Palladium(II) and platinum(II) organometallic complexes with the model nucleobase anions of thymine, uracil, and cytosine: Antitumor activity and interactions with DNA of the platinum compounds

Article abstract of DOI:10.1021/ic060374e

Pd(II) and Pt(II) complexes with the anions of the model nucleobases 1-methylthymine (1-MethyH), 1-methyluracil (1-MeuraH), and 1-methylcytosine (1-MecytH) of the types [Pd(dmba)(μ-L)]₂ [dmba = N,C-chelating 2-((dimethyl-amino)methyl)phenyl; L = 1-Methy, 1-Meura or 1-Mecyt] and [M(dmba)(L)(L′)] [L = 1-Methy or 1-Meura; L′ = PPh₃ (M = Pd or Pt), DMSO (M = Pt)] have been obtained. Palladium complexes of the types [Pd(C₆F₅)(N-N)(L)] [L = 1-Methy or 1-Meura; N-N = N,N,N′,N′-tetramethylethylenediamine (tmeda), 2,2′-bipyridine (bpy), or 4,4′-dimethyl-2,2′-bipyridine (Me₂bpy)] and [NBu₄][Pd(C₆F₅)(1-Methy)₂(H ₂O)] have also been prepared. The crystal structures of [Pd(dmba)(μ-1-Methy)]₂, [Pd(dmba)(μ-1-Mecyt)] ₂·2CHCl₃, [Pd(dmba)(1-Methy)(PPh ₃)]·3CHCl₃, [Pt(dmba)(1-Methy)(PPh₃)], [Pd(tmeda)(C₆F₅)(1-Methy)], and [NBu₄] [Pd(C₆F₅)(1-Methy)₂(H₂O)] ·H₂O have been established by X-ray diffraction. The DNA adduct formation of the new platinum complexes synthesized was followed by circular dichroism and electrophoretic mobility. Atomic force microscopy images of the modifications caused by the platinum complexes on plasmid DNA pBR322 were also obtained. Values of IC₅₀ were also calculated for the new platinum complexes against the tumor cell line HL-60. All the new platinum complexes were more active than cisplatin (up to 20-fold in some cases).

Full text of DOI:10.1021/ic060374e

Inorg. Chem. 2006, 45, 6347−6360
Palladium(II) and Platinum(II) Organometallic Complexes with the Model
Nucleobase Anions of Thymine, Uracil, and Cytosine: Antitumor
O
Activity and Interactions with DNA of the Platinum Compounds
Jose´ Ruiz,*,† Julia Lorenzo,‡ Laura Sanglas,‡ Natalia Cutillas,† Consuelo Vicente,†
Mar´ıa Dolores Villa,† Francesc X. Avile´s,‡ Gregorio Lo´pez,† Virtudes Moreno,§ Jose´ Pe´rez,| and
Delia Bautista
Departamento de Qu´ımica Inorga´nica, UniVersidad de Murcia, 30071-Murcia, Spain,
Institut de Biotecnolog´ıa i Biomedicina, Department de Bioqu´ımica i de Biolog´ıa Molecular,
UniVersitat Auto`noma de Barcelona, 08193 Barcelona, Spain, Department de Qu´ımica Inorga`nica,
UniVersitat de Barcelona, Mart´ı i Franque`s 1-11, 08028 Barcelona, Spain, Departamento de
Ingenier´ıa Minera, Geolo´gica y Cartogra´fica, AÄ rea de Qu´ımica Inorga´nica, UniVersidad
Polite´cnica de Cartagena, 30203-Cartagena, Spain, and SUIC, Edificio SACE, UniVersidad de
Murcia, 30071-Murcia, Spain
Received March 7, 2006
Pd(II) and Pt(II) complexes with the anions of the model nucleobases 1-methylthymine (1-MethyH), 1-methyluracil
(1-MeuraH), and 1-methylcytosine (1-MecytH) of the types [Pd(dmba)(
amino)methyl)phenyl; L 1-Methy, 1-Meura or 1-Mecyt] and [M(dmba)(L)(L
PPh3 (M Pd or Pt), DMSO (M
[L 1-Methy or 1-Meura; N
dimethyl-2,2 -bipyridine (Me2bpy)] and [NBu4][Pd(C6F5)(1-Methy)2(H2O)] have also been prepared. The crystal
structures of [Pd(dmba)( -1-Methy)]2, [Pd(dmba)( -1-Mecyt)]2 2CHCl3, [Pd(dmba)(1-Methy)(PPh3)] 3CHCl3, [Pt(dmba)-
(1-Methy)(PPh3)], [Pd(tmeda)(C6F5)(1-Methy)], and [NBu4][Pd(C6F5)(1-Methy)2(H2O)] H2O have been established by
µ
-L)]2 [dmba
)
N,C-chelating 2-((dimethyl-
)
′
)] [L
)
1-Methy or 1-Meura; L′ )
)
)
Pt)] have been obtained. Palladium complexes of the types [Pd(C6F5)(N
−N)(L)]
)
−N
)
N,N,N ,N -tetramethylethylenediamine (tmeda), 2,2 -bipyridine (bpy), or 4,4′-
′
′
′
′
µ
µ
‚
‚
‚
X-ray diffraction. The DNA adduct formation of the new platinum complexes synthesized was followed by circular
dichroism and electrophoretic mobility. Atomic force microscopy images of the modifications caused by the platinum
complexes on plasmid DNA pBR322 were also obtained. Values of IC50 were also calculated for the new platinum
complexes against the tumor cell line HL-60. All the new platinum complexes were more active than cisplatin (up
to 20-fold in some cases).
Introduction
with respect to the mode of action of Pt antitumor drugs.5
Pyrimidine nucleobases (Scheme 1), particularly in their
anionic forms, are versatile ligands. In fact, their several
potential donor sites enable them to form homo- and
heteropolynuclear complexes. These compounds can exhibit
metal-metal interactions which nature have been examined.6-8
Few studies have been directed toward organometallic
The faster reaction kinetics of Pd(II) electrophiles as
compared to the corresponding Pt(II) compounds (ca. 105
times) yet otherwise similar chemistry of the two metals
make the former convenient analogues for studies of Pt-
nucleobase interactions.1-4 Studies of this kind are of interest
* To whom correspondence should be addressed. E-mail: jruiz@um.es.
Fax: (+34) 968 36 41 48.
(2) Barnham, K. J.; Bauer, C. J.; Djuran, M. I.; Mazid, M. A.; Rau, T.;
Sadler, P. J. Inorg. Chem. 1995, 34, 2826.
† Departamento de Qu´ımica Inorga´nica, Universidad de Murcia.
‡ Institut de Biotecnolog´ıa i Biomedicina, Universitat Auto`noma de
Barcelona.
(3) Shen, W.-Z.; Gupta, D.; Lippert, B. Inorg. Chem. 2005, 44, 8249.
(4) Cisplatin. Chemistry and Biochemistry of a leading Anticancer Drug;
Lippert, B., Ed.; Wiley-VCH: Weinheim, Germany, 1999.
(5) Jamieson, E. R.; Lippard, S. J. Chem. Rev. 1999, 99, 2467.
(6) Mealli, C.; Pichierri, F.; Randaccio, L.; Zangrando, E.; Krumm, M.;
Holthenrich, D.; Lippert, B. Inorg. Chem. 1995, 34, 3418.
(7) Kampf, G.; Willermann, M.; Freisinger, E.; Lippert, B. Inorg. Chim.
Acta 2002, 330, 179.
§ Department de Qu´ımica Inorga`nica, Universitat de Barcelona.
| AÄ rea de Qu´ımica Inorga´nica, Universidad Polite´cnica de Cartagena.
Edificio SACE, Universidad de Murcia.
O Dedicated to Dr. Jose´-Antonio Abad on the occasion of his retirement.
(1) Micklitz, W.; Sheldrick, W. S.; Lippert, B. Inorg. Chem. 1990, 29,
211.
10.1021/ic060374e CCC: $33.50
Published on Web 07/15/2006
© 2006 American Chemical Society
Inorganic Chemistry, Vol. 45, No. 16, 2006 6347

Ruiz et al.
Scheme 1
Scheme 2
complexes with these biologically important ligands,9,10
organometallic complexes with bioligands representing new
directions for structural diversity, possible new drug discov-
eries, and the ability of these complexes to be hosts for
biologically important guests.9
The aim of this work is the synthesis of new palladium
and platinum complexes having the metal covalently bonded
to the nucleobase which can be used in biological experi-
ments as potential anticancer drugs or as metalated nucleo-
base building blocks that can be incorporated into synthetic
oligonucleotides. Oligonucleotides metalated at fixed posi-
tions could possess improved and site-specific biological
activity. Nephrotoxic side effects associated with therapies
based on metal complexes, caused by the excess of metal
used and by their uncontrolled biodistribution, can be reduced
by linking the metal to specific biological targets such as
oligonucleotides.11-13 Therefore, even today, the synthesis
of metallonucleosides is still very interesting.
On the other hand, the hydroxo complexes [Pd(dmba)(µ-
OH)]2 (dmba ) N,C-chelating 2-((dimethylamino)methyl)-
phenyl)14 and [Pd(C6F5)(N-N)(OH)] (N-N ) bpy, Me2bpy,
or tmeda with bpy ) 2,2′-bipyridine, Me2bpy ) 4,4′-
dimethyl-2,2′-bipyridine, tmeda ) N,N,N′,N′-tetramethyl-
ethylenediamine)15 have shown to be excellent precursors
in organometallic synthesis.14-19
MecytH) (Scheme 1) of the types [Pd(dmba)(µ-L)]2 (L )
1-Methy, 1-Meura, or 1-Mecyt), [M(dmba)(L)(L′)] [L )
1-Methy or 1-Meura; L′ ) PPh3 (M ) Pd or Pt), DMSO (M
) Pt)], [Pd(C6F5)(N-N)(L)] [L ) 1-Methy or 1-Meura;
N-N ) tmeda, bpy, or Me2bpy)], and [NBu4][Pd(C6F5)(1-
Methy)2(H2O)]. We also report the crystal structures of some
of these palladium or platinum complexes. To our knowl-
edge, the structures of complexes [Pd(dmba)(µ-L)]2 (L )
1-Methy, 1-Mecyt) represent the first examples of Pd(II)
organometallic complexes with two anionic pyrimidine
nucleobases acting as bridging ligands being fully character-
ized by X-ray diffraction and the structure of [NBu4]-
[Pd(C6F5)(1-Methy)2(H2O)]‚H2O is the first crystal structure
of a monomeric palladium bis(thyminate) complex.
We have also studied the interactions of the platinum
complexes with DNA by circular dichroism and electro-
phoretic mobility. Atomic force microscopy images of the
modifications caused by the platinum complexes on plasmid
DNA pBR322 were also obtained. The in vitro antiprolif-
erative activity of the new platinum complexes in HL-60
cell line has been studied.
We report here the synthesis and characterization of new
organopalladium and organoplatinum complexes with the
model nucleobase anions of 1-methyluracil (1-MeuraH),
1-methylthymine (1-MethyH), and methylcytosine (1-
(8) Ito, K.; Somozawa, R.; Matsunami, J.; Matsumoto, K. Inorg. Chim.
Acta 2002, 339, 292.
Results and Discussion
(9) Fish, R. H.; Jaouen, G. Organometallics 2003, 22, 2166.
(10) Romanelli, A.; Iacovino, R.; Piccialli, G.; Rufo, F.;. De Napoli, L.;
Pedone, C.; Di Blasco, B.; Messere, A. Organometallics 2005, 24,
3401.
(11) Navarro, J. A. R.; Romero, M. A.; Vilaplana, R.; Gonza´lez-Vilchez,
F.; Faure, R. J. Med. Chem. 1988, 31, 332.
(12) Pe´rez, J. M.; Lo´pez-Solera, I.; Montero, E. I.; Brana, M. F.; Alonso,
C.; Robinson, S. P.; Navarro-Ranninger, C. J. Med. Chem. 1999, 42,
5482.
(13) Bierbach, U.; Sabat, M.; Farrell, N. Inorg. Chem. 2000, 39, 1882.
(14) Ruiz, J.; Cutillas, N.; Rodr´ıguez, V.; Sampedro, J.; Lo´pez, G.;
Chaloner, P. A.; Hitchcock, P. J. Chem. Soc., Dalton Trans. 1999,
2939.
(15) Ruiz, J.; Mart´ınez, M. T.; Florenciano, F.; Rodr´ıguez, V.; Lo´pez, G.;
Pe´rez, J.; Chaloner, P. A.; Hitchcock, P. B. Inorg. Chem. 2003, 42,
3650.
(16) Ruiz, J.; Rodr´ıguez, V.; Cutillas, N.; Pardo, M.; Pe´rez, J.; Lo´pez, G.;
Chaloner, P. A.; Hitchcock, P. B. Organometallics 2001, 20, 1973.
(17) Ruiz, J.; Mart´ınez, M. T.; Florenciano, F.; Rodr´ıguez, V.; Lo´pez, G.;
Pe´rez, J.; Chaloner, P. A.; Hitchcock, P. B. Dalton Trans. 2004, 929.
(18) Ruiz, J.; Mart´ınez, M. T.; Rodr´ıguez, V.; Lo´pez, G.; Pe´rez, J.; Chaloner,
P. A.; Hitchcock, P. B. Dalton Trans. 2004, 3521.
Dimeric Palladium Complexes [Pd(dmba)(µ-L)]2 (L )
1-Methy, 1-Meura, 1-Mecyt). The reaction of the hydroxo-
palladium complex14 [Pd(dmba)(µ-OH)]2 in dichloromethane
with 1-methylthymine, 1-methyluracil, or 1-methylcytosine
(in a 1:2 ratio) leads to the formation of the N(3),O-bridged
(1-methylthyminato)- or (1-methyluracilato)dipalladium com-
plexes [Pd(dmba)(µ-L)]2 [L ) 1-Methy (1), 1-Meura (2)]
or the N(3),N(4)-bridged (1-methylcytosinato)dipalladium
complex (3) (Scheme 2). These reactions imply proton
abstraction from the pyrimidine nucleobase by the hydroxo
complex with the concomitant release of water. On pro-
tonation of the hydroxo complex, it is likely that an
intermediate aqua complex is formed. As far as we know,
complexes 1 and 2 represent the first examples of organo-
metallic dinuclear palladium(II) complexes with 1-Methy-
or 1-Meura- nucleobases. The analytical data are consistent
(19) Ruiz, J.; Vicente, C.; Cutillas, N.; Pe´rez, J. Dalton Trans. 2005, 1999.
6348 Inorganic Chemistry, Vol. 45, No. 16, 2006

Pd(II) and Pt(II) Organometallic Complexes
imidazol-2-yl pyridin-2-yl ketone).21 In the related complex
[Pd2(bpm)2(µ-1-Methy)2]2+ (bpm ) bis(pyridine-2-yl)-
methane), the 1-methylthyminato bridged ligands are ar-
ranged in a head-to-head fashion.22 In complex 1 both Pd
atoms are slightly out of their coordination planes. The bite
range of the 1-methylthyminato bases [N(2)‚‚‚O(2) ) 2.290
Å; N(5)‚‚‚O(3) ) 2.277 Å] is considerably smaller than the
metal-metal distance, resulting in a dihedral angle between
the two square-planar metal coordinating planes of 37.92(8)°,
giving a basket-shaped eight-membered ring. The cyclo-
metalated rings are puckered with the nitrogen atom signifi-
cantly out of the plane defined by the palladium and carbon
atoms, a feature which is quite commonly observed in
cyclometalated dmba complexes. The Pd-N(sp2) distances
(2.056 and 2.068 Å) are similar to values found for 1-Methy-
bridged compounds such as [Pd2(mipk)2(µ-1-Methy)2]2+
(2.043 and 2.045 Å).21 However the Pd-O (2.150 and 2.133
Å) distances in complex 1 are higher than the corresponding
lengths (2.010 and 2.012 Å) in the above-mentioned com-
plex.21 A number of cyclometalated dmba complexes with
nitrogen and oxygen ligands have been structurally charac-
terized. In all instances the nitrogen atoms occupy trans sites
at the metal with oxygen trans to carbon, as indeed is
observed for 1. The Pd-N(sp3) distances in 1, 2.095(2) and
2.074(3) Å, are comparable with those in [{Pd(dmba)}2(µ-
NHC(Ph)O)2] (2.079(3) and 2.087(3) Å).14 The Pd-C
distances in 1 (1.970(3) and 1.967(3) Å) also lie within the
range normal for palladium dmba complexes. Dinuclear
neutral molecules of complex 1 are stacked to give centro-
symmetric pairs, as shown in Figure 2, due to intermolecular
π-π interactions between 1-Methy ligands (C13‚‚‚C13:
3.418 Å). The planes of the ligands are parallel, the distance
between both planes being 3.403(4) Å and the distance
between centroids being 4.614 Å. The angle between the
ring normal and the centroid vector is 43°.
Figure 1. ORTEP of complex 1, showing the atom-numbering scheme.
Displacement ellipsoids are drawn at the 50% probability level.
Table 1. Selected Distances (Å) and Bond Angles (deg) for Complex 1
Bond Distances
Pd(1)-C(1)
Bond Angles
C(1)-Pd(1)-N(2)
1.970(3)
2.068(2)
2.095(2)
2.150(2)
1.967(3)
2.056(2)
2.074(3)
2.133(2)
3.008(1)
91.58(11)
82.81(12)
172.36(10)
172.01(11)
92.85(9)
92.15(10)
93.64(11)
82.76(12)
171.47(10)
172.82(11)
93.18(9)
Pd(1)-N(2)
Pd(1)-N(1)
Pd(1)-O(3)
Pd(2)-C(22)
Pd(2)-N(5)
Pd(2)-N(6)
Pd(2)-O(2)
Pd(1)‚‚‚Pd(2)
C(1)-Pd(1)-N(1)
N(2)-Pd(1)-N(1)
C(1)-Pd(1)-O(3)
N(2)-Pd(1)-O(3)
N(1)-Pd(1)-O(3)
C(22)-Pd(2)-N(5)
C(22)-Pd(2)-N(6)
N(5)-Pd(2)-N(6)
C(22)-Pd(2)-O(2)
N(5)-Pd(2)-O(2)
N(6)-Pd(2)-O(2)
90.17(10)
with the proposed formulas. The FAB mass spectra show
peaks at m/z ) 760 (for 1), 732 (for 2), and 730 (M -
CH2Cl2)+ for 3 confirming the dimeric nature of these
complexes. The IR of 1 shows an intense mode for 1-Methy
at ca. 1540 cm-1 which indicates strong metal binding to
both N(3) and the exocyclic oxygen O(4). The other intense
IR band in the double-bond stretching region, at ca. 1640
cm-1, is frequently split in 1-Methy compounds (∆ ) 15
cm-1).1
Crystal Structures of 1 and 3‚2CHCl3. Figure 1 shows
the X-ray structure of complex 1, with selected bond lengths
and angles listed in Table 1. To our knowledge, this
compound represents the first example of an organometallic
palladium(II) complex with two 1-methylthyminato nucleo-
bases acting as bridges being fully characterized by X-ray
diffraction.20 The complex consists of two {(o-C6H4CH2-
NMe2)Pd} fragments each bridged by two 1-methylthyminato
ligands through N(3) and O(4) with a head-to-tail arrange-
ment and anti structure. The intramolecular Pd‚‚‚Pd distance
in complex 1 is 3.008(1) Å, a bit longer than the shortest
value found so far (2.861 Å) for a head-to-tail 1-methyl-
thyminato-bridged palladium complex which has been
observed in [Pd2(mipk)2(µ-1-Methy)2]2+ (mipk ) 1-methyl-
The crystal structure of the dimeric complex 3‚2CHCl3 is
shown in Figure 3. Selected interatomic distances and angles
are listed in Table 2. To our knowledge, this compound
represents the first example of a (1-methylcytosinato)-
palladium(II) cyclometalated complex being fully character-
ized by X-ray diffraction. Each Pd atom is in a distorted
square-planar arrangement. The metals are bridges by two
cytosinate anions through the endocyclic N(3) and the
deprotonated amino group, N(4), in a head-to-tail arrange-
ment and anti structure. The N(2)‚‚‚N(3) and N(5)‚‚‚N(7)
bite distances (2.329 and 2.322 Å, respectively) are consider-
ably shorter than the intramolecular Pd‚‚‚Pd distance of
3.0056(6) Å, which is close to that found in [Pd2(NH3)4(µ-
1-Mecyt)2]2+ (2.948(1) Å).23 As a consequence, the coordi-
nation planes of the two metal atoms form a dihedral angle
of 31.6(2)°. The dihedral angle between the two pyrimidine
planes is 86.1(2)°. The bond lengths and angles within the
cytosinate rings do not differ significantly from published
(21) Engelking, H.; Krebs, B. J. Chem. Soc., Dalton Trans. 1996, 2409.
(22) Mock, C.; Puscasu, I.; Rauterkus, M. J.; Tallen, G.; Wolff, J. E. A.;
Krebs, B. Inorg. Chim. Acta 2001, 319, 109.
(23) Navarro-Ranninger, C.; Montero, E. I.; Lo´pez-Solera, I.; Masaguer,
J. R.; Lippert, B. J. Organomet. Chem. 1998, 558, 103.
(20) CCDC CSD version 5.27, Nov 2005, update Jan 2006.
Inorganic Chemistry, Vol. 45, No. 16, 2006 6349

Ruiz et al.
Figure 2. Centrosymmetric dimer-of-dimers arrangement of 1.
Scheme 3
Figure 3. ORTEP of complex 3‚2CHCl3, showing the atom-numbering
scheme. Displacement ellipsoids are drawn at the 50% probability level.
Table 2. Selected Distances (Å) and Bond Angles (deg) for Complex
3‚2CHCl3
both the N-methyl and the CH2 groups of the dmba are
diastereotopic, two separate signals being observed for the
former and an AB quartet for the latter. Therefore, there is
Bond Distances
Pd(1)-C(1)
Bond Angles
C(1)-Pd(1)-N(2)
1.978(5)
2.015(4)
2.097(4)
2.153(4)
1.984(5)
2.096(4)
2.167(4)
2.018(4)
3.0056(6)
93.0(2)
81.8 (2)
no plane of symmetry in the Pd coordination plane.25
A
Pd(1)-N(2)
Pd(1)-N(1)
Pd(1)-N(7)
Pd(2)-C(20)
Pd(2)-N(8)
Pd(2)-N(3)
Pd(2)-N(5)
Pd(1)‚‚‚Pd(2)
C(1)-Pd(1)-N(1)
N(2)-Pd(1)-N(1)
C(1)-Pd(1)-N(7)
N(2)-Pd(1)-N(7)
N(1)-Pd(1)-N(7)
C(20)-Pd(2)-N(5)
C(20)-Pd(2)-N(8)
N(5)-Pd(2)-N(8)
C(20)-Pd(2)-N(3)
N(5)-Pd(2)-N(3)
N(8)-Pd(2)-N(3)
folded “basket” structure related to that observed in acetato-
bridged dimers is likely.14,26 Since both C6H4CH2NMe2 and
the nucleobases anions are unsymmetrical, there are five
possible diastereomers for the dimeric complexes, two of
them with head-to-head (d and e, Scheme 3, for complexes
1 and 2) and the other three with head-to-tail arrangements
(a-c) of the bridging nucleobase anionic ligands, but only
one isomer is present in solution at room temperature,
perhaps due to the fact that the ligand dmba exerts very
172.45(18)
175.30(19)
91.39(17)
93.92(16)
93.5(2)
81.7 (2)
172.59(18)
175.83(19)
90.45(17)
94.25(16)
values.24 Furthermore, hydrogen bonding is clearly a major
factor in holding the crystal together (distance C30-
H30‚‚‚O2, 3.063 Å; distance C29-H29‚‚‚O1, 3.261 Å;
distance N5-H5A‚‚‚C4, 3.237 Å).
(24) Faggiani, R.; Lippert, B.; Lock, C. J. L.; Speranzini, R. A. J. Am.
Chem. Soc. 1981, 103, 1111.
(25) Deeming, A. J.; Mean, M. N.; Bates, P. J.; Hursthouse, M. B. J. Chem.
Soc., Dalton Trans. 1978, 1490.
(26) Deeming, A. J.; Mean, M. N.; Bates, P. J.; Hursthouse, M. B. J. Chem.
Soc., Dalton Trans. 1988, 2193.
1
NMR Data for Complexes 1-3. The H NMR spectra
of these compounds (see Experimental Section) show that
6350 Inorganic Chemistry, Vol. 45, No. 16, 2006

Pd(II) and Pt(II) Organometallic Complexes
Scheme 4
Scheme 5
Figure 4. ORTEP of complex 4‚3CHCl3, showing the atom-numbering
scheme. Displacement ellipsoids are drawn at the 50% probability level.
Table 3. Selected Distances (Å) and Bond Angles (deg) for Complex
4‚3CHCl3
Bond Distances
Bond Angles
C(1)-Pd(1)-N(2)
C(1)-Pd(1)-N(1)
N(2)-Pd(1)-N(1)
C(1)-Pd(1)-P(1)
N(2)-Pd(1)-P(1)
N(1)-Pd(1)-P(1)
Pd(1)-C(1)
2.007(7)
2.107(6)
2.149(6)
2.2385(18)
173.8(3)
82.1(3)
92.0(2)
94.0(2)
91.95(17)
175.95(17)
Pd(1)-N(2)
Pd(1)-N(1)
Pd(1)-P(1)
different trans influences through its C and N atoms.26 The
observation of a unique resonance for the MeN(1) and
MeC(5) protons of the 1-methylthyminato ligands in the 1H
NMR spectrum of complex 1 (at δ 3.32 and 1.77, respec-
tively), together with the observed C6H4CH2NMe2 reso-
nances, are consistent with only the head-to-tail structures
(b or c, Scheme 3) (the assignments were unambiguously
AgCl by addition of AgClO4 in a 1:1 molar ratio in acetone,
the solvento complexes [Pt(dmba)(L′)(Me2CO)]ClO4 gener-
ated in situ react with 1 equiv of 1-MethyH or 1-MeuraH
and [NBu4]OH to give the neutral complexes [M(dmba)-
(L′)(L)] (L ) 1-Methy or 1-Meura; 6-9).
Complexes 4-9 are white, air-stable solids that decompose
on heating above 237 °C. Evidence for the coordination of
the metal to the deprotonated imidic N3 atom comes from
the lowered CdO stretching frequencies (1705 and 1685
cm-1 in N1-methylthymine), which reflects the longer
carbonyl bonds due to deprotonation of the N3-H group.10
Crystal Structures of 4‚3CHCl3 and 6. The structure of
4‚3CHCl3 is shown in Figure 4. Selected bond distances and
bond angles are given in Table 3. Coordination at palladium
is approximately square planar, although the angles around
palladium deviate from 90° due to the bite of the cyclo-
metalated ligand. The C(1)-Pd-N(1) angle of 82.1(3)° is
within the normal range for such complexes.14,29 The PPh3
ligand is trans to the nitrogen donor due to the difficulty of
coordinating a phosphine trans to an aryl ligand in palladium
complexes (i.e. the destabilizing effect known as transpho-
bia).30 The Pd atom coordinates N3 of thyminato. The Pd-
N3 distance is 2.107 Å, suggesting a strong interaction
between the metal and the imidic nitrogen.10 The Pd-C bond
length is essentially the same as that reported in [Pd(o-
C6H4CH2NMe2)(PCy3)(TFA)].29 The complex 4‚3CHCl3 has
1
confirmed by H-13C COSY experiments for complexes
1-3). In complex 2 a unique resonance pattern for the H(6),
H(5), and MeN(1) protons of the 1-methyluracilato ligands
is observed. Furthermore, we should expect that the O atom
would be trans to the carbon atom of the C6H4CH2NMe2
chelate (an anti arrangement c) by analogy to the imidate
complexes [{(C,N)Pd(µ-NCOC2H4CO)}2] (C,N ) o-C6H4CH2-
NMe2, o-C6H4CHdNPh)27 and the amidate complex [{(C,N)-
Pd(µ-NHC(Ph)O)}2] (C,N ) o-C6H4CH2NMe2).14 All this
suggests that c is the most probable structure for the binuclear
complexes 1 and 2 in solution, which is in accordance with
the crystal structure of complex 1.
The 1H NMR spectrum of complex 3 shows the exocyclic
amine proton of 1-Mecyt as a singlet at δ 5.13. The
comparison of the chemical shifts observed for the aromatic
1-Mecyt protons in 3 (5.50 and 6.55 ppm for H(5) and H(6))
and free 1-MecytH (5.60 and 7.55 ppm for H(5) and H(6),
respectively) are also consistent with 1-Mecyt acting as a
bridged ligand through N(3) and N(4) atoms.23,28 This point
is also consistent with the X-ray determination of the crystal
structure of complex 3 described above.
Monomeric Metal Complexes [M(dmba)(L)(L′)] (M )
Pd or Pt; L ) 1-Methy or 1-Meura; L′ ) PPh3 or
DMSO). The reactions of 1 and 2 with PPh3 (in a 1:2 molar
ratio) in dichloromethane at room temperature yield the
monomeric complexes [Pd(dmba)(L)(PPh3)] [L ) 1-Methy
(4) and L ) 1-Meura (5)], respectively (Scheme 4).
The platinum analogous complexes have been prepared
from the corresponding chloro precursor [PtCl(dmba)(L′)]
(L′ ) PPh3 or DMSO) (Scheme 5). After precipitation of
(27) Adams, H.; Bailey, N.; Briggs, T. N.; McCleverty, J. A.; Colquhoun,
H. M.; Williams, D. J. J. Chem. Soc., Dalton Trans. 1986, 813.
(28) Krumm, M.; Mutikainen, I.; Lippert, B. Inorg. Chem. 1991, 30, 884.
(29) Bedford, R. B.; Cazin, C. S. J.; Coles, S. J.; Gelbrich, T.; Horton, P.
N.; Hursthouse, M. B.; Light, M. E. Organometallics 2003, 22, 987.
(30) Vicente, J.; Arcas, A.; Bautista, D.; Jones, P. G. Organometallics 1997,
16, 2127.
Inorganic Chemistry, Vol. 45, No. 16, 2006 6351

Ruiz et al.
Scheme 6
1
NMR Data for Complexes 4-9. The H NMR spectra
of complexes 4-9 show that both the N-methyl and the CH2
groups of the dmba are diastereotopic, two separate signals
being observed for the former and an AB quartet for the
later. Therefore, there is no plane of symmetry in the metal
coordination plane. In complexes 4-7 the PPh3-trans-to-
NMe2 ligand arrangement in the starting products32,33 is
preserved, after chlorine abstraction and 1-Methy or 1-Meura
coordination, as can be inferred from the small, but signifi-
cant, coupling constants 4JP-H (ranging from 1.5 to 3.0 Hz)
of the NMe2 and the CH2N protons with the phosphorus
atom.34,35 Two different signals are also observed for the
DMSO ligand in the 13C{1H} NMR spectra of complexes 8
and 9. An unique resonance is observed in the 195Pt NMR
Figure 5. ORTEP of complex 6, showing the atom-numbering scheme.
Displacement ellipsoids are drawn at the 50% probability level.
Table 4. Selected Distances (Å) and Bond Angles (deg) for Complex 6
Bond Distances
Bond Angles
C(1)-Pt(1)-N(2)
C(1)-Pt(1)-N(1)
N(2)-Pt(1)-N(1)
C(1)-Pt(1)-P(1)
N(2)-Pt(1)-P(1)
N(1)-Pt(1)-P(1)
Pt(1)-C(1)
2.014(4)
2.122(3)
2.130(3)
2.2091(9)
172.59(12)
81.79(13)
92.62(11)
95.47(11)
90.56(8)
Pt(1)-N(2)
Pt(1)-N(1)
Pt(1)-P(1)
173.71(8)
two molecules of chloroform hydrogen bonded to the
thyminate ligand (distance C34-H34‚‚‚O1, 3.016 Å; distance
C36-H36‚‚‚O2, 2.725 Å). Remarkably, there is also an
intermolecular C-Cl‚‚‚O-C interaction31 (distance O1‚‚‚Cl6,
3.153 Å; angle O1‚‚‚Cl6-C35, 161.8°; angle C28-O1‚‚‚Cl6,
114.6°). Furthermore, there is an intramolecular interaction
by phenyl-thyminato π-stacking (N2‚‚‚C22, 3.115 Å;
C23‚‚‚C28, 3.217 Å). The planes of both rings make an angle
of 21.4°, with an interplanar distance (average of the two
distances between the mean plane of one ring and the
centroid of the other) of 3.375 Å, a center-to-center distance
of 3.649 Å, and a deviation of the center-center line of the
perpendicular of the plane of 18.1(4)° (thyminate ring) and
35.1(4)° (phenyl ring).
The structure of 6 is shown in Figure 5. Selected bond
distances and bond angles are given in Table 4. The platinum
atom displays approximately square planar coordination. The
X-ray crystallographic data revealed that Pt coordinates N3
of thyminate. The plane of the Methy ligand is approximately
perpendicular to the platinum coordination plane. The PPh3
ligand is trans to the nitrogen donor. There is an intra-
molecular interaction by phenyl-thyminato π-stacking
(N2‚‚‚C41, 3.063 Å; C10‚‚‚C42, 3.270 Å; C13‚‚‚C46, 3.331
Å). The planes of both rings make an angle of 20.9°, with
an interplanar distance (average of the two distances between
the mean plane of one ring and the centroid of the other) of
3.4189 Å, a center-to-center distance of 3.566 Å, and a
deviation of the center-center line of the perpendicular of
the plane of 11.6° (thyminato ring) and 20.2° (phenyl ring).
There is also a C-H‚‚‚O bond link (distance C45-
H45‚‚‚O1#, 3.180(5) Å; angle, 143.5°).
1
spectra of complexes 6-9. A comparison of H and 13C
NMR data for complex 4 and the related complex trans-
[PdCl(1-Methy)(PPh3)2]10 suggested that the coordination of
the metal occurred on the N3 atom of the nucleobase as
observed in the solid state.
Complexes [Pd(C6F5)(N-N)(L)] (L ) 1-Methy or
1-Meura). In acetone, the hydroxo complexes [Pd(N-N)-
(C6F5)(OH)] [N-N ) bpy (2,2′-bipyridyl), Me2bpy (4,4′-
dimethyl-2,2′-bipyridyl), or tmeda (N,N,N′,N′-tetramethyl-
ethylenediamine)] react with 1 equiv of 1-MethyH or
1-MeuraH to yield the corresponding complexes [Pd(N-
N)(C6F5)(L)] (10-13) (Scheme 6) in 65-73% yields. The
structures were assigned on the basis of microanalytical, IR,
1
and H and 19F NMR data and mass spectra (positive-ion
FAB). The IR spectra show the characteristic absorptions
of the C6F5 group36 at 1630, 1490, 1450, 1050, and 950 cn-1
and a single band at ca. 800 cm-1 derived from the so-called
X-sensitive mode37 in C6F5X (X ) halogen) molecules,
which is characteristic of the presence of only one C6F5 group
in the coordination sphere of the palladium atom and behaves
like a ν(M-C) band.38
(32) Deeming, A. J.; Rothwell, I. P.; Hursthouse, M. B.; New, L. J. Chem.
Soc., Dalton Trans. 1978, 1490.
(33) Meijer, M. D.; Kleij, A. W.; Williams, B. S.; Ellis, D.; Lutz, M.; Spek,
A. L.; van Klink, G. P. M.; van Koten, G. Organometallics 2002, 21,
264.
(34) Braunstein, P.; Matt, D.; Dusausoy, Y.; Fischer, J.; Mitschler, A.;
Ricard, L. J. Am. Chem. Soc. 1981, 103, 5115.
(35) Falvello, L. R.; Ferna´ndez, S.; Navarro, R.; Urriolabeitia, E. P. Inorg.
Chem. 1997, 36, 1136.
(36) Long, D. A.; Steele, D. Spectrochim. Acta 1963, 19, 1955.
(37) Maslowski, E. Vibrational Spectra of Organometallic Compounds;
Wiley: New York, 1977; p 437.
(31) Lommerse, J. P. M.; Stone, A. J.; Taylor, R.; Allen, F. H. J. Am.
Chem. Soc. 1996, 118, 3108.
(38) Alonso, E.; Fornie´s, J.; Fortun˜o, C.; Toma´s, M. J. Chem. Soc., Dalton
Trans. 1995, 3777.
6352 Inorganic Chemistry, Vol. 45, No. 16, 2006

Pd(II) and Pt(II) Organometallic Complexes
Figure 6. ORTEP of complex 12, showing the atom-numbering scheme.
Displacement ellipsoids are drawn at the 50% probability level.
Table 5. Selected Distances (Å) and Bond Angles (deg) for
Complex 12
Bond Distances
Bond Angles
C(11)-Pd(1)-N(3)
C(11)-Pd(1)-N(1)
N(3)-Pd(1)-N(1)
C(11)-Pd(1)-N(2)
N(3)-Pd(1)-N(2)
N(1)-Pd(1)-N(2)
Figure 7. π-π interactions in complex 12.
Pd(1)-C(11)
2.012(5)
2.038(4)
2.089(4)
2.123(4)
90.11(17)
93.12(17)
176.71(15)
177.74(16)
92.14(16)
84.63(17)
Scheme 7
Pd(1)-N(3)
Pd(1)-N(1)
Pd(1)-N(2)
Crystal Structure of 12. A drawing of complex 12 is
shown in Figure 6 with selected bond lengths and angles
listed in Table 5. The Methy ligand is approximately
perpendicular to the coordination plane, with angles between
planes of 89.5(1)°. The Pd-N thyminate distance (Pd(1)-
N(3): 2.038(4) Å) is shorter than that observed in complex
4‚3CHCl3, suggesting a very strong interaction between the
metal and the imidic nitrogen.10 The different Pd-N tmeda
distances (Pd(1)-N(1), 2.089(4) Å; Pd(1)-N(2), 2.123(4)
Å) are in agreement with the higher trans influence of the
group C6F5 compared to the thyminate. The Pd-C6F5 bond
length (2.012(5) Å) is in the range found in the literature
for pentafluorophenyl-palladium complexes.39 The chelate
angle N(1)-Pd(1)-N(2) is 84.63(17)°. There are also
intermolecular π-π interactions between Methy ligands, so
molecules of complex 12 are stacked to give centrosymmetric
pairs, as shown in Figure 7. It is a slipped packing with a
deviation of the center-center line of the perpendicular of
the plane of 24.7°; the interplanar distance is 3.384 Å.
Scheme 8
Complex [NBu4][Pd(C6F5)(H2O)(1-Methy)2]. The pal-
ladium bis(thyminate)complex [NBu4][Pd(C6F5)(H2O)(1-
Methy)2] (14) has been prepared by reaction in acetone of
the halo-complex [{Pd(C6F5)(NCPh)(µ-Cl)}2] with [NBu4]OH
and 1-Methymine in the molar ratio 1:4:4, as shown in
Scheme 8.
NMR Data for Complexes 10-13. The characteristic
resonances of the chelate ligands are observed in the 1H NMR
spectra,15,17,39-41 and the assignments presented in the
Experimental Section are based on the atom numbering given
in Scheme 7. The 19F NMR spectra of complexes 10-13 at
room temperature show hindered rotation of the C6F5 ring
around the Pd-C bond, and two separate signals are
observed for the o-fluorine atoms but only one for the
p-fluorine atom.
The new complex has been characterized by elemental
analysis and spectroscopic data.
Crystal Structure of 14‚H2O. The structure of compound
14‚H2O is shown in Figure 8, and selected bond lengths and
angles are in Table 6. To the best of our knowledge this is
the first crystal structure of a monomeric palladium bis-
(thyminate) complex.20 A trans geometry around the Pd
center is observed. For example, the angle N(1)-Pd(1)-
N(3) is 177.37(14)°. The crystal structure of the related bis-
(imidato) complex trans-[[Pd(C6F5)(H2O)(succinimidate)2]‚
H2O has been recently reported.19 The Pd-OH2 distances
are similar to those found in [Pd(C6F5)(AsPh3)(H2O)2]-
[CF3SO3]42 and [NBu4][Pd(C6F5)(H2O)(succinimidate)2]‚
(39) Fornie´s, J.; Mart´ınez, F.; Navarro, R.; Urriolabeitia, E. P. J. Organomet.
Chem. 1995, 495, 185.
(40) Byers, P. K.; Canty, A. J. Organometallics 1990, 9, 210.
(41) Ruiz, J.; Cutillas, N.; Vicente, C.; Villa, M. D.; Lo´pez, G.; Lorenzo,
J.; Avile´s, F. X.; Moreno, V.; Bautista, D. Inorg. Chem. 2005, 44,
7365.
Inorganic Chemistry, Vol. 45, No. 16, 2006 6353

Ruiz et al.
Figure 8. ORTEP of complex 14‚H2O, showing the atom-numbering
scheme. Displacement ellipsoids are drawn at the 50% probability level.
Figure 9. Dimeric structure of [Pd(C6F5)(H2O)(1-Methy)2]2‚2H2O, show-
ing the hydrogen bonds.
Table 6. Selected Distances (Å) and Bond Angles (deg) for Complex
14‚H2O
Table 7. Hydrogen Bonds for 14‚H2O (Å and deg)
Bond Distances
Bond Angles
C(13)-Pd(1)-N(3)
C(13)-Pd(1)-N(1)
N(3)-Pd(1)-N(1)
C(13)-Pd(1)-O(5)
N(3)-Pd(1)-O(5)
N(1)-Pd(1)-O(5)
D-H···A
d(D-H)
d(H···A)
d(D···A)
-(DHA)
Pd(1)-C(13)
1.981(5)
2.034(4)
2.056(4)
2.140(3)
87.28(18)
90.14(18)
177.37(14)
174.62(17)
88.17(15)
94.43(15)
O(6)-H(62)···O(3)
O(6)-H(61)···O(2)
O(5)-H(51)···O(1)#1
O(5)-H(52)···O(4)#1
0.79(2)
0.79(2)
0.81(2)
0.79(2)
1.99(2)
2.00(2)
1.94(2)
2.02(2)
2.777(6)
2.790(6)
2.746(5)
2.799(5)
178(6)
177(5)
175(6)
167(5)
Pd(1)-N(3)
Pd(1)-N(1)
Pd(1)-O(5)
a Symmetry transformations used to generate equivalent atoms: (#1) -x
+ 1, -y + 1, -z + 1.
H2O.19 An interesting feature of the crystal structure is the
presence of dimeric units of [Pd(C6F5)(H2O)(1-Methy)2]2‚
2H2O (Figure 9) due to OH‚‚‚O hydrogen bonds (Table 7)
involving the thyminate ligands, the coordinated water
molecules, and the water molecules of crystallization. There
are also short intermolecular contacts (O1···N3 3.062 Å).
Table 8. CD Spectral Data for DNA for Different ri Values (Molar
Ratios)
λmax
(nm) (deg‚cm2‚dmol-1
10-3θmax
λmin
(nm) (deg‚cm2‚dmol-1
)
10-3θmin
compd
ri
)
DNA
6
275.2
1.89
1.94
1.89
1.83
1.74
1.62
1.50
2.06
2.14
2.16
2.03
2.08
2.18
246.2
246.0
247.0
246.0
245.8
247.0
247.4
246.6
248.6
247.8
246.2
246.0
247.8
-2.21
-1.94
-2.03
-1.97
-2.01
-1.62
-1.72
-1.94
-2.04
-2.02
-1.97
-2.03
-1.96
0.1 273.0
0.3 275.0
0.5 273.8
0.1 276.4
0.3 277.2
0.5 275.6
0.1 247.2
0.3 277.6
0.5 279.6
0.1 275.0
0.3 275.4
0.5 276.6
1
NMR Data for Complex 14. The H NMR spectrum of
7
8
9
complex 14 exhibits an unique set of resonances for the
thyminate ligands which suggests a trans isomer in acetone-
d6 solution. No isomerization process was observed when a
solution of 14 was kept in solution in acetone-d6 for a
prolonged period. The trans structure of 14 is in accordance
with the single-crystal X-ray diffraction data given above.
As expected, three signals with relative intensities of 2:1:2
(2Fortho:1Fpara:2Fmeta) are found in the 19F NMR spectrum of
14.
Biological Assays. Circular Dichroism Spectroscopy.
The circular dichroism (CD) spectra of calf thymus DNA
alone and incubated with the ligands 1-MethyH and 1-MeuraH
and its platinum(II) compounds at 37 °C for 24 h with several
molar ratios were recorded.
The free ligands 1-MethyH and 1-MeuraH did not
significantly modify either the ellipticity of the bands or their
position. In contrast, the changes in ellipticity and wavelength
caused by the new platinum complexes are significant (Table
8). Complexes 6 and 7 reduce the ellipticity of the positive
band with increasing values of ri. This phenomenon is
indicative of B f C transformation with increasing winding
of the DNA helix by rotation of the bases.43-46 Complexes
8 and 9 induce modifications in the secondary structure of
DNA in a way different. In this case, the two complexes
increase the ellipticity of the positive band with increasing
values of ri. These changes were similar to those produced
by cisplatin.47 These modifications are probably due to
opening of the helix upon the formation of DNA intrastrand
adducts.48
Gel Electrophoresis of Compound-pBR322 Com-
plexes. The influence of the compounds on the tertiary
structure of DNA was determined by their ability to modify
the eleectrophoretic mobility of the covalently closed circular
(ccc) and open (oc) forms of pBR322 plasmid DNA. The
(45) Cervantes, G.; Prieto, M. J.; Moreno, V. Met. Based Drugs 1997, 4,
9.
(46) Cervantes, G.; Marchal, S.; Prieto, M. J.; Pe´rez, J. M.; Gonza´lez, V.
M.; Alonso, C.; Moreno, V. J. Inorg. Biochem. 1999, 77, 197.
(47) Riera, X.; Moreno, V.; Freisinger, E.; Lippert, B. Inorg. Chim. Acta
2002, 253-264.
(48) Moradell, S.; Lorenzo, J.; Rovira, A.; Robillard, M. S.; Avile´s, S.;
Moreno, V. J. Inorg. Biochem. 2003, 96, 500.
(42) Ruiz, J.; Florenciano, F.; Vicente, C.; Ram´ırez de Arellano, M. C.;
Lo´pez, G Inorg. Chem. Commun. 2000, 3, 73.
(43) Macquet, J. P.; Butour, J. L. Biochimie 1978, 60, 901.
(44) Brabec, V.; Kleinwa¨chter, V.; Butour, J.; Johnson, M. P. Biophys.
Chem. 1990, 35, 129.
6354 Inorganic Chemistry, Vol. 45, No. 16, 2006

Pd(II) and Pt(II) Organometallic Complexes
Table 9. IC50 (µM) for Cisplatin and Complexes 6-9 for the Tumor
Cell Line HL-60
complex
24 h
72 h
cisplatin
15.61
0.692
0.674
1.614
1.537
2.15
6
7
8
9
0.729
0.643
0.935
0.782
Figure 10. Modification of the gel electrophoretic mobility of pBR322
plasmid incubated with 1-MethyH and 1-MeuraH and their platinum
compounds: lane 1, pBR; lane 2, MethyH; lane 3, MeuraH; lane 4, complex
6; lane 5, complex 7; lane 6, complex 8; lane 7, complex 9; lane 8, complex
pBR-cisplatin.
mobility, the complexes 6-9 modify the morphology of the
pBR322 DNA and the ccc forms are predominant in the
pictures (Figure 11). In all cases the complexes seem to
modify the morphology of the pBR322 DNA in mode similar
to that of cisplatin.41,45,50-52 The platinum complexes attached
to DNA cause kinks and cross-linking in the plasmid forms.
The background of the Figure 11d indicates the presence of
a layer of water molecules from the environment over the
mica surface which can be the origin of the aggregation of
the forms.
Cytotoxicity Studies. The in vitro growth inhibitory effect
of the platinum complexes 6-9 and cisplatin was evaluated
in HL-60 human tumor cell line. As listed in Table 9, both
at 24 and 72 h incubation times all of the new platinum
complexes were more active than cisplatin. In fact, at a short
incubation time (24 h) complexes 6 and 7 were about 20-
fold more active than cisplatin toward HL60 cells. After 72
h of incubation time, complexes 6-9 were also about 3-fold
more active than cisplatin.
platinum complexes and the free ligands 1-MethyH and
1-MeuraH were incubated at the molar ratio ri ) 0.50 with
pBR322 plasmid DNA at 37 °C for 24 h. Representative gel
obtained for the Pt complexes 6-9 are shown in Figure 10.
The behavior of the gel electrophoretic mobility of both
forms, ccc and oc, of pBR322 plasmid and the DNA-
cisplatin adduct is consistent with previous reports.49 No
changes were observed in sample incubated with the free
ligands 1-MethyH and 1-MeuraH. When the pBR322 was
incubated with the platinum compounds 6 (lane 4), 7 (lane
5), 8 (lane 6), and 9 (lane 7) a retard in the mobility of ccc
form was observed.
AFM Study of Compound-pBR322 Complexes. Con-
sequently to the behavior observed in the electrophoretic
Figure 11. TMAFM image corresponding to (a) pBR322, (b) pBR-cisplatin, (c) pBR-complex 6, (d) pBR-complex 7, (e) pBR-complex 8, and (f)
pBR-complex 9.
Inorganic Chemistry, Vol. 45, No. 16, 2006 6355

Ruiz et al.
Table 10. Percentages of Vital Cells, Apoptotic Cells, Dead Cells, and
Damaged Cells after Treatment of a HL-60 Cell Population with
Cisplatin and Complexes 6-9 for 24 h
The reaction of complex 6 with an excess of 9-ethylguanine
is also fast and yields the formation of [Pt(dmba)(PPh3)(9-
EtG)]+.
% vital
cells
% apoptotic
cells
% dead
cells
% damaged
cells
treatment
Conclusions
(IC50 24 h, mM)
(R1)
(R2)
(R3)
(R4)
New Pd(II) and Pt(II) organometallic complexes with the
anions of the model nucleobases 1-methylthymine, 1-methyl-
uracil, and 1-methylcytosine have been prepared. The crystal
structures of [Pd(dmba)(µ-1-Methy)]2, [Pd(dmba)(µ-1-
Mecyt)]2‚2CHCl3, [Pd(dmba)(1-Methy)(PPh3)]‚3CHCl3,
[Pt(dmba)(1-Methy)(PPh3)], [Pd(tmeda)(C6F5)(1-Methy)], and
[NBu4][Pd(C6F5)(1-Methy)2(H2O)]‚H2O, have been estab-
lished by X-ray diffraction. Dinuclear neutral molecules of
complex [Pd(dmba)(µ-1-Methy)]2 are stacked to give cen-
trosymmetric pairs due to intermolecular π-π interactions
between 1-Methy ligands. There are also intermolecular π-π
interactions between Methy ligands in [Pd(tmeda)(C6F5)(1-
Methy)]. An interesting feature of the crystal structure of
[NBu4][Pd(C6F5)(1-Methy)2(H2O)]‚H2O is the presence of
dimeric units of [Pd(C6F5)(H2O)(1-Methy)2]2‚2H2O due to
OH‚‚‚O hydrogen bonds. The DNA adduct formation of the
new platinum complexes synthesized was followed by
circular dichroism and electrophoretic mobility. Atomic force
microscopy images of the modifications caused by the
platinum complexes on plasmid DNA pBR322 were also
obtained. The changes observed are consistent with the
results of electrophoretic mobility, and they confirm the
strong interaction of the complexes with DNA. Values of
IC50 were also calculated for the new platinum complexes
against the tumor cell line HL-60. All the new platinum
complexes were more active than cisplatin (up to 20-fold in
some cases).
control
85.39
53.89
63.38
68.55
46.38
54.29
8.43
40.68
27.77
19.61
32.52
21.25
5.95
4.89
8.48
10.26
16.05
12.97
0.24
0.55
0.38
1.58
5.05
1.50
cisplatin (15.6)
6 (0.692)
7 (0.674)
8 (1.614)
9 (1.537)
In Vitro Apoptosis Assay. The most common and well-
defined form of programmed cell death is apoptosis, which
is a physiological cell suicide program that is essential for
the maintenance of tissue homeostasis in multicellular
organism. In contrast to the self-contained nature of apoptotic
cell death, necrosis is a messy, unregulated process of
traumatic cell destruction, which is followed by the release
of intracellular components. During the past decade, cell
biology as well as oncology research has focused on this
latter process of programmed cell death, or apoptosis. It is
anticipated that understanding of the basic mechanisms that
underlie apoptosis will offer potential new targets for
therapeutic treatment of diseases.53
The type of cell death induced by the new complexes was
investigated by Annexin V/PI apoptosis assay in a flow
cytometer. The results obtained for the different complexes
at 24 h of incubation time were compared with those for
cisplatin. With all drugs, apoptosis was observed and the
percentages are presented in Table 10. Exposure of cells to
cisplatin and complexes 6 and 8 resulted in a significant
induction of apoptosis (30-40% of apoptotic cells) compared
with that induced by complexes 7 and 9 (20%). However,
an additional population of cells appeared to be dying by
necrosis or late apoptosis when HL60 cells were exposed to
complexes 6-9.
Reactions of the Platinum Complexes 6 and 8 with
9-Ethylguanine and Guanosine. To gain insight into the
reactivity of the new platinum complexes, the reactions of
complex 8 with an excess of 9-ethylguanine and guanosine
were followed by 1H NMR at physiological temperature and
pH (see Experimental Section). In both cases the reactions
are very fast and in less than 5 min the formation of the
corresponding complexes [Pt(dmba)(DMSO)(L)]+ (L )
9-EtG or guanosine) is completed. No further changes were
observed after 2 h. In both cases the signal for the H(8) of
the free guanine or guanosine moves downfield upon
coordination. In the reaction of complex 8 with the model
nucleobase 9-ethylguanine two different signals were ob-
served both for the coordinated DMSO and the NMe2 of the
dmba (Experimental Section and Supporting Information).
Experimental Section
Instrumental Measurements. The C, H, N, and S analyses were
performed with a Carlo Erba model EA 1108 microanalyzer.
Decomposition temperatures were determined with a SDT 2960
simultaneous DSC-TGA of TA instruments at a heating rate of 5
°C min-1 and the solid samples under nitrogen flow (100 mL
1
min-1). The H, 13C, 31P, and 19F NMR spectra were recorded on
a Bruker AC 200E or Bruker AC 300E spectrometer, using SiMe4,
H3PO4, and CFCl3 as standards. The 195Pt NMR spectra were
recorded on a Bruker AV 400 spectrometer, using Na2[PtCl6] as
standard. Infrared spectra were recorded on a Perkin-Elmer 1430
spectrophotometer using Nujol mulls between polyethylene sheets.
Mass spectra (positive-ion FAB) were recorded on a V.G. Auto-
SpecE spectrometer and measured using 3-nitrobenzyl alcohol as
the dispersing matrix.
Materials. The starting complexes [Pd(dmba)(µ-OH)]2,14 [Pd-
(C6F5)(N-N)(OH)] (N-N ) bpy, Me2bpy, or tmeda),15 [Pt(dmba)-
Cl(L)] (L ) PPh3 and DMSO),33 and [{Pd(C6F5)(NCPh)(µ-Cl)}2]54
were prepared by procedures described elsewhere. Solvents were
dried by the usual methods. 1-Methylcytosine (1-Mecyt) was
purchased from Chemogen (Konstanz, Germany). 1-Methyluracil,
1-methylthymine, the sodium salt of calf thymus DNA, EDTA
(ethylenediaminotetracetic acid), and Tris-HCl (tris(hydroxymethyl)-
aminomethane-hydrochloride) used in the circular dichroism (CD)
(49) Ushay, H. M.; Tullius, T. D.; Lippard, S. J. Biochemistry 1981, 20,
3744.
(50) Onoa, G. B.; Cervantes, G.; Moreno, V.; Prieto, M. J. Nucleic Acids
Res. 1998, 26, 1473.
(51) Cervantes, G.; Marchal, S.; Prieto, M. J.; Pe´rez, J. M.; Gonza´lez, V.
M.; Alonso, C.; Moreno, V. J. Inorg. Biochem. 1999, 77, 197.
(52) Onoa, G. B.; Moreno, V. Int. J. Pharm. 2002, 245, 55.
(53) Martin, S. J.; Green, D. R. Curr. Opin. Oncol. 1994, 6, 616.
(54) Albe´niz, A. C.; Espinet, P.; Jeannin, Y.; Philoche-Levisalles, M.; Mann,
B. E. J. Am. Chem. Soc. 1990, 112, 6599.
6356 Inorganic Chemistry, Vol. 45, No. 16, 2006

Pd(II) and Pt(II) Organometallic Complexes
study were obtained from Sigma-Aldrich (Madrid, Spain); HEPES
(N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid) was obtained
from ICN (Madrid, Spain), and pBR322 plasmid DNA was obtained
from Boehringer-Mannheim (Mannheim, Germany).
The resulting solution was stirred at room temperature for 1 h and
then concentrated under vacuum. The addition of hexane caused
the precipitation of a white solid, which was collected by filtration
and air-dried.
Warning! Perchlorate salts of metal complexes with organic
ligands are potentially explosive. Only small amounts of material
should be prepared, and these should be handled with great caution.
Preparation of Complexes [Pd(dmba)(µ-L)]2 [L ) 1-Methy
(1) and 1-Meura (2)]. To a suspension of [Pd(dmba)(µ-OH)]2 (100
mg, 0.194 mmol) in dichloromethane (3 mL) was added 1-methyl-
thymine or 1-methyluracil (0.388 mmol). The resulting solution
was stirred at room temperature for 1 h and then concentrated under
vacuum. The addition of hexane caused the precipitation of a yellow
solid which was collected by filtration and air-dried.
Data for Complex 4. Yield: 85%. Anal. Calcd for C33H34N3O2-
PPd: C, 61.7; H, 5.3; N, 6.5. Found: C, 61.6; H, 5.5; N, 6.6. Mp:
264 °C (dec). IR (Nujol, cm-1): 1660, 1640, 1570, 1545. 1H NMR
(CDCl3): δ(SiMe4) 7.78 (m, 6H, PPh3), 7.33 (m, 9H, PPh3), 6.98
(d, 1H, aromatics of dmba, J 7.2), 6.81 (m, 1H, aromatics of dmba),
6.35 (m, 2H, aromatics dmba), 6.28 (d, 1H, H(6) of MeT, JHP 1.2),
4.15 (dd, 1H, NCH2, JHH 13.6, JHP 1.5), 3.98 (dd, 1H, NCH2, JHH
13.6, JHP 2.2), 2.93 (s, 3H, NMe of 1-Methy), 2.80 (d, 3H, NMe2,
J
HP 2.3), 2.77 (d, 3H, NMe2, JHP 2.5), 1.57 (d, 3H, CMe of 1-Methy,
J
HP 1.0). 13C{1H} NMR (CDCl3): δ(SiMe4) 138.7 (C(6) of 1-Methy
and aromatic CH dmba), 135.1, 130.2, 127.7 (aromatics CH PPh3),
124.4, 123.4, 121.9 (aromatics CH dmba), 72.6 (CH2NMe2), 51.1
(NMe2), 36.3 (NCH3 of 1-Methy), 13.7 (CH3C of 1-Methy). 13P
NMR (CDCl3): δ(H3PO4) 44.0 (s). Positive-ion FAB mass spec-
trum: m/z 643 (M + 1)+.
Data for Complex 1. Yield: 65%. Anal. Calcd for C30H38N6-
O4Pd2: C, 47.4; H, 5.0; N, 11.1. Found: C, 47.1; H, 5.0; N, 11.1.
Mp: 197 °C (dec). IR (Nujol, cm-1): 1655, 1640, 1548, 1534,
1518. 1H NMR (CDCl3): δ(SiMe4) 6.78 (m, 6H, aromatics of dmba
+ H(6) of 1-Methy), 6.65 (m, 2H, aromatics of dmba), 6.31 (d,
2H, aromatics dmba, J 7.7), 3.59 (d, 2H, NCH2, J 13.6), 3.32 (s, 6
H, NMe of 1-Methy), 3.17 (d, 2H, NCH2, J 13.6), 2.94 (s, 6H,
NMe2), 1.86 (s, 6H, NMe2), 1.77 (s, 6H, CMe of 1-Methy). 13C{1H}
NMR (CDCl3): δ(SiMe4) 139.7 (C(6) of 1-Methy), 134.0, 125.0,
123.5, 121.0 (aromatics CH dmba), 71.6 (CH2NMe2), 51.9 (NMe2),
50.6 (NMe2), 37.1 (CH3N of 1-Methy), 13.8 (CH3C of 1-Methy).
Positive-ion FAB mass spectrum: m/z 783 (M + 23)+, 760 (M)+.
Data for Complex 2. Yield: 83%. Anal. Calcd for C28H34N6-
O4Pd2: C, 46.0; H, 4.7; N, 11.5. Found: C, 46.1; H, 4.8; N, 11.2.
Mp: 195 °C (dec). IR (Nujol, cm-1): 1640, 1542. 1H NMR
(CDCl3): δ(SiMe4) 6.95 (d, 2H, H(6) of 1-Meura, JH6H5 7.3), 6.80
(m, 4H, aromatics of dmba), 6.66 (m, 2H, aromatics of dmba), 6.31
(d, 2H, aromatics dmba, J 7.7), 5.62 (d, 2H, H(5) of 1-Meura, JH6H5
7.3), 3.73 (d, 2H, NCH2, J 13.9), 3.33 (s, 6H, NMe of 1-Meura),
3.25 (d, 2H, NCH2, J 13.9), 2.96 (s, 6H, NMe2), 2.10 (s, 6 H, NMe2).
13C{1H} NMR (CDCl3): δ(SiMe4) 143.2 (C(6) of 1-Meura), 133.4,
125.1, 123.6, 121.1 (aromatics CH dmba), 101.8 (C(5) of 1-Meura),
71.9 (CH2NMe2), 52.1 (NMe2), 51.0 (NMe2), 37.8 (CH3N of
1-Meura). Positive-ion FAB mass spectrum: m/z 755 (M + 23)+,
732 (M)+.
Data for Complex 5. Yield: 80%. Anal. Calcd for C33H34N3-
Cl2O2PPd: C, 55.6; H, 4.8; N, 5.8. Found: C, 55.4; H, 4.8; N, 5.9.
Mp: 248 °C (dec). IR (Nujol, cm-1): 1640, 1564, 1548. 1H NMR
(CDCl3): δ(SiMe4) 7.80 (m, 6H, PPh3), 7.30 (m, 9H, PPh3), 6.99
(d, 1H, aromatic dmba, J 7.2), 6.82 (m, 1H, aromatic of dmba),
6.47 (d, 1H, H(6) of 1-Meura, JH6H5 7.2), 6.35 (m, 2H, aromatics
of dmba), 5.14 (d, 1H, H(5) of 1-Meura, JH6H5 7.2), 4.17 (dd, 1H,
NCH2, JHH 13.6, JHP 2.1), 3.97 (dd, 1H, NCH2, JHH 13.6, JHP 2.4),
2.90 (s, 3H, NMe of 1-Meura), 2.78 (d, 3H, NMe2, JHP 2.1), 2.76
(d, 3H, NMe2, JHP 2.4). 13C{1H} NMR (CDCl3): δ(SiMe4) 142.4
(C(6) of 1-Meura), 138.5 (aromatic CH dmba), 135.1, 130.2, 127.9
(aromatics CH PPh3), 124.4, 123.4, 121.9 (aromatics CH dmba),
102.3 (C(5) of 1-Meura), 72.6 (CH2NMe2), 50.6 (NMe2), 50.4
(NMe2), 36.5 (NCH3 of 1-Meura). 31P NMR (CDCl3): δ(H3PO4)
41.8 (s). Positive-ion FAB mass spectrum: m/z 702 (M - Meura)+.
Preparation of Complexes [Pt(dmba)(1-Methy)(L′)] (6, 8) and
[Pt(dmba)(1-Meura)(L′)] (7, 9). To a solution of [Pt(dmba)-
(Cl)(L′)] (L′ ) PPh3 or DMSO) (0.159 mmol) in acetone (20 mL)
was added AgClO4 (33.0 mg, 0.159 mmol). AgCl immediately
formed. The resulting suspension was stirred for 30 min and then
filtered through a short pad of Celite. To the filtrate was then added
20% [NBu4]OH(aq) (209 µL, 0.159 mmol) and finally either
1-MethyH or 1-MeuraH (0.159 mmol). The resulting solution was
stirred at room temperature for 6 h and then concentrated under
vacuum. The resulting white precipitate was collected by filtration
and air-dried.
Preparation of Complex [Pd(dmba)(µ-1-Mecyt)]2‚CH2Cl2 (3).
To a suspension of [Pd(dmba)(µ-OH)]2 (100 mg, 0.194 mmol) in
dichloromethane (20 mL) was added 1-methylcytosine (48.5 mg,
0.388 mmol). The resulting solution was stirred under reflux for 2
h. The addition of hexane caused the precipitation of a white solid
which was collected by filtration and air-dried.
Data for Complex 6. Yield: 82%. Anal. Calcd for C33H34N3O2-
PPt: C, 54.2; H, 4.7; N, 5.8. Found: C, 54.1; H, 4.9; N, 5.6. Mp:
303 °C (dec). IR (Nujol, cm-1): 1660, 1640, 1580. 1H NMR
(CDCl3): δ(SiMe4) 7.80 (m, 6H, PPh3), 7.33 (m, 9H, PPh3), 7.02
(d, 1H, aromatics of dmba, J 7.2), 6.82 (m, 1H, aromatics of dmba),
6.48 (m, 2H, aromatics dmba), 6.30 (d, 1H, H(6) of 1-Methy, JHP
1.0), 4.13 (dd, 1H, NCH2, JHH 13.6, JHP 2.1), 3.99 (dd, 1H, NCH2,
JHH 13.6, JHP 3.0), 2.94 (s, 3H, NMe of 1-Methy), 2.91 (d, 3H,
NMe2, JHP 2.7), 2.88 (d, 3H, NMe2, JHP 2.7), 1.57 (s, 3H, CMe of
1-Methy, JHP 0.6). 13C{1H} NMR (CDCl3): δ(SiMe4) 140.3 (C(6)
of 1-Methy), 140.2 (d, aromatic CH dmba, JHP 6.2), 137.2, 132.0,
129.4 (aromatics CH PPh3), 126.5, 124.4, 123.2 (aromatics CH
dmba), 75.3 (CH2NMe2), 52.8 (NMe2), 38.2 (NCH3 of 1-Methy),
15.4 (CH3C of 1-Methy). 31P NMR (CDCl3): δ(H3PO4) 21.2 (s,
JPt-P 4412). 195Pt NMR (CDCl3): δ(Na2[PtCl6]) -4013 (d, JPt-P
4412). Positive-ion FAB mass spectrum: m/z 730 (M)+.
Data for Complex 3. Yield: 65%. Anal. Calcd for C29H38N8-
Cl2O2Pd2: C, 42.8; H, 4.7; N, 13.8. Found: C, 42.8; H, 5.3; N,
14.1. Mp: 260 °C (dec). IR (Nujol, cm-1): 3345 ν(NH), 1650,
1615, 1538. 1H NMR (CDCl3): δ(SiMe4) 7.09 (dd, 2H, aromatics
of dmba, J 7.2, J′ 1.5), 6.88 (m, 4H, aromatics of dmba), 6.78 (d,
2H, aromatics dmba, J 7.0), 6.55 (d, 2H, H(6) of 1-Mecyt, JH6H5
7.2), 5.50 (d, 2H, H(5) of 1-Mecyt, JH6H5 7.2), 5.13 (s, 2H, NH of
1-Mecyt), 3.22 (s, 6H, NMe of 1-Mecyt), 3.05 (d, 2H, NCH2, J
13.8), 2.75 (s, 6H, NMe2), 2.74 (d, 2H, NCH2, J 13.8), 1.97 (s, 6
H, NMe2). 13C{1H} NMR (CDCl3): δ(SiMe4) 138.5 (C(6) of
1-Mecyt), 133.7, 124.0, 123.3, 121.0 (aromatics CH dmba), 100.6
(C(5) of 1-Mecyt), 71.4 (CH2NMe2), 52.0 (NMe2), 50.3 (NMe2),
36.8 (CH3N of 1-Mecyt). Positive-ion FAB mass spectrum: m/z
753 (M - CH2Cl2 + 23)+, 730 (M - CH2Cl2)+.
Preparation of Complexes [Pd(dmba)(1-Methy)(PPh3)] (4)
and [Pd(dmba)(1-Meura)(PPh3)]‚CH2Cl2 (5). To a suspension of
[Pd(dmba)(µ-L)]2 (L ) 1-Methy or 1-Meura) (0.132 mmol) in
dichloromethane (4 mL) was added PPh3 (69.1 mg, 0.254 mmol).
Data for Complex 7. Yield: 87%. Anal. Calcd for C32H32N3O2-
PPt: C, 53.6; H, 4.5; N, 5.9. Found: C, 53.8; H, 4.7; N, 5.9. Mp:
Inorganic Chemistry, Vol. 45, No. 16, 2006 6357

Ruiz et al.
1
288 °C (dec). IR (Nujol, cm-1): 1643, 1568. H NMR (CDCl3):
7.16 (d, 1H, Hâ, J(HRHâ) 6.0), 7.01 (d, 1H, H â′, J(HR′Hâ′) 6.0),
6.90 (d, 1H, H(6) of 1-Meura, JH6H5 7.3), 5.5 (d, 1H, H(5) of
1-Meura, JH6H5 7.3), 3.30 (s, 3H, NMe of 1-Meura), 2.4 (s, 6H, Me
of Me2bpy). 19F NMR (CDCl3): δ(CFCl3) -118.9 (d, 1Fo, J(FoFm)
32.2), -118.5 (d, 1Fo, J(FoFm) 39.2), -161.1 (t, 1Fp, J(FmFp) 31.0),
-163.3 (m, 2Fm). Positive-ion FAB mass spectrum: m/z 583 (M
+ 1)+.
Preparation of Complexes [Pd(tmeda)(C6F5)(L)] (12, 13). To
a solution of the hydroxo complex [Pd(tmeda)(C6F5)(OH)] (100
mg, 0.246 mmol) in acetone (15 mL) was added 1-MethyH or
1-MeuraH (0.246 mmol). The resulting mixture was stirred for 12
h at room temperature. The solvent was partially evaporated under
reduced pressure. On addition of hexane, the complexes 12 and 13
precipitated and were filtered off and air-dried.
δ(SiMe4) 7.80 (m, 6H, PPh3), 7.30 (m, 9H, PPh3), 7.02 (d, 1H,
aromatic dmba, J 7.2), 6.82 (m, 1H, aromatic of dmba), 6.50 (m,
3H, H(6) of 1-Meura + 2 H of aromatics of dmba), 5.18 (d, 1H,
H(5) of 1-Meura, JH6H5 7.3), 4.14 (dd, 1H, NCH2, JHH 13.2, JHP
2.0), 4.00 (dd, 1H, NCH2, JHH 13.2, JHP 2.8), 2.91 (s, 3H, NMe2 of
dmba), 2.88 (d, 3H, NMe2 of dmba, JHP 2.5), 2.16 (s, 3H, NMe of
1-Meura). 13C{1H} NMR (CDCl3): δ(SiMe4) 142.4 (C(6) of
1-Meura), 138.6 (d, aromatic CH dmba, JHP 6.2), 135.5, 130.5,
128.1 (aromatics CH PPh3), 125.1, 123.0, 123.0 (aromatics CH
dmba), 102.9 (C(5) of 1-Meura), 73.8 (CH2NMe2, JHP 3.1), 51.3
(NMe2, JHP 13.6), 37.2 (NMe2), 31.4 (NCH3 of 1-Meura). 31P NMR
(CDCl3): δ(H3PO4) 20.9 (s, JPt-P 4403). 195Pt NMR (CDCl3):
δ(Na2[PtCl6]) -4013 (d, JPt-P 4403). Positive-ion FAB mass
spectrum: m/z 716 (M)+.
Data for Complex 8. Yield: 86%. Anal. Calcd for C17H25N3O3-
SPt: C, 37.4; H, 4.6; N, 7.7; S, 5.9. Found: C, 37.7; H, 4.5; N,
7.5; S, 5.7. Mp: 237 °C (dec). IR (Nujol, cm-1): 1668, 1658, 1644,
1574. 1H NMR (CDCl3): δ(SiMe4) 7.93 (d, 1H, aromatics of dmba,
J 7.9, JHPt 38.0), 7.05 (m, 3H, aromatics of dmba), 6.93 (s, 1H,
H(6) of 1-Methy), 4.01 (d, 1H, NCH2, JHH 13.1), 3.92 (d, 1H, NCH2,
Data for Complex 12. Yield: 68%. Anal. Calcd for C18H23F5N4-
O2Pd: C, 40.9; H, 4.4; N, 10.6. Found: C, 41.2; H, 4.4; N, 10.3.
Mp: 285 °C (dec). IR (Nujol, cm-1): 1678, 1664, 1642, 1586,
1
ν(Pd-C6F5), 780. H NMR (CDCl3): δ(SiMe4) 6.85 (s, 1H, H(6)
of 1-Methy), 3.10 (s, 3H, NMe of 1-Methy), 2.90 (m, 4H, CH2),
2.61 (s, 3H, Me), 2.60 (s, 3H, Me), 2.58 (s, 3H, Me), 2.56 (s, 3H,
Me), 1.64 (s, 3H, CMe of 1-Methy). 19F NMR (CDCl3): δ(CFCl3)
-114.9 (m, 1Fo), -115.5 (m, 1Fo), -164.6 (t, 1Fp, J(FmFp) 20.7
Hz), -166.6 (m, 2Fm). Positive-ion FAB mass spectrum: m/z 529
(M + 1)+.
Data for Complex 13. Yield: 69%. Anal. Calcd for C17H21F5N4-
O2Pd: C, 39.7; H, 4.1; N, 10.9. Found: C, 39.9; H, 4.1; N, 10.7.
Mp: 208 °C (dec). IR (Nujol, cm-1): 1660, 1650, 1644, 1580,
ν(Pd-C6F5), 784. 1H NMR (CDCl3): δ(SiMe4) 7.01 (d, 1H, H(6)
of 1-Meura, JH6H5 7.2), 5.10 (d, 1H, H(5) of 1-Meura, JH6H5 7.2),
3.12 (s, 3H, NMe of 1-Meura), 2.90 (m, 4H, CH2), 2.61 (s, 6H,
Me), 2.60 (s, 3H, Me), 2.58 (s, 3H, Me). 19F NMR (CDCl3):
δ(CFCl3) -113.7 (m, 1Fo), -114.2 (m, 1Fo,), -163.4 (t, 1Fp,
J(FmFp) 20.7 Hz), -165.4 (m, 2Fm). Positive-ion FAB mass
spectrum: m/z 515 (M + 1)+.
Preparation of Complex [NBu4][Pd(C6F5)(1-Methy)2(OH2)]
(14). To a solution of 1-methylthymine (68 mg, 0.485 mmol) in
acetone (5 mL) was added 20% [NBu4]OH(aq) (636 µL, 0.485
mmol). To the resulting solution was added a solution of [{Pd(C6F5)-
(NCPh)(µ-Cl)}2] (100 mg, 0.121 mmol) in acetone (15 mL). The
mixture was stirred at room temperature for 24 h and then filtered
through a short pad of Celite. Solvent was partially evaporated under
reduced pressure. On addition of water the yellow complex 14
precipitated and was filtered off and air-dried.
J
HH 13.1), 3.31 (s, 3H, NMe of 1-Methy), 3.27 (s, 3H, DMSO, JHPt
30.0), 3.18 (s, 3H, DMSO, JHPt 25.5), 2.72 (s, 3 H, NMe2, JHPt
34.6), 2.70 (s, 3 H, NMe2, JHPt 33.0), 1.86 (s, 3H, CMe of 1-Methy).
13C{1H} NMR (CDCl3): δ 140.4 (C(6) of 1-Methy), 135.2, 126.3,
124.8, 122.0, (aromatics CH dmba), 75.0 (CH2NMe2), 52.0 (NMe2),
51.9 (NMe2), 47.0 (DMSO), 46.4 (DMSO), 37.1 (NCH3 of 1-
Methy), 13.9 (CH3C of 1-Methy). 195Pt NMR (CDCl3): δ(Na2[PtCl6])
-3673. Positive-ion FAB mass spectrum: m/z 546 (M)+.
Data for Complex 9. Yield: 78%. Anal. Calcd for C16H23N3O3-
SPt: C, 36.1; H, 4.4; N, 7.9; S, 6.0. Found: C, 35.8; H, 4.1; N,
7.7; S, 5.7. Mp: 238 °C (dec). IR (Nujol, cm-1): 1640, 1574. H
NMR (CDCl3): δ(SiMe4) 7.95 (d, 1 H, aromatic dmba, J 7.2, JHPt
37.6), 7.11 (m, 4 H, aromatic of dmba + H(6) of 1-Meura), 5.66
(d, 1H, H(5) of 1-Meura, JH6H5 7.5), 4.02 (d, 1H, NCH2, JHH 13.1),
3.96 (d, 1H, NCH2, JHH 13.1), 3.36 (s, 3H, NMe of 1-Meura), 3.29
(s, 3H, DMSO, JHPt 25.5), 3.20 (s, 3H, DMSO, JHPt 26.0), 2.72 (s,
6 H, NMe2, JPtH 30.8). 13C{1H} NMR (CDCl3): δ(SiMe4) 145.4
(C(6) of 1-Meura), 136.7, 127.8, 126.3, 123.5 (aromatics CH dmba),
104.9 (C(5) of 1-Meura), 76.4 (CH2NMe2), 53.5 (NMe2), 53.4
(NMe2), 48.4 (DMSO), 47.9 (DMSO), 38.9 (NCH3 of 1-Meura).
195Pt NMR (CDCl3): δ(Na2[PtCl6]) -3674. Positive-ion FAB mass
spectrum: m/z 702 (M + 1)+.
1
Preparation of Complexes [Pd(N-N)(C6F5)(L)] (N-N ) bpy
or Me2bpy) (10, 11). To a solution of the corresponding hydroxo
complex [Pd(N-N)(C6F5)(OH)] (0.243 mmol) in acetone (15 mL)
was added 1-MethyH or 1-MeuraH (0.243 mmol). The resulting
mixture was stirred for 12 h at room temperature to yield a white
suspension. The precipitate was filtered off and air-dried.
Data for Complex 10. Yield: 73%. Anal. Calcd for C22H15F5N4-
O2Pd: C, 46.5; H, 2.7; N, 9.9. Found: C, 46.6; H, 2.5; N, 9.7.
Mp: 281 °C (dec). IR (Nujol, cm-1): 1690, 1682, 1660, ν(Pd-
Data for Complex 14. Yield: 50%. Anal. Calcd for C34H52F5N5-
O5Pd: C, 50.3; H, 6.5; N, 8.6. Found: C, 50.1; H, 6.7; N, 8.3.
Mp: 202 °C (dec). IR (Nujol, cm-1): 1667, 1631,1575, ν(Pd-
1
C6F5), 776. H NMR (acetone-d6): δ(SiMe4) 7.03 (2H, H(6) of
1-Methy), 3.1 (s, 6 H, NMe of 1-Methy), 1.70 (s, 6H, CMe of
1-Methy). 19F NMR (acetone-d6): δ(CFCl3) -116.3 (m, 2Fo,
J(FoFm) 28.2 Hz), -168.0 (t, 1Fp, J(FmFp) 19.8 Hz), -169.7 (m,
2Fm). Positive-ion FAB mass spectrum: m/z 552 (M + 1 - H2O)+.
Reactions of the Platinum Complex 8 with 9-Ethylguanine
and Guanosine. Both reactions were carried out in NMR tubes
(D2O and 5% DMSO-d6 as solvents). 9-Ethylguanine or guanosine
was incubated with complex 8 in a ratio 5:1 in D2O at pH 7.0 (50
mM KD2PO4) and 37 °C. The concentration of complex 8 was 5.0
mM. Both reactions were very fast, and in less than 5 min the
formation of the corresponding complex [Pt(dmba)(DMSO)(L)]+
(L ) 9-EtG or guanosine) was completed.
1
C6F5), 780. H NMR (CDCl3): δ(SiMe4) 8.13 (m, 3H, bpy), 7.93
(m, 3H, bpy), 7.28 (m, 2H, Hâ + Hâ′), 6.80 (s, 1H, H(6) of
1-Methy), 3.31 (s, 3H, NMe of 1-Methy), 1.82 (s, 3H, CMe of
1-Methy). 19F NMR (CDCl3): δ(CFCl3) -117.9 (m, 1Fo), -118.7
(m, 1Fo), -160.9 (t, 1Fp, J(FmFp) 21.0 Hz), -163.0 (m, 2Fm).
Positive-ion FAB mass spectrum: m/z 569 (M + 1)+.
Data for Complex 11. Yield: 65%. Anal. Calcd for C23H17F5N4-
O2Pd: C, 47.4; H, 2.9; N, 9.6. Found: C, 47.1; H, 2.9; N, 9.7.
Mp: 284 °C (dec). IR (Nujol, cm-1): 1650, 1644, 1620, 1580,
ν(Pd-C6F5), 790. 1H NMR (CDCl3): δ(SiMe4) 7.95 (d, HR,
J(HRHâ) 6.0), 7.86 (s, 2H, Hδ + Hδ′), 7.70 (d, HR′, J(HR′Hâ′) 6.0),
1H NMR data for the reaction of 8 with 9-ethylguanine: δ(SiMe4)
8.66 (s, 1H, H(8) of coordinated EtG), 7.89 (s, H(8) of free
guanosine in excess), 7.76 (d, 1H, aromatics of dmba, J 7.5), 7.50
(s, 1H, H(6) of free 1-Methy), 7.29 (m, 3H, aromatics of dmba),
6358 Inorganic Chemistry, Vol. 45, No. 16, 2006

Pd(II) and Pt(II) Organometallic Complexes
Table 11. Crystal Structure Determination Details
param
1
3‚2CHCl3
4‚3CHCl3
6
12
14‚H2O
empirical formula C30H38N6O4Pd2 C28H36N8O2Pd2‚2CHCl3 C24H24.67Cl6N2O1.33P0.67Pd0.67 C33H34N3O2PPt C18H23F5N4O2Pd C34H54F5N5O6Pd
fw
759.46
monoclinic
9.9003(2)
19.3077(4)
16.9681(4)
90
106.19
90
3114.81(12)
293(2)
P21/n
968.18
monoclinic
20.1123(11)
9.7760(11)
20.7128(11)
90
92.273(5)
90
4069.3(6)
173(2)
P21/n
666.74
730.69
monoclinic
18.2589(11)
8.0212(5)
20.8978(13)
90
106.368(2)
90
2936.6(3)
100(2)
P21/c
528.80
monoclinic
21.686(2)
19.712(2)
11.9190(10)
90
108.457(7)
90
4833.0(8)
293(2)
C2/c
830.22
monoclinic
15.419(1)
16.771(1)
15.510(1)
90
97.43(1)
90
3977.1(5)
293(2)
P21/n
cryst system
a (Å)
c (Å)
triclinic
10.1410(10)
14.6570(10)
16.1070(10)
80.220(10)
72.170(10)
73.140(10)
2172.5(3)
173(2)
P1h
2
1.053
8512
c (Å)
R (deg)
â (deg)
γ (deg)
V (Å3)
temp (K)
space group
Z
4
4
4
8
4
µ (mm-1
)
1.199
21 088
7737
0.0305
0.0328
0.0829
1.315
10 388
7164
0.0368
0.0376
0.0936
4.867
31 104
5977
0.0451
0.0266
0.0580
0.825
5650
4267
0.0115
0.0411
0.1511
0.537
13 970
6990
0.0328
0.0487
0.1284
reflcns collcd
indpndt reflcns
R(int)
7571
0.0255
0.0763
0.2318
R1 [I > 2σ(I)]a
wR2 (all data)b
a R1 ) Σ||Fo| - |Fc||/Σ|Fo|, wR2 ) [Σ[w(Fo2 - Fc )2]/Σw(Fo )2]0.5
.
b w ) 1/[σ2(Fo ) + (aP)2 + bP], where P ) (2Fc2 + Fo )/3 and a and b are constants
2
2
2
2
set by the program.
4.28 (q, 4H, CH2 of coordinated EtG, J 7.2), 4.14 (q, CH2 of free
EtG in excess, J 7.2), 3.41 (s, 3H, DMSO, JHPt 19.5), 3.39 (s, 3H,
NMe of free 1-Methy), 3.16 (s, 3H, DMSO, JHPt 21.6), 2.66 (s, 3
H, NMe2, JHPt 34.0), 2.51 (s, 3 H, NMe2, JHPt 35.9), 1.93 (s, 3H,
CMe of free 1-Methy), 1.56 (t, 3H, Me of coordinated EtG, J 7.2),
1.47 (t, 3H, Me of free EtG in excess, J 7.2).
1H NMR data for the reaction of 8 with guanosine: δ(SiMe4)
8.86 (s, 1H, H(8) of coordinated guanosine), 8.05 (s, H(8) of free
guanosine in excess), 7.75 (d, 1H, aromatics of dmba, J 7.2), 7.51
(s, 1H, H(6) of free 1-Methy), 7.32 (m, 3H, aromatics of dmba),
6.08 (d, 1H, H(1)′ of coordinated guanosine, J 5.1), 5.96 (d, H(1)′
of free guanosine in excess, J 6.0).
9755 and refined anisotropically on F2.52 Hydrogen atoms were
included using a riding model, except for those of the methyl groups
in complex 6 for which a rigid model was used.
Biological Assays. Formation of Compound-DNA Com-
plexes. The platinum compounds 6-9 were dissolved in DMSO
prior to dilution with saline. The final maximum DMSO concentra-
tion in the growth medium was 2%. Stock solutions of each
compound (1 mg/mL) were stored in the dark at room temperature
until use. Compound-DNA complex formation was accomplished
by addition of CT DNA (calf thymus DNA) to aliquots of each of
the compounds at different concentrations in TE buffer (50 mM
NaCI, 10 mM Tris-HCl, 0.1 mM EDTA, pH 7.4). The amount of
compound added to the DNA solution was designated as ri (the
input molar ratio of Pt or ligand to nucleotide). The mixture was
incubated at 37 °C for 24 h.
Circular Dichroism Study. The CD spectra of the complex-
DNA compounds (DNA concentration 20 µg/mL, ri ) 0.10, 0.30,
and 0.50) were recorded at room temperature on a Applied
Photophysics Π*-180 spectrometer with a 75 W xenon lamp using
a computer for spectral subtraction and smooth reduction. Each
sample was scanned twice in a range of wavelengths between 220
and 330 nm. The drawn CD spectra are the means of two
independent scans. The data are expressed as mean residue
molecular ellipticity [θ] in deg cm2 dmol-1. The CD spectra of the
complexes were subtracted from the CD spectra of each of the
complex-DNA adducts by computer.
Electrophoretic Mobility Study. pBR322 DNA aliquots (0.25
µg/mL) were incubated in the presence of compounds in TE buffer
at the molar ratio ri ) 0.50 for electrophoresis studies. Incubation
was carried out in the dark at 37 °C for 24 h; 24 µL aliquots of
complex-DNA compounds containing 0.7 µg DNA underwent 1%
agarose gel electrophoresis for 5 h at 1.5 V/cm in 1XTBE (45 mM
Tris-borate, 1 mM EDTA pH 8.0) buffer. Gel was subsequently
stained in the same buffer containing ethidium bromide (1 µg/mL).
The DNA bands were visualized under UV light and photographed
with a digital camera.
Reaction of the Platinum Complex 6 with 9-Ethylguanine.
The reaction was carried out in a NMR tube (D2O and 5% DMSO-
d6 as solvents). 9-Ethylguanine was incubated with 6 in a ratio 5:1
in D2O at pH 7.0 (50 mM KD2PO4) and 37 °C. The reaction was
very fast, and in less than 5 min the formation of the complex
[Pt(dmba)(PPh3)(9-EtG)]+ was completed.
1H NMR data for the reaction of 6 with 9-ethylguanine: δ(SiMe4)
8.24 (s, 1H, H(8) of coordinated EtG), 7.90 (s, H(8) of free EtG in
excess), 7.81 (m, 6H, PPh3), 7.63 (m, 3H, PPh3), 7.55 (s, H(6) of
free 1-Methy), 7.42 (m, 6H, PPh3), 7.28 (d, 1H, aromatics of dmba,
J 8.0), 7.04 (m, 1H, aromatics of dmba), 6.61 (m, 1H, aromatics
of dmba), 6.53 (d, 1H, aromatics of dmba, J 7.5), 4.28 (q, CH2 of
free EtG, J 7.2), 3.39 (s, NMe of free 1-Methy), 1.93 (s, CMe of
free 1-Methy), 1.47 (t, 3H, Me of free EtG in excess, J 7.2).
Unfortunately, the aliphatic signals of the coordinated ligands of
the new complex were obscured by those of the solvents or free
ligands in excess, in part due to the low solubility of this new
compound.
X-ray Crystal Structure Analysis. Suitable crystals of 1, 3‚
2CHCl3, and 4‚3CHCl3 were grown from chloroform/hexane.
Crystals from 6, 12, and 14‚H2O were grown from acetone/hexane.
The crystal and molecular structures of the compounds 1, 3‚2CHCl3,
4‚3CHCl3, 6, 12, and 14‚H2O have been determined by X-ray
diffraction studies (Table 11).
Atomic Force Microscopy. Preparation of Adducts DNA-
Metal Complexes. pBR322 DNA (25 µg/µL) was incubated in an
appropriate volume with the required platinum concentration
The crystals were mounted onto the tip of a glass fiber, and the
data collection was performed with a Siemens P4 diffractometer
for complexes 1, 3‚2CHCl3, 4‚3CHCl3, and 12, and with a Bruker
Smart CCD diffractometer for complexes 6 and 14‚H2O. The
structures were solved by heavy atom and direct methods SHELXS-
(55) Sheldrick, G. M. SHELXL-97; Programs for Crystal Structure Analysis,
release 97-2; University of Go¨ttingen: Go¨ttingen, Germany, 1998.
Inorganic Chemistry, Vol. 45, No. 16, 2006 6359

Ruiz et al.
corresponding to the molar ratio ri ) 0.005. The complexes were
dissolved in HEPES buffer (40 mM HEPES, pH 7.4, and 10 mM
MgCl2). The different solutions as well as Milli-Q water were
passed through 0.2 nm FP030/3 filters (Schleicher & Schuell GmbH,
Germany) and centrifuged at 4000g several times to avoid salt
deposits and provide a clear background when they were imaged
by AFM. The reactions were run at 37 °C for 24 h in the dark.
Sample Preparation for Atomic Force Microscopy. Samples
were prepared by placing a drop (3 µL) of DNA solution or DNA-
metal complex solution onto green mica (Ashville-Schoonmaker
Mica Co., Newport News, VA). After adsorption for 5 min at room
temperature, the samples were rinsed for 10 s in a jet of deionized
water of 18 MΩ cm-1 from a Milli-Q water purification system
directed onto the surface with a squeeze bottle. They were then
placed into an ethanol-water mixture (1:1) five times and plunged
three times each in 100% ethanol. The samples were blow dried
with compressed argon over silica gel and then imaged in the AFM.
Imaging by Atomic Force Microscopy. The samples were
imaged in a Nanoscope III Multimode AFM (Digital Instrumentals
Inc., Santa Barbara, CA) operating in tapping mode in air at a scan
rate of 1-3 Hz. The AFM probe was a 125-mm-long mono-
crystalline silicon cantilever with integrated conical-shaped Si tips
(Nanosensors GmbH, Germany) with an average resonance fre-
quency fo ) 330 kHz and spring constant K ) 50 N/m. The
cantilever is rectangular, and the tip radius given by the supplier is
10 nm with a cone angle of 35° and high aspect ratio. In general,
the images were obtained at room temperature (T ) 23 ( 2 °C)
and the relative humidity (RH) was typically lower than 40%.
Cell Line and Culture. The cell line used in this experiment
was the human acute promyelocytic leukemia cell line HL-60
(American Type Culture Collection (ATCC)). Cells were routinely
maintained in RPMI-1640 medium supplemented with 10% (v/v)
heat-inactivated fetal bovine serum, 2 mmol/L glutamine, 100 U/mL
penicillin, and 100 µg/mL streptomycin (Life Technologies, Inc.)
in a highly humidified atmosphere of 95% air with 5% CO2 at 37
°C.
logarithmic phase were seeded into 96-well microtiter plates in 100
µL of the appropriate culture medium at plating density of 1 × 104
cells/well. Following the addition of different complex concentra-
tions to quadruplicate wells, plates were incubated at 37 °C for 24
or 72 h. Aliquots of 20 µL of MTT solution were then added to
each well. After 3 h, the color formed was quantitated by a
spectrophotometric plate reader (Bio-Tek Instruments, Inc.) at 490
nm wavelength. The percentage cell viability was calculated by
dividing the average absorbance of the cells treated with a palladium
or platinum complex by that of the control; % cell viability vs drug
concentration (logarithmic scale) was plotted to determine the IC50
(drug concentration at which 50% of the cells are viable relative
to the control), with its estimated error derived from the average
of 3 trials.
In Vitro Apoptosis Assay. Induction of apoptosis in vitro by
the platinum complexes 6-9 and cisplatin was determined by a
flow cytometric assay with Annexin V-FITC57 by using an Annexin
V-FITC apoptosis detection kit (Roche). Exponentially growing HL-
60 cells in 6-well plates (5 × 105 cells/well) were exposed to IC50
concentrations of cisplatin or complexes 6-9 for 24 h. Then the
cells were subjected to staining with the Annexin V-FITC and
propidium iodide as detailed by the manufacturer. The amount of
apoptotic cells was analyzed by flow cytometry (BD FACSCalibur).
Acknowledgment. This work was supported by the
Direccio´n General de Investigacio´n del Ministerio de Ciencia
y Tecnolog´ıa (Project No. CTQ2005-09231-C02-01/BQU
and BIO2004-05879) of Spain and the Fundacio´n Se´neca
de la Comunidad Auto´noma de la Regio´n de Murcia (Project
No. 00448/PI/04). We thank Dr. Mar´ıa Jose´ Prieto (AFM),
Dr. Francisca Garc´ıa (Cell Culture Facility), and Manuela
Costa (Cytometry Facility) for technical assistance.
Supporting Information Available: X-ray crystallography data
for the complexes 1, 3‚2CHCl3, 4‚3CHCl3, 6, 12, and 14‚H2O and
complementary figures corresponding to the NMR spectroscopic
studies of the reactions of complex 8 with 9-ethylguanine and
guanosine. This material is available free of charge via the Internet
at http://pubs.acs.org.
Cytotoxicity Assay. Growth inhibitory effect of platinum
complexes on the leukemia HL-60 cell line was measured by the
microculture tetrazolium [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide, MTT] assay.56 Briefly, cells growing in the
IC060374E
(57) Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger, C. J.
Immunol. Methods 1995, 184, 39.
(56) Mosmann, T. J. Immunol. Methods 1983, 65, 55.
6360 Inorganic Chemistry, Vol. 45, No. 16, 2006