Organometallic Palladium Complexes with a Water-Soluble Iminophosphorane Ligand As Potential Anticancer Agents

Article abstract of DOI:10.1021/om3006239

The synthesis and characterization of a new water-soluble iminophosphorane ligand TPA=N-C(O)-2BrC₆H₄ (1, C,N-IM; TPA = 1,3,5-triaza-7-phosphaadamantane) is reported. Oxidative addition of 1 to Pd₂(dba)₃ affords the orthopalladated dimer [Pd(μ-Br){C₆H₄(C(O)N=TPA-kC,N)-2}]₂ (2) as a mixture of cis and trans isomers (1:1 molar ratio) where the iminophosphorane moeity behaves as a C,N-pincer ligand. By addition of different neutral or monoanionic ligands to 2, the bridging chlorides can be cleaved and a variety of hydrophilic or water-soluble mononuclear organometallic palladium(II) complexes of the type [Pd{C₆H₄(C(O)N=TPA-kC,N)-2}(L-L)] (L-L = acac (3); S₂CNMe₂ (4); 4,7-diphenyl-1,10- phenanthrolinedisulfonic acid disodium salt C₁₂H₆N ₂(C₆H₄SO₃Na)₂ (5)), [Pd{C₆H₄(C(O)N=TPA-kC,N)-2}(L)Br] (L = P(mC ₆H₄SO₃Na)₃ (6); P(3-pyridyl) ₃ (7)), and [Pd(C₆H₄(C(O)N=TPA)-2}(TPA) ₂Br] (8) are obtained as single isomers. All new complexes were tested as potential anticancer agents, and their cytotoxicity properties were evaluated in vitro against human Jurkat-T acute lymphoblastic leukemia cells, normal T-lymphocytes (PBMC), and DU-145 human prostate cancer cells. Compounds [Pd(μ-Br){C₆H₄(C(O)N=TPA-kC,N)-2}]₂ (2) and [Pd{C₆H₄(C(O)N=TPA-kC,N)-2}(acac)] (3) (which has been crystallographically characterized) display higher cytotoxicity against the above-mentioned cancer cell lines while being less toxic to normal T-lymphocytes (peripheral blood mononuclear cells: PBMC). In addition, 3 is very toxic to cisplatin-resistant Jurkat shBak, indicating a cell death pathway that may be different from that of cisplatin. The interaction of 2 and 3 with plasmid (pBR322) DNA is much weaker than that of cisplatin, pointing to an alternative biomolecular target for these cytotoxic compounds. All the compounds show an interaction with human serum albumin faster than that of cisplatin.

Full text of DOI:10.1021/om3006239

Article
pubs.acs.org/Organometallics
Organometallic Palladium Complexes with a Water-Soluble
Iminophosphorane Ligand As Potential Anticancer Agents
Monica Carreira,† Ruben Calvo-Sanjuan,‡ Mercedes Sanau,§ Isabel Marzo,‡ and María Contel*,†
́
́
́
†Department of Chemistry, Brooklyn College and The Graduate Center, The City University of New York, Brooklyn, New York
11210, United States
‡Department of Biochemistry and Molecular and Cellular Biology, University of Zaragoza, 50009, Spain
§Departamento de Química Inorgan
́
ica, Universidad de Valencia, Burjassot, Valencia, 46100, Spain
S
* Supporting Information
ABSTRACT: The synthesis and characterization of a new water-
soluble iminophosphorane ligand TPAN-C(O)-2BrC6H4 (1, C,N-
IM; TPA = 1,3,5-triaza-7-phosphaadamantane) is reported. Oxidative
addition of 1 to Pd2(dba)3 affords the orthopalladated dimer [Pd(μ-
Br){C6H4(C(O)NTPA-kC,N)-2}]2 (2) as a mixture of cis and trans
isomers (1:1 molar ratio) where the iminophosphorane moeity
behaves as a C,N-pincer ligand. By addition of different neutral or
monoanionic ligands to 2, the bridging chlorides can be cleaved and a
variety of hydrophilic or water-soluble mononuclear organometallic
palladium(II) complexes of the type [Pd{C6H4(C(O)NTPA-
kC,N)-2}(L-L)] (L-L = acac (3); S2CNMe2 (4); 4,7-diphenyl-1,10-
phenanthrolinedisulfonic acid disodium salt C12H6N2(C6H4SO3Na)2
(5)), [Pd{C6H4(C(O)NTPA-kC,N)-2}(L)Br] (L = P-
(mC6H4SO3Na)3 (6); P(3-pyridyl)3 (7)), and [Pd(C6H4(C(O)N
TPA)-2}(TPA)2Br] (8) are obtained as single isomers. All new complexes were tested as potential anticancer agents, and their
cytotoxicity properties were evaluated in vitro against human Jurkat-T acute lymphoblastic leukemia cells, normal T-lymphocytes
(PBMC), and DU-145 human prostate cancer cells. Compounds [Pd(μ-Br){C6H4(C(O)NTPA-kC,N)-2}]2 (2) and
[Pd{C6H4(C(O)NTPA-kC,N)-2}(acac)] (3) (which has been crystallographically characterized) display higher cytotoxicity
against the above-mentioned cancer cell lines while being less toxic to normal T-lymphocytes (peripheral blood mononuclear
cells: PBMC). In addition, 3 is very toxic to cisplatin-resistant Jurkat shBak, indicating a cell death pathway that may be different
from that of cisplatin. The interaction of 2 and 3 with plasmid (pBR322) DNA is much weaker than that of cisplatin, pointing to
an alternative biomolecular target for these cytotoxic compounds. All the compounds show an interaction with human serum
albumin faster than that of cisplatin.
structural and thermodynamic analogy between platinum(II)
and palladium(II) complexes. However, the ligand-exchange
kinetics of platinum(II) and palladium(II) derivatives are quite
different; hydrolysis of palladium(II) compounds is much more
rapid, leading to reactive species unable to reach their
pharmacological targets. In addition, some Pd(II) compounds
transform into inactive trans derivatives. The stabilization of
Pd(II) compounds by strongly coordinated nitrogen ligands
and an appropriate leaving group has been exploited as a
synthetic strategy to obtain palladium(II) antitumor com-
plexes.18 Thus a variety of trans compounds containing bulky
monodentate ligands,19 complexes with bidentate ligands (N−
N; P−P, or mixed N−O, N−S),20 and organometallic
derivatives21 have been prepared. Since the seminal work by
INTRODUCTION
■
A significant fraction of currently used anticancer drugs are
platinum compounds such as cisplatin, carboplatin (Paraplatin),
and oxaliplatin (Eloxatin).1 However their effectiveness is still
hindered by clinical problems, including acquired or intrinsic
resistance, a limited spectrum of activity, and high toxicity
leading to side effects.1,2 The search for anticancer agents with
improved properties has focused on the synthesis of a new
generation of platinum compounds,3,4 which include Pt(IV)
pro-drugs,5 multimetallic derivatives,6 and the use of nano-
particles and oligonucleotides as delivery platforms.7 The
synthesis of other metallo drugs (including organometallic
complexes)8 of ruthenium,9 gold,10 titanium,11 osmium,12
copper,13 rhodium,14 iridium,15 and iron16 has also attracted
great interest. More recently, a number of heterometallic
cytotoxic complexes17 have been reported, which show a
synergic effect of two different metals with known antitumor
properties. Palladium derivatives have been explored as an
alternative18 to platinum-based compounds due to the obvious
Special Issue: Organometallics in Biology and Medicine
Received: July 4, 2012
Published: July 25, 2012
© 2012 American Chemical Society
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Navarro-Ranninger et al. on cytotoxic orthometalated palla-
dium complexes22 (some containing thiosemicarbazone
ligands), a few cyclopalladated compounds with potential
anticancer activities have been described.23−25 The higher
stability of cyclopalladated compounds in physiological media
and a lower toxicity to normal cells are promising features for
their biological applications.18b Biphosphinic palladacycles of
the type [Pd(C2,N-(S(−)dmap)(dppf)]Cl induced apoptotic cell
death in human leukemia cells by rupture of the lysosomal
membrane and release of cathepsin B in the cytoplasm.26−28
Importantly, the related compound [Pd2(S(−)C2,N-dmpa)2(μ-
dppe)Cl2] has been selected for its promising antitumor
properties against murine and cisplatin-resistant human tumor
cells both in vitro and in vivo29−31 for further preclinical
studies,30 including a gene therapy protocol in conjunction with
plasmids.31 Its mode of action seems to indicate DNA
degradation and mitochondrial damage by interaction of the
compound with thiol groups on the mitochondrial membrane
proteins.29,30
We have reported that nontoxic iminophosphorane or
iminophosphine compounds serve as stabilizing (C,N- or
N,N-) chelating ligands in the preparation of anticancer
organometallic and coordination d8 metal complexes.32−34
Organogold(III) complexes containing iminophosphorane
(C,N-IM) ligands of the type PPh3NPh displayed a high
cytotoxicity in vitro against human ovarian cancer and leukemia
cell lines32,33 while being less toxic to normal T-lymphocytes by
a non-cisplatin mode of action (involving mitochondrial
production of reactive oxygen species).33 More recently, we
have reported on the cytotoxicity of coordination Pd(II) and
Pt(II) complexes with the first water-soluble iminophosphorane
ligand, TPAN-C(O)-2-NC5H4 (N,N-IM), described to
date.34 Interestingly, we found that the Pt(II) compound was
more cytotoxic than cisplatin to leukemia cell lines (both T-
Jurkat and cisplatin-resistant Jurkat sh-Bak) by a mode of action
different from that of cisplatin.34 The coordination Pd(II)
derivative (slighty less cytotoxic than cisplatin to leukemia cell
lines) displayed a mode of action similar to cisplatin.34 We
report here on the synthesis of a related water-soluble
iminophosphorane ligand, TPAN-C(O)-2BrC6H4 (1, C,N-
IM; TPA = 1,3,5-triaza-7-phosphaadamantane), which serves as
an excellent precursor to organometallic Pd(II) derivatives with
varying hydrophilic−lipophilic ratios. All new complexes have
been tested as potential anticancer agents, and their cytotoxicity
properties were evaluated in vitro against human Jurkat-T acute
lymphoblastic leukemia cells, normal T-lymphocytes (PBMC),
and DU-145 human prostate cancer cells. The interactions of
the most cytotoxic derivatives with plasmid (pBR322) DNA
and human serum albumin (HSA) are reported.
Scheme 1. Preparation of Water-Soluble Ligand 1 by the
Pomerantz22 Method
with potential anticancer properties.34 The half-life for 1 in
D2O is 2 days, while ligand TPAN-C(O)-2-NC5H4
decomposed to 50% after 12 h.34 1 is stable in DMSO solution
for over two weeks, while in mixtures of d6-DMSO−D2O (1:1)
the half-life is 14 days as opposed to 2 days for the N,N-IM
ligand.34
Oxidative addition of 1 to Pd2(dba)3 affords the
orthopalladated dimer [Pd(μ-Br){C6H4(C(O)NTPA-
kC,N)-2}]2 (2) as a mixture of cis and trans isomers (1:1
molar ratio), where the iminophosphorane fragment behaves as
a C,N-pincer ligand (Scheme 2 and Experimental Section).
By addition of different ligands to 2 and subsequent cleavage
of the chloride bridging system, a variety of hydrophilic or
water-soluble mononuclear organometallic palladium(II) com-
plexes of the type [Pd{C6H4(C(O)NTPA-kC,N)-2}(L-L)]
(L-L = acac (3); S2CNMe2 (4); 4,7-diphenyl-1,10-phenan-
throlinedisulfonic acid disodium salt, C12H6N2(C6H4SO3Na)2
(5)), [Pd{C6H4(C(O)NTPA-kC,N)-2}(L)Br] (L = P-
(mC6H4SO3Na)3 (6); P(3-pyridyl)3 (7)), and [Pd{C6H4(C-
(O)NTPA)-2}(TPA)2Br] (8) are obtained (Scheme 2) in
moderate to high yields (see Experimental Section). All
complexes were characterized by means of NMR and IR
spectroscopy, elemental analyses, mass spectrometry, and
conductivity measurements (see Experimental Section). These
techniques confirmed that the structures proposed for
compounds 2−8 (Scheme 2) are the most plausible ones. In
the case of the reactions of 2 with one neutral phosphine (v and
vi in Scheme 2) the single isomer in which the incoming ligand
is coordinated trans to the nitrogen atom (6 and 7) is obtained
(typical reactivity of C,N-cyclopalladated halide dimers).37 The
reaction of 2 with TPA gives exclusively derivative [Pd-
{C6H4(C(O)NTPA)-2}(TPA)2Br] (8), with two TPA
phosphines and with the iminophosphorane ligand coordinated
solely through the C atom. We have observed the same effect in
the reaction of [Au{κ2-C,N-C6H4(PPh2N(C6H5)-2}Cl-
(CH3CN)]PF6 with TPA, which affords compound [Au-
{C6H4(PPh2N(C6H5)-2}(TPA)2Cl]PF6 with two TPA phos-
phines.32
Compounds 2, 3, and 4 are not soluble in water, but they are
soluble in mixtures of DMSO−H2O (1:99) in concentrations
ranging from 0.4 to 0.8 mM (which allows for their
solubilization for further biological testing). Compounds 5−8
are more hydrophilic and can be dissolved in water
(concentrations ranging from 0.5 to 1.0 mM). The stability
of the compounds in solution can be easily ascertained by
31P{1H} NMR spectroscopy in d6-DMSO or mixtures of d6-
DMSO−D2O (50:50) (see Supporting Information). Most
compounds are quite stable in d6-DMSO solution with half-
lives of several months (2−4, 6, 7), while others are stable over
2 days (5, 8). In d6-DMSO−D2O (50:50) mixtures the
compounds are also very stable, with half-lives of several
RESULTS AND DISCUSSION
■
1. Synthesis and Characterization. Ligand 1, TPAN-
C(O)-2BrC6H4, can be conveniently prepared from 2-
bromobenzamide and TPA through the Pomerantz method
(Scheme 1).35 To the best of our knowledge, ligand 1 is one of
the two examples of water-soluble iminophosphoranes
prepared to date.34 It is well known that most IMs in contact
with water or polar solvents break down to the corresponding
amine and phosphine oxide.36 We have incorporated a CO
group to stabilize the resulting water-soluble IM ligands.34
Ligand 1 (solubility in H2O 10 g/L, or 28 mM) is more stable
in solution than ligand TPAN-C(O)-2-NC5H4 prepared by
our group to synthesize coordination compounds of d8 metals
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a
Scheme 2. Preparation of Di- (2) and Mononuclear (3−8) Hydrophilic Cyclopalladated Iminophosphorane Derivatives
a
(i) Pd2(dba)3; (ii) Tl(acac); (iii) Na[S2CN(CH3)2]; (iv) C12H6N2(C6H4SO3Na)2; (v) P(mC6H4SO3Na)3; (vi) P(3-pyridyl)3; (vii) TPA.
months (3, 4, 6), 1−2 weeks (2, 7, 8), or more than 4 days (5).
The most unstable complex is that with the bathophenanthro-
line ligand (5), but this compound is stable under biological
testing conditions (24 h).
The crystal structure of 3 (determined by single-crystal X-ray
diffraction study) is depicted in Figure 1. Selected bond lengths
and angles are collected in Table 1. The geometry about the
Pd(II) centers is pseudo-square-planar with the C(18)−
Pd(1)−N(1) angle of 81.35(16)o, suggesting a rigid “bite”
angle of the chelating ligand. The Pd center is on an almost
ideal plane with negligible deviations from the least-squares
plane. The coordination plane for Pd and the metallocyclic
plane are slightly twisted, to give an angle of 5.5° between
them.
The main distances Pd−N(1) and Pd−C(18) of 2.037(4)
and 1.966(4) Å found for 3 are a little shorter than or similar to
those found in related carbonyl-stabilized iminophosphorane
complexes where the cyclometalation occurs in the benzyl ring
Figure 1. Molecular structure of the compound [Pd{C6H4(C(O)N
such as [Pd{C6H4(C(O)NP(p-tol)3-kC,N)-2}{acac-O,O′)]
TPA-kC,N)-2}(acac-O,O′)] (3) with the atomic numbering scheme.
(2.0695(16) and 1.961(2) Å),38 [Pd(μ-Cl){C6H4(C(O)N
P(m-tol)3-kC,N)-2}]2 (2.085(2) and 1.979(2) Å),38 or {Pd(μ-
chelating acac ligands.38 The P(1)−N(1) bond length in 3 of
Cl){C6H3(Me-3)(C(O)NPPh3-2}]2 (2.043(2) and 1.962(3)
Å).39 The Pd1−O1 (2.023(3) Å) and Pd1−O2 (2.094(3) Å)
1.643(4) Å is very similar to that in [Pd{C6H4(C(O)NP(p-
tol)3-kC,N)-2}{acac-O,O′)] (1.6457(18) Å),38 [Pd(μ-Cl)-
bond distances fall in the usual range of distances found for
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found for cycloaurated iminophosphorane complexes),32 but
this was not the case. In general the IC50 values in DU145 are
quite high and for 3 comparable to that of cisplatin (Table 2).
Complex 2 was found to be more cytotoxic than cisplatin for
this cell line. Complexes 2 and 3 have IC50 values in T-cell
leukemia Jurkat cells similar to those of cisplatin and very close
to those obtained by previously reported tetranuclear cyclo-
palladated compounds with a p-isopropylbenzaldehyde thio-
semicarbazone [Pd(p-is.TSCN)],22d dinuclear derivatives of the
type [Pd2L2(μ-dppe)Cl2] (L = 1-methyl-5-phenyl-1H-1,4-
benzodiazepin-2(3H)-one),25b and mononuclear [Pd-
(S(−)C2,N-dmpa)(dppf)]Cl.27 Complexes 2 and 3 were 3 and
2 times more cytotoxic to T-cell leukemia Jurkat cells than the
IM coordination complex [PdCl2(TPAN-C(O)-2-
NC5H4)].34 Complexes 2 and 3 were 26 (2) or 11 times (3)
less toxic to the normal lymphocytes (PMBC) than to the
leukemia cell line. We have found, however, that cisplatin is 70
times less toxic to PMBC than to Jurkat cell lines.34
Table 1. Selected Structural Parameters of Complex 3
Obtained from X-ray Single-Crystal Diffraction Studies
a
Pd(1)−C(18)
Pd(1)−O(1)
Pd(1)−N(1)
Pd(1)−O(2)
N(1)−P(1)
O(1)−C(2)
N(1)−C(12)
1.966(4)
2.023(3)
2.037(4)
2.094(3)
1.643(4)
1.289(6)
1.394(6)
C(18)−Pd(1)−O(1)
C(18)−Pd(1)−N(1)
O(1)−Pd(1)−N(1)
C(18)−Pd(1)−O(2)
O(1)−Pd(1)−O(2)
N(1)−Pd(1)−O(2)
C(2)−O(1)−Pd(1)
C(12)−N(1)−P(1)
C(12)−N(1)−Pd(1)
P(1)−N(1)−Pd(1)
92.08(15)
81.35(16)
173.02(13)
175.51(14)
91.49(12)
94.96(13)
123.3(3)
120.5(3)
C(18)−Pd(1)−O(1)
C(18)−Pd(1)−N(1)
92.08(15)
81.35(16)
115.8(3)
123.4(2)
a
Bond lengths in Å and angles in deg.
{C6H4(C(O)NP(m-tol)3-kC,N)-2}]2 (range 1.644−1.610
Å;28 1.644(2) Å),38 [Pd(C6H4-2-PPh2N-C(O)-2NC5H4-k-
C,N,N)Cl] (1.649(2) Å),37 or [PdCl2(TPAN-C(O)-2-
NC5H4)] (1.658(2) Å)34 but considerably longer than in
other semistabilized cyclopalladated complexes.37 This elonga-
tion of the P−N bond is due not only to the coordination of
the N atom to the palladium center but to the delocalization of
charge density through the P−N−C−O system due to the
presence of a CO group in the IM ligand.37
We also studied the activity of compounds 2 and 3 and
cisplatin against apoptosis-resistant cells (Figure 2). Multi-
2. Biological Activity. 2.1. Cytotoxicity Studies. Cytotox-
icity results for ligand 1 and the new organometallic
compounds (2−8) are collected in Table 2. The IC50 values
Table 2. IC50 (μM) of Cisplatin, Ligand 1, Phosphine Oxide
a
TPAO, and Metal Complexes 2−8 in Human Cell Lines
Jurkat
PBMC
DU-145
b
cisplatin
7.4 2.1
500 18
79.2 15.8
>500
TPAO
>500
>500
8.1 1.1
10.5 1.8
142
>500
>500
209 11
118 11
1
2
3
4
5
6
7
8
>500
49.3 3.1
96.2 12
135 27
187 16
318 20
299 25
448 38
Figure 2. Jurkat-CN or Jurkat-shBak cells were treated with 2 or 3 (50
μM) for 24 h, and PS exposure was evaluated by flow cytometry after
labeling with annexinV-PE. Co (control): cells cultured in the presence
of 1% DMSO.
9
186
>500
>500
9
89 18
322 29
101 20
374 40
298 14
>500
a
All compounds were dissolved in 1% DMSO and diluted with water
domain proapoptotic proteins Bax and Bak are essential for the
onset of mitochondrial permeabilization and apoptosis through
the intrinsic pathway. Importantly, we observed that com-
pounds 2 and, especially, 3 exhibited toxicity to cisplatin-
resistant Jurkat sh-Bak cells (Bax/Bak-deficient Jurkat cells).
We have demonstrated previously that cisplatin at 25 μM
caused the death of around 60% of Jurkat control (CN) cells,
while Jurkat-shBak cells were very resistant to cisplatin (less
than 8% death).33 Figure 2 depicts that 2 and 3 (at 50 μM)
caused the death of around 25% (2) and 75% (3) of Jurkat-
shBak cells. This could be an advantage over cisplatin, whose
activity is totally dependent on the presence of functional Bax
and Bak.
Bax-independent apoptosis has been proposed as the death
mechanism induced by a biphosphinic palladacycle complex in
K562 leukemic cells.27 Toxicity to Bax/Bak-deficient Jurkat
cells has also been found for iminophosphorane organogold-
(III)33 and coordination Pt(II) complexes.34 In contrast, the
coordination palladium compound [PdCl2(TPAN-C(O)-2-
NC5H4)] did not cause the death of Jurkat-shBak cells.34
Cyclopalladation seems to have a positive effect on the
before addition to cell culture medium for a 24 h incubation period.
Cisplatin was dissolved in water. Data are expressed as mean SD (n
b
= 3). Values obtained by the same MTT assay conditions (ref 34).
for the decomposition product of ligand 1 (TPAO) are also
collected for comparison purposes. The cytotoxicity (by a
modification of the MTT-reduction method; see Experimental
Section) was evaluated against two selected cell lines: human
Jurkat-T acute lymphoblastic leukemia cells and DU-145
human prostate cancer cells. Cells were incubated in the
presence of the compounds for 24 h. The sensitivity of T-cell
leukemia Jurkat cells to compounds cisplatin, 1−8, and TPA
O was compared to that of normal T-lymphocytes (peripheral
blood mononuclear cells: PBMC). Ligand 1 and the phosphine
oxide TPA had IC50 values greater than 500 μM in all cell lines
studied and were considered as nontoxic. The more cytotoxic
derivatives are the more lipophilic palladium complexes 2 and
3. This behavior has been also found for other cyclometalated
compounds.25a We expected that 4, with the chemoprotectant
dithiocarbamate ligand, would display a higher cytotoxicity (as
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cytotoxicity of IM-Pd complexes on cisplatin-sensitive and
cisplatin-resistant leukemia cell lines.
Human serum albumin is the most abundant carrier protein
in plasma and is able to bind a variety of substrates including
metal cations, hormones, and most therapeutic drugs. It has
been demonstrated that the distribution, the free concentration,
and the metabolism of various drugs can be significantly altered
as a result of their binding to the protein.46 HSA possesses
three fluorophores, these being tryptophan (Trp), tyrosine
(Tyr), and phenylalanine (Phe) residues, with Trp214 being
the major contributor to the intrinsic fluorescence of HSA. This
Trp fluorescence is sensitive to the environment and binding of
substrates or changes in conformation that can result in
quenching (either dynamic or static). The fluorescence spectra
of HSA in the presence of increasing amounts of ligand 1,
compounds 2−8, and cisplatin were recorded in the range
300−450 nm upon excitation of the tryptophan residue at 295
nm (Figure 4). The compounds caused a concentration-
2.2. Interactions of 2 and 3 with DNA and with Human
Serum Albumin. Since DNA replication is a key event for cell
division, it is among critically important targets in cancer
chemotherapy. Most cytotoxic platinum drugs form strong
covalent bonds with the DNA bases.40 However, a variety of
platinum compounds act as DNA intercalators upon coordina-
tion to the appropriate ancillary ligands.41 There are also
reports on palladium derivatives interacting with DNA in
covalent42,43 and noncovalent ways.22d,44 We performed
agarose gel electrophoresis studies on the effects of compounds
2 and 3 on plasmid (pBR322) DNA (Figure 3). This plasmid
has two main forms: OC (open circular or relaxed form, form
II) and CCC (covalently closed or supercoiled form, form I).
Figure 3. Electrophoresis mobility shift assays for cisplatin, ligand 1,
and compounds 2 and 3 (see Experimental Section for details). DNA
refers to untreated plasmid pBR322. a, b, and c correspond to metal/
DNA ratios of 0.25, 0.5, and 1.0, respectively.
Changes in electrophoretic mobility of both forms are usually
taken as evidence of metal−DNA binding. Generally, the larger
the retardation of supercoiled DNA (CCC, form I), the greater
the DNA unwinding produced by the drug.45 Binding of
cisplatin to plasmid DNA, for instance, results in a decrease in
mobility of the CCC form and an increase in mobility of the
OC form (see lanes a, b, and c for cisplatin in Figure 3).45
Treatment with increasing amounts of ligand 1 does not cause
any shift for either form, consistent with no unwinding or other
change in topology under the chosen conditions. Treatment
with increasing amounts of compound 3 has a similar effect.
Treatment with increasing amounts of compound 2 barely
retards the mobility of the faster-running supercoiled form
(form I) at high molar ratios (c) but to a much lesser extent
than the retardation produced by cisplatin. Organogold(III)
compounds containing iminophosphorane ligands did not
interact with DNA.32 Coordination iminophosphorane com-
pounds of platinum(II) and palladium(II) of the type
[MCl2(TPAN-C(O)-2-NC5H4)] showed a retardation of
the faster-running supercoiled form (form I) especially at high
molar ratios in a way different from that produced by
cisplatin.34
In conclusion, the experiments probing DNA−drug inter-
actions showed that the new cyclopalladated iminophosphor-
ane biologically active complexes have no (3) or very little (2)
interaction with plasmid (pBR322) DNA, pointing to an
alternative biomolecular target for these compounds. Mitocho-
drial damage by interaction of some cyclopalladated complexes
containing phosphines with membrane proteins26−28 and
inhibition of cathepsin B25,26 have been reported as the
plausible causes of their toxicity to cancer cell lines. In contrast,
orthometalated complexes with thiosemicarbazones were found
to have an enhanced capacity to form DNA interstrand cross-
links in comparison with cisplatin.22e,f
Figure 4. (a) Fluorescence titration curve of HSA with compound 2.
Arrow indicates the increase of quencher concentration. (b) Stern−
Volmer plot for quenching with ligand 1 and compounds 2, 3, and 5−
8 and cisplatin. In the measurements of the fluorescence titration curve
of HAS with compound 4 the appearance of a precipitate prevented us
from obtaining a valid reading, and therefore the Stern−Volmer plot
was not included in graph b.
dependent quenching of fluorescence without changing the
emission maximum or shape of the peaks (2, 3, 6−8), as seen
in Figure 4 for compound 3. For compound 5 a shift of the
emission maximum was observed (from 340 to 390 nm). All
these data indicate an interaction of the palladium compounds
with HSA. The fluorescence data were analyzed by the Stern−
Volmer equation. While a linear Stern−Volmer plot is
indicative of a single quenching mechanism, either dynamic
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Article
MBraun MOD System glovebox. Solvents were purified by use of a
PureSolv purification unit from Innovative Technology, Inc. The
substrates TPA and 4,7-diphenyl-1,10-phenanthrolinedisulfonic acid
disodium salt were purchased from Sigma-Aldrich, and Pd2(dba)3 was
purchased from Strem Chemicals and used without further
purification. Phosphine P(3-pyridyl)3 was prepared by a reported
method.48 Phosphine TPPTS P(mC6H4SO3Na)3 was generously
or static, the positive deviation observed in the plots of F0/F
versus [Q] of our compounds (Figure 4) is indicative of the
presence of different binding sites in the protein.47 In this graph
higher quenching by the iminophosphorane complexes was
observed compared to that of cisplatin under the chosen
conditions. This is probably an indication of the faster
hydrolysis in aqueous solution of our compounds compared
to that of cisplatin.
We have found a similar behavior for coordination
iminophosphorane complexes of d8 metals, for which we also
observed a concentration-dependent quenching.34 In the case
of [MCl2(TPAN-C(O)-2-NC5H4)] (M = Pd, Pt) isothermal
titration calorimetry34 showed two different binding inter-
actions, which explained the lack of linearity observed in the
fluorescence quenching studies, as the Stern−Volmer method
assumes all binding sites to be equivalent. We believe that a
similar behavior is observed for the cyclopalladated compounds
described here. For [PtCl2(TPAN-C(O)-2-NC5H4)] we
were able to demonstrate by circular dichroism studies that
its binding to HSA was reversed by addition of a chelating
agent (citric acid) and by a decrease in pH. In the case of the
palladium compound34 and compounds 2 and 3 described here,
the CD reversibility experiments were not conclusive. Further
analysis by a quantitative technique (atomic absorption
spectrometry) is under way since studies of the reversibility
of the binding of metallodrugs to HSA are very relevant to asses
their potential efficacy in vivo.
donated by Prof. Laszlo T. Mika (Eotvos University, Budapest,
́
́
̈
̈
Hungary). NMR spectra were recorded in a Bruker AV400 (1H NMR
at 400 MHz, 13C NMR at 100.6 MHz, 31P NMR at 161.9 MHz).
Chemical shifts (δ) are given in ppm using CDCl3, d6-DMSO, or D2O
as solvent, unless otherwise stated. H and 13C chemical shifts were
1
measured relative to solvent peaks considering TMS = 0 ppm; 31P{1H}
was externally referenced to H3PO4 (85%). Infrared spectra (4000−
250 cm−1) were recorded on a Nicolet 6700 FT-IR spectrophotometer
on KBr pellets. Elemental analyses were performed on a Perkin-Elmer
2400 CHNS/O analyzer, Series II. Mass spectra (ESI) were performed
on an Agilent analyzer or a Bruker analyzer. Conductivity was
measured in an OAKTON pH/conductivity meter in CH3CN,
acetone, or H2O solutions (10−3 M). Circular dichroism spectra
were recorded using a Chirascan CD spectrometer equipped with a
thermostated cuvette holder. Electrophoresis experiments were carried
out in a Bio-Rad Mini subcell GT horizontal electrophoresis system
connected to a Bio-Rad Power Pac 300 power supply. Photographs of
the gels were taken with an Alpha Innotech FluorChem 8900 camera.
Fluorescence intensity measurements were carried out on a PTI QM-
4/206 SE spectrofluorometer (PTI, Birmingham, NJ, USA) with right-
angle detection of fluorescence using a 1 cm path length quartz
cuvette.
Synthesis. TPAN-C(O)-2BrC6H4 (C,N-IM) (1). TPA (0.157 g, 1.0
mmol) and 2-bromobenzamide (0.200 g, 1.0 mmol) were placed in a
Schenck flask under nitrogen. Dry and degassed THF (15 mL) was
added, and to this solution was added dropwise tBuDAD (0.230 g, 1.0
mmol) in dry and degassed THF (4 mL) at 0 °C. The reaction was left
stirring to warm for 15 h. After this period the yellow reaction mixture
had become colorless. The solvent was reduced in vacuo to a minimum
(<2 mL), and Et2O (10 mL) was added, giving a white solid, which
was filtered off and dried in vacuo. Yield: 0.30 g (86%). Anal. Calcd for
C13H16BrN4OP (354.02): C, 43.96; H, 4.54; N, 15.77. Found: C,
43.81; H, 4.31; N, 15.93. MS (ESI+) [m/z]: 355.03 [M + H]+.
31P{1H} NMR: δ −31.2 (s, CDCl3), −29.8 (s, DMSO-d6), −25.1 (s,
CONCLUSION
■
We have prepared a new water-soluble iminophosphorane that
serves as an excellent precursor to iminophosphorane cyclo-
palladated complexes with varying hydrophilic:lipophilic ratios
stable in DMSO and DMSO−H2O solutions under biological
testing conditions. We found that the most lipophilic
compounds from the series, [Pd(μ-Br){C6H4(C(O)NTPA-
kC,N)-2}]2 (2) and [Pd{C6H4(C(O)NTPA-kC,N)-2}-
(acac)] (3), display higher cytotoxicity against T-Jurkat
leukemia cell while being less toxic to normal T-lymphocytes
(PBMC). The IC50 values were similar to those reported for
other cyclopalladated compounds with promising antitumor
properties and higher than an iminophosphorane coordination
palladium complex, [PdCl2(TPAN-C(O)-2-NC5H4)],34 pre-
viously described by us. Importantly, 2 and, especially, 3
showed toxicity against cisplatin-resistant Jurkat sh-Bak cells
(Bax/Bak-deficient Jurkat cells). This could be an advantage
over cisplatin, whose activity is totally dependent on the
presence of functional Bax and Bak. It was demonstrated that
these compounds have no (3) or very little (2) interaction with
plasmid (pBR322) DNA, pointing to an alternative biomo-
lecular target for their cytotoxic effect. All the compounds show
an interaction with human serum albumin faster than that of
cisplatin. We envision that an appropriate modification of IM
ligands and subsequent orthopalladation can afford new
cyclopalladated complexes with improved anticancer properties.
In addition, some of the water-soluble nontoxic organometallic
palladium complexes described here (such as [Pd{C6H4(C-
(O)NTPA-kC,N)-2}(L)Br] (L = P(mC6H4SO3Na)3 (6);
P(3-Pyridyl)3 (7)) could serve as precursors to stable
homogeneous catalysts in water.
1
3
D2O). H NMR (CDCl3): δ 7.66 (1H, d, JHH 7 Hz, H6, C6H4), 7.56
(1H, d, 3JHH 7 Hz, H33, C6H4), 7.28 (1H, dd, H5, 3JHH 8 Hz, 3JHH 3 Hz,
C6H4), 7.18 (1H, t, JHH 7 Hz, H4, C6H4), 4.34 (6H, d, JPH 4 Hz,
PCH2N), 4.40 (AB system, 6H, NCH2N). 13C{1H} NMR (CDCl3): δ
173.1 (s, CO), 133.5 (s, C6H4), 130.2 (s, C6H4), 129.8 (s, C6H4),
127.0 (s, C6H4), 72.8 (d, JPC 9 Hz, NCH2N), 52.5 (d, JPC 45 Hz,
PCH2N). IR (cm−1): ν 1574 (CO), 1340 (PN). Conductivity
(MeCN): Λ = 1.3 μS/cm. Solubility: 28 mM or 10 mg/mL (H2O).
[Pd(μ-Br){C6H4(C(O)NTPA-kC,N)-2}]2 (2). To a suspension of
Pd2(dba)3 (0.457 g, 0.50 mmol) in dry and degassed CH2Cl2 (15 mL)
was added iminophosphorane 1 (0.354 g, 1.0 mmol), and the resulting
mixture was stirred for 15 h at room temperature. The initial purple
suspension evolved to a yellowish solution with some Pd0 in
suspension, which was filtered over Celite. The clear yellow solution
was evaporated to a small volume (<2 mL), and Et2O (20 mL) was
added. By continuous stirring, 2 was obtained as a yellow solid.
Complex 2 was characterized by NMR as a mixture (1:1 molar ratio)
of the geometric cis and trans isomers. Yield: 0.170 g (82%). Anal.
Calcd for C26H32Br2N8O2P2Pd2 (922.86): C, 33.81; H, 3.49; N, 12.14.
Found: C, 34.33; H, 3.39; N, 12.13. Although these results are outside
the range viewed as establishing analytical purity, they are provided to
illustrate the best values obtained to date. The 31P, 13C, and 1H NMR
spectra of 2 appear in the Supporting Information. MS (ESI+) [m/z]:
945.09 [M + Na]+. 31P{1H} NMR: δ −17.2 and −17.3 (cis and trans
isomers, CDCl3), −16.2 and −13.0 (cis and trans isomers, DMSO-d6).
1H NMR (DMSO-d6): δ 7.68 (2H, br, H5), 7.11 (2H, br, H2), 7.01
(4H, br, H3+H4), 4.58 (12H, d, JPH 9 Hz, PCH2N), 4.30 (AB system,
12H, NCH2N). 13C{1H} NMR (DMSO-d6): δ 188.9 (s, CO), 143.3
(s, C1, C6H4), 134.8 (s, C2, C6H4), 130.5 (s, C6H4), 129.1 (s, C6H4),
EXPERIMENTAL SECTION
All manipulations involving air-free syntheses were performed using
standard Schlenk-line techniques under a nitrogen atmosphere or in a
■
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128.2 (s, C6H4), 125.4 (s, C6H4), 72.5 (d, 3JPC 9 Hz, NCH2N), 54.4 (d,
1JPC 41 Hz, PCH2N). IR (cm−1): ν 1634 (CO), 1294 (PN).
Conductivity (MeCN): Λ = 12.6 μS/cm. Solubility: 0.4 mM or 1.1
mg/mL (1:99 DMSO−H2O).
13C{1H} NMR (DMSO-d6): δ 181.2 (s, CO), 150.4 (d, J 11 Hz, C1,
C6H4), 148.6 (d, JPC 10 Hz, Ar bathoPhen), 136.7 (d, JPC 18 Hz, Ar
bathoPhen), 136.1 (d, J 14 Hz,), 133.0 (s, C2, C6H4), 132.1 (s, C6H4),
132.0 (s, Ar bathoPhen), 132.0 (s, C6H4), 131.6 (s, Ar bathoPhen),
131.3 (s, Ar bathoPhen), 130.7 (s, Ar bathoPhen), 129.8 (s, C6H4),
129.0 (s, Ar bathoPhen), 128.9 (s, C6H4), 128.9 (s, Ar bathoPhen),
128.2 (d, JPC 11 Hz, Ar bathoPhen), 124.2 (s, Ar bathoPhen), 71.5 (d,
3JPC 9 Hz, NCH2N), 54.2 (d, 1JPC 41 Hz, PCH2N). IR (cm−1): ν 1620
(CO), 1187 (SO3 anti), 1031 (SO3 syn). Conductivity (H2O): Λ =
145.3 μS/cm. Solubility: 1.0 mM or 0.90 mg/mL (H2O).
[Pd{C6H4(C(O)NTPA-kC,N)-2}(acac-O,O′)] (3). To a solution of 2
(0.060 g, 0.064 mmol) in CH2Cl2 (15 mL) was added Tl(acac) (0.039
g, 0.128 mmol). The resulting suspension was stirred for 1 h at rt and
then filtered over Celite. The clear solution was evaporated to dryness,
and the residue was treated with cold n-hexane (15 mL), giving 3 as an
off-white solid. It was filtered off and dried in vacuo. Yield: 0.034 g
(55%). Anal. Calcd for C18H23N4O3PPd (480.57): C, 44.95; H, 4.82;
N, 11.66. Found: C, 44.38; H, 4.56; N, 11.20. Although these results
are outside the range viewed as establishing analytical purity, they are
provided to illustrate the best values obtained to date. The 31P, 13C,
[Pd{C6H4(C(O)NTPA-kC,N)-2}{P(mC6H4SO3Na)3}Br] (6). TPPTS
(0.072 g, 0.128 mmol) was dissolved in MeOH (3 mL) and the
minimum amount of water to obtain a clear solution, which was then
added slowly to a solution of 2 (0.060 g, 0.064 mmol) in CH2Cl2 (7
mL). The resulting mixture was stirred for 3 h at rt, after which all
solvents were evaporated to dryness, and the residue was treated with
Et2O (15 mL), affording 6 as an off-white solid, which was filtered off
and dried in vacuo. Yield: 0.092 g (70%). Anal. Calcd for
C31H28BrN4Na3O10P2PdS3·6H2O: C, 32.72; H, 3.54; N, 4.92; S,
8.45. Found: C, 32.43; H, 3.45; N, 5.16; S, 8.07. MS (ESI+) [m/z]:
882.9577 [(M − H) − HBr] (M = C31H31BrN4O10P2PdS3). 31P{1H}
1
and H NMR spectra of 3 appear in the Supporting Information. MS
(ESI+) [m/z]: 481.0619 [M + H]+. 31P{1H} NMR (CDCl3): δ −18.6
1
3
(s). H NMR (CDCl3): δ 7.56 (1H, d, JHH 8 Hz, H6, C6H4), 7.29
(1H, d, 3JHH 8 Hz, H3, C6H4), 7.20 (1H, t, 3JHH 7 Hz, H5, C6H4), 7.06
(1H, t, JHH 8 Hz, H4, C6H4), 5.39 (s, CH, acac), 4.68 (6H, d, JPH
3
9
Hz, PCH2N), 4.44 (AB system, 6H, NCH2N), 2.06 (s, 3H, CH3, acac),
1.60 (s, 3H, CH3, acac). 13C{1H} NMR (CDCl3): δ 187.2 (s, CO,
acac), 187.1 (s, CO, acac), 146.4 (s, C1, C6H4), 139.2 (d, JPC 12 Hz,
1
NMR (DMSO-d6): δ 43.4 (s, TPPTS) and −14.0 (s, 1). H NMR
4
(DMSO-d6): δ 7.93 (3H, d, 3JHH 10 Hz, Ar TPPTS), 7.73 (3H, d, 3JHH
C2, C6H4), 130.6 (s, C6H4), 129.5 (s, C6H4), 127.2 (d, JPC 3 Hz,
3
3
C6H4), 124.4 (s, C6H4), 100.3 (s, CH, acac), 71.4 (d, JPC 10 Hz,
8 Hz, Ar TPPTS), 7.62 (3H, t, JHH 8 Hz, Ar TPPTS), 7.42 (3H, t,
1
3JHH 6 Hz, Ar TPPTS), 7.27 (1H, d, 3JHH 7 Hz, H6, C6H4), 6.84 (1H, t,
NCH2N), 52.2 (d, JPC 39 Hz, PCH2N), 29.2 (s, Me, acac), 28.0 (s,
Me, acac). IR (cm−1): ν 1617 (CO), 1570 (acac), 1518 (acac), 1302
(PN). Conductivity (MeCN): Λ = 2.1 μS/cm. Solubility: 0.8 mM
or 1.2 mg/mL (1:99 DMSO−H2O).
3
3
3JHH 7 Hz, C6H4), 6.51 (1H, t, JHH 7 Hz, C6H4), 6.32 (1H, t, JHH
7
Hz, C6H4), 4.92 (6H, d, JPH 10 Hz, PCH2N), 4.36 (6H, s, NCH2N).
13C{1H} NMR (DMSO-d6): δ 181.2 (s, CO), 150.5 (s, C1, C6H4),
148.2 (d, JPC 10 Hz, Ar TPPTS), 140.9 (d, JPC 18 Hz, C2, C6H4), 136.7
(d, JPC 18 Hz, Ar TPPTS), 136.0 (d, JPC 13 Hz, Ar TPPTS), 131.6 (s,
Ar TPPTS), 131.3 (s, C6H4), 131.2 (s, C6H4), 128.8 (s, Ar TPPTS),
128.6 (s, C6H4), 128.1 (d, JPC 16 Hz, Ar TPPTS), 124.2 (s, C6H4),
71.4 (d, JPC 15 Hz, NCH2N), 54.2 (d, JPC 41 Hz, PCH2N). IR (cm−1):
ν 1613 (CO), 1309 (PN), 1198 (SO3 anti), 1038 (SO3 syn).
Conductivity (H2O): Λ = 241.3 μS/cm. Solubility: 0.9 mM or 0.9 mg/
mL (H2O).
[Pd{C6H4(C(O)NTPA-kC,N)-2}{P(3py)3}Br] (7). To a solution of 2
(0.050 g, 0.054 mmol) in CH2Cl2 (7 mL) in a Schlenck flask under
nitrogen was added tris-3-pyridylphosphine (0.029 g, 0.109 mmol) in
CH2Cl2 (1 mL). The reaction mixture was stirred at rt for 2 h, after
which the solvent was reduced to a minimum and Et2O was added,
affording 7 as a pale brown solid that was filtered off and dried in
[Pd{C6H4(C(O)NTPA-kC,N)-2}(S2CNMe2)] (4). To a suspension of
2 (0.050 g, 0.054 mmol) in MeOH (35 mL) was added sodium
dimethyldithiocarbamate as a solid. The suspension changed
immediately from yellow to colorless. After stirring for 1 h at room
temperature, the solvent was removed in vacuo to dryness. Acetone
(35 mL) was added, and the resulting suspension was filtered through
Celite to remove the NaBr formed. The solvent was reduced in vacuo
to a minimum (∼2 mL), and Et2O (15 mL) was added, affording an
off-white solid (4), which was filtered off and dried in vacuo. Yield:
0.047 g (79%). Anal. Calcd for C16H22N5OPPdS2·2H2O: C, 35.72; H,
4.87; N, 13.02. Found: C, 36.19; H, 4.33; N, 12.88. MS (ESI+) [m/z]:
502.02 [M + H]+ (calcd for C16H22N5OPPdS2 (501.00)). 31P{1H}
NMR: δ −12.9 (s, DMSO-d6), −15.4 (s, acetone-d6). 1H NMR
(acetone-d6): δ 7.27 (1H, d, 3JHH 7 Hz, H6, C6H4), 7.09 (1H, t, 3JHH
7
3
3
Hz, C6H4), 7.02 (1H, t, JHH 7 Hz, C6H4), 7.37 (1H, d, JHH 7 Hz,
C6H4), 4.48 (6H, d, JPH 9 Hz, PCH2N), 4.37 (AB system, 6H,
NCH2N), 3.29 (3H, s, Me), 3.25 (3H, s, Me). 13C{1H} NMR
(acetone-d6): δ 207.9 (s, S2CN), 184.0 (s, CO), 151.0 (s, C1, C6H4),
138.5 (d, JPC 14 Hz, C2, C6H4), 132.2 (s, C6H4), 131.1 (s, C6H4),
v a c u o . Y i e l d : 0 . 0 7 0
g
( 8 7 % ) . A n a l . C a l c d f o r
C28H28BrN7OP2Pd·0.5Et2O: C, 47.17; H, 4.35; N, 12.84. Found: C,
47.51; H, 4.14; N, 12.90. MS (ESI+) [m/z]: 646.0867 [(M + H) −
HBr] (calcd for C28H28BrN7OP2Pd (725.0049)). 31P{1H} NMR: δ
−15.9 (s, 1) and 30.9 (br, PPy3) (CDCl3), −14.1 (s, 1) and 29.0 (br,
PPy3) (DMSO-d6). 1H NMR (CDCl3): δ 8.88 (3H, d, 3JHH 4 Hz, Py),
8.73, (3H, s), 8.07 (3H, t, 3JHH 9 Hz, Py), 7.51 (1H, d, 3JHH 8 Hz, H6,
C6H4), 7.38 (3H, t, 3JHH 4 Hz, Py), 6.96 (1H, t, 3JHH 7 Hz, C6H4), 6.60
(1H, t, 3JHH 8 Hz, C6H4),, 6.27 (1H, d, 3JHH 8 Hz, C6H4), 4.95 (6H, d,
JPH 9 Hz, PCH2N), 4.43 (6H, s, NCH2N). 13C{1H} NMR (CDCl3): δ
158.2 (s, PPy3), 154.4 (d, 1JPC 11 Hz, PPy3), 151.9 (s, PPy3), 150.5 (s,
C1, C6H4), 142.5 (s, C2, C6H4), 136.4 (s, PPy3), 131.0 (s, C6H4), 129.7
(s, C6H4), 124.7 (s, C6H4), 123.4 (s, C6H4), 123.2 (s, PPy3), 72.5 (d,
3JPC 10 Hz, NCH2N), 55.1 (d, 1JPC 42 Hz, PCH2N). IR (cm−1): ν 1640
(CO), 1297 (PN). Conductivity (MeCN): Λ = 11.7 μS/cm.
Solubility: 0.5 mM or 1.0 mg/mL (H2O).
3
128.1 (s, C6H4), 123.7 (s, C6H4), 70.9 (d, JPC 10 Hz, NCH2N), 51.6
(d, 1JPC 41 Hz, PCH2N), 39.4 (s, Me), 39.1 (s, Me). IR (cm−1): ν 1610
(CO), 1294 (PN). Conductivity (acetone): Λ = 1.7 μS/cm.
Solubility: 0.7 mM or 1.0 mg/mL (1:99 DMSO−H2O).
[Pd{C6H4(C(O)NTPA-kC,N)-2}{C12H6N2(C6H4SO3Na)2}] (5). Sulfo-
nated bathophenanthroline (0.059 g, 0.10 mmol) was dissolved in
MeOH (3 mL) and the minimum amount of water to obtain a clear
solution, which was then added slowly to a solution of 2 (0.046 g,
0.050 mmol) in CH2Cl2 (7 mL). The resulting mixture was stirred for
1 h at rt, after which all solvents were evaporated to dryness. The
resulting residue was redissolved in MeOH and filtered through Celite
to remove the byproduct NaBr. The solvent was removed in vacuo, and
the resulting pale yellow residue 5 was washed with Et2O, filtered off,
and dried in vacuo. Yield: 0.072 g (78%). Anal. Calcd for
C39H38N6NaO7PPdS2·7H2O: C, 44.47; H, 4.13; N, 7.98; S, 6.09.
Found: C, 44.63; H, 3.72; N, 7.80; S, 6.80. MS (ESI+) [m/z]: 737.20
[M + Na]+ (calcd for M = C37H32N6OPPd (714.08)). 31P{1H} NMR
[Pd{C6H4(C(O)NTPA)-2}(TPA)2Br] (8). To a solution of 2 (0.060 g,
0.065 mmol) in CH2Cl2 (7 mL) was added TPA (0.041 g, 0.261
mmol) as a solid. The reaction was left stirring at rt for 30 min, after
which the solvent was reduced to a minimum and Et2O was added,
affording an off-white solid (8), which was filtered off and dried in
1
v a c u o . Y i e l d : 0 . 0 8 2
g
( 8 2 % ) . A n a l . C a l c d f o r
(DMSO-d6): δ −26.0 (s). H NMR (DMSO-d6): δ 9.29 (2H, s, Ar
C25H40BrN10OP3Pd·CH2Cl2 (774.0818): C, 36.28; H, 4.92; N,
16.27. Found: C, 36.74; H, 5.18; N, 16.04. MS (ESI+) [m/z]:
776.08 [M + H]+ (calcd for C25H40BrN10OP3Pd (775.8918)). 31P{1H}
NMR: δ −30.5 (s, 1) and −68.7 (br, TPA) (CDCl3). 1H NMR
bathoPhen), 8.21 (2H, s, Ar bathoPhen), 8.07 (2H, s, Ar bathoPhen),
7.99 (2H, s, Ar bathoPhen), 7.91 (2H, s, Ar bathoPhen), 7.70 (2H, s,
Ar bathoPhen), 7.41 (1H, d, 3JHH 7 Hz, H6, C6H4), 7.14 (1H, d, 3JHH
Hz, H3, C6H4), 7.03 (1H, t, 3JHH 5 Hz, H5, C6H4), 6.93 (1H, t, 3JHH
7
5
3
Hz, H4, C6H4), 4.47 (6H, d, JPH 9 Hz, PCH2N), 4.42 (6H, s, NCH2N).
(CDCl3): δ 7.87 (1H, dd, JHH 8, JPH 2 Hz, H6, C6H4), 8.22 (1H, d,
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3
3JHH 7 Hz, H3, C6H4), 7.14 (1H, td, JHH 7 Hz, JPH 2 Hz, H5, C6H4),
7.03 (1H, d, 3JHH 8 Hz, H4, C6H4), 4.45 (AB system, 6H, NCH2N 1),
4.39 (6H, d, JPH 8 Hz, PCH2N 1), 4.35 (12 H, virtual t, PCH2N TPA),
3.89 (12H, s, NCH2N TPA). 13C{1H} NMR (CDCl3): δ 180.7 (d, JPC
9 Hz, CO), 148.8 (s, C1, C6H4), 141.3 (s, JPC 16 Hz, C2, C6H4),
137.3 (s, C6H4), 130.7 (s, C6H4), 130.5 (s, C6H4), 123.7 (s, C6H4),
72.1 (s, NCH2N, TPA), 72.7 (d, 3JPC 9 Hz, NCH2N), 53.2 (d, 1JPC 44
Hz, PCH2N), 51.1 (s, PCH2N, TPA). IR (cm−1): ν 1575 (CO),
1321 (PN). Conductivity (MeCN): Λ = 22.0 μS/cm. Solubility: 0.8
mM or 1.2 mg/mL (H2O).
(10 μM) in phosphate buffer (pH = 7.39), the fluorescence being
measured 240 s after each addition. The data were analyzed using the
classical Stern−Volmer equation: F0/F = 1 + KSV[Q].
ASSOCIATED CONTENT
* Supporting Information
■
S
1H and 13C{1H} NMR spectra of compounds 2 and 3. Studies
on the stability of ligand 1 and compounds 2−8 in d6-DMSO
solution and d6-DMSO−D2O (50:50) mixtures over time by
31P{1H} NMR spectroscopy and selected 31P{1H} NMR
spectra of the decomposition of ligand 1 and compound 5
over time. Table with the crystal data and structure refinement
for complex 3. CIF file for the X-ray crystal structure of
compound 3. This material is available free of charge via the
Internet at http://pubs.acs.org.
X-ray Crystallography. Single crystals of 3 (see details in Table
S2 in the Supporting Information) were mounted on a glass fiber in a
random orientation. Data collection was performed at room
temperature on a Kappa CCD diffractometer using graphite-
monochromated Mo Kα radiation (λ = 0.71073 Å). Space group
assignments were based on systematic absences, E statistics, and
successful refinement of the structures. The structures were solved by
direct methods with the aid of successive difference Fourier maps and
were refined using the SHELXTL 6.1 software package. All non-
hydrogen atoms were refined anisotropically. Hydrogen atoms were
assigned to ideal positions and refined using a riding model. Details of
the crystallographic data are given in Table 2. These data can be
obtained free of charge from The Cambridge Crystallographic Data
Center via www.ccdc.cam.ac.uk/data_request/cif (CCDC 887963) or
in the Supporting Information. 3: Crystals of 3 (colorless prisms with
approximate dimensions 0.14 × 0.16 × 0.22 mm) were obtained from
a solution of 3 in CHCl3−n-hexane by slow diffusion of Et2O at rt.
Determination of Compound Cytotoxicity. The human T-cell
leukemia Jurkat (clone E6.1) cells from the ATCC collection and the
prostate carcinoma DU-145 cells were routinely cultured in RPMI
1640 medium supplemented with 5% FCS, L-glutamine, and
penicillin/streptomycin (hereafter, complete medium). Blood samples
from healthy donors were obtained from the Banco de Sangre y
AUTHOR INFORMATION
Corresponding Author
*Phone: +1-7189515000, ext 2833. Fax: +1-7189514607. E-
mail: mariacontel@brooklyn.cuny.edu.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the financial support of a grant from the National
Institute of General Medical Sciences (NIGMS,
SC2GM082307, M.C.) and a grant from the Spanish Ministry
of Science and Innovation (SAF2010-14920, I.M.). We thank
Dr. Esteban P. Urriolabeitia for useful discussions on the
synthesis of ligand 1 and undergraduate LSAMP student Farrah
Benoit for her assistance with some of the fluorescence
Tejidos de Aragon
́
. All subjects gave written informed consent, and the
approved the study. Normal T-
Ethical Committee of Aragon
́
lymphocytes (peripheral blood mononuclear cells, PBMC) were
freshly isolated by Ficoll-Paque density centrifugation. After isolation,
normal PBMC were kept in RPMI 1640 medium supplemented with
10% decomplemented FCS, L-glutamine, and penicillin/streptomycin.
Jurkat cells lacking Bak were generated using RNA interference
techniques, as described previously.49 For toxicity assays, Jurkat cells
(5 × 105 cells/mL), DU145 cells (1 × 105 cells/mL), or normal T-
lymphocytes PBMC (3 × 106 cells/mL) were seeded in flat-bottom
96-well plates (100 μL/well) in complete medium. DU145 cells were
allowed to attach prior to addition of cisplatin or tested compounds.
Compounds were added at different concentrations in triplicate. Cells
were incubated with cisplatin or compounds for 24 h, and then cell
proliferation was determined by a modification of the MTT-reduction
method.50 Total cell number and cell viability were determined by the
Trypan-blue exclusion test.
Interaction of 1 and Metal Complexes 2 and 3 with Plasmid
(pBR322) DNA by Electrophoresis (Mobility Shift Assay).
Aliquots (10 μL) of pBR322 plasmid DNA (20 μg/mL) in buffer (5
mM Tris/HCl, 50 mM NaClO4, pH = 7.39) were incubated with
molar ratios between 0.25 and 4.0 of the compounds (1−3) at 37 °C
for 20 h in the dark. Samples of free DNA and cisplatin-DNA adduct
were prepared as controls. After the incubation period, 2 μL of loading
dye was added to the samples, and 7 μL of these mixtures was finally
loaded onto the 1% agarose gel. The samples were separated by
electrophoresis for 1.5 h at 80 V in Tris-acetate/EDTA buffer.
Afterward, the gel was stained for 30 min with a solution of GelRed
nucleic acid stain.
́ ́
spectroscopy measurements. We thank Prof. Laszlo T. Mika for
his generous donation of phosphine TPPTS
P(mC6H4SO3Na)3.
DEDICATION
■
This paper is dedicated to Prof. Paul G. Pringle on the occasion
of his 55th birthday.
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