Exploring the scope of [Pt2(4-FC6H4) 4(μ-SEt2)2] as a precursor for new organometallic platinum(II) and platinum(IV) antitumor agents

Article abstract of DOI:10.1021/om5000908

The new compound [Pt₂(4-FC₆H₄) ₄(μ-SEt₂)₂] (A) was prepared and fully characterized. The reactions of compound A with ligands ArCH=NCH ₂CH₂NMe₂ (Ar = 2-BrC₆H₄, 1a; 2,6-Cl₂C₆H₃, 1b; 4-ClC₆H ₄, 1c; 2-Cl,6-FC₆H₃, 1d) were studied under different conditions and produced platinum(II) compounds [Pt(4-FC ₆H₄)₂(ArCH=NCH₂CH ₂NMe₂)] (2b-2d), containing a bidentate [N,N′] ligand, as well as cyclometalated platinum(IV) or platinum(II) compounds such as [PtBr(4-FC₆H₄)₂(C₆H ₄CH=NCH₂CH₂NMe₂)] (4a) or [PtCl{(3-FC₆H₃)(2-XC₆H₃)CH=NCH ₂CH₂NMe₂}] (5b: X = Cl; 5d: X = F), containing a tridentate [C,N,N′] ligand and either a five (4a) or a seven (5b, 5d) membered metallacycle. These compounds exhibit a great antiproliferative activity against non-small lung cancer cells (A549), and the best result was obtained for compound 2c (IC₅₀ = 0.3 ± 0.1 μM). While compounds 5 alter the mobility of plasmid DNA in a similar way to cisplatin, compound 4 was less efficient in removing the supercoils from DNA. In spite of the very low IC₅₀ value obtained for compound 2c, this compound does not interact with DNA, and it is neither an intercalator nor a topoisomerase I inhibitor.

Full text of DOI:10.1021/om5000908

Article
pubs.acs.org/Organometallics
Exploring the Scope of [Pt2(4-FC6H4)4(μ-SEt2)2] as a Precursor for New
Organometallic Platinum(II) and Platinum(IV) Antitumor Agents
Anna Escola,† Margarita Crespo,*,†,‡ Josefina Quirante,*,‡,§ Roldan Cortes,⊥ Anusha Jayaraman,⊥
̀
́
́
Josefa Badía,‡,∥ Laura Baldoma,‡,∥ Teresa Calvet,# Merce Font-Bardía,#,△ and Marta Cascante‡,⊥
̀
̀
†Departament de Química Inorgan
‡Institut de Biomedicina (IBUB), Universitat de Barcelona, Barcelona, Spain
§Laboratori de Química Organ
̀
ica, Facultat de Farmacia, Universitat de Barcelona, Avenida Joan XXIII, s/n, 08028 Barcelona, Spain
̀
ica, Facultat de Química, Universitat de Barcelona, Diagonal 645, 08028 Barcelona, Spain
̀
⊥Department of Biochemistry and Molecular Biology, Faculty of Biology, Universitat de Barcelona, and IDIBAPS, Unit Associated
with CSIC, Diagonal 643, 08028 Barcelona, Spain
∥Departament de Bioquímica i Biologia Molecular, Facultat de Farmac
̀
ia, Avenida Joan XXIII s/n, 08028 Barcelona, Spain
#Departament de Cristal·lografia, Mineralogia i Dipos
̀
its Minerals, Facultat de Geologia, Universitat de Barcelona, Martí i Franques
̀
s/n, 08028 Barcelona, Spain
△Unitat de Difraccio
Barcelona, Spain
́ ̀ ́
de Raigs-X, Centre Científic i Tecnologic de la Universitat de Barcelona (CCiTUB), Sole i Sabarís, 1-3, 08028
S
* Supporting Information
ABSTRACT: The new compound [Pt2(4-FC6H4)4(μ-SEt2)2] (A) was
prepared and fully characterized. The reactions of compound A with
ligands ArCHNCH2CH2NMe2 (Ar = 2-BrC6H4, 1a; 2,6-Cl2C6H3, 1b;
4-ClC6H4, 1c; 2-Cl,6-FC6H3, 1d) were studied under different
conditions and produced platinum(II) compounds [Pt(4-
FC6H4)2(ArCHNCH2CH2NMe2)] (2b−2d), containing a bidentate
[N,N′] ligand, as well as cyclometalated platinum(IV) or platinum(II)
compounds such as [PtBr(4-FC6H4)2(C6H4CHNCH2CH2NMe2)]
(4a) or [PtCl{(3-FC6H3)(2-XC6H3)CHNCH2CH2NMe2}] (5b: X =
Cl; 5d: X = F), containing a tridentate [C,N,N′] ligand and either a five
(4a) or a seven (5b, 5d) membered metallacycle. These compounds
exhibit a great antiproliferative activity against non-small lung cancer cells
(A549), and the best result was obtained for compound 2c (IC50 = 0.3
0.1 μM). While compounds 5 alter the mobility of plasmid DNA in a similar way to cisplatin, compound 4 was less efficient in
removing the supercoils from DNA. In spite of the very low IC50 value obtained for compound 2c, this compound does not
interact with DNA, and it is neither an intercalator nor a topoisomerase I inhibitor.
very promising outcomes. In these compounds, the presence of
a σ(Pt−C) bond increases the stability of the complexes, thus
allowing them to reach the cell unaltered. Furthermore, the
aromatic groups in the cyclometalated ligand might favor
intercalative binding to DNA through π−π stacking,7 while the
labile positions in the coordination sphere of the platinum atom
favor covalent coordination to DNA as for cisplatin. Therefore
a high cytotoxic activity may result from the combined effect of
intercalation and platination operating for cyclometalated
platinum compounds.5,6a On the other hand, platinum(IV)
complexes, able to produce Pt(II) species by reductive
elimination8 or photoactivation,9 offer several potential
advantages. They are stable enough to be administered orally,
their stability should result in diminished side effects, and they
INTRODUCTION
■
Nowadays platinum(II) complexes (cis-, carbo-, and oxalipla-
tin) dominate the field of metal-based chemotherapy in
worldwide cancer treatment protocols.1 However, major
limitations of these drugs are (i) dose-limiting severe toxicities,
(ii) poor bioavailability, and (iii) intrinsic or acquired
resistance.2,3 As a consequence, different approaches have
emerged to improve the cytotoxic profile of these anticancer
platinum compounds.4 Relevant strategies are focused on (i)
the stabilization of the Pt(II) ion in the complexes, (ii) the
design of Pt(IV) complexes as prodrugs, and (iii) the
exploitation of the promising properties associated with
organometallic compounds based not only on platinum but
also on other metal ions such as palladium, ruthenium, gold,
copper, or iron. Cyclometalated platinum(II) complexes
containing either bidentate [C,N]5 or terdentate [C,N,N′]6
ligands have been recently screened against tumor cells with
Received: January 24, 2014
Published: March 18, 2014
© 2014 American Chemical Society
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are amenable to structural modifications via the axial ligands,
which can be used to improve their pharmacological properties.
In recent years we have been involved in the use of
diarylplatinum(II) complexes as precursors in the synthesis of
[C,N,N′] cyclometalated platinum(II) and platinum(IV)
compounds10,11 and the study of the mechanisms involved in
these processes.12,13 In particular, along these studies, a novel
class of seven-membered platinacycles has been obtained in a
reaction involving formation of a Caryl−Caryl bond, and these
compounds were shown to display a remarkable antiprolifer-
ative activity, even greater than cisplatin, in several human
cancer cell lines.6a In order to further explore this area, we
undertook a project aimed at the preparation of a new
precursor, [Pt2(4-FC6H4)4(μ-SEt2)2] (A), analogous to com-
pound [Pt2(4-MeC6H4)4(μ-SEt2)2] previously used as metal-
ating agent.11 Binuclear compound A, upon reaction with
adequate dinitrogen ligands, should produce new series of
organometallic platinum complexes potentially useful as
antitumor agents. In addition, the presence of a fluoro
substituent in the aryl ligands of compound A should allow
an analysis on the importance of the electronic effects in the
subsequent reactions. Interestingly, several examples of bio-
logically active platinum fluoroaryl compounds have been
Figure 1. Molecular structure of compound A. Hydrogen atoms have
been omitted for clarity. Selected bond lengths (Å) and angles (deg)
with estimated standard deviations: Pt(1)−C(7): 2.006(8); Pt(1)−
C(1): 2.015(7); Pt(1)−S(2): 2.3504(16); Pt(1)−S(1): 2,3581(19);
Pt(2)−C(19): 2,029(6); Pt(2)−C(13): 2,039(7); Pt(2)−S(2):
2.3545(18); Pt(2)−S(1): 2.3705(16); C(7)−Pt(1)−C(1): 89.8(3);
C(7)−Pt(1)−S(2): 96.11(11); C(1)−Pt(1)−S(1): 93.6(2); S(2)−
Pt(1)−S(1): 80.45(6); C(19)−Pt(2)−C(13): 91.2(3); C(19)−
Pt(2)−S(2): 94.1(2); C(13)−Pt(2)−S(1): 94.53(18); S(2)−Pt(2)−
S(1): 80.11(6).
14
previously reported.5l‑o, Moreover, the presence of the NMR-
active 19F nucleus will provide an additional spectroscopic
handle to characterize the obtained compounds.15,16 An
additional interest in this system relies on the fact that fluoro
substituents may enhance the binding efficacy and selectivity in
pharmaceuticals.17
para-fluorophenyl groups form dihedral angles of 85.9(3)°,
78.6(3)°, 66.3(3)°. and 84.9(3)° with the Pt2S2 plane.
Ligands ArCHNCH2CH2NMe2 (Ar = 2-BrC6H4, 1a; 2,6-
Cl2C6H3, 1b; 4-ClC6H4, 1c; 2-Cl,6-FC6H3, 1d) were selected
for this study since the presence of two nitrogen atoms allows
formation of a [N,N′]-chelate upon coordination to platinum
and further reaction should produce cyclometalated platinum-
(II) or platinum(IV) compounds in agreement with previous
studies for similar systems.20 The presence of different
substituents in the ortho positions of the aryl ring (Br, Cl, or
H) is determinant in the reaction pathway and the nature of the
final products of such reactions.
The results presented here include the synthesis of the new
dimer A, which was found to be an adequate precursor for the
synthesis of several organoplatinum compounds such as
diarylplatinum(II) compounds containing a bidentate [N,N′]
ligand (2b−2d) and cyclometalated platinum(II) (5b, 5d) and
platinum(IV) (4a) compounds containing a terdentate
[C,N,N′] ligand. Their antiproliferative activity against the
A549 human lung cancer cell line has been investigated by
means of the MTT colorimetric assay. Additionally, electro-
phoretic DNA migration studies, in the absence and in the
presence of topoisomerase I, have been performed, in order to
get further insights into the biological behavior of the
synthesized compounds.
The reactions of compound A with ligands 1a−1d are
summarized in Scheme 1. The reaction of [Pt2(4-FC6H4)4(μ-
SEt2)2] (A) with imines 1b−1d carried out in toluene at room
temperature produced compounds of general formula [Pt(4-
FC6H4)2(ArCHNCH2CH2NMe2)] (2b−2d). For 1a, the
C−Br bond in the ortho position is easily activated at room
temperature to produce cyclometalated platinum(IV) com-
pound [PtBr(4-FC6H4)2(C6H4CHNCH2CH2NMe2)] (4a).
For this reason, compound 2a was not isolated, although the
reaction is expected to proceed through this species in
agreement with previous mechanistic studies for related
systems.13 Compounds 2b−2d were characterized by usual
techniques, and in addition, 2d was also characterized
RESULTS AND DISCUSSION
■
Synthesis and Characterization of Platinum Com-
pounds. Although the synthesis of compound cis-[Pt(4-
FC6H4)2(SMe2)2] has been recently reported,18 our target
compound A has not been described so far. The dinuclear
compound [Pt2(4-FC6H4)4(μ-SEt2)2] (A) was obtained from
the reaction of cis-[PtCl2(SEt2)2] with 4-fluorophenyllithium,
prepared in situ, and was fully characterized including single-
crystal X-ray diffraction analysis (Figure 1). Suitable crystals of
compound A were grown from dichloromethane−methanol at
room temperature as the solvate A·CH2Cl2. The crystal
structure is composed of discrete molecules held together by
van der Waals interactions. Both platinum atoms display
square-planar coordination geometry, while the sulfur atoms are
tetrahedrically bonded to both platinum centers and two ethyl
groups. Bond parameters are similar to those found for the
analogous compound [Pt2(4-MeC6H4)4(μ-SEt2)2], whose
structure has been previously reported.19 In particular, the
Pt(1)···Pt(2) separation is 3.605 Å. The mean planes of the
1
crystallographically. The H NMR spectra of compounds 2b
and 2c display only one group of signals, which was assigned, in
3
agreement with the observed J(H−Pt) values for the imine
hydrogen (ca. 50 Hz), to the E isomer. In contrast, compound
2d was obtained as a mixture of Z and E isomers, as readily
deduced from the 1H and 19F NMR spectra. This result
prompted us to monitor by 1H NMR spectroscopy the stability
of compounds 2b and 2c in CDCl3 solution. While compound
2c was stable in solution as the E isomer, compound 2b gave
after several hours at room temperature a mixture of three
compounds, as evidenced by the three resonances observed in
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a
Scheme 1. Synthesis of Coordination and Cyclometalated Platinum Compounds from Precursor A
a
Conditions: (i) toluene, room temperature, 4 h; (ii) toluene, reflux, 6 h.
the imine region. These were assigned, based on the chemical
shifts and the J(H−Pt) values, to isomer E of compound 2b
reported for analogous compounds. In particular, the angles
CPtC and CPtN are close to 90°, while the bite angle NPtN is
80.62°. The chelate ring is nearly coplanar with the
coordination plane (dihedral angle 10.5(2)°), and the para-
fluorophenyl groups trans to the imine and to the amine are
tilted 89.6(3)° and 64.4(3)°, respectively, to that plane. This
arrangement allows for an intramolecular π−π stacking21
between the C7−C12 and the C16−C21 rings, the distance
being 3.663(4) Å.
3
initially present, to the Z isomer (δ = 8.17 ppm, 3J(H−Pt) = 28
Hz), and to the platinum(IV) species 4b (δ = 9.29 ppm, 3J(H−
Pt) = 48 Hz) arising from intramolecular C−Cl bond
activation. After several days at room temperature, 4b was
the major component, the ratio E:Z was 1:2.2, and a new peak
corresponding to 5b was also observed. Formation of the Z
isomer releases the steric congestion by placing the aryl group
away from the platinum atom; however, formation of
compound 4b takes place from the E isomer. These results
support the reaction sequence shown in Scheme 2, in
agreement with previously reported mechanisms for analogous
systems.12,13
As stated above, the reaction of precursor A with ligand 1a
produced a cyclometalated platinum(IV) compound containing
a terdentate [C,N,N′] cyclometalated ligand. Compound 4a
was fully characterized including X-ray molecular structure
determination of the crystals grown in dichloromethane−
methanol. Multinuclear (1H, 19F, 13C, and 195Pt) NMR spectra
indicated formation of a single isomer, and bidimensional
1H−1H COSY and NOESY experiments allowed the assign-
Suitable crystals of compound 2d were grown in dichloro-
methane−methanol at room temperature. The crystal structure
is composed of discrete molecules held together by van der
Waals forces. The molecular structure (Figure 2) consists of the
E isomer, and bond distances and angles are similar to those
1
ment of all signals observed in the H NMR spectrum. The
presence of eight cross-peaks in the aromatic region of the
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Scheme 2. Isomerization and Reactivity of Compound 2b
Figure 3. Molecular structure of compound 4a. Hydrogen atoms have
been omitted for clarity. Selected bond lengths (Å) and angles (deg)
with estimated standard deviations: Pt−C(1): 2.001(6); Pt−C(12):
2.051(6); Pt−C(18): 2.060(7); Pt−Br: 2.5919(6); Pt−N(1):
2.082(5); Pt−N(2): 2,337(5); N(1)−C(7): 1.272(8); N(1)−C(8):
1.455(8); N(2)−C(9): 1.506(8); C(8)−C(9): 1.515(9); C(1)−Pt−
C(12): 95.6(2); C(1)−Pt−N(1): 81.1(2); C(12)−Pt−N(2):
104.9(2); N(1)−Pt−N(2): 78.6(2); C(1)−Pt−C(18):92.9(2);
C(12)−Pt−C(18): 87.3(2); C(18)−Pt−N(1): 90.7(2); C(18)−Pt−
N(2): 93.0(2); C(1)−Pt−Br: 84.76(17); C(12)−Pt−Br: 91.54(17);
N(1)−Pt−Br: 90.29(15); N(2)−Pt−Br: 89.66(13).
toluene. As reported for analogous systems,10,11 compound 2c
is expected to produce under these conditions a five-membered
cyclometalated platinum(II) compound, 3c. In the present case,
1
this compound could only be characterized in solution by H
and 19F NMR spectra, but could not be isolated in a pure form.
Residual amounts of the coordination compound 2c were
present after a reaction time of six hours under reflux. Attempts
to achieve full conversion of 2c into 3c using longer reaction
times as well as attempts to purify compound 3c were
unsuccessful and result in decomposition with formation of
metallic platinum. Formation of compounds analogous to 3c
has been reported, along with reductive elimination of either
benzene or toluene, from complexes such as cis-
[PtPh2(SMe2)2]10,25 or [Pt2(4-MeC6H4)4(μ-SEt2)2].11 In addi-
tion several compounds of general formula [PtAr2L2] (L =
dmso or SMe2) have been used as metalating agents in the
synthesis of cycloplatinated compounds.26 The failure to obtain
pure 3c could be related to the low nucleophilic character of A
that renders the intramolecular C−H bond activation more
difficult than for the previously reported precursors. In this
sense, it has been previously reported that only intramolecular
C−Br bond activation, and not C−Cl or C−H bond activation,
took place when cis-[Pt(C6F5)2(SMe2)2] was used as starting
material in analogous reactions.27
When toluene solutions of 2b or 2d were heated at reflux
temperature for six hours, compounds 5b and 5d, depicted in
Scheme 1, were obtained as pure solids. These [C,N,N′]
cyclometalated platinum(II) compounds containing a seven-
membered metallacycle are formed from the corresponding
compounds 2 in a process involving intramolecular C−Cl bond
activation to produce a platinum(IV) cyclometalated com-
pound, which further reacts to produce compound 5 and
eliminates fluorobenzene, as depicted in Scheme 2 for 2b.
Figure 2. Molecular structure of compound 2d. Hydrogen atoms have
been omitted for clarity. Selected bond lengths (Å) and angles (deg)
with estimated standard deviations: Pt−C(7): 2.007(6); Pt−C(1):
2.025(6); Pt−N(1): 2.096(4); Pt−N(2): 2,214(5); N(1)−C(15):
1.260(7); N(1)−C(14): 1.501(8); N(2)−C(13): 1.464(8); C(13)−
C(14): 1.525(9); C(7)−Pt−C(1): 88.4(2); C(7)−Pt−N(1): 96.0(2);
C(1)−Pt−N(2): 94.9(2); N(1)−Pt−N(2): 80.62(18).
1H−13C HSQC is also consistent with the proposed structure.
All observed coupling constants are in the range expected for
monofluorinated aromatic compounds22 and in good agree-
ment with the values reported for cyclometalated compounds.23
In addition, the chemical shift of the observed signal in the
195Pt NMR spectra (δ = −1929.6 ppm) is consistent with the
presence of a platinum(IV) compound.24
The crystal structure is composed of discrete molecules held
together by van der Waals interactions. The molecular structure
(Figure 3) confirmed the proposed structure in which the
platinum atom displays an octahedral coordination with the
three Pt−C bonds in a fac arrangement. The Pt−C and Pt−N
distances are in the expected range, and the coordination angles
involving the mer-[C,N,N′] are smaller than 90° (C(1)−Pt−
N(1) = 81.1(2)° and N(1)−Pt−N(2) = 78.6(2)°).
As indicated above, the reactions of ligands 1b−1d with
precursor A in toluene at room temperature gave compounds
2b−2d, in which the ligands act as bidentate [N,N′] ligands;
further reaction of these compounds was tested in refluxing
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Alternatively, compounds 5 could also be obtained in a one-pot
procedure after stirring for four hours a toluene mixture of
compound A and the corresponding ligand. Compounds 5b
and 5d were characterized by NMR spectra (1H, 19F, and
195Pt). The δ(195Pt) values are in the range expected for
confirms the geometry predicted by NMR spectroscopy. The
platinum atom displays an approximately square-planar
coordination with a terdentate [C,N,N′] and a chloro ligand.
As expected, the seven-membered platinacycle includes a biaryl
fragment, and the position of the fluoro substituent (F1) in
para position to the newly formed Caryl−Caryl bond and meta to
the platinum atom supports the mechanism previously
suggested for analogous reactions.13 The process shown in
Scheme 3 takes place through reductive coupling of one para-
fluorobenzene ligand and the aryl ring of the imine ligand to
produce a biaryl moiety, which is consequently cyclometalated
with elimination of fluorobenzene.
Biological Studies. In this work, a set of compounds with
different properties (2b, 2c, 2d, 4a, 5b, and 5d) and the
corresponding free ligands (1a−1d) were evaluated in vitro to
assess their activity on the inhibition of A549 human lung
cancer cell proliferation, using cisplatin as positive control.
Compounds 2b−2d are organometallic platinum(II) com-
pounds with one labile position (the dimethylamino fragment),
compounds 5b and 5d are cyclometalated platinum(II)
compounds containing two labile positions (both the chloro
ligand and the dimethylamino fragment), and compound 4a is a
cyclometalated platinum(IV) compound with a fac-PtC3
arrangement and a meridional [C,N,N′] terdentate ligand,
thus leaving one bromide and one aryl ring as axial ligands.
Their effect on the growth of the selected cell line was
evaluated after 72 h, and the results are displayed in Figure 5.
The obtained IC50 values resulting from an average of two
experiments are shown in Table 1.
platinum(II) compounds,24 and in the H NMR spectra the
1
nonequivalence of the protons in both the methyl and the
methylene groups indicates that the molecule deviates from
planarity. In addition, two-dimensional 1H−1H COSY and
NOESY were also carried out for 5d in order to achieve a
complete assignment. Moreover, a 1H−13C HSQC hetero-
correlation evidenced the presence of six cross-peak signals in
the aromatic region for 5d.
Crystals of 5d were grown in dichloromethane−methanol at
room temperature. In spite of the disorder problems
encountered, the obtained molecular structure (Figure 4)
It can be seen from Table 1 that compounds 5b and 5d
exhibit a great antiproliferative activity and lower IC50 values
than cisplatin itself. These compounds show little difference in
their cytotoxic effectiveness among them and when compared
with similar seven-membered platinacycles previously describe-
d.6a Although the presence of fluorine substituents could favor
DNA binding, no increase in potency is observed for
compounds 5b and 5d, containing fluoro substituents. As
previously reported,6a the seven-membered metallacycles are
not planar, the tilt angle between both aryl rings contained in
the seven-membered ring is in the range 50.6−54.2°, and
consequently, intercalative binding to DNA is not expected.
Compounds 2b and 2d show a notable antiproliferative activity
with lower IC50 values than that of cisplatin. With regard to the
same standard reference, compound 2c exhibited a ca. 50-fold
increase in potency. Interestingly compounds 2b−2d have a
very similar structure and only differ in the substitution pattern
Figure 4. Molecular structure of compound 5d. The disordered
moiety and the hydrogen atoms have been omitted for clarity. Selected
bond lengths (Å) and angles (deg) with estimated standard deviations:
Pt(1)−C(2): 1.986(7); Pt(1)−N(1): 1.992(7); Pt(1)−N(2):
2.183(6); Pt(1)−Cl(1): 2.293(2); C(1)−C(2): 1.407(8); C(1)−
C(7):1.513(8); N(1)−C(13): 1.272(11); N(1)−C(14): 1.483(11);
N(2)−C(15): 1.505(13); C(7)−C(8): 1.406(11); C(8)−C(13):
1.463(11); C(14)−C(15): 1.505(14); C(2)−Pt(1)−N(1): 92.1(3);
N(1)−Pt(1)−N(2): 82.9(3); C(2)−Pt(1)−Cl(1): 93.1(2); N(2)−
Pt(1)−Cl(1): 92.0(2).
Scheme 3. Formation of Compound 5d
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On the other hand, there are no structure−activity rules for
platinum(IV) complexes per se, except that the platinum(II)
congeners used for constructing a platinum(IV) complex must
be active.8a Furthermore, to the best of our knowledge, no
cytotoxicity has been evaluated so far for [C,N,N′]-cyclo-
metalated Pt(IV) complex. Therefore we intended to determine
within this project if it is possible that unsymmetrical
monomeric Pt(IV) complexes, featuring halide and 4-
fluorophenyl as axial ligands, exhibit cytotoxicity versus the
cell line selected (A549 human lung cancer). Interestingly the
Pt(IV) complex 4a synthesized in this study exhibited IC50
values in non-small lung cancer cells (A549) very close to that
of the standard reference cisplatin.
The effect of binding of the compounds investigated in this
study on supercoiled DNA was determined by their ability to
alter the electrophoretic mobility of pBluescript plasmid DNA:
supercoiled closed circular (ccc) and open circular (oc) forms.
Figure 6 shows the electrophoretic mobility of native
Figure 5. Inhibition of cell growth proliferation in the A549 human
lung cancer cell line, after 72 h of exposure to coordination
compounds (2b−2d) (top), cyclometalated compounds (4a, 5b, 5d)
(bottom), and cisplatin.
Table 1. Cytotoxic activities on the A549 Lung Human
Cancer Cell Line for Studied Compounds and Cisplatin
a
compound
IC50 (μM)
1a
>100
1b
>100
1c
>100
1d
>100
2b
6.5 2.0
0.3 0.1
12.1 0.8
11.6 2.2
2.8 0.5
2.5 0.1
14.1 1.3
2c
2d
4a
5b
5d
cisplatin
a
Data are shown as the mean values of two experiments performed in
triplicate with the corresponding standard deviation (SD).
in the imine aryl group. However, the presence of two
substituents in the ortho positions for 2b and 2d could favor E−
Z isomerization around the imine bond or even formation of a
platinum(IV) compound as depicted in Scheme 2 for 2b.
Complex 2c (without an ortho substituent) turned out to be ca.
20-fold more active than 2b and ca. 40-fold more active than
2d.
Figure 6. Interaction of pBluescript SK+ plasmid DNA (0.8 μg) with
increasing concentrations of compounds 2, 4, and 5, cisplatin, and
ethidium bromide. Lane 1: DNA only. Lane 2: 2.5 μM. Lane 3: 5 μM.
Lane 4: 10 μM. Lane 5: 25 μM. Lane 6: 50 μM. Lane 7: 100 μM. Lane
8: 200 μM. ccc = supercoiled closed circular DNA; oc = open circular
DNA.
Most evidence to date indicates that platinum(IV) complexes
exhibiting symmetrical axial ligands (Cl, OH, and OAc) are
reduced under physiological conditions by biologically relevant
reducing agents (ascorbic acid, glutathione, metallothionein) to
release two axial ligands and yield the cytotoxic platinum(II)
species.8a Investigations with a series of three model Pt(IV)
complexes with axial chloro, acetato, and hydroxo ligands
revealed that they have reduction potentials such that the ease
of reduction follows the trend Cl > OAc > OH.8j In addition,
the difficulty of reduction of Pt(IV) analogues exhibiting OH−
ligands has been correlated with good in vivo biological
activity.8k However it was found recently that redox potential
does not always correlates with the rate of reduction of the
platinum(IV) complexes, and also the precise mechanisms of
reduction are not always fully understood.8e,f
pBluescript DNA incubated with the synthesized compounds
(2b, 2c, 2d, 4a, 5b, and 5d) at increasing amounts ranging from
0 to 200 μM. To provide a basis for comparison, incubation of
DNA with cisplatin and ethidium bromide (EtB) was also
performed. As expected, cisplatin greatly altered the electro-
phoretic mobility of pBluescript DNA at all concentrations
tested. At concentrations up to 50 μM none of the assayed
compounds produced a significant effect on the electrophoretic
mobility of native pBluescript DNA. Compounds 5b and 5d
greatly alter the mobility of plasmid DNA at 50 μM, and at 100
μM concentration, the rate of migration of the supercoiled
band (ccc) decreases even more and tends to approach that of
the nicked relaxed band (oc). Platinum(IV) compound 4a
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displayed a much lower effect on plasmid DNA mobility, while
compounds 2b−2d did not modify the DNA migration in spite
of their low IC50 values. These results indicated that
compounds 5 alter the electrophoretic mobility of pBluescript
plasmid DNA and hence interact with DNA like the standard
reference, cisplatin. However, compounds 2 and 4 showed a
weak effect on DNA electrophoresis, pointing out another
mechanism of action or another biomolecular target.
Since compound 2c was found to be very active (IC50 = 0.3
μM) against A549 lung cancer cells, and π−π stacking
interactions are plausible for these types of compounds, as
observed in the crystal structure of compound 2d, we
hypothesized that compounds 2 might behave as intercalating
agents. Although intercalation has been traditionally associated
with molecules containing fused bi- or tricyclic ring structures,
atypical intercalators might be more prevalent than originally
thought.28 In order to ascertain whether compound 2c could be
a DNA intercalator, a topoisomerase-based gel assay was
performed upon this compound.29,30 Figure 7 shows the
metalated platinum(IV) compound 4a. However, for com-
pounds 5 the fluoro group is now para to the formed Caryl−Caryl
bond and meta to the platinum center. This result is consistent
with a process involving Caryl−Caryl coupling between the
carbon atoms bound to platinum of the metalated aryl ring and
the para-fluoroaryl ligand, as expected for a biaryl reductive
elimination from a platinum(IV) compound. On the other
hand, the failure to obtain pure 3c could be related to the
presence of electron-withdrawing fluorosubstituents that reduce
the nucleophilic character of compound A compared to
previously reported precursors.
The new compounds exhibited notable to great antiprolifer-
ative activities against the A549 human lung cancer cell line.
The behavior of compounds 5 is very similar to that obtained
for analogous seven-membered platinacycles, which suggests
that the presence of fluoro substituents is not relevant to their
biological properties. In spite of the very high potency of
compound 2c (ca. 50-fold greater than the standard reference
cisplatin), electrophoretic studies carried out for this compound
do not show any evidence of either covalent binding or
intercalation with DNA, nor a topoisomerase I inhibitor
behavior. The results presented here constitute the first step
of current work centered on (i) mechanistic studies for
elucidating the mode of action of compounds 2b−2d and 4a in
terms of cell cycle arrest, induction of apoptosis, etc., and (ii)
structure−activity relationship analysis upon platinum(IV)
complex 4a and structurally related analogues, previously
synthesized in our group, in order to elucidate the structural
requirements for activity. These studies may provide valuable
information for the design of new organometallic compounds
with improved potency and pharmacokinetic properties.
Figure 7. Analysis of 2c as a putative DNA intercalator or
topoisomerase I inhibitor. Conversion of supercoiled pBluescript
plasmid DNA (0.8 μg) to relaxed DNA by the action of topoisomerase
I (3 units) in the absence or in the presence of increasing amounts of
compound 2c was analyzed by agarose gel stained with ethidium
bromide (EtBr). Also shown are the negative and positive intercalator
controls, etoposide (Etop, 100 μM) and ethidium bromide (EtBr, 10
μM). Lanes 1, DNA only, lane 2, 0 μM compound; lanes 3, 10 μM;
lane 4, 25 μM, lane 5, 50 μM; lanes 6, 100 μM. Except for lane 1, all
the lanes included topoisomerase I. ccc = supercoiled closed circular
DNA form; oc = open circular DNA form.
EXPERIMENTAL SECTION
■
General Procedures. Microanalyses were performed at the
́
Centres Cientifics i Tecnolog
̀
ics (Universitat de Barcelona). Mass
spectra were performed at the Unitat d’Espectrometria de Masses
(Universitat de Barcelona) in an LC/MSD-TOF spectrometer using
H2O−CH3CN (1:1) to introduce the sample. NMR spectra were
performed at the Unitat de RMN d’Alt Camp de la Universitat de
Barcelona using a Mercury-400 (1H, 400 MHz; 1H−1H COSY;
1
1H−1H NOESY; H−13C HSQC; 13C, 100.6 MHz; 19F, 376.5 MHz)
or a Varian VNMRS-400 (195Pt, 85.68 MHz) spectrometer and
referenced to SiMe4 (1H, 13C), CFCl3 (19F), or H2PtCl6 in D2O
electrophoretic mobility of supercoiled DNA treated with
topoisomerase I in the presence of compound 2c at increasing
amounts ranging from 10 to 100 μM. To provide a basis for
comparison, unwinding assays with etoposide (100 μM) and
ethidium bromide (10 μM) as examples of nonintercalative and
intercalative drugs, respectively, were also performed. Results
presented in Figure 7 showed that 2c does not prevent
unwinding of DNA by the action of topoisomerase I, indicating
that this compound is not an intercalator nor an inhibitor of
topoisomerase I.
(
195Pt). δ values are given in ppm, and J values in Hz. Abbreviations
used: s = singlet; d = doublet; t = triplet; m = multiplet; br = broad.
X-ray Diffraction. Suitable crystals were grown in dichloro-
methane−methanol at room temperature. For 2d, X-ray diffraction
data were collected on a Mar 345 diffractometer with image plate
detector at 293 K, and the structure was solved by direct methods
using the SHELX97 software package and refined by the full-matrix
least-squares method with the SHELX97 software package.31 For A,
4a, and 5d X-ray diffraction data were collected on a D8 VENTURE
system equipped with a multilayer monochromator and a Mo high
brilliance Incoatec Microfocus Source (λ = 0.71073 Å) at 100 K (A
and 5d) or 90 K (4a), and the structures were solved and refined using
the Bruker SHELXTL software package.31 For 5d, the compound
displays molecular disorder for all atoms except for Pt, C1, and C17,
which lie on a mirror plane. CIFs for all four structures and a table of
crystallographic data are included in the Supporting Information.
Preparation of the Complexes. Ligands 1a−1d19,32 and
compound cis-[PtCl2(SEt2)2]33 were prepared as reported elsewhere.
Compound [Pt2(4-FC6H4)4(μ-SEt2)2] (A) was prepared using the
following procedure: 3.5 mL (37.15 mmol) of n-butyllithium in hexane
was added under N2 to 30 mL of diethyl ether, and the solution was
cooled to 0 °C. 4-Fluoroiodobenzene (1.225 g; 5.52 mmol) was slowly
added, and the mixture was stirred for 30 min at 0 °C After this time,
CONCLUSION
■
The new compound [Pt2(4-FC6H4)4(μ-SEt2)2] (A) appears to
be a suitable precursor for the synthesis of several types of
organometallic species such as diarylplatinum(II) compounds
containing a bidentate [N,N′] ligand (2b−2d) and cyclo-
metalated platinum compounds containing a terdentate
[C,N,N′] ligand with either a five-membered platinum(IV)
(4a) or a seven-membered platinum(II) (5b−5d) metallacycle.
The fluoro substituent in the aryl group remains para to the
platinum in coordination compounds 2 and in the cyclo-
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3
4
[PtCl2(SEt2)2] (0.502 g; 1.23 mmol) was added, and the mixture was
stirred for 2 h at room temperature. After cooling to 0 °C, water (5
mL) was added, the aqueous layer was extracted with dichloromethane
(3 × 15 mL), and the combined organic layers were dried over
magnesium sulfate, filtered, and evaporated to give an oily residue. The
solid obtained upon addition of hexane was filtered and dried under
vacuum. Yield: 314 mg (53.8%). 1H NMR (400 MHz, CDCl3): δ 7.20
NMR (376.5 MHz, CD3COCD3): δ −126.5 (tt, JH−F = 10.2, JH−F
7.2, 1F), −124.8 (tt, 3JH−F = 10.2, 4JH−3F = 6.8, 1F), −124.5 (tt, 3JH−F
=
=
4
4
10.2, JH−F = 7.2, 1F), −124.2 (tt, JH−F = 10.2, JH−F = 6.8, 1F),
3
4
3
−108.5 (dd, JH−F = 9.0, JH−F = 6.0, 1F), −107.5 (dd, JH−F = 8.6,
4JH−F = 6.0 Hz, 1F). 195Pt NMR (85.68 MHz, CDCl3): δ (ppm)
−3359.0 (s). HRMS-ESI-(+) {H2O−CH3CN (1:1)}: m/z 518.0774
(calcd for C17H18ClF2N2Pt 518.0768) [M − FC6H4]+; 614.1133 (calcd
for C23H23ClF3N2Pt 614.1146) [M + H]+. Anal. Found (calcd for
C23H22ClF3N2Pt): C: 45.3 (45.0); H: 4.0 (3.6); N: 4.3 (4.6).
Compound [PtBr(4-FC6H4)2{Me2NCH2CH2NCHC6H4}] (4a)
was obtained as a white solid following the same procedure as for
compounds 2b−2d from 0.100 g (0.105 mmol) of cis-[Pt(4-
FC6H4)2(μ-SEt2)]2 and 0.054 g (0.212 mmol) of ligand 1a for 4 h.
Yield: 77.5 mg (57.8%).
(dd, 3JH−H = 8.8, 4JH−F = 6.4, 8H, Hortho), 6.72 (dd, 3JH−H = 8.8, 3JH−F
=
3
3
9.2, 8H, Hmeta), 2.51 (q, JH−H = 7.2, 8H, CH2), 1.82 (t, JH−H = 7.2,
12H, CH3). 19F NMR (376.5 MHz, CDCl3): δ −121.6 (tt, 4JF−H = 6.4,
3JF−H = 9.4). HRMS-ESI-(+) {H2O−CH3CN (1:1)}: m/z 968.1805
(calcd for C32H40F4NPt2S2 968.1828) [M + NH4]+. Anal. Found
(calcd) for C32H36F4Pt2S2: C: 40.3 (40.4); H: 3.9 (3.8); S: 6.9 (6.7).
Compound [Pt(4-FC6H4)2{Me2NCH2CH2NCH(2,6-
Cl2C6H3)}] (2b) was obtained after stirring for 4 h a mixture
containing 0.100 g (0.105 mmol) of cis-[Pt(4-FC6H4)2(μ-SEt2)]2 and
0.055 g (0.224 mmol) of ligand 1b in toluene at room temperature.
The solvent was evaporated, and the residue was treated with diethyl
ether. The yellow solid was filtered and dried under vacuum. Yield:
NMR labeling for 4a:
1
3
106 mg (80.2%). H NMR (400 MHz, CDCl3): δ 8.71 (s, JH−Pt
54.4, 1H, CHN), 7.26 (m, 2H, Hmeta), 6.97 (br s, 3H, HAr), 6.77 (m,
2H, Hmeta), 6.62 (m, 2H, Hortho), 6.14 (m, 2H, Hortho), 4.18 (t, 3JH−H
=
=
3
3
5.6, 2H, CH2), 2.81 (t, JH−H = 5.6, 2H, CH2), 2.60 (s, JH−Pt = 18.8,
6H, NMe2). 19F NMR (376.5 MHz, CDCl3): δ −126.7 (m, 1F),
−124.9 (m, 1F). HRMS-ESI-(+) {H2O−CH3CN (1:1)}: m/z
534.0475 (calcd for C17H18Cl2FN2Pt 535.0473) [M − C6H4F]+;
652.0697 (calcd for C23H22Cl2F2N2NaPt 652,0668) [M + Na]+;
1276.1858 (calcd for C46H48Cl4F4N5Pt2 1276.1890) [2M + NH4]+.
Anal. Found (calcd for C23H22Cl2F2N2Pt): C: 44.0 (43.8); H: 3.6
(3.5); N: 4.2 (4.4).
3
1H NMR (400 MHz, CD3COCD3): δ 8.95 (s, JH−Pt= 47.6, 1H,
H1), 7.69 (t, 3JH−Pt = 32.0, 3JH−H = 4JH−F = 6.8, 2H, H8), 7.51 (d, 3JH−H
= 6.8, 1H, H13), 7.21 (d, JH−H = 7.6, 1H, H10), 7.15 (t, JH−H = 6.8,
3
3
Compound [Pt(4-FC6H4)2{Me2NCH2CH2NCH(4-ClC6H4)}]
(2c) was obtained using the same procedure from 1c. Yield: 108 mg
(86.4%).
1H, H11), 7.05 (t, 3JH−H = 6.8, 1H, H12), 6.88 (dd, JH−Pt= 52.0, JH−H
= 8.0, 4JH−F = 6.0, 2H, H6), 6.88 (t, 3JH−F = 3JH−H = 8.7, 2H, H7), 6.62
(m, 2H, H9), 4.70 (m, 1H, H2), 4.45 (m, 2H, H3′, H2′), 3.08 (m, 1H,
3
3
NMR labeling for 2c:
H3), 2.93 (s, 3JH−Pt = 11.2, 3H, H4), 2.64 (s, 3JH−Pt = 15.2, 3H, H5). 19
F
=
=
5
3
NMR (376.5 MHz, CD3COCD3): δ −123.2 (tt, JF−Pt = 12.8, JF−H
9.0, 4JF−H = 6.0, 1F, F2), −122.4 (tt, 5JF−Pt = 18.8, 3JF−H = 9.4, 4JF−H
6.0, 1F, F1). 13C NMR (100 MHz, CD3COCD3): δ 171.9 (C1), 161.7,
147.3, 139.0 (d, JH−F = 5.0, C8), 135.5 (d, JC−F = 6.4, C6), 134.86,
4
3
131.75, 131.6 (C10), 131.5 (C11), 129.9 (C13), 128.8, 128.1, 124.1
2
2
(C12), 113.1 (d, JC−F = 19.6, C7), 112.9 (d, JC−F = 19.1, C9), 66.0
(C2), 52.6 (C3), 50.0 (C4), 47.9 (C5). 195Pt NMR (85.68 MHz,
CDCl3): δ −1929.6 (s). HRMS-ESI-(+) {H2O−CH3CN (1:1)}: m/z
464.1092 (calcd for C17H18FN2Pt 464.1096) [M − FC6H4 − H −
Br]+; 544.0354 (calcd for C17H19BrFN2Pt 544.0357) [M − FC6H4]+;
560.1469 (calcd for C23H23F2N2Pt 560.1471) [M − Br]+; 657.1006
(calcd for C23H27BrF2N3Pt 657.0998) [M + NH4]+; 1199.2099 (calcd
for C46H46BrF4N4Pt2 1199.2132) [2M − Br]+; 1279.1362 (calcd for
C46H47Br2F4N4Pt2 1279.1393) [2M + H]+. Anal. Found (calcd for
C23H23BrF2N2Pt): C: 43.2 (43.2); H: 3.8 (3.6); N: 4.4 (4.4).
Compound [PtCl{(3-FC6H3)(2-ClC6H3)CHNCH2CH2NMe2}]
(5b) was obtained after stirring under reflux for 6 h a solution
containing 0.075 g (0.119 mmol) of compound 2b. The solvent was
evaporated, and the residue was treated with diethyl ether. The yellow
solid was filtered and dried under vacuum. Yield: 29 mg (45.9%).
NMR labeling for 5b:
3
1H NMR (400 MHz, CD3COCD3): δ 9.11 (s, JH−Pt = 47.2, 1H,
H1), 8.20 (d, 3JH−H = 8.8, 2H, H2), 7.38 (dd, JH−H = 8.4, JH−F = 6.8,
3
4
2H, H7), 7.09 (d, 3JH−H = 8.4, 2H, H3), 6.84 (dd, 3JH−H = 8.8, 4JH−F
=
6.8, 2H, H9), 6.67 (dd, JH−H = 8.8, JH−F = 10, 2H, H8), 6.13 (dd,
3
4
3JH−H = 8.8, JH−F = 10, 2H, H10), 4.36 (t, JH−H = 5.6, 2H, H4), 2.96
3
3
(t, JH−H = 5.6, 2H, H5), 2.67 (s, JH−Pt = 21.2, 6H, H6). 19F NMR
3
3
3
4
(376.5 MHz, CD3COCD3): δ −126.9 (tt, JF−H = 10.2, JF−H = 6.8,
1F), −126.1 (tt, JF−H = 10.2, JF−H = 6.8, 1F). 195Pt NMR (85.68
MHz, CDCl3): δ −3388.6 (s). HRMS-ESI-(+) {H2O−CH3CN (1:1)}:
m/z 404.0473 (calcd for C11H14ClN2Pt 404.0487) [M − 2FC6H4 −
H]+; 613.1484 (calcd for C23H27ClF2N3Pt 613.1503) [M + NH4]+;
1208.2616 (calcd for C46H50Cl2F4N5Pt2 1208.2669) [2M + NH4]+.
Anal. Found (calcd for C23H23ClF2N2Pt·C4H10O): C: 48.5 (48.4); H:
4.6 (5.0); N: 4.3 (4.2).
3
4
Compound [Pt(4-FC6H4)2{Me2NCH2CH2NCH(2-Cl-6-F-
C6H3)}] (2d) was obtained using the same procedure from 1d.
Yield: 107 mg (82.9%). 1H NMR (400 MHz, CD3COCD3): δ 8.74 (s,
3JH−Pt = 55.6, 1H, CHN, E isomer), 8.27 (s, 3JH−Pt = 26.0, 1H, CHN, Z
isomer), 7.43 (m, 2H), 7.34 (t, 3JH−H = 8.0, 4H), 7.08−7.02 (m, 2H),
3
1H NMR (400 MHz, CDCl3): δ 9.24 (s, JH−Pt = 144.0, 1H, H4),
7.51 (t, JH−H = 7.6, 1H, H2), 7.38 (m, 1H, H8), 7.31 (d, JH−H = 7.6,
2H, H1,3), 6.92 (m, 1H, H9), 6.70 (m, 1H, H10), 4.52 (m, 1H, H5),
3.98 (d, 2JH−H = 10.8, 1H, H5′), 3.01 (s, 3H, H7), 2.73 (m, 4H, H7′,6′),
2,61 (m, 1H, H6). 19F NMR (376.5 MHz, CDCl3): δ (ppm) −116.6
3
3
3
3
6.87 (m, 2H), 6.78−6.71 (m, 10H), 6.14 (dd, JH−F = 12.0, JH−H
=
3
8.0, 2H), 4.20 (m, 4H, CH2), 2.81 (t, JH−H = 8.0, 2H, CH2), 2.78 (t,
3JH−H = 8.0, 2H, CH2), 2.62 (s, 6H, NMe2), 2.61 (s, 6H, NMe2). 19F
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(ddd, 3JF−H = 10.5, 3JF−H = 7.9, 4JF−H = 6.0). 195Pt NMR (85.68 MHz,
CDCl3): δ −3075.1 (s). HRMS-ESI-(+) {H2O−CH3CN (1:1)}: m/z
534.0466 (calcd for C17H18Cl2FN2Pt 534.0473) [M + H]+; 551.0734
(calcd for C17H21Cl2FN3Pt 551.0738) [M + NH4]+; 1084.1108 (calcd
for C34H38Cl4F2N5Pt2 1084.1139) [2M + NH4]+. Anal. Found (calcd
for C17H17Cl2FN2Pt): C: 37.9 (38.2); H: 3.2 (3.2); N: 4.8 (5.2).
Compound [PtCl{(3-FC6H3)(2-FC6H3)CHNCH2CH2NMe2}]
(5d) was prepared using the same procedure from 2d. Alternatively,
5d was prepared as a yellow solid from 0.150 g (0.158 mmol) of
compound cis-[Pt(4-FC6H4)2(μ-SEt2)]2 and 0.074 g (0.324 mmol) of
ligand 1d in toluene with continuous stirring at room temperature for
4 h followed by heating under reflux for 6 h. The reaction mixture was
evaporated, and the yellow oily residue was treated with dichloro-
methane−methanol. After cooling the mixture, a solid was produced,
filtered, and dried under vacuum. Yield: 124 mg (75.9%).
The assay was performed by a variation of the MTT assay described by
Mosmann et al.34 as specified by Matito and co-workers,35 which is
based on the ability of live cells to cleave the tetrazolium ring of the
MTT, thus producing formazan, which absorbs at 550 nm. In brief, 2.5
× 103 A549 cells/well were cultured in 96-well plates for 24 h prior to
the addition of different compounds at different concentrations, in
triplicate. After further incubation for 72 h, the supernatant was
aspirated, and 100 μL of filtered MTT (0.5 mg/mL) was added to
each well. Following 1 h of incubation with the MTT, the supernatant
was removed, and the precipitated formazan was dissolved in 100 μL
of DMSO. Relative cell viability, compared to the viability of untreated
cells, was measured by absorbance at 550 nm on an ELISA plate reader
(Tecan Sunrise MR20-301, TECAN, Salzburg, Austria). Concen-
trations that inhibited cell growth by 50% (IC50) after 72 h of
treatment were subsequently calculated.
DNA Migration Studies. Compounds were dissolved in high-purity
DMSO at 10 mM as stock solution. Then, serial dilutions were made
in Milli-Q water (1:1). Plasmid pBluescript SK+ (Stratagene) was
obtained using QIAGEN plasmid midi kit as described by the
manufacturer. Interaction of drugs with pBluescript SK+ plasmid DNA
was analyzed by agarose gel electrophoresis following a modification of
the method described by Abdullah et al.36 In brief, plasmid DNA
aliquots (40 μg mL−1) were incubated in TE buffer (10 mM Tris-HCl,
1 mM EDTA, pH 7.5) with different concentrations of the compounds
(ranging from 0 to 200 μM) at 37 °C for 24 h. Final DMSO
concentration in the reactions was always lower than 1%. For
comparison, cisplatin and ethidium bromide were used as controls.
Aliquots of 20 μL of compound−DNA complexes containing 0.8 μg of
DNA were subjected to 1% agarose gel electrophoresis in TAE buffer
(40 mM Tris-acetate, 2 mM EDTA, pH 8.0). The gel was stained in
the same buffer containing ethidium bromide (0.5 mg mL−1) and
visualized and photographed under UV light.
NMR labeling for 5d:
3
1H NMR (400 MHz, CD3COCD3): δ 9.38 (s, JH−Pt = 144.4, 1H,
3
4
3
H1), 7.69 (td, JH−H = 8.0, JH−F = 6.0, 1H, H10), 7,27 (dd, JH−F
=
10.8, JH−H = 2.8, 1H, H6), 7.21 (d, JH−H = 8.0, 1H, H9), 7.20 (ddd,
4
3
3JH−F = 12.0, 3JH−H = 8.0, 4JH−H = 0.8, 1H, H11), 6.94 (dd, 3JH−H = 8.4,
4JH−F = 6.0, 1H, H8), 6.65 (td, JH−H = JH−F = 8.4, JH−H = 2.8, 1H,
3
3
4
H7), 4.56 (dtd, 2JH−H = 12.8, 3JH−H= 3JH−H = 4.4, 4JH−H = 1.2, 1H, H2),
Topoisomerase I-based experiments were performed as described
previously.29 Supercoiled pBluescript DNA, obtained as described
above, was treated with topoisomerase I in the absence or presence of
increasing concentrations of compound 2c. Assay mixtures contained
supercoiled pBluescript DNA (0.8 μg), calf thymus topoisomerase I (3
units), and complex 2c (0−100 μM) in 20 μL of Tris-HCl buffer (pH
7.5) containing 175 mM KCl, 5 mM MgCl2, and 0.1 mM EDTA.
Ethidium bromide (10 μM) was used as a control of intercalating
agents. Reactions were incubated for 30 min at 37 °C and stopped by
the addition of 2 μL of agarose gel loading buffer. Samples were then
subjected to electrophoresis and DNA bands stained with ethidium
bromide as described above.
4.31 (ddd, JH−H = 11.6, JH−H = 3.6, JH−H = 0.4, 1H, H2′), 2.99 (s,
2
3
3
3H, H5), 2.85 (m, 2H, H3, H3′), 2.65 (s, 3H, H4). 19F NMR (376.5
MHz, CD3COCD3): δ −120.0 (ddd, 3JF−H = 11.3, 3JF−H = 8.7, 4JF−H
=
6.4, 1F, F2), −118,3 (ddd, JF−H = 10.2, JF−H = 6.0, JF−H = 2.3, 1F,
3
4
5
F1). 13C NMR (100 MHz, CD3COCD3): δ 159.8 (C1), 133.1 (d, 3JC−F
= 10.4, C10), 130.1 (d, JC−F = 8.2, C8), 128.8 (d, JC−F = 2.8, C9),
125.8 (d, 2JC−F = 17.5, C6), 112.9 (d, 2JC−F = 22.2, C11), 109.4 (d, 2JC−F
= 23.0, C7), 67.4 (C2), 64.8 (C3), 50.1 (C4), 47.2 (C5). 195Pt NMR
(85.68 MHz, CD3COCD3): δ −3259.0 (s). HRMS-ESI-(+) {H2O−
CH3CN (1:1)}: m/z 518.0759 (calcd for C17H18ClF2N2Pt 518.0768)
[M + H]+; 535.1006 (calcd for C17H21ClF2N3Pt 535.1034) [M +
NH4]+; 1052.1709 (calcd for C34H38Cl2F4N5Pt2 1052.1730) [2M +
NH4]+. Anal. Found (calcd for C17H17ClF2N2Pt): C: 39.7 (39.4); H:
3.4 (3.3); N: 5.3 (5.4).
3
4
ASSOCIATED CONTENT
* Supporting Information
■
S
Compound [Pt{(4-FC6H4){Me2NCH2CH2NCH(3-ClC6H3)}]
(3c) was obtained as an impure solid from 0.150 g (0.158 mmol) of
compound cis-[Pt(4-FC6H4)2(μ-SEt2)]2 and 0.066 g (0.313 mmol) of
ligand 1c in toluene with continuous stirring at room temperature for
4 h followed by heating under reflux for 6 h. The reaction mixture was
evaporated, and the yellow oily residue was treated with dichloro-
methane−methanol. After cooling the mixture, a solid was produced,
filtered, and dried under vacuum. Analogous results were obtained
when 2c was refluxed in toluene for 6 h. 1H NMR (400 MHz, CDCl3):
δ 8.38 (s, 3JH−Pt = 57.2, CHN), 3.97 (t, 3JH−H = 4.0, 2H, CH2), 3.12 (t,
CIFs giving crystallographic data for the four complexes
characterized by XRD and a table of crystallographic data are
included. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*Phone: +34 934039131. Fax: +34 934907725. E-mail:margar
ita.crespo@qi.ub.es (M. Crespo).
■
3
3JH−H = 4.0, 2H, CH2), 2.67 (s, JH−Pt = 20.0, 6H, NMe2). 19F NMR
*E-mail: quirantese@ub.edu (J. Quirante).
Notes
The authors declare no competing financial interest.
3
4
(376.5 MHz, CDCl3): δ (ppm) −123.4 (tt, JF−H = 10.2, JF−H = 6.8,
1F).
Biological Studies. Cell Culture. Human lung carcinoma A549
cells were grown as a monolayer culture in minimum essential medium
(DMEM with L-glutamine, without glucose and without sodium
pyruvate) in the presence of 10% heat-inactivated fetal calf serum, 10
mM D-glucose, and 0.1% streptomycin/penicillin, in standard culture
conditions (humidified air with 5% CO2 at 37 °C).
Cell Viability Assay. For A549 cell viability assays, compounds were
suspended in DMSO at 20 mM as stock solution. To obtain final assay
concentrations, they were diluted in DMEM (final concentration of
DMSO was the same for all final dilutions and always lower than 1%).
ACKNOWLEDGMENTS
We are grateful to the Ministerio de Ciencia y Tecnologia for
■
́
financial support (Grants CTQ2009-11501/BQU and
SAF2011-25726), to the AGAUR, Generalitat de Catalunya
(Grants 2009-SGR-1111, 2009-SGR-1308, and 2009 CTP
0026), and to the Icrea Academia award 2010 (to M.
Cascante).
1748
dx.doi.org/10.1021/om5000908 | Organometallics 2014, 33, 1740−1750

Organometallics
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