Cyclopalladated benzophenone imines: Synthesis, cytotoxicity against human breast adenocarcinoma cell lines and DNA interaction

Article abstract of DOI:10.1016/j.jorganchem.2012.11.034

Treatment of benzophenone imine with the stoichiometric amount of Pd(OAc)₂ in acetic acid at 60 °C produced the corresponding acetato-bridged five-membered ortho-cyclopalladated dimer [Pd{C ₆H₄CPhNH}(μ-OAc)]₂ (1), which was isolated in pure form in a 79% yield. Reaction of 1 with an excess of LiCl in acetone gave rise to the corresponding chlorido-bridged cyclopalladated dimer [Pd{C ₆H₄CPhNH}(μ-Cl)]₂ (2) in a 78% yield. Compounds 1-2 reacted with an excess of py-d₅ or the stoichiometric amount of PPh₃ to give the mononuclear compounds trans-N,L-[Pd{C ₆H₄CPhNH}(X)(L)] [3 (X = OAc, L = py-d₅); 4 (X = Cl, L = py-d₅); 5 (X = OAc, L = PPh₃) and 6 (X = Cl, L = PPh₃)]. Compounds 2-3 were prepared in CDCl₃/py-d ₅ solution and were studied by ¹H NMR, but were not isolated. In contrast, compounds 5-6 were prepared in acetone and were isolated in pure form in 43 and 79% yields, respectively. Compounds 1, 2, 5 and 6 were characterized by elemental analyses, mass spectrometry, IR, NMR and electronic spectroscopy. Compounds 1, 2, 5 and 6 showed high antiproliferative activity against MDA-MB231 and MCF7 human breast cancer cell lines, especially, compounds 5-6. These two latter compounds presented greater antiproliferative activity than cisplatin and produced IC₅₀ values in the range 1-5 μM. The interaction of compounds 1, 2, 5 and 6 with DNA was also studied by the DNA electrophoretic migration, DNA-ethidium bromide fluorescence quenching and viscometry techniques.

Full text of DOI:10.1016/j.jorganchem.2012.11.034

Journal of Organometallic Chemistry 724 (2013) 289e296
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Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
Cyclopalladated benzophenone imines: Synthesis, cytotoxicity against human
breast adenocarcinoma cell lines and DNA interaction
,
e
,
a
a,
Joan Albert a , Silvia García , Jaume Granell e, Albert Llorca a, María Victoria Lovelle a,
*
Virtudes Moreno a, Andreu Presa a, Laura Rodríguez a, Josefina Quirante b e, Carme Calvis c,
,
Ramon Messeguer c, Josefa Badía d e, Laura Baldomà d
,
,e
a Departament de Química Inorgànica, Facultat de Química, Universitat de Barcelona, Martí i Franquès 1-11, 08028 Barcelona, Spain
b Laboratori de Química Orgànica, Facultat de Farmàcia, Av. Joan XXIII, s/n, 08028 Barcelona, Spain
c Biomed Division LEITAT Technological Center, Parc Científic, Edifici Hèlix, C/ Baldiri Reixach, 15-21, 08028 Barcelona, Spain
d Departament de Bioquímica i Biología Molecular, Facultat de Farmàcia, Universitat de Barcelona, Av. Joan XXIII s/n, 08028 Barcelona, Spain
e Institut de Biomedicina (IBUB), Universitat de Barcelona, Barcelona, Spain
a r t i c l e i n f o
a b s t r a c t
Treatment of benzophenone imine with the stoichiometric amount of Pd(OAc)2 in acetic acid at 60 ꢀC
Article history:
Received 16 October 2012
Received in revised form
16 November 2012
produced the corresponding acetato-bridged five-membered ortho-cyclopalladated dimer [Pd
{C6H4CPh]NH}(
of LiCl in acetone gave rise to the corresponding chlorido-bridged cyclopalladated dimer [Pd{C6H4CPh]
NH}( -Cl)]2 (2) in a 78% yield. Compounds 1e2 reacted with an excess of py-d5 or the stoichiometric
m-OAc)]2 (1), which was isolated in pure form in a 79% yield. Reaction of 1 with an excess
Accepted 22 November 2012
m
amount of PPh3 to give the mononuclear compounds trans-N,L-[Pd{C6H4CPh]NH}(X)(L)] [3 (X ¼ OAc,
L ¼ py-d5); 4 (X ¼ Cl, L ¼ py-d5); 5 (X ¼ OAc, L ¼ PPh3) and 6 (X ¼ Cl, L ¼ PPh3)]. Compounds 2e3 were
prepared in CDCl3/py-d5 solution and were studied by 1H NMR, but were not isolated. In contrast,
compounds 5e6 were prepared in acetone and were isolated in pure form in 43 and 79% yields,
respectively. Compounds 1, 2, 5 and 6 were characterized by elemental analyses, mass spectrometry, IR,
NMR and electronic spectroscopy. Compounds 1, 2, 5 and 6 showed high antiproliferative activity against
MDA-MB231 and MCF7 human breast cancer cell lines, especially, compounds 5e6. These two latter
compounds presented greater antiproliferative activity than cisplatin and produced IC50 values in the
Keywords:
Cyclopalladated
Imine
Anticancer
Activity
DNA
Interaction
range 1e5
mM. The interaction of compounds 1, 2, 5 and 6 with DNA was also studied by the DNA
electrophoretic migration, DNA-ethidium bromide fluorescence quenching and viscometry techniques.
Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction
It has been reported that the organopalladium(II) compounds
AeF shown in Fig. 1, either alone or combined with other therapies,
Despite the tremendous success of cisplatin e cis-[PtCl2(NH3)2]
e as an anticancer drug, this compound suffers from two main
disadvantages; it is inefficient against cisplatin-resistant tumours,
and it has severe side effects such as nephrotoxicity [1]. Therefore,
the modification of cisplatin or the development of new anticancer
drugs based on other transition metal compounds other than those
of platinum(II) are themes that arouse great interest in medicinal
chemistry [2].
give positive results for the treatment of primary and metastatic
cancers in animal models [3]. Organopalladium(II) compounds
with a structural formula related to that of compounds A, D, E and F
are under a WO patent because of their possible role as active
inhibitors of enzymes, such Catepsin B, and their ability to modu-
late the immunological system due to their action on these
enzymes and their interaction with DNA [4]. These findings [3,4]
add to a few more [5], which point to the use of cyclopalladated
compounds as alternative chemotherapeutic drugs for cisplatin in
view of their in vitro cytotoxicity against different cancer cell lines.
The cell killing mechanism of the diphosphane-organo-
dipalladium(II) compound A has been investigated [3a,5f,m].
These studies showed that this compound interacted with thiol
groups from proteins of the mitochondrial membrane. This
* Corresponding author. Departament de Química Inorgànica, Facultat de Quí-
mica, Universitat de Barcelona, Martí i Franquès 1-11, 08028 Barcelona, Spain.
Tel.: þ34 93 4039131; fax: þ34 93 4907725.
E-mail address: joan.albert@qi.ub.es (J. Albert).
0022-328X/$ e see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jorganchem.2012.11.034

290
J. Albert et al. / Journal of Organometallic Chemistry 724 (2013) 289e296
Fig. 1. Organopalladium(II) compounds used as chemotherapeutic drugs with positive results for the treatment of different cancers in animal models.
interaction caused dissipation of the mitochondrial membrane
potential and Bax translocation from the cytosol to the mitochon-
dria. Compound A also produced an increase in cytosolic calcium
concentration, a decrease in ATP levels, and the activation of cas-
pases, which induced chromatin condensation and DNA degrada-
tion. These results suggested that compound A induced the
activation of the apoptotic intrinsic pathway in murine melanoma
B16F10-Nex2 cells. Interestingly, the cell killing mechanism of
organopalladium(II) compounds may be highly dependent on their
structure [3f].
should be noted that no previous studies on the cytotoxic activity of
simple cyclopalladated imines such as those shown in Fig. 2 or in
Scheme 1 have been reported. Cyclopalladated a-benzoyl-N-ben-
zylideneamines are the compounds most closelystructurally related
with this work for which the antiproliferative activity has been
studied [5p,tev].
Finally, it should be noted that the cyclopalladation of a few
benzophenone imines derived from aniline, benzylamine, cyclo-
hexylamine, 3,3-bis(isopropylthio)prop-2-en-1-amine,
acid and peptide esters has been reported [9f,h,14].
a-amino-
In our ongoing work on the cyclopalladation of imines [6], we
realized that the cyclopalladation reaction of benzophenone imine
(Ph2CH]NH) had not been reported even though it is a relatively
non-expensive commercial chemical. Its cyclometallation with
platinum(II) compounds has recently been described and its
cyclometallated platinum(II) derivatives were found to be lumi-
nescent in solution and in solid state at room temperature [7].
Fe(II), Ir(III), Rh(III), Ru(II), Os(II) and Os(IV) metallic centres have
also been bonded to benzophenone imine by cyclo-ortho-metal-
lation reactions [8].
The precedent studies [3e14] prompted us to investigate: i) the
preparation and characterization of several new cyclopalladated
compounds derived from benzophenone imine (Ph2C]NH),
including their photophysical properties, ii) their cytotoxicity
towards MDA-MB231 and MCF7 human breast adenocarcinoma
The cyclopalladation reaction of N-benzylideneamines of
formula AreCH]NeR (R ¼ aryl, benzyl, .) has been extensively
studied [9] and their cyclopalladated derivatives have found appli-
cation in organic synthesis [10] and as precatalysts for Heck and
Suzuki reactions [11]. Furthermore, some cyclopalladated N-ben-
zylideneamines with adequate substituents at the imine ligand and
coligands at the palladium atom form mesophases [12] or present
non-linear optical properties [13]. Fig. 2 shows the most usual
structural formulae for cyclopalladated N-benzylideneamines. It
Scheme 1. i) Pd(OAc)2 (stoichiometric), HOAc, 60 ꢀC, 24 h; ii) LiCl (excess), acetone, r.t.,
2 h; iii) 3 and 4: py-d5 (excess), CDCl3, r.t.; 5 and 6: PPh3 (stoichiometric), acetone, r.t.,
Fig. 2. Most usual structural formulae of cyclopalladated N-benzylideneamines.
2 h.

J. Albert et al. / Journal of Organometallic Chemistry 724 (2013) 289e296
291
cell lines and iii) their interaction with DNA using the DNA elec-
trophoretic migration, DNA-ethidium bromide fluorescence
quenching and viscometry techniques.
proton appeared as a broad singlet centred at 8.38 ppm and the
aromatic protons produced three groups of signals in the interval
7.61e7.02 ppm integrating six, one and two protons respectively.
Interestingly, the aromatic protons of compound 1 appeared in
the interval 7.41e6.70 ppm at lower frequencies than those of
compound 2. This shift to lower frequencies of the aromatic
protons of compound 1 in relation to compound 2 indicates
2. Results and discussion
2.1. Organopalladium compounds: synthesis and characterization
a
quite important change in the structure on going from
Scheme 1 shows the structural formula of the new compounds
prepared in this study and the numbering of their protons for the
discussion that follows.
compound 1 to compound 2. Overall, comparison of the 1H NMR
data of compounds 1 and 2 suggests that compound 1 adopted
a trans-folded (open book) structure and compounds 2 a trans-
planar structure (Fig. 3). Most of the acetato and chlorido bridged
cyclopalladated dimers adopt these structures in CDCl3 solution
and in the solid state [16].
Compounds 1e2 reacted with an excess of py-d5 or a stoichio-
metric amount of PPh3 giving the mononuclear compounds 3e6.
Compounds 3e4 were prepared in CDCl3/py-d5 solution and
studied by 1H NMR, but were not isolated. The 1H NMR of the
solution resulting from the reaction of dinuclear cyclopalladated
compounds with acetato or halogeno bridging ligands with py-d5 in
CDCl3 is a fast test for determining their cyclopalladated structure
[6a]. Compounds 3e4 were highly soluble in CDCl3/py-d5.
Compounds 5e6 were prepared in acetone and isolated in pure
form as pale yellow solids in 43% and 79% yields, respectively, and
were quite soluble in CDCl3. These latter CDCl3 solutions and those
of compounds 3e4 in CDCl3/py-d5 were stable on contact with air.
Compounds 5e6 produced satisfactory elemental analysis, mass
spectra, IR and 1H and 31P{1H} NMR.
The LDI-TOF (þ) mass spectrum for compound 5 produced
intense peaks corresponding to [M þ Na]þ and [M ꢁ OAc]þ and the
MALDI-TOF(þ) of compound 6 produced an intense signal at
[M ꢁ Cl]þ in accordance with their proposed structures [6a]. For
compound 5 the NeH stretching appeared at 3319 cmꢁ1, and the
asymmetric and symmetric stretching of the carboxylate function
of the acetato ligand produced intense signals at 1574 and
1374 cmꢁ1, in agreement with its unidentate coordination mode
[15]. The C]N stretching of compound 5 was not observed since it
was occluded inside the broad and strong band corresponding to
the asymmetric stretching of the carboxylate function of 5. For
compound 6, the NeH and C]N stretching produced sharp signals
Treatment of Ph2CH]NH (compound a in Scheme 1) with the
stoichiometric amount of Pd(OAc)2 in acetic acid at 60 ꢀC produced
the corresponding acetato-bridged five-membered ortho-cyclo-
palladated dimer 1. This compound was easily converted by
a metathesis reaction with LiCl into the chlorido-bridged cyclo-
palladated dimer 2 (Scheme 1). Compounds 1e2 were isolated in
pure form in 79% and 78% yields respectively. The details of the
preparation and isolation of these compounds are given in the
Experimental section. Compound 1 was a deep yellow solid which
was highly soluble in CDCl3 and compound 2 was a pale yellow
solid which was moderately soluble in CDCl3. The CDCl3 solutions of
compounds 1e2 were stable on contact with air. Compounds 1e2
produced satisfactory elemental analyses, ESI-(þ) mass spectrum,
IR and 1H NMR spectra.
The ESI-(þ) mass spectrum produced intense peaks for the
cations [(M/2) þ Na]þ for compound 1 and [M ꢁ Cl]þ and [(M/2)e
Cl þ CH3CN]þ for compound 2. These results were in agreement
with their proposed dinuclear structure with acetato- and chlorido-
bridged ligands respectively [6a].
The asymmetric and symmetric stretching of the carboxylate
functions of 1 produced broad intense bands at 1565 and 1431 cmꢁ1
respectively, indicating that the acetato ligands of compound 1
presented a bridging coordination mode [15]. The NeH st and the
C]N stretching of compound 1 were not observed since they were
occluded inside the broad and strong signals corresponding to the
water OeH stretching and to the asymmetric stretching of the
carboxylate functions of 1 respectively. For compound 2, the NeH
and the C]N stretching were assigned to sharp signals of
medium intensity at 3309 and 1584 cmꢁ1, and were shifted 43 and
26 cmꢁ1, respectively, to lower wave numbers in relation to the Ne
H and C]N stretching of free benzophenone imine. The 26 cmꢁ1
shift of the C]N stretching of compound 2 to lower wave numbers
in relation to free benzophenone imine was an indication of its k1-N
coordination to the palladium(II) centre [9h].
The principal feature of the 1H NMR at 400 MHz of compound 1
in CDCl3 solution was the clear separation between non-palladated
and ortho-palladated phenyl proton signals. Thus, the non-
palladated phenyl produced a triplet of triplets for proton 7 (the
para proton) at 7.86 ppm, a broad triplet for protons 8 and 6 (the
meta protons) at 7.24 ppm, and a doublet of doublets for protons 9
and 5 (the ortho protons) at 6.83 ppm. The protons of the ortho-
palladated phenyl appeared in the interval 7.21 and 6.83 ppm and
afforded the expected multiplicity pattern for a 1,2-disubstituted
phenyl ring. Thus, protons 1 and 4 afforded doublets of doublets
at 7.21 and 6.83 ppm, while protons 2 and 3 afforded triplets of
doublets at 7.11 and 6.88 ppm. In addition, the protons of the ace-
tato ligands of 1 produced a singlet at 2.22 ppm. This result indi-
cated that the dinuclear compound 1 with acetato ligands bridging
the cyclopalladated units adopted a trans configuration. Finally,
proton 10 (the NH proton) of compound 1 afforded a broad singlet
centred at 7.86 ppm. This proton for free benzophenone imine in
CDCl3 solution produced a broad signal at 8.4 ppm.
The 1H NMR at 400 MHz of compound 2 in CDCl3 solution was
less informative than that of compound 1. For compound 2, the NH
Fig. 3. A) trans folded and B) trans planar structures proposed for compounds 1 and 2
respectively. CeN]C6H4PhC]NH.

292
J. Albert et al. / Journal of Organometallic Chemistry 724 (2013) 289e296
of medium intensity at 3310 and 1592 cmꢁ1, respectively. Further-
more, the q X-sensitive mode of the coordinated PPh3 appeared at
1103 and 1098 cmꢁ1 for compounds 5 and 6 respectively [17].
1H NMR data for the mononuclear compounds 3e6 were
consistent with their proposed stereochemistry, in which the L
ligand is located trans to the iminic nitrogen and the X ligand is
trans to the palladated carbon atom (see Scheme 1). We refer to this
arrangement as trans-N,L stereochemistry. Accordingly to this
stereochemistry, the aromatic protons of the ortho-palladated ring
of compounds 3e6 appeared at lower frequencies (between 7.20
and 6.40 ppm) in relation to the other aromatic protons, because
they were located in the shielding zone of the aromatic ring of the
py-d5 ligand for compounds 3e4 and in that of the phenyl
substituents of the PPh3 ligand for compounds 5e6 [6a]. Protons of
the ortho-palladated ring of compounds 3e6 afforded the expected
multiplicity pattern for an 1,2 disubstituted phenyl. In addition, for
compounds 5e6, the 1 protons were coupled with the phosphorus
nuclei. The value of this coupling constant of ca. 8 Hz was also an
indication of their trans-N,P stereochemistry [6a,17]. The NH proton
for compounds 3, 4, 5 and 6 appeared at 10.18 (broad singlet), 8.73
3
(broad singlet), 9.44 (broad doublet, JHP z 4) and 9.21 (broad
3
doublet, JHP z 4) respectively. In addition, the protons of the
unidentate acetato ligands of compounds 3 and 5 afforded a singlet
at 1.98 and 1.44 ppm respectively.
A
further indication of the trans-N,P stereochemistry of
compounds 5e6 was given by the chemical shift of their 31P-{1H}
NMR, which were 40.73 and 41.34 ppm, respectively. These
chemical shifts were in the range expected for mononuclear
cyclopalladated compounds of general formula trans-N,P-[Pd(Ce
N)(X)(PPh3)] (X ¼ OAc, Cl, Br, I) that contain a five-membered
palladacycle and a phenyl or a naphthyl metallated carbon atom
(43e40 ppm) [6a,18].
In order to complete the characterization of compounds 1, 2, 5
and 6, their absorption and emission electronic spectra were
recorded. The absorption spectra of the complexes in dichloro-
Fig. 4. Inhibition of cell growth proliferation in MDA-MB231 and MCF7 breast human
cancer cell lines, after 72 h of exposure to compounds 1, 2, 5, 6 and cisplatin.
methane at 298 K showed high intensity superimposed
p / p*
intraligand transitions in the range 280e350 nm. In addition,
a lower energy band at 466 nm was observed for compound 1. This
band was attributed to a MLCT [4d(Pd) / p* C]N] transition. This
Finally, it should be noted that attempts to grow crystals suitable
for X-ray diffraction analysis for compounds 1, 2, 5 and 6 were not
successful.
assignation is based on similarities in energy and on theoretical
calculations carried out with analogous platinum cyclometallated
complexes [7]. This transition is probably occluded under the
intraligand transitions and displayed as a shoulder for the other
compounds. The absorption observed at longer wavelength for
compound 1 is also present in the electronic spectrum of other
acetato bridged cyclopalladated complexes [19].
Compounds 1, 2, 5 and 6 were not luminescent in solution but
a weak emission was observed in the solid state for the acetato
compounds 1 and 5 upon excitation of their powders at the lower
energy absorption band. No emission was observed when the
samples were excited at the intraligand transitions. This is consis-
tent with an involvement of the MLCT on this emission band. This
transition is more favoured for compound 1 and give rise to a red-
shifted emission band. The large Stokes’ shift observed is in
agreement with a triplet origin (3MLCT). Table 1 gives a summary of
the absorption and emission electronic spectra of compounds 1, 2, 5
and 6.
2.2. Antitumour activity
Compounds 1, 2, 5 and 6 were evaluated in vitro for inhibition of
cell proliferation against the MDA-MB231 and MCF7 human breast
cancer cell lines, using cisplatin as a positive control. The effects of
the assayed palladacycles on the growth of the selected cell lines
were assessed after 72 h and the results are displayed in Fig. 4. The
IC50 values of compounds 1, 2, 5 and 6 resulting from an average of
two experiments are listed in Table 2.
Table 2 and Fig. 4 show that compounds 5e6 exhibit a high
antiproliferative activity, showing IC50 values in the range 1e5 mM
below those of cisplatin in both cellular lines. Compounds 5e6 were
Table 2
IC50
(mM) values for compounds 1, 2, 5 and 6. Data are shown as the mean values of
two experiments performed in triplicate with the corresponding standard deviation.
Compound
Tumour cell line
MDA-MB231
Table 1
MCF-7
Absorption and emission electronic spectra for compounds 1, 2, 5 and 6.
1
2
5
6
15 ꢂ 1.2
13 ꢂ 1
14 ꢂ 4.2
11 ꢂ 1.5
4 ꢂ 0.5
Compound
l
absorption in nm (ε$10ꢁ3, Mꢁ1 cmꢁ1
)
l emission in nm (solid)
1
2
5
6
275 (22.6), 322 (12.4), 466 (3.0)
285 (31.1), 311 (19.9), 330 (19.3)
280 sh (14.8), 313 sh (7.1), 336 sh (3.3) 615 (lexc ¼ 370 nm)
287 sh (10.7), 312 sh (6.32), 340 sh (3.1)
670 (lexc ¼ 450 nm)
1.1 ꢂ 0.3
1.1 ꢂ 0.1
6.5 ꢂ 2.4
e
4.1 ꢂ 0.9
19 ꢂ 4.5
cisplatina
e
a
cis-[PtCl2(NH3)2] is taken as reference compound.

J. Albert et al. / Journal of Organometallic Chemistry 724 (2013) 289e296
293
Fig. 5. Aqua cations I and II.
Fig. 7. Relative Calf thymus DNA-ethidium bromide fluorescence. ri ¼ molar ratio
between drug/DNA nucleotides. Red rhombus compound 1, green
square ¼ compound 2, blue cross ¼ compound 5, black triangle ¼ compound 6. (For
interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)
¼
approximately 6- and 4-fold times more potent than cisplatin in the
MDA-MB231 and MCF7 human breast cancer cells respectively. In
addition, the IC50 of compounds 1e2 was on the low side of the
interval reported for the IC50 values of cyclopalladated compounds
towards different cancer cell lines, which varies between 0.5 and
The rate of migration of the supercoiled band (ccc) decreased and
tended to approach that of the nicked relaxed band (oc). At high
concentrations of compounds 1e2, a strong unwinding of negative
to positive supercoiled DNA was displayed in the electrophoreto-
gram (Compounds 1e2, Lane 7). The same effect was observed for
cisplatin (Fig. 6, cisplatin, Lanes 3 and 4).
On the basis of the alteration of the electrophoretic mobility of
pBluescript plasmid DNA it is hypothesized that compounds 1e2
and 5e6 alter the DNA tertiary structure by the same mechanism as
the standard reference, cisplatin. In addition, the interaction of
compounds 5e6 with supercoiled plasmid DNA seems to be
stronger than that of compounds 1e2 since Lanes 6e7 for
compounds 5e6 are less brilliant than Lanes 6e7 for compounds
1e2 [5j].
450 mM for these compounds [3e5]. In addition, compound 2 was
more potent than cisplatin against the MCF7 tumour cell line.
The similarity in the IC50 values of compounds 1e2 and 5e6 for
the MDA-MB231 and MCF7 cell lines (see Table 2) can be under-
stood if we consider that in the biological media these compounds
may be present as the aqua cations shown in Fig. 5. The aquatiza-
tion of the PdeOAc and PdeCl bonds of compounds 1e2 should
give the aqua cation I and that of compounds 5e6 the aqua
cation II [20].
2.3. Interaction with DNA
DNA-ethidium bromide fluorescence quenching and viscometry
experiments were performed in order to explore an intercalating
mode of binding to DNA for compounds 1e2 and 5e6. In the DNA-
ethidium bromide fluorescence quenching technique, the quench-
ing of the emission maximum of the fluorescence of ethidium
bromide intercalated between DNA base pairs can be taken as an
indication of a competition between the drug and the ethidium
bromide for the intercalating binding sites in DNA [21]. This exper-
iment (Fig. 7) suggested that compounds 1e2 could be intercalating
agents since theirDNA-ethidiumbromide fluorescencewas 0.63 and
0.72 respectively, at ri ¼ 1 (ri ¼ molar ratio between drug/DNA
nucleotides [22]) relative to the DNA-ethidium bromide initial
fluorescence, taken as1. However, viscometryexperiments ruledout
this possibility since no increase in viscosity was observed when
compounds 1e2 were incubated with DNA [23].
The interaction of compounds 1e2 and 5e6 with DNA was
studied by their ability to modify the electrophoretic mobility of the
supercoiled closed circular (ccc) and the open circular (oc) forms of
pBluescript SK þ plasmid DNA. The ccc form usually moves faster
due to its compact structure. When the test compounds were
incubated with plasmid DNA at 37 ꢀC, they coordinated to DNA
molecule, which in some extend was cleaved into fragments, and
the brightness of band diminished in gel. In spite of their high
antiproliferative activity, compounds 1e2 and 5e6 were less effi-
cient than cisplatin for removing the supercoils from pBluescript
SK þ plasmid DNA, suggesting that the unwinding of the DNA is not
the key factor responsible of their cytotoxicity [3a,5f,m].
An unwinding experiment was performed with increasing
concentration of compounds 1e2 and 5e6 ranging from 2.5
mM to
200 M and 40 g/mL pBluescript (Fig. 6). At 50 M, only a slight
m
m
m
The DNA-ethidium bromide fluorescence quenching for
compounds 1e2 and 5e6 followed the SterneVolmer equation:
I0=I ¼ 1 þ Kq½Qꢃ. Where I0 and I represent the emission intensity in
the absence and presence of quencher respectively, is a linear
decrease in the rate of migration of the supercoiled closed circular
form was observed for the dinuclear compound 2 (compound 2,
Lane 5) and at 100
mM all the compounds greatly altered the
mobility of the plasmid DNA (compounds 1e2 and 5e6, Lane 6).
Fig. 6. Interaction of pBluescript SK þ plasmid DNA (40
m
g/mL) with increasing concentrations of compounds 1, 2, 5, 6, cisplatin and ethidium bromide. Lane 1: DNA only. Lane 2:
M drug. Lane 6: 100 M drug. Lane 7: 200
M drug. ccc ¼ supercoiled closed circular DNA. oc ¼ open circular DNA.
2.5 M drug. Lane 3: 5 M drug. Lane 4: 25 M drug. Lane 5: 50
m
m
m
m
m
m

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J. Albert et al. / Journal of Organometallic Chemistry 724 (2013) 289e296
SterneVolmer quenching constant and [Q] is the quencher
concentration, the concentration of cyclopalladated compound in
our study. The I0/I vs. [Q] followed a linear pattern from which Kq
was calculated. Kq was 1.1 ꢄ104 Mꢁ1 and 7.0 ꢄ 103 Mꢁ1 for 1 and 2,
6.88 (td, 1H, 3JHH z 8, 4JHH z 2), 6.83 (dd, 1H, 3JHH z 8, 4JHH z 2),
6.70 (dd, 2H, H9 and H5, 3JHH z 8, 4JHH z 1), 2.22 (s, 3H, OAc).
4.3. Preparation of compound 2
respectively, and 1.3 ꢄ 103
M
ꢁ1 for 5 and 6.
A suspension formed by 100 mg (0.15 mmol) of 1, 25 mg
(0.59 mmol) of LiCl and 30 mL of acetone was stirred at room
temperature for 2 h. The resulting pale yellow solution was
concentrated under vacuum and the residue was eluted through
a silica-60 gel column chromatography with a solution of methanol
in chloroform in a 4e100 volume ratio. The yellow band was
collected and concentrated under vacuum. Addition of diethyl ether
(5 mL) to the residue led to the precipitation of 2 as a pale yellow
solid, which was filtered and dried under vacuum. Yield: 73 mg
(78%). Characterization data: Anal. Calc for C26H26Cl2Pd2: C, 48.48;
H, 3.13; N, 4.35%. Found: C, 48.52; H, 3.18; N, 4.57%. ESI-(þ): intense
3. Conclusions
Four new cyclopalladated benzophenone imines (1e2 and 5e6)
were prepared by cyclopalladation, metathesis and splitting
reactions and were fully characterized by the standard
techniques. These compounds showed a high antiproliferative
activity against the MDA-MB231 and MCF7 human breast cancer
cell lines. Compounds 5e6 were approximately 6- and 4-fold times
more potent than cisplatin in the MDA-MB231 and MCF7 human
breast cancer cells and presented IC50 values in the range 1e5 mM.
peaks at [M
ꢁ
Cl]þ
¼
608.9392 (calc. 608.9395), [(M/2)e
The study of the interaction of compounds 1e2, and 5e6 with DNA
by agarose gel electrophoresis showed that they modify the helicity
of the DNA as the alkylating agent cisplatin, although in a less
extensive way. In addition, DNA-ethidium bromide fluorescence
quenching and viscometry experiments ruled out an intercalating
binding mode to DNA for these kind of compounds.
Cl
þ
CH3CN]þ
¼
327.0114 (calc. 327.0120) and [(M/2)e
Cl]þ ¼ 285.9851 (calc. 285.9853). IR (cmꢁ1): 3309 st NeH, 1584 st
C]N. 1H NMR (400 MHz, CDCl3): 8.38 (br s, 1H, H10), 7.61e7.52 (m,
6H), 7.15 (td, 1H, 3JHH z 8, 4JHH z 2), 7.09e7.02 (m, 2H).
4.4. NMR tube reactions
4. Experimental
ca. 10 mg of compound 1e2 were treated with 0.7 mL of CDCl3
and two drops of py-d5. The formation of a pale yellow solution was
an indication of the quantitative transformation of compounds 1e2
into the mononuclear compounds 3e4. Characterization data:
Compound 3: 1H NMR (400 MHz, CDCl3 þ py-d5): 10.18 (br s, 1H,
H10); 7.47e7.57 (m, 5H, H5eH9); 7.16 (m, 1H, H4); 7.02 (m, 2H, H2 -
H3); 6.47 (m, 1H, H1); 1.98 (s, 3H, OAc). Compound 4: 1H NMR
(400 MHz, CDCl3 þ py-d5): 8.73 (br s, 1H, H10); 7.51e7.60 (m, 5H,
H5eH9); 7.17 (dd, 1H, H4, 1H, 3JHH z 8, 4JHH z 2); 7.04e7.08 (m, 2H,
4.1. Instruments and reagents
Elemental analyses of C, H and N were performed with an Eager
1108 microanalyzer. Infrared spectra were recorded on a Nicolet
Impact-400 spectrophotometer using pressed discs of dispersed
samples of the compounds in KBr. 1H NMR at 400 MHz and 31P{1H}
NMR at 121.4 MHz spectra were recorded in Varian 400 and 300
instruments, respectively. Chemical shifts are reported in
d
values
3
H2eH3); 6.44 (d, 1H, H1, JHH z 8).
(ppm) relative to SiMe4
(d
¼ 0.00) for 1H NMR and to PO(OMe)3
(d
¼ 2.39 ppm) for 31P{1H} NMR, and coupling constants are giving
4.5. Preparation of compound 5
in Hz. MALDI-TOF (þ) and LDI-TOF (þ) spectra were registered on
a VOYAGER-DE-RP spectrometer. 2,5-dihydroxybenzoic acid (DHB)
was used as matrix for the MALDI-TOF (þ) experiment. ESI-(þ)
spectra were acquired on a LC/MSD-TOF mass spectrometer, using
1:1H2O:CH3CN as eluent. The mass of the most abundant isotopic
peak is given for the different molecular fragments [24]. UVevis
were recorded at 298 K with a Cary 100 scan 388 Varian UV
spectrometer. The fluorescence emission spectra were carried out
on a Horiba-Jobin-Yvon SPEX Nanolog-TM at 25 ꢀC. All chemicals
and solvents were of commercial grade and used as received.
Viscosity experiments were carried out with an AND-SV-1
viscometer in a water bath using a water jacket accessory and
maintained at a constant temperature of 25 ꢀC.
A suspension formed by 100 mg (0.14 mmol) of 1, 76 mg of PPh3
(0.29 mmol) and 10 mL of acetone was stirred at room temperature
for 2 h. The resulting solution was concentrated under vacuum and
the residue was eluted trough a silica-60 gel column chromatog-
raphy with a solution of methanol in chloroform in a 4e100 volume
ratio. The second coloured band was collected and concentrated
under vacuum. Addition of diethyl ether (5 mL) to the residue led to
the precipitation of 5 as a white solid, which was filtered and dried
under vacuum. Yield: 59 mg (43%). Characterization data: Anal. Calc
for C33H28NO2PPd: C, 65.19; H, 4.64; N, 2.30%. Found: C, 65.08; H,
4.62; N, 2.31%. LDI-TOF(þ): Intense peaks at [M þ Na]þ ¼ 630.1
(calc. 630.0803) and [M ꢁ OAc]þ ¼ 548.1 (calc. 548.0771). IR
(cmꢁ1): 3319 st NeH, 1574 (st as carboxylate function, acetate), 1374
(st s carboxylate function, acetate), 1103 q X-sensitive mode of the
4.2. Preparation of compound 1
coordinated PPh3. 1H NMR (400 MHz, CDCl3): 9.44 (br d, 1H, H10
,
A suspension formed by 497 mg (2.21 mmol) of Pd(OAc)2,
400 mg (2.21 mmol) of benzophenone imine and 20 mL of acetic
acid was stirred at 60 ꢀC for 24 h. The resulting brown suspension
was concentrated under vacuum. Addition of acetone (10 mL) to the
residue produced the precipitation of 1 as a yellow powder, which
was filtered and dried under vacuum. Yield: 79% (605 mg). Char-
acterization data: Anal. Calc for C30H26N2O4Pd2: C, 52.12; H, 3.79; N,
4.05%. Found: C, 52.06; H, 3.72; N, 4.02%. ESI-(þ): Intense peak at
[(M/2) þ Na]þ ¼ 368.0395 (calc. 367.9885). IR (cmꢁ1): 3145 NeH st,
1565 (st as carboxylate function, acetate), 1431 (st s carboxylate
function, acetate). 1H NMR (400 MHz, CDCl3): 7.86 (br s, 1H, H10),
7.41 (tt, 1H, H7, 3JHH z 8, 4JHH z 1), 7.24 (t, 2H, H6 and H8, 3JHH z 8),
3JHP z 4), 7.82e7.77 (m, 6H, o-PPh3), 7.57e7.37 (m, 14 H, m- and p-
PPh3 and H5eH9), 7.10 (dd, 1H, H4, 3JHH z 8, 4JHH z 2), 6.86 (td, 1H,
H3, 3JHH z 8, 4JHH z 1), 6.62 (td, 1H, H1, 4JHP z 3JHH z 8, 4JHH z 2),
6.55 (td, 1H, H1, 3JHH z 8, 4JHH z 2), 1.44 (s, 3H, OAc). 31P{1H} NMR
(121.4 MHz, CDCl3): 40.73.
4.6. Preparation of compound 6
A suspension formed by 51 mg (0.08 mmol) of 2, 43 mg of PPh3
(0.16 mmol) and 30 mL of acetone was stirred at room temperature
for 2 h. The resulting white suspension was concentrated under
vacuum and the residue was eluted trough a silica-60 gel column
chromatography with a solution of methanol in chloroform in a 4e
3
4
3
4
7,21 (dd, 1H, JHH z 8, JHH z 1), 7.11 (td, 1H, JHH z 8, JHH z 2),

J. Albert et al. / Journal of Organometallic Chemistry 724 (2013) 289e296
295
100 volume ratio. The yellowish band was collected and concen-
trated under vacuum. Addition of diethyl ether (5 mL) to the
residue led to the precipitation of 6 as a white solid, which was
filtered and dried under vacuum. Yield: 74 mg (79%). Character-
ization data: Anal. Calc for C31H25ClNPPd: C, 63.71; H, 4.31; N,
2.40%. Found: C, 62.92; H, 4.04; N, 2.50%. MALDI-TOF(þ): Intense
peak at [M ꢁ Cl]þ ¼ 548.3 (calc. 548.0771). IR (cmꢁ1): 3310 st NeH,
1592 st C]N, 1098 q X-sensitive mode of the coordinated PPh3. 1H
8.0). The gel was stained in TAE buffer containing ethidium bromide
(0.5 mg mLꢁ1) and visualized and photographed under UV light.
4.10. DNA-ethidium bromide fluorescence quenching
To a 50
mM Calf thymus (CT) DNA solution in Milli-Q water (3 mL
total volume), 30
mL of a 5 mM ethidium bromide solution was
added to get a 3:1 DNA:ethidium bromide molar ratio. The mixture
was incubated for 30 min at 37 ꢀC. Increasing amounts of a 1.5 mM
DMSO/Milli-Q water (1:1) stock solutions of compounds 1, 2, 5 and
6 were added to reach the following final concentrations of the
compounds in the DNA:ethidium bromide solution: 0, 10, 20, 30, 40
NMR (400 MHz, CDCl3): 9.21 (d, 1H, H10
,
3JHP z 4); 7.75e7.80 (m,
3
5H, H5eH9); 7.37e7.57 (m, 15H, PPh3); 7.14 (dd, 1H, H4, JHH z 8,
3
4
4JHH z 2), 6.90 (td, 1H, H3, JHH z 8, JHH z 1), 6.62 (td, 1H, H2,
3JHH z 8, 4JHH z 2), 6.54 (td, 1H, H1, 4JHP z 3JHH z 8, 4JHH z 2). 31
P
{1H} NMR (121.4 MHz, CDCl3): 41.34.
and 50
emission spectra
temperature in the wavelength range 530e670 nm. For each
compound, three blanks were prepared and measured: 1) 100
of 1.5 mM of compound solution in 3 mL TE buffer (to rule out
autofluorescence of the compound), 2) 100 L of 1.5 mM solution
of compound in 3 mL of the CT DNA solution (to verify that the
compound does not fluoresce with DNA), and 3) 100 L of the
1.5 mM solution of compound and 30 L of ethidium bromide
m
M. After incubation for 4e5 h at 37 ꢀC, fluorescence
lex 514 nm) were recorded at room
(
¼
4.7. Cell culture
mL
Breast cancer MCF-7 and MBA-MD-231 cells were grown as
a monolayer culture in minimum essential medium (DMEM with
glutamine, without glucose and without sodium pyruvate) in the
presence of 10% heat-inactivated fetal calf serum, 10 mM -glucose
L-
m
D
m
and 0.1% streptomycin/penicillin, in standard culture conditions
m
(humidified air with 5% CO2 at 37 ꢀC).
5 mM solution in TE buffer (to check any fluorescent interaction
between the compound and ethidium bromide). FluorEssence
software for Windows was used in data treatment.
4.8. Cell viability assay
A stock solution (50 mM) of each compound was prepared in
high purity DMSO. Then, serial dilutions were made with DMSO
(1:1) and finally a 1:500 dilution of the diluted solutions of
compounds on cell media was prepared. In this way DMSO
concentration in cell media was always the same. The assay was
performed as described by Givens et al. [25]. MDA-MB231 and
4.11. Viscometry
All compounds were dissolved in high purity DMSO (1 mg
compound/1 mL). The stock solutions were freshly prepared, just
before use. The samples were prepared by addition of aliquots of
these stock solutions to the appropriate volume of Calf thymus DNA
in a TE buffer solution (50 mM NaCl, 10 mM tris-(hydroxymethyl)
aminomethane hydrochloride (TriseHCl), 0.1 mM H4edta, pH 7.4)
(3 mL). The amount of complex added to the DNA solution was
designated as ri. As a blank, a solution in TE of free native DNA was
used. The viscosity spectra of DNA in the presence or absence of
MCF7 cells were plated at 5000 cells/well, respectively, in 100
media in tissue culture 96-well plates (Cultek). After 24 h, media
was replaced by 100 L/well of drug serial dilutions. Control wells
mL
m
did not contain compounds 1, 2, 5 and 6. Each point concentration
was run in triplicate. Reagent blanks, containing media and color-
imetric reagent without cells were run on each plate. Blank values
were subtracted from test values and were routinely 5e10% of the
control values. Plates were incubated 72 h. Hexosaminidase activity
was measured according to the following protocol. The media was
complexes (DNA concentration 20
mM, molar ratios ri ¼ 0.2, 0.6 and
1) were recorded at 25 ꢀC, after 5 h incubation at 37 ꢀC.
Acknowledgements
removed and cells were washed once with PBS. 60
mL of substrate
solution (p-nitrophenol-N-acetyl- -glucosamide 7.5 mM,
b-D
We are grateful to the Ministerio de Ciencia y Tecnología for
financial support (Grant CTQ2009-11501/BQU and CTQ2009-
07021/BQU) and to the AGAUR, Generalitat de Catalunya (Grant
2009-SGR-1111).
sodium citrate 0.1 M at pH 5.0, and 0.25% Triton X-100) was
added to each well and incubated at 37 ꢀC for 1e2 h. After this
incubation time, a bright yellow appeared. Then, the plates were
developed by adding 90 mL of developer solution (Glycine 50 mM,
pH 10.4; EDTA 5 mM) and the absorbance was recorded at 410 nm.
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