
Insert Table 2
Table 3 lists the ligands and complexes concentrations that produce 50% of growth
inhibition (IC50, µM) against DU-145 (human prostate cancer cells), MDA-MB-231
(human breast cancer cells) and against the L929 cell line (health cell line from mouse).
The seven new palladium(II) complexes, the free ligands and the precursor
[PdCl2(PPh3)2], were tested against the tumor cells and L929 cells. For comparison the
cytotoxicity of cisplatin and of [PdCl2(PPh3)2] were also evaluated under the same
experimental conditions. The IC50 values, calculated from the dose-survival curves
generated by the MTT assays obtained after drug treatment, are shown in Table 3. It is
worth pointing out that the most promising complex is (5), with the N, N-diethyl-N´-
furoylthiourea ligand, which had very low IC50 and was selective for the MDA-MB-231
cells. Additionally, its IC50 against the L929 cell is very high. It is interesting to observe
that the smaller volume of ligand in complex (5), when compared with the other
complexes, may be the steric factor affecting the enhanced cytotoxicity of the
compound. In this case the low steric effect of the complex can facilitate its contact with
the DNA molecule, while the presence of the oxygen atom in the furoyl thiourea group
allows its interaction through hydrogen bonding. These preliminary results are very
15

ACCEPTED MANUSCRIPT
encouraging in that they support our hypothesis that the non-bonded atoms present in
the ligands can play an important role in the biological activity of the complex. We are
observing this behavior for a series of ruthenium and platinum complexes and detailed
studies are currently underway in our laboratory to investigate their biological activity
and to try to establish a structure-activity relationship.
Insert Table 3
3. Conclusions
The preparation of a novel series of Pd(II) complexes with N,N-disubstituted- N´-acyl
thiourea bidentate ligands is here reported. The X-ray crystallographic characterization
shows that these ligands coordinate with the metal through the oxygen and sulfur atoms.
The complexes exhibit a slightly distorted square–planar geometry corresponding to
stoichiometry metal:ligand in 1:1 ratio. In this work there are two series of complexes:
for the first one, the complexes (1-4), R1 is the phenyl group and for the other one it is
the furoyl one (5-7). The most promising complex as antitumor agent against the MDA-
MB-231 from the two series here reported is the one where R1 is the furoyl group and
R2 is some of the ethyl groups (5), probably due the lower steric hindrance caused by
these R2 groups in the contact of the complex with the DNA molecule. Additionally,
the furoyl oxygen atom allows the electrostatic interaction of the complex with the
biological molecule.
Acknowledgements
This work was supported by CAPES (Project Oficio/CSS/CGCI/23038009487/2011-
25/DRI/CAPES, AUX CAPES-MES-Cuba, 339/2011), CNPq and FAPESP of Brazil.
16

ACCEPTED MANUSCRIPT
References:
[1] S.J. Lippard, M.J. Berg, Principles of Bioinorganic Chemistry, University Science
Books, Mll Valley, CA. 1994
[2] D. Kovala-Demertzi, M. A. Demertzis, J.R. Miller, C. Papadopoulou, C. Dodorou,
G. Filousis, J. Inorg. Biochem. 86(2-3) (2001) 555-563.
[3] D. Kovala-Demertzi, A. Boccarelli, M.A. Demertzis, M. Coluccia, Chemotherapy
53 (2007) 148-152.
[4] A. Rodger, K. K. Patel, K. J. Sanders, M. Datt, C. Sacht, M. J. Hannon, J. Chem.
Soc., Dalton Trans. (2002) 3656-3663.
[5] A.D. Burrows, M.D. Coleman, M.F. Mahon, Polyhedron 18 (1999) 2665-2671.
[6] W. Henderson, B.K. Nicholson, M.B. Dinger, R.L. Bennett, Inorg. Chim. Acta 338
(2002) 210-218.
[7] A.A. Aly, E.K. Ahmed, K.M. El-Mokadem, M.A.F. Hegazy, J. Sulfur Chem. 28
(2007) 73-93.
[8] A.A. Aly, A.M. NourEl-Din, A.M. Gomaa, A.B. Brown, M.S. Fahmi, J. Chem.Res.
(2007) 439-441.
[9] M.K. Rauf, I. Din, A. Badshah, M. Gielen, M. Ebihara, D. Vos, S.J. Ahmed, J.
Inorg. Biochem. 103(8) (2009) 1135-1144.
[10] A. Saeed, N. Abbas, H. Rafique, S. Rashid, A. Hameed, Khimiya (Sofiya
Bulgaria) 18 (2009) 152-158.
[11] J. Müller, C. Limban, B. Stadelmann, A.V. Missir, I.C. Chirita, M.C. Chifiriuc,
G.M. Nitulescu, Parasitol. Int. 58 (2009) 128-135.
[12] H.M. Faidallah, K.A. Khan, A.M. Asiri, J. Chem. Sci. (Bangalore, India) 124
(2012) 625-631.
17

ACCEPTED MANUSCRIPT
[13] L.P. Duan, J. Xue, L.L. Xu, H.B. Zhang, Molecules 15 (2010) 6941-6947.
[14] Z.M. Zhong, R. Xing, R., S. Liu, S., L. Wang, S.B. Cai, P.C. Li, Carbohydr. Res.
343 (2008) 566-570.
[15] Z.Y. Wang, S. Wang, X.J. Song, Y.G. Wang, Chin. J. Pestic. Sci. 7 (2005) 282-
284.
[16] C. Sacht, M. S. Datt, S. Otto, A. Roodt, J. Chem. Soc., DaltonTrans. (2000) 727-
733.
[17] C. Sacht, M. S. Datt, S. Otto, A. Roodt, J. Chem. Soc., Dalton Trans. (2000) 4579-
4586.
[18] C. Sacht, M. S. Datt, Polyhedron 19 (2000) 1347-.1354.
[19] K.R. Koch, Coord. Chem. Rev. 473 (2001) 216-217.
[20] W. Zhou, Y. Wen, L. Qiu, Y. Zhang, Z. Yu, J. Mol. Struct. 749 (2005) 89-95.
[21] J.C. Bruce, N. Revaprasadu, K.R. Koch, New J. Chem. 31 (2007) 1647-1653.
[22] A.N. Westra, S.A. Bourne, C. Esterhuysen, K.R. Koch, Dalton Trans. (2005) 2162
- 2172.
[23] M. Gielen, E Tiekink,. Metallotherapeutic Drugs and Metal Based Diagnostic
Agents, West Sussex (England): John Wiley and Sons Ltd., 2005.
[24] A. Plutin, H. Márquez, M. Morales, M. Sosa, L. Morán, Y. Rodríguez, M. Suárez,
C. Seoane, N. Martín, Tetrahedron 56 (2000) 1533 - 1594.
[25] B. O’Reilly, A. M. Plutín, H. Pérez, O. Calderón, R. Ramos, R. Martínez, R. A.
Toscano, J. Duque, H. Rodríguez-Solla, R. Martínez-Alvarez, M. Suárez, N.
Martín, Polyhedron 36 (2012) 133 - 140.
[26] Enraf-Nonius, Collect, Nonius BV, Delft, The Netherlands, 1997–2000.
[27] Z. Otwinowski, W. Minor, Processing of X-ray diffraction data collected in
oscillation mode, Macromol. Crystallogr. Part A 276 (1997).
18

ACCEPTED MANUSCRIPT
[28] R.H. Blessing, Acta Crystallogr. Sect. A 51 (1995) 33 - 38.
[29] G.M. Sheldrick, ShelXS-97 Program for Crystal Structure Resolution, University
of Göttingen, Göttingen, Germany, 1997.
[30] G.M. Sheldrick, ShelXL-97, Program for Crystal Structures Analysis, University
of Göttingen, Göttingen, Germany, 1997.
[31] L.J. Farrugia, J. Appl. Crystallogr. 30 (1997) 565 -566.
[32] T. Mosmann, J. Immunol. Methods, 65 (1983) 55 - 63.
[33] M. Mikami, I. Nakagawa, T. Shimanouchi, Spectrochim. Acta, Part A 25 (1969)
365 - 374.
[34] A.E. Graminha, C. Rodrigues, A.A. Batista, L. R. Teixeira, E. S. Fagundes, H.
Beraldo, Spectrochim. Acta Part A, 69 (2008) 1073 - 1076.
[35] P. I. da S. Maia, A. E. Graminha, F. R. Pavan, C. Q. F. Leite, A. A. Batista, D. F.
Back, E. S. Lang, J. Ellena, S. de S. Lemos, H. S. Salistre-de-Araujo, V. M. Deflon,
J. Braz. Chem. Soc., 21 (2010) 1177 - 1186.
[36] A. P. Rebolledo, M. Vieites, D. Gambino, O. E. Piro, E. E. Castellano, C. L. Zani ,
E.M. Souza-Fagundes, L. R. Teixeira, A. A. Batista, H. Beraldo, J. Inorg. Biochem.
99 (2005) 698 - 706.
[37] A. Pérez-Rebolledo, L. R. Teixeira, A.A. Batista, A. S. Mangrich, G. Aguirre, H.
Cerecetto, M. González, P. Hernández, A.M. Ferreira, N.L. Speziali, H. Beraldo,
Eur. J. Med. Chem. 43 (2008) 939 - 948.
[38] K. Nakamoto, Infrared and Raman Spectra of Inorganic and Coordination
Compounds, fourth ed., Wiley, New York, 1986.
[39] A. Pérez-Rebolledo, O. E. Piro, E.E. Castellano, L. R. Teixeira, A. A. Batista, H.
Beraldo, J. Mol. Struct. 794 (2006) 18 - 23.
19

ACCEPTED MANUSCRIPT
[40] S. I. Orysyk, L. N. Rybachuk, V. I. Pekhnyo, V. V. Orysyk, Russian J. of Inorg.
Chem. 56 (2011) 1747 - 1751.
[41] W.J. Geary, Coord. Chem. Rev. 7 (1971) 81 - 122.
[42] S. Ahmad., A.A. Isab., S. Ali., Trans. Met. Chem., 31 (2006) 1003 - 1016.
[43] S. Ahmad., Chem. & Biodiv., 7 (2010) 543 - 566.
[44] H.H. Nguyen, J. J. Jegathesh, P.I. da S. Maia, V.M. Deflon, R. Gust, S.
Bergemann, U. Abram, Inorg. Chem. 48 (2009) 9356 - 9364.
Captions to Scheme and Figures
Scheme 1. Pathways for the synthesis of N, N-disubstituted-N´-acylthioureas Pd (II)
complexes, and the R1 and R2 groups present in the ligands.
Figure 1: Structures of the N,N-disubstituted-N´-acylthioureas used as ligands in this
work.
Figure 2. ORTEP view of (1), (3), (4), (5), and (6) complexes showing 50% probability
ellipsoids except for (4) which is shown at a 30% level. Hydrogen atoms, the (PF6)-
anion and some labels of the ligands are omitted for clarity.
20

ACCEPTED MANUSCRIPT
+
PPh3
S
Ph3P
O
Pd
R2
R1
N
N
O
R2
S
(2 - 7)
1) KPF6
2) MeOH
R2
PdCl2(PPh3)2
+
R1
N
H
N
R2
Cl
Ph3P
L (1-7)
R2 = R2
Pd
O
S
R2
R1
N
N
R2
(1)
R1
R2
R1
R2
Ligands
L1
Ligands
-
-
CH3
CH3-CH2
L5
L6
O
-
CH3-CH2
L2
L3
O
-
L7
CH2
O
-
L4
CH2
Scheme 1
21

ACCEPTED MANUSCRIPT
S
O
S
S
O
O
S
O
CH3
Ph
Ph
N
H
N
CH3
CH3
N
H
N
N
H
N
N
H
N
CH3
Ph
Ph
L4
L1
L2
L3
S
O
S
O
S
O
Ph
Ph
N
H
N
N
H
N
CH3
CH3
N
N
O
O
O
Ph
H
Ph
L7
L6
L5
Figure 1
22

ACCEPTED MANUSCRIPT
Figure 2
23

ACCEPTED MANUSCRIPT
Table 1. Crystal data and refinement parameters for (1), (3), (4), (5) and (6) complexes.
Compound
(1)
(3)
(4)
(5)
(6)
Empirical formula
Formula weight
Crystal system
Space group
a (Å)
C28H26ClN2OPPdS
611.39
C56H45F6N2OP3PdS
1107.31
C58 H49F6N2OP3PdS C46H43F6N2O2P3PdS C54H43F6N2O2P3PdS
1135.36
Monoclinic
P21/n
1001.19
Monoclinic
P21/a
1097.27
Triclinic
P-1
Monoclinic
P21/c
Triclinic
P-1
14.3350(5)
11.1240 (5)
18.1440 (5)
110.608 (2)
2708.15 (17)
4
11.2530(7)
11.7780(8)
20.7010(14)
89.442 (4)
2559.4 (3)
2
15.7849(8)
13.2553(7)
25.2766(11)
102.264(3)
5168.0(4)
4
11.2500(4)
29.0220(16)
14.1490(7)
92.472 (3)
4615.3 (4)
4
11.2080(2)
11.89.00(2)
20.5390(3)
87.7960 (10)
2554.94 (7)
2
b (Å)
c (Å)
 ( 0)
V (Å3)
Z
D.calc (mg/m3)
1.500
1.437
1.459
1.441
1.426
Absorption
0.944
0.561
0.557
0.615
0.562
coefficient(mm-1)
F(000)
1240
1128
2320
2040
1016
 range for data
2.89 to 26.00
2.61 to 24.82
2.78 to 26.00
2.75 to 22.49
2.62 to 26.00
collection (0)
Index ranges
-17 ≤h ≤ 19
-15 ≤ k ≤ 16
-29 ≤ l ≤ 31
37665
-17 h17
-13 k10
-22 i22
16636
-12 h13
-13 k13
-23 l18
16727
-10 h12
-29 k31
-15 l13
21137
-13 h12
-14 k 14
-25 l25
45097
Reflections collected
Independent
reflections
(Rint)
Goodness-of-fit on F2
5140(0.0760)
1.058
7823 (0.1018)
1.090
9539(0.1124)
0.945
6001(0.1218)
0.981
9513(0.980)
1.059
Final R índices
[I > 2 (I)]
R1= 0.0429,
wR2=0.1070
R1= 0.0806,
wR2=0.2093
R1 = 0.0506,
wR2 = 0.1113
R1= 0.0578,
wR2=0.1171
R1= 0.051,
wR2=0.1624
Largest diff. peak and
0.343 and -0.669
1.008 and -1.107
842053
0.821 and -0.921
931560
0.430 and -0.395
842056
0.998 and -0.736
842055
hole (eÅ)_3
CCDC deposit number 842052
24

ACCEPTED MANUSCRIPT
Table 2. Selected bond lengths (Å) and angles (0) for the complexes (1), (3), (5-6).
bond lengths
(3)
(1)
(4)
(5)
(6)
Pd-O1
Pd-P1
Pd-P2
Pd-S
2.081(2)
2.052(5)
2.076(2)
2.018 (5)
2.069 (2)
2.2509(8) 2.2571 (19) 2.2580(10) 2.2612 (18) 2.2611 (8)
2.3516(19) 2.3403(11) 2.3512 (19) 2.3389 (8)
2.2474(10) 2.3026 (18) 2.3108(11) 2.2945 (19) 2.3067 (8)
-
Pd-Cl
S-C
C1-O1
N2-C
N2-C1
N1-C
2.3269(9)
1.733(4)
1.262(4)
1.327(5)
1.316(5)
1.330(5)
-
-
-
-
1.755 (8)
1.248 (9)
1.300(9)
1.314 (10)
1.349(9)
1.748(4)
1.268(4)
1.333(5)
1.320(5)
1.344(4)
1.727 (8)
1.267 (8)
1.344(9)
1.315 (9)
1.334 (8)
1.746 (3)
1.244 (4)
1.322(4)
1.310 (5)
1.348 (4)
Angles (o)
O1Pd-S
91.30(7)
91.19 (15)
93.12(7)
91.55 (15)
91.52 (8)
O1-Pd-P1
O1-Pd-P2
S-Pd-P2
S-Pd-P1
P2-Pd-P1
175.94(7) 175.79 (18) 176.77(8)
177.75 (15) 175.69 (8)
-
81.96 (15)
169.70 (7)
90.54(7)
82.15(7)
175.06(3)
89.07(4)
95.72(4)
83.35 (15)
174.73 (7)
89.85 (7)
95.29 (7)
81.95 (8)
169.62 (3)
90.29 (3)
96.79 (3)
-
92.52 (4)
-
96.82 (7)
Table 3. Cytotoxic effect of (L1-L7) ligands and the (1-7) complexes against DU-145,
MDA-MB-231 and L929 cell line, after 48 h of incubation.
IC50 (µmolL-1)
Complexes
DU-145
MDA-MB-231
L929
4.25 ± 0.92
7.02 ± 0.81
>200
2.15 ± 0.97
1.12 ± 0.52
>200
<0.8
<0.8
>200
(1)
(2)
(3)
>200
>200
>200
(4)
31.85 ± 1.41
68.97 ± 1.64
47.85 ± 1.15
>200
<0.8
>200
>200
>200
>200
>200
>200
>200
>200
(5)
(6)
(7)
(L1)
(L2)
(L3)
(L4)
(L5)
48.70 ± 13.07
37.12 ± 9.87
79.11 ± 22.12
95.71 ± 30.49
>200
>200
>200
>200
>200
68.02 ± 1.95
>200
71.36 ± 1.50
45.57 ± 1.51
>200
>200
>200
>200
>200
82.85 ± 25.88
>200
(L6)
(L7)
PPh3
2.00 ± 0.47
>200
2.43 ± 0.20
>200
16.53 ± 2.38
>200
Cisplatin
[PdCl2(PPh3)2]
25

ACCEPTED MANUSCRIPT
The synthesis, characterization and the cytotoxicity of Pd(II)
complexes of N, N-disubstituted -N´-acyl thioureas against DU-
145 (human prostate cancer cells), MDA-MB-231 (human breast
cancer cells) and the toxicity against the L929 cell line (health cell
line from mouse) is reported in this article.
26

ACCEPTED MANUSCRIPT
Highlights
1) New Pd(II)/N, N-disubstituted -N´-acyl thioureas complexes were synthesized.
2) The new complexes present high activity against DU-145 and MDA-MB-231
tumour cells.
3) The new palladium(II) complexes present low toxicity against L-929.
27