In vitro antiproliferative activity of palladium(ii) thiosemicarbazone complexes and the corresponding functionalized chitosan coated magnetite nanoparticles

Article abstract of DOI:10.1039/c5nj02429c

This work reports the synthesis and characterization of palladium(ii) complexes Pd(L¹)₂ (1), Pd(L²)₂ (2), Pd(L³)₂ (3) and Pd(L⁴)₂ (4), where L¹H: 1-naphthal

Full text of DOI:10.1039/c5nj02429c

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Received 00th January 20xx,
In Vitro Antiproliferative Activity of Palladium(II) Thiosemicarbazone Complexes and the
Corresponding Functionalized Chitosan Coated Magnetite Nanoparticles
Wilfredo Hernándeza, Abraham. J. Vaisbergb, Mabel Tobarc,d, Melisa Álvarez c,d, Jorge
Manzur c,d*, Yuri Echevarríad,e*, Evgenia Spodine d,e*
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
This work reports the synthesis and characterization of palladium(II) complexes Pd(L1)2 (1), Pd(L2)2
(2), Pd(L3)2 (3) and Pd(L4)2 (4), where L1H: 1‐naphthaldehyde thiosemicarbazone; L2H: 4‐phenyl‐(1‐
naphthaldehyde) thiosemicarbazone; L3H: (2‐hydroxy‐1‐naphthaldehyde) thiosemicarbazone; L4H: 4‐
phenyl‐1‐(2‐hydroxy‐1‐naphthaldehyde) thiosemicarbazone. All four complexes show in vitro
antiproliferative activity against the following human tumor cell lines: H460, DU145, MCF‐7, M14,
HT‐29, K562, HuTu 80. In particular Pd(L1)2 has the most potent activity for all the studied cell lines
(IC50 ~ 1 μM), with the exception of H460. Pd(L2)2 is a promising candidate as pharmacological agent,
since it presents a significant activity and is more innocuous than cisplatin against mouse fibroblast
normal cells, 3T3. Pd(L4)2 is the complex which exhibits the lowest activity against the same cell lines
(IC50 ~ 11 μM), being ten times lower than that of Pd(L1)2. These complexes were used to
functionalize chitosan coated superparamagnetic magnetite nanoparticles with a metallic core of 11‐
13 nm, and the activity of these functionalized nanoparticles (NPs) against diverse human tumor cell
lines was also tested. The nanoparticles, functionalized with Pd(L1)2, Pd(L3)2 and Pd(L4)2 show
antiproliferative activity against DU‐145, while those with Pd(L2)2, Pd(L3)2 and Pd(L4)2 against
HuTu80.
Introduction
complexes M(L)Cl and ML2 with (N,N,S) ligands derived
from thiosemicarbazone against different human
Thiosemicarbazones derivatives and their transition
metal complexes have been shown to possess a wide
variety of biological activities as antiviral, antimicrobial,
anticancer, and antitumor agents1‐8. Recently, in vitro
studies of the platinum(II) and palladium(II) bis‐chelate
tumor cell lines showed that these metal complexes
are more cytotoxic than their respective ligands9‐11. The
nature of the heteroatomic ring and the presence of
the imine group (‐N=CH‐) are important factors in the
inhibitory action against tumor cell growth12. Probably,
the high cytotoxicity of these complexes may be
related to the intercalation between pairs of DNA
aFacultad de Ingeniería Industrial, Universidad de Lima, Lima 33, Perú.
bLaboratorios de Investigación y Desarrollo, Facultad de Ciencias y
Filosofía, Universidad Peruana Cayetano Heredia, Lima 31, Perú. cFacultad
de Ciencias Físicas y Matemáticas, Universidad de Chile, Santiago, Chile.
dCentro para el Desarrollo de la Nanociencia y la Nanotecnología
bases, or to the breaking of DNA strands13,14
.
In cancer treatment a targeted controlled delivery of
the anticancer drugs is desirable to avoid the damage
of the normal tissue. For biomedical applications
superparamagnetic nanoparticles are preferred
because they can be guided by an external magnetic
field and do not retain any magnetism after removal of
the magnetic field; thus preventing the spontaneous
aggregation of the nanoparticles. For this purpose,
(CEDENNA). Santiago, Chile. eFacultad de Ciencias Químicas
Farmacéuticas, Universidad de Chile, Santiago, Chile.
y
e‐mail: jmanzur@ing.uchile.cl, espodine@uchile.cl, yuriech@yahoo.es
Supplementary information. CCDC 966873 contains the supplementary
crystallographic data for this paper. These data can be obtained free of
charge at www.ccdc.cam.ac.uk/conts/retrieving.html [or from the
Cambridge Crystallographic Data Centre (CCDC), 12 Union Road,
Cambridge CB2
1EZ, UK;
fax: +44(0)1223‐336033; email:
deposit@ccdc.cam.ac.uk].
This journal is © The Royal Society of Chemistry 20xx
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superparamagnetic iron oxides are emerging as spectra were obtained on a Bruker AMX‐300 NMR
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DOI: 10.1039/C5NJ02429C
promising candidates because they can be obtained at spectrometer. X‐ray powder diffraction was performed
nanoscale size and their biocompatibility has been using a Bruker D‐8 Advance diffractometer, with a
proved15‐17. Iron oxide nanoparticles have a great CuKα1 radiation in the 5° < 2θ < 60° range. The
potential for several applications in medicine16,17 magnetic properties of the nanoparticles were
including drug delivery18,19 and hyperthermia20. For the measured at room temperature, on a vibrating
improvement of targeting and biocompatibility the magnetometer applying an external field from ‐10 T to
nanoparticles are coated with different species, which 10 T. The average size of the core of the chitosan
also allow further functionalization. Among others, coated nanoparticles was estimated by the Debye‐
chitosan is an interesting choice due to many Scherrer formula26. The hydrodinamic average size of
significant biological (biodegradable, biocompatible, the functionalized nanoparticles was determined using
bioactive) and chemical properties (polycationic, a Zeta Sizer Nano ZS Malvern Instrument. A Milestone
hydrogel, contains reactive groups such as OH and (Microwave Laboratory Systems) model LAVIS‐1000
NH)20,21. Kim et al.22 reported chitosan‐coated magnetic multiQUANT equipment was used to functionalize the
nanoparticles for application in magnetic targeted coated nanoparticles. UV‐visible spectra were obtained
hyperthermia, where a temperature rise of 23°C under with a Lambda 11 Perkin Elmer spectrophotometer.
an alternating magnetic field can be obtained. The TEM images were acquired on a FEI, Tecnai ST F20
nanoparticles were biocompatible and exhibited higher microscope. Scanning electron microscopy (SEM) was
affinity for KB carcinoma cells than L929 normal cells. done using a Jeol Scanning Microscope (JSM‐5410),
The development of an embolic material capable of with an Oxford Link Isis energy dispersive X‐ray
MRI contrast enhancement, based on microspheres detector (EDXS).
obtained
from
superparamagnetic
magnetite
X‐Ray Diffraction
nanoparticles and chitosan has also been reported23.
Besides, chitosan is characterized by its high affinity for
X‐ray data collection was made on a Bruker Smart Apex
diffractometer, using separations of 0.3° between
frames and 10 s by frame, at 293 K. Data integration
was made using SAINTPLUS27. The structure of Pd(L2)2 ∙
4 (C3H6O) (2) was solved by direct methods using XS in
SHELXTL28 and completed (non‐H atoms) by Fourier
difference synthesis. Refinement until convergence was
obtained using XL SHELXTL and SHELXL9729. All
hydrogen atoms were calculated in idealized positions
on geometric basis, and refined with restrictions.
Crystal data collection and refinement parameters for 2
are given in Table 1.
metal ions and also can bind metal complexes24,25
.
In this work we describe the synthesis and
characterization of thiosemicarbazone based
palladium(II) complexes, based on ligands with shown
anticancer properties, and the adsorption of
compounds (PdL1‐4)2 on superparamagnetic magnetite
nanoparticles coated with chitosan. A study of the
antiproliferative activity of the above mentioned
palladium(II) complexes, and that of the corresponding
functionalized nanoparticles with Pd(L1‐4)2 is reported.
Experimental
Synthesis of the ligands.
Equipment
All reagents and solvents were Sigma‐Aldrich of
Palladium analyses for the complexes were carried analytical quality, and were used without further
out on an AAnalyst 700 Perkin Elmer equipment, using purification. The ligands and complexes were prepared
an acetylene/air flame. The analyses of C, N, and H according to reported methods30,31. A solution of
were done on a Thermo Fisher Flash 2000 Elemental naphthaldehyde or 2‐hydroxy‐naphthaldehyde (20
Analyzer. FT‐IR spectra were registered using KBr mmol) in 40 mL of methanol was added to a hot
1
pellets, on a Bruker Vector 22 equipment. H‐NMR solution of thiosemicarbazide (20 mmol) or 4‐phenyl
2 | J. Name., 2012, 00, 1‐3
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thiosemicarbazide (20 mmol) in ethanol (100 mL). The Table 1 Crystal data and structure refinement for 2
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DOI: 10.1039/C5NJ02429C
reaction mixture was refluxed for 4 h and then stirred
for 24 h at room temperature (scheme 1). The solid
product was filtered, washed several times with cold
ethanol or methanol, and dried in vacuo.
Empirical formula
Formula weight
Temperature
Wavelength
Crystal system
Space group
C48 H52 N6 O4 S2 Pd
947.50
297(2) K
0.71073 Å
Triclinic
P‐1
1‐naphthaldehyde thiosemicarbazone (L1H). Light
yellow microcrystalline solid. Yield: 93%. Anal. Exp. %
(calc. for C12H11N3S): C: 62.7 ( 62.9 ), H: 4.7 (4.8 ), N:
18.4 (18.3 ). FTIR (KBr); ν(cm‐1): 3442, 3265 (NH2), 3150
Unit cell dimensions
a = 7.957(5) Å
b = 9.112(5) Å
c = 16.833(10) Å
α= 80.061(9)°
β= 80.638(9)°
γ = 82.591(9)°
1179.7(12) Å3
1
1
(NHCS), 1600 (C=N), 1110 (C=S). H‐NMR; (DMSO‐d6):
Volume
Z
Density (calculated)
Absorption coefficient
F(000)
Crystal size
Theta range for data collection
Index ranges
11.48 δ (s, 1H: =N‐NH); 8.9 δ (s, 1H: N=C‐H); 8.35 δ (d:
1H, H8); 8.3 δ (s, br: 1H, NH2); 8.2 δ (d, 1H: H7); 7.95 δ
(m, 3H: H4, H5; 1H, NH2); 7.6 δ (m, 3H: H3, H6, H7).
1.334 Mg/m3
0.530 mm‐1
492
0.40 x 0.31 x 0.11 mm3
2.28 to 27.96°
10<=h<=10, ‐11<=k<=11,
‐21<=l<=21
9945
4‐phenyl‐(1‐naphthaldehyde)-thiosemicarbazone (L2H).
Light yellow microcrystalline solid. Yield: 90%. Anal.
Exp. % (calc. for C18H15N3S): C: 70.5 (70.8), H: 4.9 (5.0),
N: 13.7 (13.8). FTIR (KBr); ν(cm‐1): 3327 (NHPh), 3165
Reflections collected
Independent reflections
Completeness to theta =
Absorption correction
Max. and min. transmission
Refinement method
Data / restraints / parameters
Goodness‐of‐fit on F2
Final R indices [I>2sigma(I)]
R indices (all data)
Largest diff. peak and hole
5103 [R(int) = 0.0337]
26.00°
98.7 %
1
(NHCS), 1600 (C=N), 1088 (C=S). H NMR (DMSO‐d6):
Semi‐empirical from equivalents
0.943 and 0.821
11.9 δ (s, 1H: =N‐NH); 10.2 δ (s, 1H: NH‐Ph); 9.05 δ (s,
1H: N=C‐H); 8.45 δ (d, 1H: H8); 8.3 (d, 1H: H2): 8.0 δ (m,
2H: H4, H5); 7.57 δ (m, 5H: H3, H6, H7; 2Hortho, Ph); 7.38 δ
(t, 2H: 2Hmeta, Ph); 7.24 δ (t, 1H: Hpara, Ph).
Full‐matrix least‐squares on F2
5103 / 1 / 285
1.035
R1 = 0.0594, wR2 = 0.1462
R1 = 0.0674, wR2 = 0.1525
2.037 and ‐1.218 e.Å‐3
(2‐hydroxy‐1‐naphthaldehyde)thiosemicarbazone (L3H).
Yellow microcrystalline solid. Yield: 91%31. Anal. Exp. %
(calc. for C12H11N3SO): C: 58.5 (58.8), H: 4.4 (4.5), N:
16.9 (17.1). FTIR (KBr); ν (cm‐1): 3445, 3248 (NH2), 3160
Synthesis of complexes.
General method.
1
(NHCS), 1609 (C=N), 1114 (C=S). H NMR (DMSO‐d6):
11.41 δ (s, 1H: =N‐NH); 10.53 δ (s, br: 1H, OH); 9.05 δ (s,
1H: N=C‐H); 8.51 δ (d: 1H, H8); 8.22 δ (s, br: 1H, NH2);
7.85 δ (m, 2H: H4, H5; 1H, NH2); 7.56 δ (t, 1H: H7); 7.36 δ
(t, 1H, H6); 7.19 δ (d, 1H, H3).
A solution of Pd(acac)2 (0.153 g, 0.5 mmol) in acetone‐
ethanol (2 : 1, 90 mL), was added dropwise to a stirred
hot solution of LnH (1.0 mmol) in ethanol (60 mL).
Sodium acetate (0.082 g, 1 mmol) in 5 mL of water was
then added. The solution was refluxed for 2 h and then
stirred for 24 h at room temperature. (scheme 1). The
precipitate was collected by filtration, washed three
times with cold ethanol (50 mL) and dried under
vacuum.
4‐Phenyl‐1‐(2‐hydroxy‐1‐naphthaldehyde)
thiosemi‐
carbazone, (L4H). Intense yellow micro‐crystalline solid.
Yield: 90%. Anal. Exp. % (calc. for C18H15N3SO): C: 67.5
(67.3), H: 4.6 (4.7), N: 12.8 (13.1). FTIR (KBr); ν (cm‐1):
1
3380 (NHPh), 3150 (NHCS), 1620 (C=N), 1084 (C=S). H
NMR (DMSO‐d6): 11.77 δ (s, 1H: =N‐NH); 10.63 δ (s br:
Bis[(1‐naphthaldehyde)‐thiosemicarbazonato]
1H, OH); 10.08 δ (s, 1H: NH‐Ph); 9.17 δ (s, 1H: N=C‐H); palladium(II), Pd(L1)2 (1). Orange microcrystalline solid.
8.49 δ (d, 1H: H8); 7.89 δ (m, 2H: H4, H5); 7.57 δ (m, 3H:
Yield: 91.8%. Anal. Exp. % (calc. for C24H20N6S2Pd):
H7; 2Hortho, Ph); 7.38 δ (m, 3H, H6; 2Hmeta, Ph); 7.24 δ (m,
C:50.2 (51.2); N: 14.5 (14.9); H: 3.7 (3.6); Pd: 18.7
(18.9). FTIR (KBR); ν (cm‐1): 3440, 3330 (NH2), 1600
2H, H3; Hpara, Ph).
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(C=N), 1080 (C=S). H‐NMR; (DMSO‐d6): 8.15 δ (s, 1H:
N=C‐H); 8.60 δ (d: 1H, H8); 8.05 δ (s, br: 2H, NH2); 8.01
(d, 1H; H4); 87.93 δ (d, 1H: H2); 7.65 δ (various signals,
4H: H3, H5, H6, H7).
Bis[4‐phenyl‐(2‐hydroxy‐1‐naphthaldehyde)tVhieiwosAretimcleiO‐nline
DOI: 10.1039/C5NJ02429C
carbazonato] palladium(II), Pd(L4)2 (4): Orange micro‐
crystalline solid. Yield: 90.8%. Anal. Exp. % (calc. for
C36H28N6S2O2Pd): C:58.1 (57.9); N: 11.5 (11.3.); H: 3.8
(3.8); Pd: 14.2 (14.3). FTIR (KBr); ν (cm‐1): 3400 (broad,
OH, NH2), 1592 (C=N), 1085 (C=S) cm‐1. 1H‐NMR;
(DMSO‐d6): 9.35, 9.15 δ (s, 2H: N=C‐H); 8.65, 8.35 δ (d:
2H; H8; H8’); 10.67, 9.50 δ (s, br: 2H; NH‐Ph); 13.01,
10.95 δ (2H; OH; OH’); 7.85, 7.50, 7.30 δ (m, 18 H; arom
protons); 6.95 δ (t, 2H; H3, H3’).
Bis[4‐phenyl‐(1‐naphthaldehyde)thiosemicarbazonato]‐
palladium(II), Pd(L2)2∙4 C3H6O (2). Orange micro‐
crystalline solid. Yield: 90.3%. Single crystals suitable
for structure determination were obtained by
recrystallization from acetone. Anal. Exp. % (calc. for
C48H52N6S2O4Pd): C:60.3 (60.8); N: 9.0 (8.9); H: 5.4 (5.5);
Pd: 11.3 (11.2). FTIR (KBR); ν (cm‐1): 3380 (NHPh),
1594 (C=N), 1076 (C=S). 1H‐NMR; (DMSO‐d6): 8.46 δ (s,
1H: N=C‐H); 8.27 δ (d: 1H, H8); 9.90 δ (s, br: 1H, NH‐Ph);
8.08; 8.12 δ (dd, 2H; H4; H5); 7.93 δ (d, 1H: H2); 7.66 δ
(various signals, 3H: H6, H7, o‐H (Ph)); 7.26 δ (d, 1H; o‐H
(Ph)); 7.02 δ (t, 2 H; m-H (Ph); 6.96 δ (t, 1H, H3); 6.86 δ
(t, 1H; p‐H(Ph)).
Chitosan coated magnetite nanoparticles.
A mixture of FeCl3 ∙ 6 H2O (2.7 g, 10 mmoles) and FeCl2 ∙
4 H2O (1.0 g, 5 mmoles) in 50 mL of H2O were stirred
under nitrogen for 15 min. An aliquot of 12.5 mL of a
degassed 1% solution of chitosan in 2% acetic acid was
added, followed by the dropwise addition of 11.5 mL of
a degassed 5 M NaOH solution, during 10 min. The
nanoparticles were separated with a magnet, washed
with water, acetone and dried under vacuum. Yield ca.
1.4 g.
Bis[(2‐hydroxy‐1‐naphthaldehyde)thiosemicarbazon‐
ato] palladium(II), Pd(L3)2 (3). Orange micro‐crystalline
solid. Yield: 91.1%. Anal. Exp.
%
(calc. for
C24H20N6S2O2Pd): C:48.2 (48.4); N: 14.5 (14.1.); H: 3.5
Adsorption of PdII complexes on chitosan coated
magnetite nanoparticles.
(3.4); Pd: 17.7 (17.9). FTIR (KBr); ν (cm‐1): 3420, 3330
1
(broad, OH, NH2), 1615 (C=N), 1092 (C=S) cm‐1. H‐
NMR; (DMSO‐d6): 9.10, 8.95 δ (s, 2H: N=C‐H); 8.80, 8.75
δ (d: 2H; H8; H8’); 10.25, 7.85, 6,70 δ (various signals, br:
4H; NH2); 12.5, 10.75 δ (2H; OH; OH’) 8.15, 7.95 δ (d,
2H; H4, H4’); 7.85 (m, 2H; H5, H5’),7.6, 7,5 δ (t, 2H; H7,
H7’); 7.4 δ (t, 1H; H3); 7.25 δ (m, 3H; H6, H6’, H3’).
50 mg of coated magnetite nanoparticles and 5 mg of
Pd(II) complex were dispersed in 20 mL of DMF by
sonication at ambient temperature for 10 min.; the
suspension was then irradiated with microwave (800
W) for 10 min. The particles were separated with a
magnet and washed with 2‐propanol and acetone and
dried under vacuum. The adsorbed palladium(II) was
determined by atomic absorption analyses, previous
digestion of the particles in HNO3. The calculated
adsorption values are given in Table 2 as mg of
adsorbed palladium complex for 100 mg of particles.
S
S
R
H
R
N
H
H
N
N
H
N
N
O
H
NH2
R`
8
1
R`
Ethanol, reflux, 4 hrs.
-H2O
7
6
2
3
4
5
H
S
R
3
R
R`
N
2
1
H
4
N
N
N
N
7`
Pd(acac)2
S
H
N
6`
5`
8`
1`
5
6
Pd
Biological activity
Cell culture
acetone-ethanol
CH3COONa
8
R`
N
S
7
reflux, 2 h.
N
4`
2`
R`
R
N
3`
H
R = H; R`= H (L1H)
R = phenyl; R`= H (L2H)
R = H; R`= OH (L3H)
The BALB/3T3 (non‐transformed mouse embryonic
fibroblasts), were cultured in Dulbecco´s Modified
Eagle Medium (DMEM) supplemented with 10 %
newborn calf serum and 50 µg/mL gentamycin. H460
R = phenyl; R`= OH (L4H)
Scheme 1. Syntheses of ligands and complexes.
4 | J. Name., 2012, 00, 1‐3
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Table 2. Adsorption percentage of palladium(II) In the case of the cells treated with the functionalized
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complexes on chitosan coated magnetite nanoparticles nanoparticles with the palladium(IDI)OIc: o10m.10p3l9e/Cxe5NsJ,02a4f2t9eCr
(100 mg of NPs).
the 48 h of incubation and before adding the
trichloroacetic acid to the cell monolayers, the growth
media was removed and the plates were carefully
washed twice with Hank’s balanced salt solution in
order to remove the nanoparticles that would
otherwise interfere with the colorimetric assay . Then
Complex
Pd(L1)2 Pd(L2)2 Pd(L3)2 Pd(L4)2
0.5 0.6 0.6
Adsorption (%) 0.4
(human lung large cell carcinoma), DU145 (human
prostate carcinoma) and HuTu 80 (human duodenum
adenocarcinoma) cell lines were cultured in Eagle’s
Minimum Essential Medium (MEM) supplemented with
10 % fetal bovine serum and 50 µg/mL gentamycin. M‐
14 (human amelanotic melanoma), MCF‐7 (human
breast adenocarcinoma), HT‐29 (human colon
adenocarcinoma) and K562 (human chronic
myelogenous leukemia) were cultured in RPMI 1640
Medium (RPMI) supplemented with 7.5 % fetal bovine
serum and 50 µg/mL gentamycin in humidified 5 % CO2
/95 % air at 37 °C.
the cell monolayers were fixed with 10
%
trichloroacetic acid and stained for 20 minutes, using
the sulforhodamine B dye. The excess dye was
removed by washing repeatedly with 1 % acetic acid.
Then the protein‐bound dye was solubilized with 10
mM Tris buffer (pH 10.5) and the absorbance values
were obtained at 510 nm, using a microplate reader.
Results and discussion
The thiosemicarbazone ligands were obtained
straightforwardly by reaction of the corresponding
semicarbazide (R= H, phenyl) and substituted
naphthaldehyde (R’=H, OH), while PdII complexes were
Assessment of cytotoxicity
synthesized
acetylacetonate
by
reaction
with
of
palladium(II)
corresponding
The assays were performed as described previously30.
Briefly, 3000‐5000 cells were inoculated in each well of
96‐well tissue culture plates and incubated at 37 °C
with their corresponding culture medium during 24 h.
The palladium(II) complexes (0.01‐10 µM) or cisplatin
(1‐10 µM) in DMSO were then added and incubated for
48 h at 37 °C with a highly humidified atmosphere, 5 %
CO2 and 95 % air. After the incubating period, cell
monolayers were fixed with 10 % trichloroacetic acid
and stained for 20 minutes, using the sulforhodamine B
dye. Then, the excess dye was removed by washing
repeatedly with 1 % acetic acid. The protein‐bound dye
was solubilized with 10 mM Tris buffer (pH 10.5) and
the absorbance values were obtained at 510 nm, using
a microplate reader. The IC50 value was defined as the
concentration of a test sample resulting in a 50 %
reduction of absorbance as compared with untreated
controls, in other words 50% reduction in the growth of
the cells, and was determined by linear regression
analysis.
the
thiosemicarbazone ligand. FTIR spectra of the
complexes show the absorption bands corresponding
to the C=N (ca. 1600 cm‐1) and C=S (ca. 1080 cm‐1)
1
groups. For the HNMR spectra, the =N‐NH signal
observed for the ligands at ca. 11‐12 δ, is absent in the
corresponding complexes due to the deprotonation on
complexation. Besides, all complexes show the typical
signal due to the N=C‐H proton. The experimental
results are in agreement with the expected
compounds.
Structural description.
Recrystallization of the synthesized complexes in
several solvents gave microcrystalline products. Only
complex with HL2, recrystallized from acetone, gave
suitable crystals for X‐ray diffraction studies. Complex
Pd(L2)2 (2) crystallizes in the triclinic space group P‐1
with one complex molecule and four acetone solvate
molecules in the unit cell (Fig. 1).
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0.6
0.3
0.6
0.3
0.0
a
DOI: 10.1039/C5NbJ02429C
0.0
300
400
wavelength (nm)
500
300
400
500
wavelength (nm)
Figure 2. a) UV‐Visible spectrum of L1H (red) and Pd(L1)2 (black); b)
L4H (red) and Pd(L4)2 (black).
Figure 1. Graphical representation of 2. Hydrogen atoms have been
omitted for clarity.
UV‐visible spectra
The deprotonated ligand coordinates bidentately
through the S and N atoms. The C7‐N2 and C7‐S1 bond
distances are modified with respect to the free ligand,
due to coordination30. The coordination of the PdII
atom is square planar with a trans arrangement of the
coordinating atoms. The angle formed by this plane
with the mean phenyl plane is 20.24°, while the angle
with the naphthalene mean plane is 66.60°. The phenyl
and the naphthalene moieties are almost
perpendicular, forming an angle of 78.69°. The N1‐C1‐
N2‐N3a‐C8a moiety is planar with angles close to 120°
(N1‐C1‐N2 = 120.0(3); C1‐N2‐N3a = 113.7(3) and N2‐
N3a‐C8a = 114.8(3)°), according to the sp2 character of
the deprotonated N2 amine nitrogen. The Pd‐S and the
Pd‐N distances are in the range of previously reported
thiosemicarbazone derived complexes30‐33. Selected
bond lengths and angles are listed in Table 3.
Figure 2 shows the spectra of the selected ligands and
the corresponding palladium(II) complexes. The spectra
of the four ligands and the corresponding complexes
can be classified in two groups: those that have the
naphthaldehyde ring
naphthaldehyde) thiosemicarbazonato]palladium(II),
Pd(L1)2
and bis[4‐phenyl‐(1‐naphthaldehyde)‐
unsubstituted,
bis
[(1‐
thiosemicarbazonato]palladium(II), Pd(L2)2 ∙ 4 C3H6O (2),
and those which present a naphthaldehyde moiety
substituted by a hydroxyl group,bis[(2‐hydroxy‐1‐
naphthaldehyde) thiosemicarbazonato]palladium(II),
Pd(L3)2
(3)
and
bis[4‐phenyl‐(2‐hydroxy‐1‐
naphthaldehyde) thiosemicarbazonato]palladium(II),
Pd(L4)2(4). As can be observed in the corresponding
spectra, 1 and 2 are characterized by an absorption
band that has a maximum at 370 nm and 354 nm,
respectively, with shoulders at both higher and lower
energies. When the spectra of 3 and 4 are analyzed it
becomes evident that new absorption bands appear,
making the spectra to have a more complex profile. At
higher energies a double absorption band in the 325‐
350 nm region is observed for both 3 and 4. At lower
energies, in the 425 to 450 nm region another double
absorption band is present. A similar effect has been
reported for the spectra of naphthalene and 2‐
Table 3. Selected bond lengths, (Å), and bond angles,
(º), for Pd(L2)2 (2).
Bond lengths
Å
Bond angles
(°)
Pd1‐N3
Pd1‐S1
S1‐C1
2.029(3)
2.2792(13)
1.732(4)
1.308(4)
1.349(5)
1.387(4)
1.287 (4)
N3‐Pd1‐N3a
N3‐Pd1‐S1
N3a‐Pd1‐S1
S1‐Pd1‐S1a
C1‐S1‐Pd1
N1‐C1‐N2
180.00(15)
96.89(9)
83.11(9)
180
C1‐N2
C1‐N1
N3a‐N2
C8‐N3
hydroxynaphthalene.
The
spectrum
of
2‐
96.52(12)
120.0(3)
113.7(3)
114.8(3)
114.9(3)
124.0(2)
121.2(2)
125.1(3)
hydroxynaphthalene is characterized by a series of
absorption maxima which are absent for naphthalene,
compound that only absorbs below 300 nm34. Thus, it is
possible to infer that the substitution of the naphtyl
ring produces a set of new absorption bands, both at
higher and lower energies, as compared with the
intense band at 370 nm (1) or 350 nm (2), 375 nm (3)
C1‐N2‐N3a
N2‐N3a‐C8a
N1‐C1‐S1
C8‐N3‐Pd1
N2‐N3a‐Pd1
N2‐C1‐S1
6 | J. Name., 2012, 00, 1‐3
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and 380 nm (4). This band has been assigned to the n→ From the reported values of measured cytotoxic
View Article Online
π* of the thiosemicarbazone group35‐37. A detail of the activities (Table 4), it is possible toDiOnIf:e10r.1t0h39a/tC5cNoJm02p42le9Cx
bands and the corresponding log ϵ is given in Table S1.
Pd(L1)2, (L1H:1‐naphtaldehyde thiosemicarbazone) has
better activity in all cell lines than the original ligand.
The same behavior is not observed for complex Pd(L2)2
since for both M14 and HuTu80 the activity of the
complex and the ligand is similar (L2H: 4‐phenyl‐(1‐
naphthaldehyde) thiosemicarbazone). Complex Pd(L3)2
(L3H: (2‐hydroxy‐1‐naphtaldehyde) thiosemicarbazone)
has a better activity than that of the ligand, except in
two cases: M14, where the activity in both is similar,
and K562 where the activity of ligand is better than the
corresponding complex. For complex Pd(L4)2, (L4H: 4‐
phenyl‐(2‐hydroxy‐1‐naphthaldehyde)thiosemicarbaz‐
one) the activity of the complex and the ligand is
similar for DU145, MCF7, HuTu80 and H460. When
analyzing the data for M14 and K562, the activity of the
ligand is better than that of the complex. Only for HT29
the activity of the complex becomes greater than that
of the ligand. Even though Pd(L1)2 shows a better
activity than Pd(L2)2 for most of the studied cell lines,
Pd(L2)2 can be considered as the best of all the
complexes according to the above cited cytotoxic
activities, since it is more innocous than Pd(L1)2 against
the studied normal cells. Moreover, when we compare
the IC50 of this complex with that of cisplatin for all the
studied cell lines, Pd(L2)2 is better than cisplatin with
the exception of lines DU145, H460 and K562 cases
where they have similar cytotoxic activities. Thus, this
palladium(II) complex could be considered as a future
candidate to be investigated as a pharmacological
agent. As stated above, complex Pd(L4)2 is the least
active of all the complexes, including the normal cells.
However, when the activities between the complex and
cisplatin are compared; in two cases the activity of the
complex is better than that of cisplatin (MCF7 and
HT29); is worse in four cases (DU145, H460, HuTu80
and K562), while Pd(L4)2 and cisplatin have similar
activities for M14 line. So, striking differences in
cytotoxicity were observed across the studied cell lines
for the palladium species. All cell lines were
consistently resistant to Pd(L4)2, while most were
sensitive to Pd(L1)2, (Pd(L2)2 and Pd(L3)2.
The stability of the complexes in DMSO and
DMSO/water solutions was controlled in order to have
complete certainty that the species whose
antiproliferative activity was recorded was the initial
complex, and not a species derived from the
decomposition of the initial complex. All recorded
spectra at 24, 48, and 72 hours after the preparation of
the solutions were similar to those obtained for the
freshly prepared solutions.
Antiproliferative activity of the PdII complexes.
The cytotoxic potential of the synthesized palladium(II)
complexes was investigated in the following six human
tumor cell lines: DU145, MCF‐7, H460, M14, HT‐29,
HuTu80 and K562. For comparison purposes, the
cytotoxicity of the studied complexes was contrasted
with that of the ligands and cisplatin, which were
evaluated under the same experimental conditions38.
The results of the cytotoxic activity of the ligands,
palladium(II) complexes and cisplatin are expressed as
IC50 values (micromolar concentration inhibiting 50 %
cell growth), as shown in Table 4, and figure 3. The
reported values are an average of three independent
measurements, with a standard deviation of 10%.
60
50
40
30
20
10
0
Figure 3. Citotoxic activity of complexes and the corresponding free
ligans againts various tumor cell lines: Pd(L1)2;
HL1; Pd(L2)2;
HL2; Pd(L3)2; HL3; Pd(L4)2; HL4.
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DOI: 10.1039/C5NJ02429C
Journal Name
ARTICLE
Table 4 Antiproliferative activity at 48 h, expressed in IC50 values. Values in parenthesis correspond to IC50 for
pure ligands.
Human tumor cell
Pd(L1)2 (L1H)
Pd(L2)2 (L2H)
Pd(L3)2 (L3H)
Pd(L4)2 (L4H)
Cisplatin
lines / complex
DU145
MCF7
1.1 (8.0)
1.1 (7.7)
4.3 (15.8)
1.1 (6.4)
1.1 (30.3)
1.1 (15.0)
4.4 (16.2)
2.4 (26.5)
2.1 (29.9)
2.1 (39.7)
2.3 (>250)
2.4 (>250)
1.8 (24.7)
5.2 (7.0)
4.1 (10.4)
<4.1 (11.8)
4.1 (19.3)
4.1 (7.3)
10.4 (11.2)
10.4(12.9)
11.3 (13.9)
11.3 (6.4)
2.0
19.8*
1.5
H460
M14
8.5
HT29
4.1 (15.4)
1.0 (0.3)
12.5 (24.7)
12.5 (0.6)
26.7
1.1
K562
HuTu80
1.1 (18.5)
13.7 (14.82)
7.6
3T3 mouse
embryonic
1.2 (28.7)
5.2 (10.3)
1.1 (16.0)
54.7(18.5)
2.1
fibroblast cells
*Palaniandavar et al39 reported IC50 values for cisplatin using MCF‐7.
These results permit to infer that the presence of
the hydroxy substituent on the naphthaldehyde or
the phenyl substituent on the thiosemicarbazone
lowers the activity of the complexes by a factor of
four and two approximately, as compared to the
activity of the complex derived from the
unsubstituted ligand.
A special reference has to be made in relation to the
antiproliferative activity of Pd(L1)2 and Pd(L2)2 when
tested with the MCF7 cell line (breast cancer). The
activity of the studied complexes is much better
than that of two recently reported PdII complexes
with substituted diacetyl bis(4N‐tolylthiosemicarbaz‐
one) ligands40.
In contrast, the presence of both substituents in the
ligand decreases dramatically by a factor of ten the
biological activity against all the studied tumor cell
lines. With the exception of Pd(L4)2, all the studied
complexes show similar or better biological activity
than cisplatin. Thus, Pd(L4)2 shows the lowest
biological activity as compared with the complex
with the unsubstituted ligand or the ones which
have either only the hydroxyl or the phenyl
substituent.
The reported data does not permit to deduce a clear
structure‐activity
correlation,
since
the
antiproliferative activity of the complexes was
shown to be sensitive to the type of cancer cell line
used in the probes. While Pd(L1)2 has a planar
naphtyl derived ligand, which would favour the
intercalative interactions, the presence of phenyl
rings does not enhance the antiproliferative activity
of the studied complexes Pd(L2)2 and Pd(L4)2. This
fact can be rationalized assuming that the presence
of bulky phenyl groups, perpendicular to the
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coordination plane, can sterically hinder the possible
metal‐DNA intercalation, thus lowering these
interactions as compared with those of the
palladium(II) complex with the unsubstituted, 1‐
naphthaldehyde thiosemicarbazone ligand, Pd(L1)2.
Besides, apparently the hydrophobic phenyl groups
present in the ligands of complexes 2 and 4 also do
not enhance the permeability of these across the cell
membrane, as could be expected. As the migration
of the complexes into the cells by a concentration
gradient is not increased, these complexes cannot
increase their interference with the cellular function
of DNA by binding to it.
View Article Online
DOI: 10.1039/C5NJ02429C
a)
b)
c)
Characterization
of
chitosan
coated
and
functionalized magnetite nanoparticles.
Figure 5. a) TEM image of coated nanoparticles, b) Diffraction
pattern, c) Size distribution of the nanoparticles.
Chitosan coated magnetite nanoparticles (NPs) were
functionalized with the obtained palladium(II)
complexes in order to investigate the possibility to
use them as carriers for the studied drugs. These NPs
can be directed to a specific site, where neoplastic
cells exist, by an external magnetic field, since they
can become superparamagnetic if a specific size is
attained.
The cristallinity and size of these nanoparticles was
corroborated by transmission electron microscopy
(TEM), as shown in Fig. 5. Scanning electron
microscopy images (SEM) are given for the chitosan
coated nanoparticles as supplementary material (Fig.
S2).The mean size permits to infer that the observed
spherical particles correspond to agglomerates of
the coated nanoparticles observed by TEM.
The average diameter of the metallic core of the
coated nanoparticles was estimated by the Debye
Scherrer formula, using the middle width of the
highest peak of the corresponding diffraction
pattern. The estimated average had a mean value of
10 nm (Fig. 4).
Figure 6 shows the magnetic behaviour of both the
magnetite nanoparticles, and the coated magnetite
ones. The curve corresponds to
a
typical
superparamagnetic behaviour, since the coercivity is
zero. The saturation magnetization is 50 emu/g for
the pristine nanoparticles, while a lower value of 15
emu/g is obtained for the chitosan coated
nanoparticles. These values are similar to those
reported by Gupta et al. of 45 to 50 emu/g for
nanoparticles of 12.92 nm41.
300
250
35,48°
200
150
100
50
From the obtained data the effect of the coating on
the magnetic properties becomes evident. Several
other authors have reported the decrease of the
saturation magnetization of studied nanoparticles
when these are coated, especially with organic
polymers, since a non magnetic surface affects the
magnetic behavior of the nanoparticles of size from
0
0
20
40
2 theta (o)
60
80
Figure 4. Powder X‐raydiffractogram of coated magnetite
1 to 20 nm42‐46
.
nanoparticles.
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DOI: 10.1039/C5NJ02429C
60
40
Moreover, the electrokinetic potential changes for
the chitosan coated nanoparticles as compared to
that of the functionalized ones, from a negative
value (‐20 mV) to a positive one (range between 25
to 30 eV). This change can be attributed to the
adsorption of the palladium complex on the chitosan
surface. The electrokinetic potential for the coated
nanoparticles is negative due to the fact that the
amine groups of the chitosan are not protonated in
the range of pH = 6; this value being the pH of the
solutions where the measurements were done. This
pH was larger than the isoelectric point for chitosan
(pH = 5.45). The obtained results are similar to those
reported by Zhang et al47.
Magnetite uncoated
Magnetite coated
with chitosan
20
0
-20
-40
-60
-10 -8
-6
-4
-2
0
2
4
6
8
10
Applied Field (kOe)
Figure 6. Magnetic properties of magnetite and coated
magnetite nanoparticles.
The studied nanoparticles were then functionalized
with the corresponding palladium(II) complexes. The
hydrodynamic
nanoparticles was determined by the Dynamic Light
Scattering technique (DLS). While the measured
mean size of the chitosan coated nanoparticles was
300 nm, the functionalized nanoparticles were
slightly smaller in size. The measured size for the
Pd(L1)2, Pd(L2)2, Pd(L3) and Pd(L4)2 functionalized
nanoparticles was in the range of 200 to 240 nm.
In
vitro
antiproliferative
activity
of
size
of
the
functionalized
functionalized chitosan coated magnetite
nanoparticles.
All four complexes were used to functionalize the
nanoparticles coated with chitosan. The NPs, whose
palladium content was approximately 0.5 mg
complex/100 mg of coated nanoparticles, were
suspended in the same biological medium used to
study the antiproliferative activity of the original
thiosemicarbazone derived palladium(II) complexes.
All experimental conditions were maintained
constant, in order to obtain an estimation of the
activity of the functionalized NPs. The results
permitted to assess that the functionalized NPs
present some activity against the DU145, K562 and
HuTu80 tumor cell lines (Table 5).
Table 5. In vitro antiproliferative activity of
functionalyzed
nanoparticles, expressed as percentages of remnant
cancer cells.
chitosan
coated
magnetite
Cell line Pd(L1)2 (1) Pd(L2)2 (2) Pd(L3)2(3) Pd(L4)2 (4)
DU‐145 60
w/a
w/a
w/a
w/a
w/a
76
75
60
MCF7
H460
M14
w/a*
w/a
w/a
w/a
50
w/a
w/a
w/a
w/a
75
The data of Table 5 indicate that the observed
activity of the functionalized nanoparticles is not
related to that of the free complexes. The
adsorption process probably prevents the
intercalation of the complex, thus reducing its
activity. Moreover, it is possible to infer from the
obtained data that the complexes are not liberated
from the surface of the nanoparticles, during the
study. The observed activity would be intrinsic to
that of the functionalized nanoparticles.
w/a
w/a
w/a
K562
HuTu80 w/a
3T3
25
w/a
w/a
w/a
w/a
* Without significant activity. (0.5 mg of nanoparticles/ml
equivalent to ca. 4 μmoles of complex/ml).
10 | J. Name., 2012, 00, 1‐3
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Conclusions
5 M. Jagadeesh, H. K. Rashimi, Y. S. Rao, A. S. Reddy,
B. Prathima, P. U. Maheswari, A. V. Reddy,
Spectrochim. Acta A, 2013, 115, 583.
View Article Online
DOI: 10.1039/C5NJ02429C
In this work the antiproliferative activity of four
ligands derived from thiosemicarbazone and the
corresponding palladium(II) complexes was studied
and compared to cisplatin.
6 C. Santini, M. Pellei, V. Gandin, M. Porchia, F.
Tisato, C. Marzano, Chem. Rev., 2014, 114, 815.
Even though the activity of Pd(L1)2 is better than that
of Pd(L2)2 the last one is more inocuous to normal
7 S. A. Elsayed, I. S. Butler, F. R. Gilson, B. J. Jean‐
Claude, S. I. Mostafa, J. Coord. Chem., 2014, 67,
2711.
cells, thus being
a
better candidate for
pharmacological use. Both complexes have similar
activity as compared to cisplatin, with the exception
of MCF7, M14, HT29 cell lines. For these cell lines,
8 M. Jagadeesh, M. Lavanya, S. K. Kalangi, Y. Sarala,
C. Ramachandraiah, A. V. Reddy, Spectrochim. Acta
A, 2015, 135, 180.
the two studied complexes have
antiproliferative activity.
a
better
9 W. Hernández, J. Paz, J. Vaisberg, E. Spodine, R.
Richter, L. Beyer, Bioinorg. Chem. Appl., 2008, ID
690952.
The immobilization of the complexes on the surface
of magnetite nanoparticles reduces the activity of
the complexes. Some exceptions can be mentioned,
for example, nanoparticles functionalized with
Pd(L3)2 show activity against HuTu80, K562 and
DU145.
10 D. Kovala‐Demertzi, A. Papageorgiou, L.
Papathanasis, A. Alexandratos, P. Dalezis, J. R. Miler,
M. A. Demertzis, Eur. J. Med. Chem., 2009, 44, 1296.
11 Z. Iakovidou, A. Papageorgiou, M. A. Demertzis, E.
Mioglou, D. Mourelatos, A. Kotsis, P. Nath Yadav, D.
Kovala‐Demertzi, Anti‐Cancer Drugs, 2001, 12, 65.
Acknowledgements
W. H. thanks Universidad de Lima Scientific Research
Institute for financial support. E. S., M. T., M.A. and J.
M. thank Basal Project FB0807 (CEDENNA), and Y.E.
thanks FONDECYT Project 3130418.
12 D. Kovala‐Demertzi, M. A. Demertzis, J. R. Miller,
C. Papadopoulou, C. Dodorou, G. Filousis, J. Inorg.
Biochem., 2001, 86, 555.
13 I. H. Hall, C. B. Lackey, T. D. Klister, R. W.
Durhman, E. M. Jouad, M. Khan , X. D. Thanh , S.
Djebbar–Sid , O. Benali‐Baitich, G. M. Bouet,
Pharmazie, 2000, 55, 937.
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DOI: 10.1039/C5NJ02429C
In Vitro Antiproliferative Activity of Palladium(II) Thiosemicarbazone Complexes and the
Corresponding Functionalized Chitosan Coated Magnetite Nanoparticles
Wilfredo Hernandez, Abraham J.Vaisberg, Mabel Tobar, Melisa Alvarez,
Jorge Manzur, Yuri Echevarría, Evgenia Spodine
Magnetite functionalized nanoparticles with Pd(L3)2 and Pd(L4)2 show antiproliferative activity
against DU-145 and HuTu80; Pd(L2)2 appears as promising pharmacological agent.