The thermal effects of platinum(II) and palladium(II) complexes with 2-acetyl pyridine and pyridine-2-carbaldehyde N(4)-ethyl-thiosemicarbazones in membrane bilayers

Article abstract of DOI:10.1016/j.tca.2004.05.031

Platinum(II) and palladium(II) complexes with 2-acetyl pyridine and pyridine-2-carbaldehyde N(4)-ethyl-thiosemicarbazones, HAc4Et and HFo4Et respectively were synthesized and found to exhibit a cytotoxic potency in a very low micromolar range and to be able to overcome the cisplatin resistance of A2780/Cp8 cells. The biologically active complexes Pd(Fo4Et)₂ (1), Pd(Ac4Et)₂ (2), Pt(Fo₄Et)₂ (3) and Pt(Ac4Et)₂ (4) were tested for their perturbation in model membrane bilayers. The aim was to investigate if there is a possible relation between their mechanism of action in membranes with their biological activity. Indeed, it was found that complexes of deprotonated HAc4Et, (2) and (4), are more perturbing than complexes of deprotonated HFo4Et, (1) and (3).

Full text of DOI:10.1016/j.tca.2004.05.031

Thermochimica Acta 424 (2004) 53–58
The thermal effects of platinum(II) and palladium(II) complexes with
2-acetyl pyridine and pyridine-2-carbaldehyde
N(4)-ethyl-thiosemicarbazones in membrane bilayers
D. Kovala-Demertzia, C. Vidjeluca, M.A. Demertzisa, E. Siapib, T. Mavromoustakosb,∗
a
Inorganic and Analytical Chemistry, Department of Chemistry, University of Ioannina, 45110 Ioannina, Greece
b
National Hellenic Research Foundation, Institute of Organic and Pharmaceutical Chemistry, Vas. Constantinou 48, 11635 Athens, Greece
Received 16 January 2004; received in revised form 21 May 2004; accepted 26 May 2004
Available online 2 July 2004
Abstract
Platinum(II) and palladium(II) complexes with 2-acetyl pyridine and pyridine-2-carbaldehyde N(4)-ethyl-thiosemicarbazones, HAc4Et and
HFo4Et respectively were synthesized and found to exhibit a cytotoxic potency in a very low micromolar range and to be able to overcome
the cisplatin resistance of A2780/Cp8 cells. The biologically active complexes Pd(Fo4Et)2 (1), Pd(Ac4Et)2 (2), Pt(Fo4Et)2 (3) and Pt(Ac4Et)2
(4) were tested for their perturbation in model membrane bilayers. The aim was to investigate if there is a possible relation between their
mechanism of action in membranes with their biological activity. Indeed, it was found that complexes of deprotonated HAc4Et, (2) and (4),
are more perturbing than complexes of deprotonated HFo4Et, (1) and (3).
© 2004 Elsevier B.V. All rights reserved.
Keywords: Platinum(II) and palladium(II) complexes; DSC; X-ray structure
1. Introduction
ical ones [1–5]. Dipalmitoylphosphatidylcholine (DPPC)
bilayers spontaneously form lipid bilayers in aqueous en-
vironments and have been utilized extensively to study
drug:membrane interactions. These phospholipid bilayers
are convenient to use because they undergo phase transi-
tions close to ambient temperatures [6]. For some classes
of pharmacologically important molecules (i.e. anesthetic
steroids flavonoids, anti-inflammatory etc.) a relationship
has been revealed between the degree these drugs affect the
thermodynamic parameters described above (perturbation)
and pharmacological activity [7–10].
Differential scanning calorimetry is a fast and relatively
inexpensive technique that allows the study of the ther-
motropic properties of the membranes in the absence and
presence of bioactive molecules.
The presence of an additive in membrane bilayers affects
the thermodynamic parameters that govern a thermogram
such as the maximum of the main-phase transition or the
pre-transition (Tm), the heat capacity of the peaks (Cp) and
the line-width (Tm ). The nature of the DSC thermograms
1/2
can be understood if the total intermolecular effects (i.e.
interfacial, hydrogen bonds and nonspecific hydrophobic
and electrostatic interactions) between the additive and the
phospholipid bilayers are considered.
Phospholipid bilayers are widely used as model mem-
branes to study drug:membrane interactions since they have
found to have similar dynamic properties as the biolog-
Thiosemicarbazones (tsc’s) have aroused considerable
interest in chemistry and biology due to their antibacterial,
antimalarial, antineoplastic and antiviral activities and are
among the most potent inhibitors of ribonucleotide reduc-
tase (RR). The group of compounds catalyses the synthesis
of deoxyribonucleotides from their ribonucleotide precur-
sors and as such is responsible for maintaining a balanced
supply of the deoxyribonucleotides required for DNA syn-
thesis and repair. Strong positive correlation has been es-
tablished between RR activity and the rate of replication of
cancer cells [11]. The chemistry of transition metal com-
plexes of tsc’s has been receiving considerable attention
∗
Corresponding author. Tel.: +30 210 7273869;
fax: +30 210 7273831.
E-mail addresses: dkovala@cc.uoi.gr (D. Kovala-Demertzi),
tmavro@eie.gr (T. Mavromoustakos).
0040-6031/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.tca.2004.05.031

54
D. Kovala-Demertzi et al. / Thermochimica Acta 424 (2004) 53–58
largely because of their pharmacological properties [12].
The combination of tsc’s with agents like platinum(II) or
palladium(II) that damage DNA produces synergistic in-
hibition of tumor growth and may lead to improvements
in the effectiveness of cancer chemotherapy [13–15]. The
complexes of palladium(II) with 2-acetylpyridine 4N-ethyl
thiosemicarbazone, HAc4Et, were investigated against
Leukemia P388 [16]. A high correlation between potency
for Sister Chromatid Exchange, (SCE) induction, effective-
ness in cell division delay (P < 0.01) in normal human
lymphocytes in vitro and in vivo established antitumour
activity in P388 leukemia bearing mice was found. All
the complexes of palladium(II) were less cytotoxic and
almost, all were found more effective than the parent lig-
and, HAc4Et, acting synergistically [16]. The complexes of
platinum(II), with HAc4Et were found to exhibit a cyto-
toxic potency in a very low micromolar range, and are able
to overcome the cisplatin resistance of A2780/Cp8 cells;
these cells are characterized by a marked intracellular glu-
tathione content and a reduced cisplatin uptake with respect
to the parental A2780 cells [17]. These complexes may be
endowed with important anticancer properties since they
elicit IC50 values in the ␮M range as does the clinically
used drug cis-DDP (cis-diamminedichloro-platinum(II)),
and, moreover, they display cytotoxic activity in tumor
lines resistant to cis-DDP. cis-DDP has for a long time
been of major significance in cancer therapy. There are
two major limitations to cis-DDP therapy; the toxic side
effects and the acquired resistance [18]. The goal of re-
ducing toxic side effects, while maintaining therapeutical
efficacy, can be accomplished by improving the solubility
of the complexes, by slowing down degradation processes
through shielding of the platinum with bulky ligands, and
by increasing membrane permeability with more lipophilic
ligands.
2.2. Preparation of the complexes
Solvents were purified and dried according to stan-
dard procedures. The heterocyclic thiosemicarbazones,
pyridine-2-carbaldeheyde HFo4Et, HAc4Et, 4N-ethyl
thiosemicarbazone, 2-acetyl pyridine 4N-ethyl thiosemicar-
bazone, were prepared as described by Klayman [19].
• 1 [Pd(Fo4Et)2]. To a solution of HFo4Et (3.2 mmol)
in methanol (10 mL) was added a solution of K2PdCl4
(1.5 mmol) in distilled water (10 mL). The pH of the so-
lution was adjusted to 8.0–9.0 by the addition of aqueous
1.0 M NH3 and the reaction mixture was stirred for 5 h
at room temperature at constant pH. The powder was fil-
tered off, washed with cold methanol and ether and dried
in vacuo over silica gel, finally redried at 70 ◦C in vacuo
over P4O10 d.p. 165–166 ◦C, yield 50%. IR: v = 3444
br [v(OH)], 3284 and 3121 [v(NH)], 1587, 1555sh, 1539
=
=
[ν(C N)], 769 749 [ν(C S−)]1, 741 [ν(C–S)], 437 and
418 [ν(Pd–N)], 389, 375 cm [ν(Pd–S)]; UV-Vis for 1
(DMF) λ/nm (ε/L mol−1 cm−1) 479 (1650), 380 (8730)
and 324 (68000). Elemental analyses are consistent with
C18H22N8S2Pd (Found: C, 41.8; H, 4.3; N, 21.3; S, 12.1;.
Calcd: C, 41.5; H, 4.2; N, 21.5; S, 12.3 %).
• 2 [Pd(Ac4Et)2] was prepared according to published
procedure by reaction of HAc4Et and K2PdCl4 [20].
D.p. 192–193 ◦C. Elemental analyses were consistent
with the stoichiometry with C20H26N8S2Pd (Found: C,
44.1; H, 4.3; N, 20.3; S, 12.0;. Calcd: C, 43.8; H, 4.8; N,
20.4; S, 11.7 %).
• 3 [Pt(Fo4Et)2] and 4 [Pt(Ac4Et)2] were prepared accord-
ing to published procedure by reaction of HAc4Et and
[PtCl4]2− [17]. D.p. 182 and 185 ◦C for 3 and 4 respec-
tively. Elemental analyses were consistent with the sto-
ichiometry C18H22N8S2Pt (Found: C, 35.6; H, 4.0; N,
18.7; S, 10.6; Pt; 31.5. Calcd: C, 35.3; H, 3.6; N, 18.3; S,
10.5; Pt, 31.8%) and C20H26N8S2Pt (Found: C, 37.4; H,
4.5; N, 17.5; S, 9.8; Pt; 30.3. Calcd: C, 37.7; H, 4.11; N,
17.6; S, 10.0; Pt, 30.4%) for 3 and 4 respectively.
The aim of this study was to investigate any existing
relationship between the biological activity of the com-
plexes Pd(Fo4Et)2 (1), Pd(Ac4Et)2 (2), Pt(Fo4Et)2 (3) and
Pt(Ac4Et)2 (4) and their thermal effects related to mem-
brane perturbation by DSC.
2.3. Differential scanning calorimetry
Appropriate amounts of the phospholipid with HAc4Et
or HFo4Et or their complexes with Pd or Pt were dissolved
in spectroscopic grade chloroform. The solvent was then
evaporated by passing a stream of O2-free nitrogen over
the solution at 50 ◦C and the residue was placed under
vacuum (0.1 mmHg) for 12 h. For measurements this dry
residue was dispersed in appropriate amounts of bidistilled
water by vortexing. After dispersion in water (50% w/w),
portions of the samples (ca. 5 mg) were sealed in stainless
steel capsules (7.54 mm diameter and 2.79 mm height) ob-
tained from Perkin-Elmer. Thermograms were obtained on
a Perkin-Elmer DSC 7 calorimeter. Prior to scanning, the
samples were held above their phase transition tempera-
ture for 1–2 min to ensure equilibration. All samples were
2. Materials and methods
2.1. Materials
Dipalmitoyl-glycero-sn-3-phosphatidylcholine (DPPC)
was obtained from Avanti Polar Lipids Inc., AL, USA. Sol-
vents were purified and dried according to standard proce-
dures. For the platinum(II) compounds a stock [PtCl4]2− so-
lution was prepared by dissolving of PtCl2 (2.66 g, 10 mmol)
in conc. HCl under reflux, filtering to remove a turbidity
of undissolved material, neutralizing with Na2CO3 and di-
luting with distilled water up to 250 ml (pH = 6.0–6.5) to
yield a solution of 0.04 M [PtCl4]2−
.

D. Kovala-Demertzi et al. / Thermochimica Acta 424 (2004) 53–58
55
scanned with a scanning rate of 2.5 ◦C/min. The tempera-
ture scale of the calorimeter was calibrated using indium
(Tm = 156.6 ◦C) as standard sample.
3. Results and discussion
3.1. Preparation of complexes
The complexes of Pd(II) and Pt(II) were prepared from
the reaction of ligands, HFo4Et, HAc4Et (Fig. 1) and the
appropriate metal salt in aqueous methanol solutions in basic
solution, pH 8–9 and molar ratio 1:2, and according to the
reactions (1)–(2)
[MCl4]2− + 2HFo4Et + 2NH4OH
Fig. 2. Structural representation of 4 [22].
C−−H−3O−H−/−H→2O M(Fo4Et)2 + 2Cl− + 2NH4Cl + 2H2O
with (N,N,S) donation, the other being monodentate using
only the sulfur atom to coordinate to the metal. It might
be possible that there is a weak ‘agostic’ bond between
Pt and the amido-nitrogen, and its attached hydrogen, the
metal-hydrogen distance being 2.755 Å. The crystal packing
is determined by ␲–␲, and Pt–C interactions.
(1)
[MCl4]2− + 2HAc4Et + 2NH4OH
C−−H−3O−H−/−H→2O M(Ac4Et)2 + 2Cl− + 2NH4Cl + 2H2O
(2)
where M = Pt(II) and Pd(II).
3.2. Differential scanning calorimetry
The stoichiometry of the complexes indicates that Pd(II)
and Pt(II) are connected with the deprotonated form (an-
ion Fo4Et− and Ac4Et−) in the complexes [M(L)2]. The
complexes are moderately soluble in H2O and alcohols and
more soluble in polar solvents, such as DMF and DMSO.
The structure of the complex 4 has been solved [22] (Fig. 2).
The platinum atom is in a square planar environment sur-
rounded by two cis nitrogen atoms and two cis sulfur
atoms. The ligands are not equivalent, one being tridentate
Fully hydrated DPPC bilayers show a characteristic ther-
mogram consisting of a broad low enthalpy transition at
35.3 ◦C and a sharp enthalpy main transition at 41.2 ◦C.
The DPPC bilayer exists in the gel phase (L␤ꢀ ) for temper-
atures lower than 33 ◦C, and in the liquid crystalline phase
for temperatures higher than 42 ◦C (L␣ꢀ ). In between 33
and 42 ◦C the phospholipid bilayer exists in P␤ꢀ or ripple
phase [5,6,23]. The obtained DSC scan of fully hydrated
Fig. 1. (a) The fully PM3 optimized geometry of the neutral HFo4Et (ꢀHf = 102.2 kcal mol−1; dipole moment = 6.85 Debye) in the gas state. (b) The
fully PM3 optimized geometry of the neutral Hac4Et (ꢀHf = 90.0 kcal mol−1; dipole moment = 7.68 Debye) in the gas state (Hyperchem 6.0) [21].

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D. Kovala-Demertzi et al. / Thermochimica Acta 424 (2004) 53–58
Table 1
Diagnostic DSC parameters of the free ligands HAc4Et and HFo4Et or
in a complex form with Pt or Pd in DPPC bilayers
Sample
ꢀH (kcal/mol)
Tm
Tm
1/2
DPPC
7.6
7.3
7.3
7.1
7.1
7.5
7.4
7.4
7.3
7.6
7.5
7.5
7.4
7.4
7.8
7.7
8.0
7.6
7.8
8.0
7.5
7.4
7.5
7.6
7.8
1.2
0.9
1.3
1.2
1.6
1.3
1.3
1.4
1.2
1.3
1.4
1.8
6.6
1.2
1.2
1.5
1.6
1.1
2.8
5.4
6.6
1.2
1.3
1.6
1.8
41.2
41.0
40.8
40.8
40.4
41.1
40.7
40.9
41.0
41.1
40.8
40.7
40.4
41.0
40.2
39.3
39.9
39.0
37.4
39.8
40.6
41.3
40.4
39.8
39.9
DPPC + x = 0.01 HAc4Et
DPPC + x = 0.05 HAc4Et
DPPC + x = 0.1 HAc4Et
DPPC + x = 0.2 HAc4Et
DPPC + x = 0.01 HFo4Et
DPPC + x = 0.05 HFo4Et
DPPC + x = 0.1 HFo4Et
DPPC + x = 0.2 HFo4Et
DPPC + x = 0.01 Pd Pd(Ac4Et)2
DPPC + x = 0.05 Pd Pd(Ac4Et)2
DPPC + x = 0.1 Pd(Ac4Et)2
DPPC + x = 0.2 Pd(Ac4Et)2
DPPC + x = 0.01 Pd(HFo4Et)2
DPPC + x = 0.05 Pd(HFo4Et)2
DPPC + x = 0.1 Pd(HFo4Et)2
DPPC + x = 0.2 Pd(HFo4Et)2
DPPC + x = 0.01 Pt(Ac4Et)2
DPPC + x = 0.05 Pt(Ac4Et)2
DPPC + x = 0.1 Pt(Ac4Et)2
DPPC + x = 0.2 Pt(Ac4Et)2
DPPC + x = 0.01 Pt(HFo4Et)2
DPPC + x = 0.05 Pt(HFo4Et)2
DPPC + x = 0.1 Pt(HFo4Et)2
DPPC + x = 0.2 Pt(HFo4Et)2
Fig. 3. DSC scan of DPPC bilayers containing different concentrations
of the ligands HFo4Et (left) and HAc4Et (right).
DPPC multibilayers shows a pre-transition centered at
35 ◦C and a peak maximum at 41.2 ◦C. The main-phase
transition is accompanied by several structural changes
in the lipid molecules as well as systematic alterations
in the bilayer geometry, but the most prominent feature
is the trans-gauche isomerization-taking place in the acyl
chain conformation. The average number of gauche con-
formers indicates the effective fluidity, which depends not
only on the temperature, but also on perturbations due to
the presence of a drug molecule intercalating between the
lipids.
most obliterated it (Fig. 3). The lowering of the main-phase
transition although not very significant (<1 ◦C) it paralleled
with increasing concentration of HAc4Et. ꢀH remained al-
most constant in all preparations.
3.2.3. Complexation of HFo4Et with Pd
When HFo4Et was complexed with Pd (Pd(HFo4Et)2)
and was incorporated in DPPC bilayers its effects were aug-
mented (Fig. 4). At x = 0.01 the complex affected the
DPPC bilayers as HFo4Et. At higher concentration, how-
ever, the presence of complex in DPPC bilayers resulted in
the obliteration of the pre-transition. The obliteration of the
pre-transition was obvious at concentrations with x = 0.05.
Tm and ꢀH were remained almost constant in all prepara-
tions (see Table 1).
3.2.1. Incorporation of HFo4Et in DPPC bilayers
The presence of HFo4Et in DPPC bilayers at low concen-
tration of 1:99 molar ratio (x = 0.01) affects only marginally
the thermal scan by broadening the pre-transition (Fig. 3).
At higher concentration of 5:95 molar ratio (x = 0.05)
it further causes broadening of the pre-transition. At x =
0.10 and 0.20 the effect is surprisingly depressed. Thus,
the pre-transition is shifted towards higher temperatures. Tm
and ꢀH were not affected significantly by the presence of
HFo4Et (see Table 1).
3.2.4. Complexation of HAc4Et with Pd
Similarly, when HAc4Et was complexed with Pd
(Pd(Ac4Et)2) and was incorporated in DPPC bilayers its
effects were augmented (Fig. 4). Already, from x = 0.01
the complex almost caused abolish of the pre-transition
while when was not in complex form only broadened it
slightly. When x = 0.01 was used pre-transition was almost
barely observed and the main-phase transition was signifi-
cantly broadened. It is evident from these results that com-
plexation of ligands HAc4Et and HFo4Et with palladium
resulted in the enhancement of the membrane perturbing
effects.
3.2.2. Incorporation of HAc4Et in DPPC bilayers
In contrast to HFo4Et, increase of the HAc4Et concentra-
tion resulted in a progressive effect. More particularly, at x =
0.01 HAc4Et caused slight broadening of the pre-transition,
at x = 0.05 caused further broadening and at x = 0.20 al-

D. Kovala-Demertzi et al. / Thermochimica Acta 424 (2004) 53–58
57
concentration of x = 0.01 caused a significant broadening
of the pre-transition while at higher concentrations caused
abolishment of it and broadening of the main-phase transi-
tion.
3.2.6. Complexation of HAc4Et with Pt
HAc4Et when complexed with Pt (Pt(Ac4Et)2) exerted
more significant effects than when it is complexed with Pd.
The presence of x = 0.01 Pt(Ac4Et)2 caused abolishment
of the pre-transition. At higher concentration of x = 0.05
the incorporation of the complex resulted in significant
broadening of the main-phase transition. At x = 0.1 the
observed peak was significantly broadened consisting of
different components. The same applies when x = 0.2 is
used. The quality of these thermal scans may be interpreted
as a reversible transition from a vesicular suspension to an
extended peak bilayer network. In particular, Schneider and
his collaborators published an article in which they combine
calorimetry, viscosity and electron microscopy methods
to explain similar thermal profile [24,25]. They state that
these structural transitions arise from two effects: (i) the
enhanced membrane elasticity accompanying the lipid state
fluctuations on chain melting and (ii) solvent-associated
interactions (including electrostatics) that favor a change
in membrane curvature. Other authors explain similar
thermal behavior of bioactive molecules as the “inherent
inhomogeneity” of the membrane bilayer. Thus, this prepa-
ration may contain domains consisting mainly of pure
DPPC bilayers and other domains rich with the ligand
[26,27].
Fig. 4. DSC scan of DPPC bilayers containing different concentrations
of the complexes Pd(Ac4Et)2 (left) and Pd(HFo4Et)2 (right).
3.2.5. Complexation of HFo4Et with Pt
HFo4Et when complexed with Pt (Pt(HFo4Et)2) exerted
similar effects as with Pd (Fig. 5). More specifically, at the
4. Conclusions
HAc4Et is a more perturbing agent than HFo4Et as it
is evident that causes the most significant broadening of
the pre- and main-phase transitions and lowering of the
main-phase transition temperature at identical concentra-
tions. When both ligands are complexed they augment their
perturbing effects in membrane bilayers. Overall, depro-
tonated HAc4Et complexes Pd(Ac4Et)2 and Pt(Ac4Et)2
are more perturbing than complexes Pd(HFo4Et)2 and
Pt(HFo4Et)2. Pt(Ac4Et)2 is the most perturbing complex
observed.
The different perturbation observed between the drugs un-
der study may related to their possible differences in drug de-
livery and consequently in part explains their distinct bioac-
tivity.
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