Platinum(II) and palladium(II) complexes with (N,N′) and (C,N,N′)- ligands derived from pyrazole as anticancer and antimalarial agents: Synthesis, characterization and in vitro activities

Article abstract of DOI:10.1016/j.jinorgbio.2011.09.021

The study of the reactivity of three 1-(2-dimethylaminoethyl)-1H-pyrazole derivatives of general formula [1-(CH₂)₂NMe ₂}-3,5-R₂-pzol] {where pzol represents pyrazole and RH (1a), Me (1b) or Ph (1c)} with [MCl

Full text of DOI:10.1016/j.jinorgbio.2011.09.021

Journal of Inorganic Biochemistry 105 (2011) 1720–1728
Contents lists available at SciVerse ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Platinum(II) and palladium(II) complexes with (N,N′) and (C,N,N′)− ligands derived
from pyrazole as anticancer and antimalarial agents: Synthesis, characterization and
in vitro activities
c
Josefina Quirante a, Daniel Ruiz a, Asensio Gonzalez a, Concepción López b, , Marta Cascante ,
⁎
Roldán Cortés c, Ramon Messeguer d, Carme Calvis d, Laura Baldomà e, Aurélie Pascual f, Yann Guérardel g,h
,
Bruno Pradines f, Mercè Font-Bardía i, Teresa Calvet j, Christophe Biot g,h,⁎⁎
Laboratori de Química Orgànica, Facultat de Farmàcia, Institut de Biomedicina, (IBUB), Universitat de Barcelona, Av. Joan XXIII, s/n, 08028 Barcelona, Spain
a
b
Departament de Química Inorgànica, Facultat de Química, Universitat de Barcelona, Martí i Franquès 1-11, 08028 Barcelona, Spain
c
Department of Biochemistry and Molecular Biology, Faculty of Biology, Institute of Biomedicine of University of Barcelona (IBUB) and IDIBAPS, Unit Associated with CSIC, Diagonal 645,
08028 Barcelona, Spain
d
Leitat Tecnological Center, Parc Cientific de Barcelona, Edifici Hèlix, C/Baldiri Reixach, 15-21, 08028 Barcelona, Spain
e
Departament de Bioquímica i Biologia Molecular, Facultat de Farmàcia, Institut de Biomedicina de la Universitat de Barcelona (IBUB), Av. Joan XXIII s/n, 08028 Barcelona, Spain
Institut de Recherche Biomédicale des Armées, Antenne de Marseille, Unité de Parasitologie, URMITE-UMR 6236, Allée du Médecin Colonel Jamot, Parc le Pharo, BP 60109,
f
13262 Marseille Cedex 07, France
g
Université Lille Nord de France, Université Lille 1, Unité de Glycobiologie Structurale et Fonctionnelle, 59650 Villeneuve d'Ascq, France
UMR CNRS 8576, France
Unitat de Difracció de Raig-X, Centre Científici Tecnològic de La Universitat de Barcelona, Solé I Sabarís 1-5, 08028 Barcelona, Spain
Departament de Cristal.lografia, Mineralogia I Dipòsits Minerals, Facultat de Geologia, Universitat de Barcelona, Martí i Franquès s/n. 08028 Barcelona, Spain
h
i
j
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 May 2011
Received in revised form 12 September 2011
Accepted 14 September 2011
Available online 22 September 2011
The study of the reactivity of three 1-(2-dimethylaminoethyl)-1H-pyrazole derivatives of general formula
[1-(CH2)2NMe2}-3,5-R2-pzol] {where pzol represents pyrazole and R_H (1a), Me (1b) or Ph (1c)} with
[MCl2(DMSO)2] (M_Pt or Pd) under different experimental conditions allowed us to isolate and character-
ize cis-[M{κ2-N,N′-{[1-(CH2)2NMe2}-3,5-R2-pzol])}Cl2] {MM_PtPt (2a–2c) or Pd (3a–3c)} and two cyclo-
metallated complexes [M{κ3-C,N,N′-{[1-(CH2)2NMe2}-3-(C5H4)-5-Ph-pzol])}Cl] {M_Pt(II) (4c) or Pd(II)
(5c)}. Compounds 4c and 5c arise from the orthometallation of the 3-phenyl ring of ligand 1c. Complex
2a has been further characterized by X-ray crystallography. Ligands and complexes were evaluated for
their in vitro antimalarial against Plasmodium falciparum and cytotoxic activities against lung (A549) and
breast (MDA MB231 and MCF7) cancer cellular lines. Complexes 2a–2c and 5c exhibited only moderate an-
timalarial activities against two P. falciparum strains (3D7 and W2). Interestingly, cytotoxicity assays
revealed that the platinacycle 4c exhibits a higher toxicity than cisplatin in the three human cell lines
and that the complex 2a presents a remarkable cytotoxicity and selectivity in lung (IC50 =3 μM) versus
breast cancer cell lines (IC50 N20 μM). Thus, complexes 2c and 4c appear to be promising leads, creating a
novel family of anticancer agents. Electrophoretic DNA migration studies in presence of the synthesized
compounds have been performed, in order to get further insights into their mechanism of action.
© 2011 Elsevier Inc. All rights reserved.
Keywords:
Cancer
Malaria
Palladium
Platinum
1. Introduction
(NH3)2] cisplatin [2] (Fig. 1) in 1965 was rapidly followed by clinical trials
and finally in 1978, FDA granted approval. Platinum(II) complexes such as
Platinum drugs have played a key role among the metal-based anti-
cancer agents [1]. The discovery of the antitumor properties of cis-[PtCl2
cisplatin and carboplatin (Fig. 1) are widely used to treat cancers such as
testicular, ovarian, urinary bladder, melanoma, etc. The cytotoxicity of Pt-
based drugs is mainly attributed to their ability to bind DNA and to induce
DNA damage leading then to apoptosis [3–5].
Unfortunately, the use of cisplatin is restricted due to dose-limiting
toxicity, including nephrotoxicity, neurotoxicity and ototoxicity [6,7].
Additional side effects such as blood pressure increase, severe nausea,
vomiting and diarrhea have also been reported. Moreover, the biochem-
ical resistance mode limits the clinical utility of the Pt-based drugs in cur-
rent use [1].
⁎
Corresponding author. Tel.: +34 934039134; fax: +34 934907725.
⁎⁎ Correspondence to: C. Biot, Université Lille Nord de France, Université Lille 1, Unité
de Glycobiologie Structurale et Fonctionnelle, 59650 Villeneuve d'Ascq, France. Tel.:
+33 3 20436941; fax: +33 3 20436555.
E-mail addresses: conchi.lopez@qi.ub.es (C. López), christophe.biot@univ-lille1.fr
(C. Biot).
0162-0134/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2011.09.021

J. Quirante et al. / Journal of Inorganic Biochemistry 105 (2011) 1720–1728
1721
H2
N
Pt
Me
Cl
O
O
O
Pt
O
Cl
Cl
O
H2 N
H2 N
H2 N
H2N
N
Pt
Pt
O
N
NH3
H2
Cl
cisplatin
carboplatin
oxaliplatin
picoplatin
R
R
Cl
N
N
N
R
N
N
M
M
Cl
R
N
Cl
NMe2
NMe2
NMe2
2,3
1
M=Pt(4c)orPd(5c)
M=Pt,R=H(2a),Me(2b)orPh(2c)
M=Pd,R=H(3a),Me(3b)orPh(3c)
R = H(1c),Me(1b)orPh(1a)
Fig. 1. Chemical structure of some platinum(II) based anticancer agents and compounds used in this study.
In the search of new metallodrugs avoiding toxicity and resistance,
special attention has been paid to the replacement of one or both NH3
ligands of cisplatin by other N-donor ligand(s). This strategy gave rise
to oxaliplatin and picoplatin [8] (Fig. 1). In oxaliplatin, which is active
in patients with colorectal cancer, both NH3 units have been replaced
by (1R,2R)-cyclohexane-1,2-diamine (R,R-dach); while picoplatin, that
is in clinical development for the treatment of patients with solid tu-
mors, contains a 2-methylpyridine instead of one NH3 ligand.
The search of novel N-donor ligands (i.e. amines, oximes, imines or
azoles) for the synthesis of optimized platinum(II) and palladium(II)
drugs is still in progress [9,10]. In addition, it is well-known that azoles
are valuable reagents in coordination chemistry and that their binding
to a transition metal ion affects their properties and activities. Few com-
plexes with pyrazole ligands showing an antitumor activity similar to
that of cisplatin have been reported [11–20], but the effect produced
by the substituents on the heterocycle has not been clarified so far.
Within this therapeutic context, and in order to clarify this point and
to elucidate the influence of mode of binding of this family of ligands
in the biological activity of the complexes, we decided to synthesize
the new pyrazole derivatives 1a–1c (Fig. 1) and to study their reactivity
with Pt(II) and Pd(II). Due to the relative disposition of the two nitrogen
atoms, compounds 1a–1c may bind to the M(II) center as a neutral (N)
or (N,N′) ligand. Moreover, for 1c, the presence of the phenyl ring on po-
sition 3 of the heterocycle may also allow the formation of metallacycles
containing 1c as a mer-terdentate (C,N,N′)− ligand.
Cyclometallated complexes derived from N-donor ligands have
attracted great interest during the last decade due to their properties
and applications in a wide variety of fields [10,20–36] and compounds
of this kind containing Pd(II), Pt(II), Ru(II), Ir(III) Rh(III) and Au have
shown promising cytotoxic activities [10,20,27–36]. Few cyclopalla-
dated complexes derived from pyrazole are known [37–41] and, to
the best of our knowledge, their cytotoxicity has only been reported
once [41]. On the other hand despite the interest for Pt(II) complexes,
platinacycles with pyrazolyl ligands are scarce and studies on their bio-
logical activity have not been performed so far.
In this work, we present the study of the reactivity of ligands 1a–1c
with syntheses of cis-[MCl2(DMSO)2], the new compounds 2a–2c and
3a–3c and the cyclometallated complexes 4c and 5c, together with a
comparative study of their antineoplastic activity against lung (A549)
and breast (MDA MB231 and MCF7) human cancer cell lines. As our in-
terests lie also in the area of antiparasitic drugs [42,43] and since a few
Pt(II) [44] and Pd(II) based complexes [45] with antimalarial activity (in
the micromolar range) have been reported, we also tested the potential
of the free ligands (1a–1c) and the complexes 2a–5c against the
chloroquine-susceptible strain (3D7) and the chloroquine-resistant
strain (W2) of Plasmodium falciparum.
2. Experimental
2.1. Chemistry
2.1.1. Materials and methods
Reagents were obtained from commercial sources and used as re-
ceived. Cis-[MCl2(DMSO)2] (M_Pd or Pt) were prepared according to
literature protocols [46,47]. All reactions were carried out with dry and
freshly distilled solvents. Column chromatography refers to flash chro-
matography and was carried out on SiO2 (silica gel 60, SDS, 230–240
mesh) or Al2O3 (neutral alumina, 63–200 μm). Analytical TLC was per-
formed on SiO2 (Merck silica gel 60 F254) plates. Elemental analyses
were carried out at the Serveis Cientifico-Tècnics (Universitat Barcelona).
Mass spectra (electrospray ionization, ESI+) were performed at the Ser-
vei d'Espectrometria de Masses (Universitat de Barcelona). Infrared spec-
tra were obtained with a Nicolet 400FTIR instrument using KBr pellets.
Only noteworthy IR absorptions are listed. Unless otherwise noted 1H
and 13C NMR spectra were recorded in CDCl3 at 298 K with a Mercury-
400 MHz. 1H and 13C chemical shifts (δ) are reported in ppm downfield
and referred to SiMe4 and to the resonance of CDCl3, respectively and
coupling constants (J) are given in Hz. All NMR assignments were
Fig. 2. ORTEP plot of complex 2a. Hydrogen atoms have been omitted for clarity. Selected
bond lengths (in Å) and angles (in deg.) for 2a: Pt\N(1), 2.010(3); Pt\N(3), 2.091(4);
Pt\Cl(1), 2.3056(13); Pt\Cl(2), 2.3058(13); N(1)\C(1),1.339(6); N(1)\N(2), 1.370(6);
N(3)\C(7), 1.499(5); N(3)\C(6), 1.499(7); N(3)\C(8), 1.506(6); C(1)\C(2), 1.409(8); C
(2)\C(3), 1.367(10); C(3)\N(2), 1.349(7); N(2)\C(5), 1.453(7); C(5)\C(6), 1.483(7); N
(1)\Pt\N(3), 93.57(15); N(1)\Pt\Cl(1), 176.60(11), N(3)\Pt\Cl(1), 89.61(12);
N
(1)\Pt\Cl(2), 89.32(12); N(3)\Pt\Cl(2), 177.03(12); Cl(1)\Pt\Cl(2), 87.52(5); C(1)\
N(1)\N(2), 105.3(4); C(1)\N(1)\Pt, 129.5(3); N(2)\N(1)\Pt, 125.2(3); C(7)\N(3)\C
(6), 108.2(4); C(7)\N(3)\C(8), 108.3(4); C(6)\N(3)\C(8), 106.6(4); C(7)\N(3)\Pt,
109.7(3); C(6)\N(3)\Pt, 113.5(3); C(8)\N(3)\Pt, 110.4(3); N(1)\C(1)\C(2), 111.0
(5); C(3)\C(2)\C(1), 104.7(5); N(2)\C(3)\C(2), 108.5(6); C(3)\N(2)\N(1), 110.6(4);
C(3)\N(2)\C(5), 129.1(5); N(1)\N(2)\C(5), 120.1(4); N(2)\C(5)\C(6), 111.5(4) and
C(5)\C(6)\N(3), 113.8(4).

1722
J. Quirante et al. / Journal of Inorganic Biochemistry 105 (2011) 1720–1728
Cl(1)
H(4A)
N(3)
Pt
N(2)
N(1)
H(3)
Cl(2)
Fig. 3. Assembly of molecules of 2a in the crystal by multiple weak C\H···Cl intermolecular interactions involving: a) the two chlorides {Cl(1) and Cl(2)} the unit at (x, y, z) (repre-
sented as the central unit) and the H(4B) and H(3) atoms, respectively of another and close molecule at (x, 1+y, z) and b) these two hydrogen atoms and the two Cl− ligands of the
molecule located at (−1+x, y, z).
made on the basis of two dimensional NMR experiments (gradient cor-
relation spectroscopy gCOSY, gradient heteronuclear single quantum
correlation gHSQC, and gradient Heteronuclear Single Quantum Correla-
tion gHMBC). In all cases the atom labeling scheme for the assignment of
the signals corresponds to that shown in Scheme 1. If not specified, 195Pt
{1H} NMR spectra of 2a, 2c and 4c were recorded in CDCl3 with a Bruker
250DXR instrument using CDCl3 as solvent and H2[PtCl6] {δ195Pt(H2
[PtCl6])=0.0 ppm} as reference. The splitting of proton resonances in
the reported 1H NMR spectra is defined as s = singlet, d = doublet,
dd = doublet of doublet, t = triplet, td = triplet of doublet and m =
multiplet.
with stirring for 2.5 h. The mixture was cooled, 2-dimethylaminoethyl
chloride hydrochloride (2.6 g, 2.8×10−3 mol) was added, and the mix-
ture was heated under reflux for 4 h. Water (20 mL) was added, the tol-
uene layer was separated and the aqueous phase was extracted with
toluene (20 mL). The joined organic phases was extracted with aqueous
0.1 M HCl solution, basified with aqueous saturated Na2CO3, extracted
with CH2Cl2, dried with Na2SO4 and finally evaporated to dryness to
give 1a as a colorless oil (2.6 g, 67%). IR (cm−1): 3423, 2946, 2772,
1461, 1397, 1283, 1092, 1054, 751. 1H NMR (gCOSY): 2.27 (s, 6 H,
NMe2), 2.76 (t, J=7 Hz, 2 H, \CH2\b), 4.24 (t, J=7 Hz, 2 H, \
CH2\a), 6.24 (m, 1 H, H4), 7.45 (d, J=2 Hz, 1 H, H5), 7.50 d,
J=1.6 Hz, 1 H, H3). 13C NMR (gHSQC, gHMBC): 45.6 (NMe2), 50.2
(Ca), 59.2 (Cb), 105.3 (C4), 129.3 (C5), 139.2 (C3). EM (ESI+):
m/z=140.12 (Calc. 140.11) {[M]+H}+. C7H13N3 (FW=139.11).
2.1.2. Synthesis of ligands [1-(CH2)2NMe2}-3,5-R2-pzol] {R_H (1a), Me
(1b) or Ph (1c)}
2.1.2.2. Preparation of 1-(2-dimethylaminoethyl)-3,5-dimethyl-1H-pyrazole
(1b). This product 1b was obtained as a colorless oil following the same
procedure as described for 1a. Yield 75%. IR (cm−1): 3423, 2945, 2771,
1554, 1461, 1425, 1386, 1042, 1018, 773. 1H NMR (gCOSY): 2.21 (s, 3H,
Me), 2.21 (s, 3H, Me), 2.28 (s, 6H, \NMe2), 2.68 (t, J=3.2, 2H, \
CH2\b), 4.06 (t, J=3.4, 2H, \CH2\a), 5.77 (s, 1H, H4). 13C NMR
(gHSQC, gHMBC): 11.0 (Me), 22.0 (Me), 45.7 (\NMe2), 46.0 (Ca), 59.1
(Cb), 104.9 (C4), 140.0 (C5), 147.4 (C3). EM (ESI+): m/z=168.15 (Calc.
168.14) {[M]+H}+. C9H17N3 (FW=167.14).
2.1.2.1. Preparation of 1-(2-dimethylaminoethyl)-1H-pyrazole (1a). A
mixture of 1H-pyrazole (1.92 g, 2.8×10−3 mol) and sodamide (1.56 g,
4×10−3 mol) in dry toluene (20 mL) was heated at reflux temperature
2.1.2.3. 1-(2-dimethylaminoethyl)-3,5-diphenyl-1H-pyrazole (1c). To a
suspension of 3,5-diphenylpyrazole (1.26 g, 5.74×10−3 mol) in tolu-
ene (100 mL) Aliquat 336 (1 g), 40% aqueous NaOH solution (50 mL)
and ClCH2CH2NMe2·HCl (2.5 g, 23.04×10−3 mol) were added and the
resulting mixture was stirred at 95 °C for 24 h. After this period water
(50 mL) was added, the organic phase was extracted, dried with
Na2SO4 and finally evaporated to dryness. The crude material was chro-
matographed on silica gel (Et2O to Et2O:CH2Cl2 1:1), to yield the ligand
1c as a clear viscous oil (1.28 g, 77%). An analytical sample was recrys-
tallized from hexane/CH2Cl2 to obtain colorless crystals. IR (cm−1):
3463, 3040, 2945, 2819, 2775, 1483, 1460, 1438, 1299, 763, 694. 1H
NMR (gCOSY): 2.17 (s, 6H, NMe2), 2.79 (t, J=7.2, 2H, \CH2\b), 2.25
(t, J=7.2, 2H, \CH2\a), 6.57 (s, 1H, H4), 7.25–7.48 (m, 8H, ArH), 7.83
(dd, J=8 and 1.4, 2H, H2′ and H6′). 13C NMR (gHSQC): 45.6 (NMe2),
47.8 (Ca), 59.1 (Cb), 103.4 (C4), 125.6, 127.5, 128.5, 128.7, 129.0 (Ar)
130.8, 135.5 (C1′ and C1″), 145.1 (C5), 150.7 (C3). EM (ESI+):
Fig. 4. DNA unwinding assay of supercoiled pBluescript SK+ (40 μg/mL) by platinum
and palladium drugs at 5 μM (lanes 1) or 50 μM (lanes 2). A, 2c; B, 5c; C, 2a; D, 4c; E,
3b; F, 3a; G, 2b, H, 1c. Cisplatin (CP) was analyzed in parallel for comparison. The mo-
lecular mass marker was lambda DNA digested with HindIII. cc, closed circular DNA
form (supercoiled form); oc, open circular DNA form.
m/z=292.18 (Calc. 292.17) {[M]+H}+
.
Anal (%) Calc. for
C19H21N3.1/4 H2O: C, 77.12; H, 7.32; N, 14.20. Found: C, 77.5; H, 7.4;
N, 14.4. C19H21N3 (FW=291.17).
2.1.3. Synthesis of compounds
cis-[Pt{κ2-N,N′-[1-(CH2)2NMe2]-3,5-R2-pzol}Cl2] (2)
Fig. 5. (A) Interaction of pBluescript SK+ plasmid DNA (40 μg/mL) with increasing
concentrations of the platinum(II) complex 2a and the palladium(II) complex 5c.
Lanes 1, DNA only; lanes 2, 5 μM drug; lanes 3, 10 μM drug, lanes 4, 25 μM drug;
lanes 5, 50 μM drug; and lanes 6, 100 μM drug. (B) Unwinding assay with cisplatin
(CP) at the indicated concentrations for comparison. cc, closed circular DNA form; oc,
open circular DNA form.
2.1.3.1. Cis-[Pt{κ2-N,N′-[1-(CH2)2NMe2]pzol}Cl2] (2a). A suspension of
cis-[PtCl2(DMSO)2] (52.8 mg, 125×10−3 mol) and ligand 1a (17.4 mg,
125×10−3 mol) in toluene (10 mL) was heated at reflux temperature
for 2 h. The reaction mixture was evaporated to dryness and the obtained

J. Quirante et al. / Journal of Inorganic Biochemistry 105 (2011) 1720–1728
1723
R
NHMe2
Cl
+
N
Cl
R
N
H
i)
3
4
R
N2
R
N
1
5
NMe2
a
1
b
ii)
iv)
iii)
6'
6'
3
4
1'
1'
4
3
5'
4'
5'
4'
3
R
6""
4
6""
3"
5
5
N 2
N 2
5"
5"
4"
2'
N2
2'
N
N1
Cl
3'
1"
Cl 3'
Pt
Pd
1"
2"
1
5
a
R
N
1
M
4"
3"
Cl
a
a
2"
Cl
NMe2
NMe2
b
NMe2
b
b
M = Pt, 2 or Pd, 3
4c
5c
Scheme 1. Synthesis of ligands (1a–1c) and their palladium(II) and platinum(II) derivatives with 1 acting as a bidentate (N,N′) ligand (2,·3) and the cyclometallated compounds 4c
and 5c. In order to ease the visualization of the reactions, letters a, b and c refer to the substituents on positions 3 and 5 of the pyrazole {R_H (a), Me (b) or Ph (c)}. Reagents and
conditions: i) in toluene, in the presence of sodamine (for R_H or Me) or using Aliquat 336 and NaOH (40%) for R_Ph. ii) Cis-[MCl2(DMSO)2] (M_Pt or Pd for 2 and 3, respec-
tively) (1:1) toluene, reflux; iii) Cis-[PtCl2(DMSO)2] and NaOAc in toluene:MeOH (12:1) mixture, reflux 3 h. iv) Cis-[PdCl2(DMSO)2] and NaOAc in toluene:MeOH (12:1) mixture,
reflux 1 h.
residue was purified by column chromatography (Al2O3, hexane: acetone
50:50 to 25:75) to yield 33 mg (65%) of the title complex as a yellow solid.
An analytical sample was recrystallized from CH2Cl2/MeOH. IR (cm−1):
3430, 3085, 2921, 1740, 1627, 1517, 1447, 1422, 1156, 1020, 756. 1H
NMR (DMSO-d6, 500 MHz, gCOSY): 2.85 (m, 2H, \CH2\b), 2.94 (s, 6H,
NMe2), 4.53 (m, 2H, \CH2\a), 6.45 (m, 1H, H4), 8.07 (m, 2H, H3 and
H5). 13C NMR (DMSO-d6, 125.9 MHz, gHSQC, gHMBC): 47.5 (Ca), 52.4
(\NMe2), 62.6 (Cb), 105.8 (C4), 133.8 (C5), 141.3 (C3). 195Pt{1H} NMR
(DMSO-d6, 54 MHz): −2178 (s).1 EM (ESI+): m/z=422.05 (Calc.
422.05) {[M]+NH4}+. Anal (%) Calc. for C7H13Cl2N3Pt (FW=404.01):
C, 20.75; H, 3.23; N, 10.37. Found: C, 20.9; H, 3.3; N, 10.3.
14.7 (Me), 45.4 (Ca), 53.1 (NMe2), 64.1 (Cb), 107.8 (C4), 143.5
(C5), 153.1 (C3). EM (ESI+): m/z=450.10 (Calc. 450.08) {[M]
+NH4}+. Anal (%) Calc. for C9H17Cl2N3Pt (FW=432.04): C, 24.95;
H, 3.96; N, 9.70. Found: C, 25.0; H, 4.0; N, 9.4.
2.1.3.3. Cis-[Pt{κ2-N,N′-[1-(CH2)2NMe2]-3,5-Ph2-pzol}Cl2] (2c). A sus-
pension of cis-[PtCl2(DMSO)2] (26.4 mg, 62.5×10−3 mol) and ligand 1c
(18.2 mg, 62.5×10−3 mol) in toluene (5 mL) was heated at reflux for
1.5 h. The solid formed was filtered out and purified by column chroma-
tography {Al2O3, hexane: CH2Cl2 (25:75)} to yield 23 mg (66%) of the title
complex as a yellow solid. IR (cm−1): 3430, 2925, 1628, 1480, 1023, 768,
698. 1H NMR (gCOSY): 2.66 (b m, 2H, \CH2\b), 3.18 (s, 6H, \NMe2), 4.6
(b m, 2H, \CH2\a), 6.65 (s, 1H, H4), 7.45 (m, 2H, H2″ and H6″), 7.44–7.54
(m, 6H,), 8.30 (d, J=8.4, 2H, H2′ and H6′). 13C NMR (gHSQC, gHMBC):
48.7 (\NMe2), 54.0 (Ca), 64.8 (Cb), 107.3 (C4′), 127.6 (C4), 128.4 (C3″
and C″), 128.4 (C2″ and C6″), 128.8 (C2′ and C6′), 129.3 (C1′), 129.4 (C3′
and C5′), 130.4 (C4″), 131.5 (C1″), 148.8 (C5), 157.1 (C3). 195Pt{1H} NMR
(54 MHz): −2165 (s). EM (ESI+): m/z=521.11 (Calc. 520.58) {[M]-
Cl}+. Anal (%) Calc. for C19H21Cl2N3Pt.1/2 H2O: C, 40.29; H, 3.92; N, 7.42.
Found: C, 40.25; H, 3.8; N, 7.4. C19H21Cl2N3Pt (FW=556.08).
2.1.3.2. Cis-[Pt{κ2-N,N′-[1-(CH2)2NMe2]-3,5-Me2pzol}Cl2] (2b). A sus-
pension of cis-[PtCl2(DMSO)2] (52.8 mg, 125×10−3 mol) and ligand 1b
(20.9 mg, 125×10−3 mol) in toluene (10 mL) was heated at reflux tem-
perature for 3 h. The reaction mixture was evaporated to dryness and the
residue was treated with MeOH (10 mL). The obtained suspension was
stirred for 2 h and filtered out. The solid was washed with two 5 mL por-
tions of CH2Cl2 to remove the MeOH, and finally dried under vacuum to
yield 32.5 mg (60%) of complex 2b as a yellow solid. IR (cm−1): 3427,
2916, 2342, 1553, 1465, 1450, 1156, 987, 808. 1H NMR (DMSO-d6,
500 MHz, gCOSY): 2.33 (s, 3H, Me), 2.43 (s, 3H, Me), 2.45 (m, 2H,
\CH2\b), 2.83 (s, 6H, NMe2), 4.40 (m, 2H, \CH2\a), 6.09 (s, 1H,
H4). 13C NMR (DMSO-d6, 125.9 MHz, gHSQC, gHMBC): 11.1 (Me),
2.1.4. Synthesis of compounds
cis-[Pd{κ2-N,N′-[1-(CH2)2NMe2]-3,5-R2-pzol}Cl2] (3)
2.1.4.1. Cis-[Pd{κ2-N,N′-[1-(CH2)2NMe2]pzol}Cl2] (3a). A suspension of
cis-[PdCl2(DMSO)2] (41.7 mg, 125×10−3 mol) and ligand 1a (17.4 mg,
125×10−3 mol) in toluene (10 mL) was heated at reflux temperature
for 2 h. The reaction mixture was evaporated to dryness and the obtained
residue was purified by column chromatography {Al2O3, CH2Cl2: MeOH
1
During the acquisition time (more than 24 h) of the 195Pt{1H} NMR data the pres-
ence of an additional signal at δ=−2982 ppm was also observed in the spectrum. Its
intensity increased with time, thus suggesting the formation of a new species in
solution.

1724
J. Quirante et al. / Journal of Inorganic Biochemistry 105 (2011) 1720–1728
(from 2% to 5%)} to yield 28 mg (71%) of the title complex as an orange
solid. IR (cm−1): 3447, 3086, 2920, 1628, 1421, 1277, 1109, 1078,
796, 760. 1H NMR (acetone-d6, 500 MHz, gCOSY): 2.82 (masked
m, 2H, \CH2\b), 2.91 (s, 6 H, NMe2), 4.70 (m, 2H, \CH2\a), 6.39
(t, J=2.5, 1H, H4), 7.91 (dd, J=2.5, 1H, H5), 8.17 (d, J=2.5, 1H,
H3). 13C NMR (acetone-d6, 125.9 MHz, gHSQC, gHMBC): 48.4 (Ca),
52.0 (NMe2), 63.2 (Cb), 106.5 (C4), 134.2 (C5), 143.9 (C3). EM
(ESI+): m/z=321.0 (320.45) {[M]-Cl+CH3CN}+. Anal (%) Calc.
for C7H13Cl2NPd·1H2O·1/4C7H8: C, 29.40; H, 4.76; N, 11.70.
Found: C, 29.5; H, 4.9; N, 11.0.2 C7H13Cl2NPd (FW=314.95).
NMR (gCOSY): 2.77 (s, 6H, NMe2), 2.99 (m, 2H, \CH2\b), 4.25 (m,
2H, \CH2\a), 6.45 (s, 1 H, H4), 6.95 (td, J=7.6 and 1.6, 1H, H4′), 7.01
(td, J=7.2 and 1.2, 1H, H5′), 7.23(dd, J=7.6 and 1.6, 1H, H3′), 7.40–
7.46 (m, 2H, H2″ and H6″), 7.50–7.56 (m, 3H, H3″, H4″ and H5″), 7.87 (d,
J=7.2, 1H, H6′). 13C NMR (gHSQC, gHMBC): 45.2(Ca), 48.5 (\NMe2),
60.9 (Cb), 101.0 (C4), 122.1 (C3′), 124.5 (C5′), 127.1 (C4′), 128.9 (C2″
,
C4″ and C6″), 129.2 (C3″ and C5″), 129.8 (C1″), 136.6 (C6′), 137.5 (C1′),
145.1 (C5), 148.4 (C2′), 160.5 (C3). EM (ESI+): m/z=396.07 {[M]-Cl}+
.
Anal (%) Calc. for C19H20ClN3Pd (FW=431.04): C, 52.80; H, 4.66; N,
9.72. Found: C, 52.55; H, 4.6; N, 9.9.
2.1.4.2. Cis-[Pd{κ2-N,N′-[1-(CH2)2NMe2]-3,5-Me2-pzol}Cl2] (3b). A sus-
pension of cis-[PdCl2(DMSO)2] (41.7 mg, 125×10−3 mol) and ligand
1b (20.9 mg, 125×10−3 mol) in toluene (10 mL) was heated at reflux
temperature for 2 h. The reaction mixture was evaporated to dryness
and the obtained residue was purified by column chromatography
(Al2O3, CH2Cl2: MeOH 1% to 8%) to yield 30 mg (70%) of complex 3b
as an orange solid. IR (cm−1): 3449, 3003, 2916, 2860, 1151, 1465,
1430, 1310, 1091, 1026, 991, 810. 1H NMR (acetone-d6, 500 MHz,
gCOSY): 2.36 (s, 3H, Me), 2.52 (s, 3H, Me), 2.56 (m, 2H, \CH2\b),
2.80 (s, 6H, NMe2), 4.68 (m, 2H, \CH2\a), 6.09 (s, 1H, H4). 13C NMR
(acetone-d6, 125.9 MHz, gHSQC, gHMBC): 11.1 (Me), 15.6 (Me), 48.9
(Ca), 53.1 (NMe2), 64.1 (Cb), 106.5 (C4), 134.0 (C5), 142.6 (C3).
EM (ESI+): m/z=377 (Calc. 375.0) {[M]+MeOH}+. Anal (%) Calc. for
C9H17Cl2N3Pd·3/2H2O·1/4C7H8: C, 32.71; H, 5.51; N, 10.65. Found: C,
32.7; H, 5.2; N, 10.7. C9H17Cl2N3Pd (FW=342.98).
2.2. Crystallography
A prismatic crystal of 2a (sizes in Table 1) was selected and mounted
on a MAR345 diffractometer with an image plate detector. Unit-cell pa-
rameters were determined from 5843 reflections (3°bθb31°) and re-
fined by least-squares method. Intensities were collected with graphite
monochromatized Mo Kα radiation. 9754 reflections were measured
(in the range 2.39°≤θ≤32.38°) of which 2861 were non-equivalent by
symmetry {Rint(on I)=0.072} and 2741 reflections were assumed as ob-
served applying the condition IN2σ(I). Lorentz-polarization and absorp-
tion corrections were made.
The structure was solved by Direct methods using SHELXS computer
program [48] and refined by full-matrix least-squares method with
SHELX97 computer program [49] using 9754 reflections, (very negative
intensities were not assumed). The function minimized was Σw| |Fo|2 −
|Fc|2 |2, where w=[σ2(I)+(0.0518P)2+0.5576P]−1, and P=(|Fo|2+2
|Fc|2)/3; ƒ, ƒ′ and ƒ″ were taken from International Tables of X-Ray Crystal-
lography [50]. All H atoms were computed and refined, using a riding
model, with an isotropic temperature factor equal to 1.2 times the
equivalent temperature factor of the atom to which is linked. The final
R(on F) factor was 0.033, wR(on |F|2)=0.085 and goodness of
fit=1.062 for all observed reflections. Number of refined parameters
was 118. Max. shift/esd=0.00, Mean shift/esd=0.00. Max. and min.
2.1.5. Synthesis of the cyclometallated compounds
2.1.5.1. [Pt{κ2-C,N,N′-{[1-(CH2)2NMe2}-3-(C5H4)-5-Ph-pzol])}Cl2] (4c).
To a suspension of cis-[PtCl2(DMSO)2] (52.8 mg, 125×10−3 mol) and li-
gand 1c (18.2 mg, 125×10−3 mol) in toluene (5 mL), a MeOH (1 mL) so-
lution of NaOAc (11.2 mg, 138×10−3 mol). The resulting mixture was
heated at reflux for 3 h and later on concentrated to dryness. The solid
formed was dissolved in CH2Cl2 and filtered very slowly through a short
pad of celite. The filtrate was evaporated and the obtained residue was
submitted to column chromatography {Al2O3, hexane:CH2Cl2 (75:25)},
to yield 47 mg (72%) of the title complex as a yellow solid. IR (cm−1):
3438, 3046, 2923, 1506, 1448, 1335, 1022, 799, 759, 695. 1H NMR
(gCOSY): 2.95 (s, 6H, NMe2), 3.27 (m, 2H, \CH2\b), 4.31 (m, 2H, \
CH2\a), 6.50 (s, 1 H, H4), 6.97–7.08 (m, 2H, H4′ and H5′), 7.24 (m, 1H,
H3′), 7.40–7.47 (m, 2 H, H2″ and H6″), 7.48–7.57 (m, 2H, H3″ and H5″),
7.71 (dd, J=5.6, 3.2 Hz, 1H, H4″), 7.89 (dd, J=7.2 and 1.6 Hz, 1H,
H6′). 13C NMR (gHSQC, gHMBC): 45.7 (Ca), 49.2 (\NMe2), 61.7 (Cb),
101.3 (C4), 121.7 (C3′), 123.7 (C5′), 127.5 (C4′), 128.8 (C2″, C4″ and C6″),
129.3 (C3″ and C5″), 129.8 (C1″), 135.0 (C6′), 137.5 (C1′), 144.6 (C5),
160.5 (C3 and C2′). 195Pt{1H} NMR (54 MHz): _3555 (s). EM (ESI+):
m/z=521.11 (Calc. 521.10){[M]+H}+. Anal (%) Calc. for C19H20ClN3Pt
(FW=520.10): C, 43.81; H, 3.87; N, 8.07. Found: C, 43.81; H, 3.84; N, 7.77.
peaks in final difference synthesis were 2.767 and −2.054 eÅ−3
respectively.
,
Table 1
Crystal data and details of the structure refinement for 2a. Standard deviations are
given in parentheses.
Empirical formula
Formula weight
Temperature (K)
λ (Å)
Crystal size (mm×mm×mm)
Crystal system
Space group
a (Å)
C7H13Cl2N3Pt
405.19
293 (2)
0.71073
0.2×0.1×0.1
Monoclinic
P21/c
7.162 (3)
8.725 (3)
17.478 (4)
90
102.76 (2)
1065.2 (6)
4
2.527
13.633
752
From 2.39 to 32.38
9754
2861[0.0721]
118
2.1.5.2. [Pd{κ2-C,N,N′-{[1-(CH2)2NMe2}-3-(C5H4)-5-Ph-pzol])}Cl2] (5c).
To a suspension of cis-[PdCl2(DMSO)2] (41.7 mg, 125×10−3 mol) and li-
gand 1c (36.4 mg, 125×10−3 mol) in toluene (12 mL), a MeOH (1 mL)
solution of NaOAc (11.2 mg, 138×10−3 mol) was added. The resulting
mixture was heated at reflux for 1 h. Afterwards the solvent was evapo-
rated to dryness and the residue formed was dissolved in CH2Cl2 and fil-
tered very slowly through a short pad of celite. The filtrate was
evaporated and the obtained residue was passed through a Al2O3 col-
umn using CH2Cl2:MeOH (100:1) mixture as eluent. The band collected
gave, after concentration to dryness, 5c as a white solid (38 mg, 70%). IR
(cm−1): 3448, 3038, 2924, 2856, 1528, 1449, 1387, 796, 758, 698. 1H
b (Å)
c (Å)
α=γ (deg.)
β (deg.)
Volume (Å3)
Z
Dcalc. (Mg×m−3
)
μ (mm−1
)
F(000)
Θ for data collection (deg.)
N. of reflections collected
N. of unique reflections, [R(int)]
N. of parameters
Completeness to Θ=25.00°
94.2%
1.062
Goodness-of-fit on F2
2
Compounds 3a and 3b retain solvents. Evidence of the presence of small amounts
Final R indices [IN2σ(I)]
R indices (all data)
Largest diff. peak and hole
R1 =0.0327, wR2 =0.0850
R1 =0.0338, wR2 =0.0859
2.767 and −2.054 e.Å−3
of toluene is in the 1H NMR spectra (a singlet at 2.3 ppm). Despite the fact that the two
compounds were dried in the vacuum for a week, it was impossible to evaporate the
solvents. The best analytical results are presented above.

J. Quirante et al. / Journal of Inorganic Biochemistry 105 (2011) 1720–1728
1725
2.3. Biological studies
CO2 and 85% N2 at 37 °C with a humidity of 95%. All strains were syn-
chronized twice with sorbitol before use. Clonality was verified using
PCR genotyping of polymorphic genetic markers, msp1, msp2 and mi-
crosatellite loci [55,56].
Chloroquine (CQ) diphosphate was purchased from Sigma (Saint
Louis, MO). CQ was suspended in water in concentrations ranging
from 5 to 3200 nM. Compounds 1–5 were suspended in methanol and
then diluted in RPMI to obtain final concentrations ranging from
0.01 μM to 500 μM.
For in vitro isotopic microtests, 25 μL/well of antimalarial drug and
200 μL/well of the parasitized red blood cell suspension (final parasite-
mia, 0.5%; final hematocrit, 1.5%) were distributed into 96 well plates.
Parasite growth was assessed by adding 1 μCi of tritiated hypoxanthine
with a specific activity of 14.1 Ci/mmol (Perkin-Elmer, Courtaboeuf,
France) to each well at time zero. The plates were then incubated for
48 h in controlled atmospheric conditions. Immediately after incubation,
the plates were frozen and thawed to lyse erythrocytes. The contents of
each well were collected on standard filter microplates (Unifilter GF/B;
Perkin-Elmer) and washed using a cell harvester (Filter-Mate Cell Har-
vester; Perkin-Elmer). Filter microplates were dried, and 25 μL of scintil-
lation cocktail (Microscint O; Perkin-Elmer) was placed in each well.
Radioactivity incorporated by the parasites was measured with a scintil-
lation counter (Top Count; Perkin-Elmer).
2.3.1. Cell culture
Human lung carcinoma A549 cells (from the American Type Culture
Collection), MBA MD231 and MCF7 (from the European Collection of
Cell Cultures — ECACC) were used in all the experiments. Cells were
grown as a monolayer culture in minimum essential medium (DMEM
with L-glutamine, without glucose and without sodium pyruvate) in the
presence of 10% heat-inactivated fetal calf serum, 10 mM of D-glucose
and 0.1% streptomycin/penicillin in standard culture conditions.
2.3.2. Cell proliferation assay
The assay was performed by a variation of the method described by
Mosmann et al. [51] as specified by Matito and coworkers [52]. In brief,
3×103 A549 cells/well were cultured in 96 well plates. Concentrations
that inhibited cell growth by 50% (IC50) after 72 h of treatment were cal-
culated based on the survival rate compared with untreated cells. Relative
cell viability was measured by the absorbance on an ELISA (enzyme-
linked immunosorbent assay) plate reader (Tecan Sunrise MR20-301,
TECAN, Salzburg, Austria) at 550 nm.
2.3.3. Cell viability assay
The compounds were dissolved in 100% DMSO at 50 mM as stock so-
lution. Then, serial dilutions have been done in DMSO (1:1), in this way
DMSO concentrations in cell media were always the same. Finally, 1:500
dilutions of the serial dilutions of compounds on cell media were done.
The assay was performed as described by Givens et al. [53]. In brief,
MDA MB231 and MCF7 cells were plated at 5000 and 10,000 cells/well,
respectively, in 100 μL media in tissue culture 96 well plates (Cultek).
After 24 h, media was replaced by 100 μL/well of serial dilution of
drugs. Control wells did not contain compounds. Each point concentra-
tion was run in triplicate. Reagent blanks, containing media plus colori-
metric reagent without cells were run on each plate. Blank values were
subtracted from test values and were routinely 5–10% of uninhibited
control values. Plates were incubated 72 h. Hexosaminidase activity
was measured according to the following protocol: the media containing
was removed and cells were washed once with PBS 60 μL of sub-
strate solution (p-nitrophenol-N-acetyl-β-D-glucosamide 7.5 mM
[Sigma N-9376], sodium citrate 0.1 M, pH 5.0, 0.25% Triton X-100)
was added to each well and incubated at 37 °C for 1–2 h; after this
incubation time, a bright yellow appears; then, plates could be de-
veloped by adding 90 μL of developer solution (glycine 50 mM, pH
10.4; EDTA 5 mM), and absorbance was recorded at 410 nM.
The IC50, the drug concentration able to inhibit 50% of parasite
growth, was assessed by identifying the drug concentration corre-
sponding to 50% of the uptake of tritiated hypoxanthine by the parasite
in the drug-free control wells. The IC50 value was determined by non-
linear regression analysis of log-based dose–response curves (Rias-
martTM, Packard, Meriden, USA). IC50 are expressed as means of 3 to 4
experiments standard deviation.
3. Results and discussion
3.1. Synthesis and characterization
The preparation of ligands 1a and 1b was carried out by a modifi-
cation of former literature protocols [57,58] consisting in the alkyl-
ation of 1H-pyrazole and 3,5-dimethyl-1H-pyrazole respectively with
2-dimethylaminoethyl chloride using sodamide as a base (Scheme 1).
The best yield for the synthesis of 1c was obtained by alkylation of
3,5-diphenyl-1H-pyrazole using Aliquat 336 as a phase transfer catalyst.
The three ligands were fully characterized by NMR, IR and mass spec-
troscopy. To the best of our knowledge, these data were found to be ab-
sent or incomplete in the literature.
2.3.4. DNA migration studies
The reactivity of ligands 1a–1c with cis-[MCl2(DMSO)2] {M_Pt(II)
or Pd(II)} was studied under different experimental conditions. Treat-
ment of equimolar amounts of the corresponding ligand (1a–1c) and
of cis-[PtCl2(DMSO)2] in toluene under reflux gave after work up yel-
low solids that were identified as the corresponding cis-[Pt{κ2-N,N′-
{[1-(CH2)2NMe2}-3,5-R2-pzol])}Cl2] (2a–2c in Scheme 1). The palla-
dium(II) analogs (3a–3c) were isolated using the same strategy.
However, treatment of 1c with cis-[PtCl2(DMSO)2] in a toluene:
methanol (12:1) mixture in the presence of a slight excess (~10%)
of NaOAc, produced the activation of the ortho σ(C\H) bond of the
phenyl ring on position 3 giving the platinacycle [Pt{κ3-C,N,N′-[1-
{(CH2)2NMe2}-3-(C6H4)-5-Ph-pzol]}Cl] 4c. When cis-[PtCl2(DMSO)2]
was replaced by its palladium(II) analog, the palladacycle [Pd{κ3-C,
N,N′-[1-{(CH2)2NMe2}-3-(C6H4)-5-Ph-pzol]}Cl] 5c was achieved. It
should be noted that this product could also be isolated in the ab-
sence of the base in the reaction medium.
Plasmid pBluescript SK+ was obtained using a QIAGEN plasmid midi
kit as described by the manufacturer. Interaction of drugs with pBlue-
script SK+ plasmid DNA (Stratagene) was analyzed by agarose gel elec-
trophoresis following a modification of the method described by
Abdullah et al.[54]. In brief, plasmid DNA aliquots (40 μg/mL) were incu-
bated with different concentrations of the platinum and palladium com-
pounds (ranging from 5 μM to 100 μM) at 37 °C for 24 h. For comparison,
cisplatin was used as a positive control. Aliquots of 20 μL of compound:
DNA complexes containing 0.8 μg of DNA were subjected to 1% agarose
gel electrophoresis in TAE buffer (40 mM Tris–acetate, 2 mM EDTA, pH
8.0). The gel was stained in the same buffer containing ethidium bromide
(0.5 mg.mL−1) and visualized and photographed under UV light.
2.3.5. In vitro antimalarial assay
The 3D7 chloroquine-susceptible P. falciparum clone (Africa) and the
W2 chloroquine resistant clone (Indochina) were maintained in culture
in RPMI 1640 (Invitrogen, Paisley, United Kingdom), supplemented
with 10% human serum (Abcys S.A., Paris, France) and buffered with
25 mM HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
and 25 mM NaHCO3. Parasites were grown in A-positive human blood
under controlled atmospheric conditions that consisted of 10% O2, 5%
The new complexes were characterized by elemental analyses, mass
spectrometry, infrared spectroscopy and 1H, 13C and two-dimensional
homo (gCOSY) and heteronuclear (gHSQC and gHMBC) correlations. In
the 13C{1H} NMR spectra of cyclometallated compounds 4c and 5c the in-
tensity of the signal due to the C2′ atom decreased substantially and was
low-field shifted when compared with that of the free ligand 1c. In

1726
J. Quirante et al. / Journal of Inorganic Biochemistry 105 (2011) 1720–1728
addition, no evidence of cross-peak between the resonance of the C2′ nu-
clei and those of the aromatic protons was detected in the [1H–13C]-HSQC
spectra of compounds 4c and 5c. According to previous studies [59,60],
these observations suggested the existence of a σ(M–C2′) bond and the
presence of five membered ring metallacycle in 4c and 5c. 195Pt{1H}
NMR spectra not only provided convincing evidence of the coordination
sphere of the platinum and structure of 2c and 4c, but also explained
the variations produced by the different mode of binding of ligand 1c.
The spectrum of 2c showed a singlet at −2165 ppm, the position of
which is consistent with the values reported for related complexes with
a “(N,N′)Cl2” environment around the platinum(II) [60–62]. For 2c the
signal appeared at higher fields (δ=−3555 ppm); this trend agrees
with those reported for complexes containing R\CH=N\(CH2)nNMe2
(R = phenyl or ferrocenyl moieties) as bidentate (N,N′) or terdentate
(C,N,N′)− ligands [61,62]. The 195Pt NMR spectrum of 2b was not
recorded due to the low solubility of this product and for 2a, two singlets
of relative intensities (1.0:0.3) centered at δ=−2178 and −2982 ppm
were observed. The chemical shift of the former one is similar to that of
2c and the presence of the second and less intense signal suggested
that this product is less stable in CDCl3 than its analog 2c.
Table 2
Cytotoxic activities on A549 human lung carcinoma and MDA-MB231 and MCF7 breast
cancer cell lines for the free ligands and their platinum(II) or palladium(II) complexes,
using cisplatin as reference. Data are shown as the mean SD of two or more experi-
ments performed in triplicate.
IC50 values (μM)
A549
MDA-MB231
MCF/
A) Free ligands
1a
1b
1c
N100
N100
55 14
N100
N100
64 24
N100
N100
52 10
B) Platinum(II) complexes
2a
2b
2c
4c
3
1
57 8.5
62 9.3
14 1.7
6.2 1.3
20 3.6
51 10.4
17 2.2
9.3 3.4
12
5
13 5.8
2.8
7
C) Palladium(II) complexes
3a
3b
5c
N100
N100
N100
16.2 4.6
6.5 2.4
N100
N100
38.4 16.5
19 4.5
73.5 2.1
38.5 4.9
9.3 3.0
Cisplatin
Complex 2a was also characterized by X-ray diffraction. Its molecu-
lar structure and the atom numbering scheme are presented in Fig. 2.
The crystal contains molecules of [Pt{κ2-N,N′-[1-(CH2)2NMe2]-Ph2-
pzol}Cl2] (2a) in which the platinum(II) atom is bound to the two nitro-
gen atoms [N(1) and N(3)] of the pyrazolyl ligand and to two chlorides
[Cl(1) and Cl(2)], in a slightly distorted square-planar environment thus
confirming the mode of binding of the ligand. The Pt\Cl(1) and Pt\Cl
(2) bond lengths are practically identical (the differences do not clearly
exceed 3σ) and fall in the range found in Pt(II) complexes having cis-
coordinated chlorido ligands trans to N-donor ligands.[63–68] The
Pt\N(1) [2.010(3)Å] and Pt\N(3) [Pt\N(3) 2.091(4)Å] bond
lengths are within the normal distances (1.98–2.06 Å) reported for
the same type of complexes and the differences may be attributed
to the different basicities of the two donor atoms {N(amine) versus
N(heterocycle)}[69].
For the purely organic ligands 1a–1c, a moderate activity was ob-
served for the 3,5 diphenyl substituted compound 1c (IC50 values be-
tween 52 and 64 μM). In all cases, the Pd(II) and Pt(II) complexes were
more potent than their corresponding parent ligand and compound 2a
exhibited in human lung A549 cancer cell line the highest potency
(IC50 =3 μM) of the synthesized complexes and a notable selectivity for
lung cancer cell line versus the two breast cancer cell lines (MDA MB
231 and MCF7) selected. Interestingly, 2a turned out to be three times
more potent than cisplatin in lung A549 cancer cell line. In the three can-
cer cell lines, the platinacycle 4c exhibited the highest cytotoxic activity
with IC50 values in the range of 6.2–9.3 μM. It was more effective against
human lung carcinoma (A549) cells and breast cancer (MDA MB231 and
MCF7) cells than the reference drug cisplatin. Cytotoxicity effectiveness of
4c was approximately twice that of the coordination complex 2c in the
three cancer cell lines. Compound 4c arises from the coordination of the
two nitrogen atoms and the formation of a σ(Pt\C) bond, this leads to
a [6.5.5.6] tetracyclic system. Consequently, it has a higher degree of rigid-
ity and planarity than 2c. It is well-known that square-planar metal com-
plexes with aromatic ligands bind to DNA by intercalation [71–74] and
consequently compound 4c may behave not only as alkylating but also
as an intercalating agent. This potential dual behavior may account for
the increased potency of the platinacycle 4c when compared with 2c.
Although compounds 4c and 5c only differ by the nature of the M(II)
ion, the platinum(II) complex 4c is (2.6–5.5 times depending on the cell
line) more potent than its Pd(II) analog 5c. This may be due to the great-
er lability and faster hydrolysis rate of palladium complexes compared
to their platinum equivalents [75]. Therefore, higher cytotoxicity of 5c
could be also connected to slower hydrolysis of Pt\Cl bond and to the
stability of this compound compared to the palladium(II) derivative 5c.
The effect of binding of the compounds investigated in this study
on supercoiled DNA was determined by their ability to alter the elec-
trophoretic mobility of pBluescript plasmid DNA: supercoiled closed
circular (cc) and open circular (oc) forms.
Each molecule of cis-[Pt{κ2-N,N′-[1-(CH2)2NMe2]-3,5-Ph2-pzol}Cl2]
(2a) contains a [5.6] bicyclic system formed by the pyrazolyl unit that
shares the N(2)\N(3) bond with the six membered chelate ring generat-
ed by the coordination of the platinum(II) to the N(1) and N(2) atoms of
the ligand. Bond lengths and angles of the pyrazolyl unit agree with those
reported for most metal complexes containing this azole. The heterocycle
is planar3 and it forms an angle of ca. 22.3° with the coordination plane of
the platinum(II). The puckering analyses of the six-membered chelate
ring is formed by the set of atoms Pt, N(1), N(2), C(4), C(5) and N(3)
[θ=106.6(4)° and ϕ=30.3 (4)° indicates that it adopts a twisted-boat
conformation.
In each molecule of cis-[Pt{κ2-N,N′[1-{(CH2)2NMe2}pzol]}Cl2], the
distances Cl(1)····H(7B) [2.642 Å], Cl(1)····H(6C) [2.699 Å], and Cl
(2)···H(1) [2.721 Å] are smaller than the sum of the van der Waals
radii of these atoms (Cl, 1.7 Å and H, 1.0 Å) [70] thus suggesting weak
C\H···Cl intramolecular interactions.
In the crystal a molecule at (x, y, z) is connected to two different
and vicinal ones [at (x, 1+y, z) and (−1+x, y, z)] by four weak inter-
molecular C\H···Cl interactions (Fig. 3) forming chains.
3.2. Biological studies
Fig. 4 shows the electrophoretic mobility of native pBluescript DNA
incubated with the synthesized compounds (1c and 2–5) at 5 μM or
50 μM concentration. To provide a basis for comparison, incubation of
DNA with cisplatin was also performed using the same concentrations
and conditions. As expected, at both 5 μM and 50 μM concentrations, cis-
platin greatly altered the electrophoretic mobility of pBluescript DNA. At
5 μM concentration, none of the assayed compounds produced a signifi-
cant effect on the electrophoretic mobility of native pBluescript DNA. At
this concentration, the mobility of the supercoiled closed circular form
was only slightly decreased by the platinum(II) compound 2a and the
A human lung carcinoma cell line (A549) and two human breast
cancer cell lines (MDA MB231 and MCF7) were used to test the cytotox-
ic activity of the synthesized compounds. Cisplatin was used as a posi-
tive control, showing a value of IC50 below 20 μM in the three cancer
cellular lines (Table 2).
3
Deviations from the mean plane: N(1), −0.005(4); N(2), 0.008(5); C(1) 0.001(5),
C(2), 0.004(6) and C(3), −0.008(7)Å.

J. Quirante et al. / Journal of Inorganic Biochemistry 105 (2011) 1720–1728
1727
palladium(II) compound 5c. Consistently, at 50 μM, both compounds
greatly alter the mobility of plasmid DNA. For these two compounds
(2a and 5c), an unwinding assay was performed with increasing
amounts of drugs ranging from 5 μM to 100 μM (Fig. 5). For complex
2a, the migration rate of supercoiled band decreases until it comigrates
with the nicked relaxed band. In this titration experiment of 40 μg/mL
pBluescript, the coalescence point, defined as the amount of platinum
complex that is necessary for complete removal of all supercoils from
DNA, occurs with 50 μM concentration of 2a. The lowest efficiency of
complex 2a than cisplatin in removing the supercoils from DNA could
be related to the shorter incubation time (24 h) of the experiments com-
pared to the incubation time in the experiments with cancer cell lines
(72 h). Under these conditions, hydrolysis of the platinum complex
should not occur.
W2). The cyclopalladated complex 5c was 10-times more potent than
its platinum(II) analog 4c against P. falciparum. Compound 2c showed
similar cytotoxic activity on P. falciparum parasites and cancer cells.
Compound 5c was 2 to 3-times more potent against P. falciparum para-
sites than against cancer cells.
In vivo efficiency of platinum derivatives has been already demons-
trated in malaria. Cisplatin (Fig. 1) cured mice infected with Plasmodium
berghei at a dose of 6 mg/kg body weight [76]. The Pd-cyclometallated
derivative 5c was more efficient than Pt-protoporphyrin against P. falci-
parum (38 μM) [77]. However, these compounds were less potent
against the two P. falciparum clones 3D7 and W2 than Fe-derivatives,
such as ferroquine and analogs [78,79], ferrocenic derivatives from cipro-
floxacin [80], or Ru, such as ruthenoquine and analogs [42].
For the palladium(II) compound 5c, the rate of migration of super-
coiled band also decreases as drug concentration increases up to 10 μM.
At higher concentrations the migration rate begins to increase again in
parallel to a decrease in the bands intensity. At higher concentrations of
5c (more than 100 μM), DNA is no longer visible.
Regarding the other assayed complexes (Fig. 4), at 50 μM concentra-
tion, small changes on the migration rate of the supercoiled closed circu-
lar plasmid DNA were observed for most of them, with the exception of
the free ligand 1c and the platinum(II) complex 4c. When assayed at
higher concentrations (500 μM), the synthesized compounds dramati-
cally altered plasmid DNA mobility (data not shown). Again, compounds
1c and 4c did not produce any effect. Paradoxically, as it was mentioned
before, complex 4c exhibited a great cytotoxicity in the three selected
human cancer cell lines.
Overall, these results indicated that most of the compounds investi-
gated in this study interact with DNA, 2a and 5c exhibiting the highest
effect on plasmid DNA mobility. These two compounds may behave as
alkylating agents acting by the same mechanism as cisplatin. However,
compound 4c is hypothesized to act on tumor cells through a different
mechanism than cisplatin.
We also evaluated the potential activities of products 1–5 against the
chloroquine-susceptible strain (3D7) and the chloroquine-resistant
strain (W2) of P. falciparum (Table 3). The free ligands exhibited poor
(for 1a–1b) to moderate (for 1c) antimalarial activities. In contrast with
these results, the platinum(II) complexes (2a–2c) with a (N,N′)Cl2 envi-
ronment demonstrated higher in vitro activity against P. falciparum
chloroquine-susceptible and chloroquine-resistant clones than their
corresponding parent ligand. The toxicity of platinacycle 4c was low
on P. falciparum parasites (IC50 of 111.7 μM on 3D7 and 138.0 μM on
4. Conclusions
The study of the reactivity of the three pyrazolyl ligands [1-
(CH2)2NMe2]-3,5-R2-pzol] {with R_H (1a), Me (1b) or Ph (1c)} with
[MCl2(DMSO)2] (M_Pd or Pt) under different experimental conditions
reveals that they may act as a bidentate (N,N′) (in 2a–2c and 3a–3c) or
as terdentate (C,N,N′)− ligand (in 4c and 5c). It is noteworthy that 4c
and 5c are the first examples of platina- and palladacycles with (C,N-
pyrazole,N′)− pincer ligands reported so far. The in vitro antimalarial activity
was evaluated against one chloroquine susceptible strain 3D7 and three
chloroquine-resistant strains W2 of P. falciparum. Compounds 2a–2c
and 5c were found to exhibit significant in vitro activity (with IC50 in
the microM range). On the contrary, compound 4c was inactive in similar
experimental conditions. The evaluation of the in vitro cytotoxic activity
of complexes 2c–5c revealed that they exhibit growth inhibitory activity
against lung (A549) and breast (MDA MB 231 and MCF7) human cancer
cell lines. The comparison of the results obtained for the three types of
complexes in human carcinoma A549 cell line indicates that the obtained
IC50 values follow the trend 2ab4cbcisplatinb2bb2cb5cb3bb3a. This
means that: a) the platinum(II) derivatives are more potent than their
palladium(II) analogs, b) for the platinum(II) complexes the change of
binding mode of the ligand from (N,N′) in 2c to (C,N,N′) in 4c increases
the cytotoxic activity, and c) compound 2a is specially relevant due to re-
markable potency in lung cancer A549 cell line (twice that of 4c and three
times more potent than cisplatin). It is noteworthy that complex 4c, the
first example of a pyrazole containing platinacycle, shows greater antitu-
moral in vitro activity than cisplatin. The platinum and palladium com-
plexes evaluated in this study more or less exhibit an effect on DNA
electrophoretic mobility. Complexes 2a and 5c are those exhibiting the
strongest interaction with DNA, and both display moderate to good cyto-
toxic activities towards different cancer cell lines. In particular complex 2a
is the most potent of the new synthesized compounds in human lung car-
cinoma A549 cell line (IC50 value of 3 μM). An exception occurs in the case
of compound 4c that exhibits a considerable cytotoxicity in several cancer
cell lines but has no effect on plasmid DNA mobility. These results under-
score the importance of additional factors in predicting anticancer
activity.
Table 3
In vitro activity of ligands 1, compounds 2–5 and chloroquine against chloroquine sus-
ceptible P. falciparum 3D7 clone and the chloroquine resistant W2 clone. IC50 are
expressed as means of 3 to 4 experiments standard deviation.
IC50 values (μM)
3D7
W2
New Pt- and Pd-cyclometallated derivatives will be synthetized to
improve antimalarial activity. Finally it should be noted that the
methods and strategies described here, especially those concerning
complex 4c, constitute the first step toward the development of new
but closely related platinum(II) complexes with pincer (C,N;N′)− li-
gands derived from pyrazole. These compounds may exhibit improved
biological activities owing to the conversion of 4c into ionic prod-
ucts of the type [Pt(C,N,N′)(L)]X (L = neutral ligand and X =
monoanion), which may be more soluble in polar solvents, or [Pt
(C,N,N′)(CCPh)] that have an additional interest in view of their po-
tential luminescence. The rich chemistry of pyrazole ligands also
permits to introduce changes on the pendant arm of the nitrogen
N1 or on the pyrazole ring. These strategies open up a vast array of
possibilities.
A) Free ligands
1a
1b
1c
182.3 19.6
248.0 15.5
38.4 4.2
196.3 13.8
183.3 16.6
20.2 4.3
B) Platinum(II) complexes
2a
2b
2c
4c
18.9 1.2
18.3 0.7
13.8 4.5
111.7 18.0
22.5 0.8
24.9 2.4
16.1 1.1
138.0 6.2
B) Palladium(II) complexes
3a
3b
5c
133.3 11.6
132.0 10.1
10.3 5.5
111.0 10.6
85.8 3.1
19.2 0.8
0.56 0.09
Chloroquine
0.020 0.004

1728
J. Quirante et al. / Journal of Inorganic Biochemistry 105 (2011) 1720–1728
[39] P.K. Basu, A. Gonzalez, C. López, M. Font-Bardia, T. Calvet, Journal of Organometal-
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Université Lille Nord de France, and CNRS for financial support.
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