New palladium(II) and platinum(II) complexes with the model nucleobase 1-methylcytosine: Antitumor activity and interactions with DNA

Article abstract of DOI:10.1021/ic0502372

Palladium and platinum complexes with the model nucleobase 1-methylcytosine (1-Mecyt) of the types [Pd(N-N)-(C₆F₅)(1-Mecyt)]ClO ₄ [N-N = bis(3,5-dimethylpyrazol-1-yl)methane (bpzm*), bis(pyrazol-1-yl)methane (bpzm), N,N,N′,N′- tetramethylethylenediamine (tmeda), or 2,2′-bipyridine (bpy)] and [M(dmba)(L′)(1-Mecyt)]CIO₄ [dmba = N,C-chelating 2-(dimethylaminomethyl)phenyl; L′ = PPh₃ (M = Pd or Pt), DMSO (M = Pt)] have been obtained. Palladium and platinum complexes of the types cis-[M(C₆F₅)₂(1-Mecyt)₂] (M = Pd or Pt) and cis-[Pd(L′)(C₆F₅)(1-Mecyt) ₂]ClO₄ (L′ = PPh₃ or t-BuNC) have also been prepared. The crystal structures of [Pd(bpzm*)(C₆F ₅)(1-Mecyt)]ClO₄, [Pt(dmba)(DMSO)(1-Mecyt)]ClO ₄, cis-[Pd(C₆F₅)₂(1-Mecyt) ₂], and cis-[Pd(t-BuNC)(C₆F₅)(1-Mecyt) ₂]ClO₄ have been established by X-ray diffraction. There is extensive hydrogen bonding (N-H...O, C-H...F or C-H...O) in all the compounds. There are also intermolecular π-π interactions between pyrimidine rings of adjacent chains in [Pd(C₆F₅) ₂(1-Mecyt)₂]. DNA adduct formation of the new complexes synthesized was followed by circular dichroism and electrophoretic mobility. Atomic force microscopy images of the modifications caused by the complexes on plasmid DNA pBR322 were also obtained. Values of IC₅₀ were also calculated for the new complexes against the tumor cell line HL-60. At a short incubation time (24 h) almost all new complexes were more active than cisplatin.

Full text of DOI:10.1021/ic0502372

Inorg. Chem. 2005, 44, 7365−7376
New Palladium(II) and Platinum(II) Complexes with the Model
Nucleobase 1-Methylcytosine: Antitumor Activity and Interactions with
DNA
Jose´ Ruiz,* Natalia Cutillas, Consuelo Vicente, Mar´ıa Dolores Villa, and Gregorio Lo´pez
Departamento de Qu´ımica Inorga´nica, Facultad de Qu´ımica, UniVersidad de Murcia,
30071 Murcia, Spain
Julia Lorenzo and Francesc X. Avile´s
Institut de Biotecnolog´ıa i Biomedicina, Department de Bioqu´ımica i de Biolog´ıa Molecular,
UniVersitat Auto`noma de Barcelona, 08193 Barcelona, Spain
Virtudes Moreno
Department de Qu´ımica Inorga`nica, UniVersitat de Barcelona, Mart´ı i Franque`s 1-11,
08028 Barcelona, Spain
Delia Bautista
S.U.I.C., Edificio S.A.C.E., UniVersidad de Murcia, 30071-Murcia, Spain
Received February 15, 2005
Palladium and platinum complexes with the model nucleobase 1-methylcytosine (1-Mecyt) of the types [Pd(N
(C6F5)(1-Mecyt)]ClO4 [N bis(3,5-dimethylpyrazol-1-yl)methane (bpzm*), bis(pyrazol-1-yl)methane (bpzm),
N,N,N ,N -tetramethylethylenediamine (tmeda), or 2,2 -bipyridine (bpy)] and [M(dmba)(L )(1-Mecyt)]ClO4 [dmba
N,C-chelating 2-(dimethylaminomethyl)phenyl; L′ ) PPh3 (M Pd or Pt), DMSO (M Pt)] have been obtained.
Palladium and platinum complexes of the types cis-[M(C6F5)2(1-Mecyt)2] (M Pd or Pt) and cis-[Pd(L )(C6F5)(1-
−N)-
−N )
′
′
′
′
)
)
)
)
′
Mecyt)2]ClO4 (L′ ) PPh3 or t-BuNC) have also been prepared. The crystal structures of [Pd(bpzm*)(C6F5)(1-
Mecyt)]ClO4, [Pt(dmba)(DMSO)(1-Mecyt)]ClO4, cis-[Pd(C6F5)2(1-Mecyt)2], and cis-[Pd(t-BuNC)(C6F5)(1-Mecyt)2]ClO4
have been established by X-ray diffraction. There is extensive hydrogen bonding (N−H‚‚‚O, C−H‚‚‚F or C−H‚‚‚O)
in all the compounds. There are also intermolecular π−π interactions between pyrimidine rings of adjacent chains
in [Pd(C6F5)2(1-Mecyt)2]. DNA adduct formation of the new complexes synthesized was followed by circular dichroism
and electrophoretic mobility. Atomic force microscopy images of the modifications caused by the complexes on
plasmid DNA pBR322 were also obtained. Values of IC50 were also calculated for the new complexes against the
tumor cell line HL-60. At a short incubation time (24 h) almost all new complexes were more active than cisplatin.
Introduction
cepted as the main pharmacological target of cisplatin-
induced cytotoxicity.3,4 The major product of cisplatin
interaction with DNA is a 1,2-intrastrand cross-link, which
induces a kink on a DNA double helix.3,5,6 However,
Today platinum complexes are some of the most promising
drugs in chemotherapy.1,2 Genomic DNA is generally ac-
* To whom correspondence should be addressed. Fax: (+34) 968 36
41 48. E-mail: jruiz@um.es.
(1) Cisplatin-Chemistry and Biochemistry of a leading Anticancer Drug;
Lippert, B., Ed.; Wiley-VCH: Weinheim, 1999.
(2) Wong, E.; Giandomenico, C. M. Chem. ReV. 1999, 99, 2451.
(3) Reedjik, J. Chem. Commun. 1996, 801.
(4) Takahara, P. M.; Frederick, C. A.; Lippard, S. J. J. Am. Chem. Soc.
1996, 118, 12309.
10.1021/ic0502372 CCC: $30.25
Published on Web 09/13/2005
© 2005 American Chemical Society
Inorganic Chemistry, Vol. 44, No. 21, 2005 7365

Ruiz et al.
Scheme 1
significant problems still exist, including side effects, toxicity,
cancer specificity, and specially acquired resistance. Con-
sequently, antitumor drug research has been moving toward
the development of new compounds outside the usual
coordination sphere, e.g., organometallic complexes7 or
complexes with metallic elements different from platinum.
Because of the lanthanoid contraction, platinum and pal-
ladium both have nearly the same ionic radius (Pt(II) 0.74
Å, Pd(II) 0.78 Å). Accordingly, a very large similarity of
both metals within their coordinative behavior can be
observed, although their kinetic behavior with respect to
ligand substitution is very different.
In the present study our initial aim was to synthesize
organopalladium and organoplatinum complexes derived
from the N,C-chelating 2-(dimethylaminomethyl)phenyl (dmba)
and pentafluorophenyl groups with a representative model
nucleobase, namely, 1-methylcytosine (1-Mecyt), which
display rather versatile metal-ion binding patterns.8 To our
knowledge, the complexes reported herein represent the first
examples of palladium and platinum complexes containing
neutral 1-Mecyt and a σ-metal carbon bond. Some of these
complexes have been characterized by X-ray diffraction. We
also studied the interactions of these complexes with DNA
by circular dichroism and electrophoretic mobility. Atomic
force microscopy images of the modifications caused by the
complexes on plasmid DNA pBR322 were also obtained.
The in vitro antiproliferative activity of the new complexes
in HL-60 cell line has been studied.
characteristic absorptions of the C6F5 group11 at 1630, 1490,
1450, 1050, and 950 and a single band at ca. 800 cm-1
derived from the so-called X-sensitive mode12 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.13 The
characteristic resonances of the chelate ligands are observed
Results and Discussion
Complexes [Pd(N-N)(C6F5)(1-Mecyt)]ClO4. In acetone,
the solvento complexes [Pd(N-N)(C6F5)(Me2CO)]ClO4 [N-N
) bis(3,5-dimethylpyrazol-1-yl)methane (bpzm*), bis(pyra-
zol-1-yl)methane (bpzm), N,N,N′,N′-tmeda (tetramethyleth-
ylenediamine), or bpy (2,2′-bipyridyl)] (prepared by reaction
of the corresponding chloride derivatives [PdCl(C6F5)(N-
N)] with AgClO4 in a 1:1 molar ratio in acetone at room
temperature) react with 1 equiv of 1-Mecyt to yield the
corresponding cationic complexes [Pd(N-N)(C6F5)(1-Mecyt)]-
ClO4 (1-4) (Scheme 1) in 55-85% yields. The structures
1
in the H NMR spectra,14-17 and the assignments presented
in the Experimental Section are based on the atom numbering
1
given in Scheme 2. In complexes 1 and 2 two sets of H
resonances are observed for the C3HMe2N2 or C3H3N2 rings
corresponding to one pyrazolate ligand trans to 1-Mecyt and
one pyrazolate ligand trans to C6F5. The proton resonances
of the bridging methylene of the bis(pyrazol-1-yl)methane
ligands in complexes 1 and 2 show that the boat-to-boat
inversion usually observed in this type of complex14 is frozen
at room temperature; the methylene protons are magnetically
nonequivalent and give an NMR pattern corresponding to
1
were assigned on the basis of microanalytical, IR, and H
and 19F NMR data. Complexes 1-4 are all air-stable solids,
and thermal analysis shows that they decompose above 230
°C in a dynamic N2 atmosphere. Their acetone solutions
show conductance values corresponding to 1:1 electrolytes
(ΛM in the range 132-152 S cm2 mol-1).9 Two IR bands
are observed at ca. 1090 and 1050 cm-1; one is assigned to
the ν3 mode of free perchlorate (Td symmetry) and the other
to a vibrational mode of the C6F5 group. The observation of
an additional band at ca. 623 cm-1 for the ν4 mode confirms
the presence of free perchlorate.10 The IR spectra show the
2
two diastereotopic protons: two doublets with JH H ≈ 15
A
B
(11) Long, D. A.; Steele, D. Spectrochim. Acta 1963, 19, 1955.
(12) Maslowski, E. Vibrational Spectra of Organometallic Compounds;
Wiley: New York, 1977; p 437.
(13) Alonso, E.; Fornie´s, J.; Fortun˜o, C.; Toma´s, M. J. Chem. Soc., Dalton
Trans. 1995, 3777.
(5) Guo, Z. J.; Sadler, P. J. Angew. Chem., Int. Ed. Engl. 1999, 38, 1513.
(6) Jamieson, E. R.; Lippard, S. J. Chem. ReV. 1999, 99, 2467.
(7) Fish, R. H.; Jaouen, G. Organometallics 2003, 22, 2166.
(8) Lippert, B. Coord. Chem. ReV. 2000, 200-202, 487.
(9) Geary, W. J. Coord. Chem. ReV. 1971, 7, 81.
(10) Nakamoto, K. Infrared and Raman Spectra of Inorganic and Coor-
dination Compounds, 5th ed.; Wiley-Interscience: New York, 1997;
p 199.
(14) 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.
(15) 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.
(16) Byers, P. K.; Canty, A. J. Organometallics 1990, 9, 210.
(17) Fornie´s, J.; Mart´ınez, F.; Navarro, R.; Urriolabeitia, E. P. J. Organomet.
Chem. 1995, 495, 185.
7366 Inorganic Chemistry, Vol. 44, No. 21, 2005

New Palladium(II) and Platinum(II) Complexes
Scheme 2
Hz. Figure 1 shows the variable-temperature 1H NMR spectra
of complex 1 in the N-CH2-N region and the boat-to-boat
inversion of the metallacycle Pd(N-N)2C. In complexes 1-4
the H(5) and H(6) resonances of the 1-Mecyt ligand (Scheme
2) are shifted downfield by ∼0.1-0.2 ppm due to Pd binding,
although the coupling constant between H(5) and H(6) was
Figure 2. ORTEP of complex 1 showing the atom-numbering scheme.
Displacement ellipsoids are drawn at the 50% probability level.
Table 1. Selected Bond Lengths and Angles for Complex 1
bond lengths, Å
bond angles, deg
molecule 1 Pd(1)-C(1)
Pd(1)-N(1)
1.988(4) C(1)-Pd(1)-N(1)
2.031(3) C(1)-Pd(1)-N(5)
2.044(3) N(1)-Pd(1)-N(5)
2.131(3) C(1)-Pd(1)-N(3)
N(1)-Pd(1)-N(3)
91.21(13)
88.01(12)
178.02(12)
167.48(13)
88.31(11)
92.86(11)
Pd(1)-N(5)
Pd(1)-N(3)
N(5)-Pd(1)-N(3)
C(22)-N(5)-C(18) 121.8(3)
2.004(4) C(31)-Pd(2)-N(8)
2.015(3) C(31)-Pd(2)-N(12) 90.90(12)
2.034(3) N(8)-Pd(2)-N(12) 177.17(12)
2.090(3) C(31)-Pd(2)-N(11) 168.72(13)
molecule 2 Pd(2)-C(31)
Pd(2)-N(8)
90.83(13)
Pd(2)-N(12)
Pd(2)-N(11)
N(8)-Pd(2)-N(11)
N(12)-Pd(2)-N(11) 93.06(11)
C(48)-N(12)-C(52) 121.8(3)
84.83(12)
unaffected (7.2 Hz). On the other hand, an interesting
observation is that while the NH2 of the free 1-Mecyt base
is a broad singlet as is typically found for amino resonances
which normally undergo free rotation on the NMR time scale,
two NH resonances are observed for the coordinated 1-Mecyt
ligand in complexes 1-4. Both of these NH resonances are
strongly shifted downfield (∼1.5-1.0 ppm) relative to the
free base. The observation of two resonances is consistent
with the intermolecular steric effects and hydrogen bonding
observed in the solid state18 (vide infra). The 19F NMR
spectra of complexes 1 and 3 reveal the presence of a freely
rotating pentafluorophenyl ring which gives the expected
three resonances (in the ratio 2:1:2) for the o-, p-, and
m-fluorine atoms, respectively. The 19F NMR spectra of
complex 4 at room temperature show hindered rotation of
the C6F5 ring around the Pd-C bond, and two separate
signals are observed for the o- and m-fluorine atoms but only
one for the p-fluorine atom.
Crystal Structure of 1. Figure 2 shows the X-ray structure
of complex 1 with selected bond lengths and angles listed
in Table 1. The crystal structure of compound 1 shows two
independent molecules in the asymmetric unit with the
palladium atoms in slightly distorted square-planar geom-
etries. The different Pd-N (bpzm) distances (Pd(1)-N(1)
Figure 1. Variable-temperature 1H NMR spectra of complex 1 in the
N-CH2-N region, and the boat-to-boat inversion of the metallacycle Pd-
(N-N)2C.
(18) Orbell, J. D.; Marzilli, L. G.; Kistenmacher, T. J. J. Am. Chem. Soc.
1981, 103, 5126.
Inorganic Chemistry, Vol. 44, No. 21, 2005 7367

Ruiz et al.
Table 2. Hydrogen Bonds for Complex 1a
<(DHA),
deg
D-H‚‚‚A
d(D-H), Å d(H‚‚‚A), Å d(D‚‚‚A), Å
N(6)-H(06B)‚‚‚O(5)
0.89(3)
0.89(3)
0.98
0.98
0.95
0.98
0.98
0.98
2.05(3)
2.32(4)
2.38
2.33
2.48
2.54
2.50
2.40
2.938(4)
2.962(5)
3.250(5)
3.297(5)
3.422(5)
3.461(5)
3.472(5)
3.383(5)
171(4)
129(3)
147.7
167.7
169.2
155.9
171.9
176.8
N(13)-H(01A)‚‚‚O(4)#1
C(53)-H(53A)‚‚‚F(35)#2
C(46)-H(46B)‚‚‚F(35)#3
C(50)-H(50)‚‚‚O(4)#4
C(23)-H(23C)‚‚‚O(6)#5
C(15)-H(15B)‚‚‚O(5)#6
C(23)-H(23B)‚‚‚O(1)#7
a Symmetry transformations used to generate equivalent atoms: #1 x -
1/2, -y + 1/2, z + 1/2; #2 x + 1/2, -y + 1/2, z + 1/2; #3 -x + 3/2, y -
1/2, -z + 3/2; #4 x, y, z + 1; #5 x - 1/2, -y + 1/2, z - 1/2; #6 -x + 3/2,
y - 1/2, -z + 1/2; #7 -x + 1, -y, -z.
) 2.031(3) Å, Pd(1)-N(3) ) 2.131(3) Å in molecule 1 and
(Pd(2)-N(8) ) 2.015 (3) Å, Pd(2)-N(11) ) 2.090(3) Å in
molecule 2) are in agreement with the higher trans influence
of the C6F5 group compared with the 1-Mecyt ligand. The
Pd-N(1-Mecyt) bond distance (Pd(1)-N(5) ) 2.044(3) Å
in molecule 1, Pd(2)-N(12) ) 2.034(3) Å in molecule 2) is
similar to the mean value of 2.040(7) Å found in the cation
[Pd(1-Mecyt)4]2+ 19 and slightly longer than 2.028(7) Å found
in [(trpy)Pd(1-Mecyt)]2+.20 Bond distances and angles within
the 1-Mecyt molecule are in agreement with values previ-
ously reported for analogous ligands.21 The 1-Mecyt ligand
is planar, and the highest displacement from the least-squares
plane defined by the six endocyclic atoms is 0.0323 Å for
C(22) in molecule 1 and 0.0037 Å for C(51) in molecule 2.
The 1-Mecyt plane is almost perpendicular to the coordina-
tion plane (dihedral angle between the planes is 91.9° in
molecule 1 and 105.1° in molecule 2). The two C-N(5)-
Pd angles are significantly different. The angle on the side
of the NH2 group (123.8(2)°) is larger than that on the O
side (114.3(2)°). The two C-N(12)-Pd angles are also
significantly different. The angle on the side of the NH2
group (125.7(2)°) is larger than that on the O side (121.6-
(2)°). The perchlorate anion conects two molecules through
classical NH‚‚‚O bonds. The nonclassical CH‚‚‚O bonds link
these molecules to form a three-dimensional network (Table
2). The chelate angle N(1)-Pd(1)-N(3) (88.31(11)°) in
molecule 1 is larger than that found in molecule 2, N(8)-
Pd(2)-N(3) (84.83(12)°).
Figure 3. ORTEP of complex 7 showing the atom-numbering scheme.
Displacement ellipsoids are drawn at the 50% probability level.
Table 3. Selected Bond Lengths and Angles for Complex 7
bond lengths, Å
bond angles, deg
Pt(1)-C(1)
Pt(1)-N(1)
Pt(1)-N(2)
Pt(1)-S(1)
2.015(3)
2.105(3)
2.142(3)
2.2064(9)
C(1)-Pt(1)-N(1)
C(1)-Pt(1)-N(2)
N(1)-Pt(1)-N(2)
C(1)-Pt(1)-S(1)
N(1)-Pt(1)-S(1)
N(2)-Pt(1)-S(1)
81.89(13)
169.30(12)
90.60(11)
94.02(10)
175.87(8)
93.53(8)
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 (some broadening being observed
for complexes 5 and 7). Therefore, there is no plane of
symmetry in the palladium coordination plane. In complexes
5 and 6 the PPh3-trans-to-NMe2 ligand arrangement in the
starting products22,23 is preserved after chlorine abstraction
and 1-Mecyt coordination, as can be inferred from the small
4
but significant coupling constants JP-H (ranging from 2.4
to 3.8 Hz) of the NMe2 and CH2N protons with the
phosphorus atom24,25 in complex 6. In complexes 5-7 the
H(5) and H(6) resonances of 1-Mecyt ligand are also shifted
downfield due to the metal binding. Two NH resonances
are observed for the coordinated 1-Mecyt ligand in complex
7, which are also strongly shifted downfield relative to the
free base.
Complexes [M(dmba)(L′)(1-Mecyt)]ClO4. The dmba
analogues of complexes 1-4 have been prepared from the
corresponding precursor [MCl(dmba)(L′)] [L′ ) PPh3 (M
) Pd or Pt), DMSO (M ) Pt)]. After precipitation of AgCl
by addition of AgClO4 in a 1:1 molar ratio in acetone, the
solvento complexes [[M(dmba)(L′)(Me2CO)]ClO4 generated
in situ react with 1 equiv of 1-Mecyt to give the cationic
complexes [M(dmba)(L′)(1-Mecyt)]ClO4 (5-7) (Scheme 1).
Complexes 5-7 are white, air-stable solids that decompose
on heating above 239 °C. Their acetone solutions show
conductance values corresponding to 1:1 electrolytes.9 The
1H NMR spectra of complexes 5-7 show that both the
Crystal Structure of 7. A drawing of the cationic complex
of 7 is shown in Figure 3 with selected bond lengths and
angles listed in Table 3. The platinum atom is located in a
slightly distorted square-planar environment, surrounded by
the C and N atoms of the cyclometalated dmba ligand (with
a C,N-bite angle of 81.89(13)°), the DMSO ligand positioned
trans to the NMe2 group, and the 1-Mecyt ligand trans to
the aromatic ring. The cyclometalated ring is puckered with
the nitrogen atom significantly out of the plane defined by
(22) Deeming, A. J.; Rothwell, I. P.; Hursthouse, M. B.; New, L. J. Chem.
Soc., Dalton Trans. 1978, 1490.
(23) 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.
(24) Braunstein, P.; Matt, D.; Dusausoy, Y.; Fischer, J.; Mitschler, A.;
Ricard, L. J. Am. Chem. Soc. 1981, 103, 5115.
(19) Krumm, M.; Mutikainen, I.; Lippert, B. Inorg. Chem. 1991, 30, 884.
(20) Cosar, S.; Janik, M. B. L.; Flock, M.; Freisinger, E.; Farkas, E.; Lippert,
B. J. Chem. Soc., Dalton Trans. 1999, 2329.
(21) Navarro, J. A. R.; Lippert, B. Coord. Chem. ReV. 2001, 222, 219 and
(25) Falvello, L. R.; Ferna´ndez, S.; Navarro, R.; Urriolabeitia, E. P. Inorg.
Chem. 1997, 36, 1136.
references therein.
7368 Inorganic Chemistry, Vol. 44, No. 21, 2005

New Palladium(II) and Platinum(II) Complexes
Table 4. Hydrogen Bonds for Complex 7a
<(DHA),
deg
D-H‚‚‚A
d(D-H), Å d(H‚‚‚A), Å d(D‚‚‚A), Å
N(4)-H(0A)‚‚‚O(90)#1
N(4)-H(0B)‚‚‚O(6)#1
C(14)-H(14)‚‚‚O(1)#1
C(16)-H(16A)‚‚‚O(1)#1
C(11)-H(11B)‚‚‚O(6)#1
C(3)-H(3)‚‚‚O(6)#2
C(92)-H(92B)‚‚‚O(4)#3
C(10)-H(10C)‚‚‚O(3)#4
C(92)-H(92A)‚‚‚O(1)#5
0.88(3)
0.88(3)
0.95
0.98
0.98
0.95
0.98
0.98
0.98
2.10(3)
2.45(4)
2.44
2.35
2.23
2.49
2.57
2.53
2.41
2.949(4)
3.107(5)
3.267(4)
3.280(4)
3.190(5)
3.283(5)
3.540(6)
3.477(5)
3.231(5)
161(4)
132(4)
145.8
157.8
164.8
140.6
169.7
161.7
141.1
a Symmetry transformations used to generate equivalent atoms: #1 x,
-y + 1/2, z - 1/2; #2 -x + 1, y - 1/2, -z + 3/2; #3 x, -y + 3/2, z - 1/2;
#4 -x, y - 1/2, -z + 1/2; #5 -x, y + 1/2, -z + 1/2.
the platinum and carbon atoms, a feature which is quite
commonly observed in cyclometalated dmba complexes. The
Pt-C(1) (dmba) and Pt-N(1) (dmba) distances are similar
to those found in other Pt(dmba) complexes.23 The 1-Mecyt
plane is almost perpendicular to the coordination plane
(dihedral angle between the planes ) 88.4°). The Pt-N(1-
Mecyt) bond distance, 2.142(3) Å, is longer than the values
of 2.031(6) and 2.040(6) Å found in the cation cis-[(NH3)2-
Pt(1-Mecyt)2]2+ 18 or 2.056(8) Å found in [(trpy)Pt(1-
Mecyt)]2+.20 In the crystal a rather complex three-dimensional
macromolecular network structure is observed built up by
extensive hydrogen bonding which involves the 1-Mecyt
ligand, the perchlorate anion, the coordinated DMSO, and
the acetone molecule of crystallization (Table 4).
Figure 4. ORTEP of complex 8 showing the atom-numbering scheme.
Displacement ellipsoids are drawn at the 50% probability level.
Scheme 3
Complexes cis-[M(C6F5)2(1-Mecyt)2]. The complexes cis-
[NBu4]2[Pd2(C6F5)4(µ-Cl)2]26 and cis-[Pt(C6F5)2(THF)2]27 are
good precursors for the synthesis of complexes 8 and 9,
respectively (Scheme 3). The reactions take place without
isomerization, and the reaction products are the cis isomers.
Their IR spectra show the characteristic absorptions of the
C6F5 group (1630 m, 1490 vs, 1050 s, and 950 vs cm-1)11
and a split band at ca. 800 cm-1 assigned to the cis-M(C6F5)2
moiety.12,13 The 19F NMR spectra of 8 and 9 show the
expected three signals for two equivalent C6F5 rings with
relative intensities of 4Fo:2Fp:4Fm. The broad resonance
observed at room temperature for the ortho fluorine atoms
suggests a hindered rotation of the C6F5 ring around the M-C
bond. The 1H NMR spectra of complexes 8 and 9 consist of
five resonances with relative intensities of 2:2:2:2:6; two NH
resonances are observed for the coordinated 1-Mecyt ligands
and a unique set of resonances for the H(5), H(6), and Me
protons, indicating the presence of only one type of 1-Mecyt
group.
Crystal Structure of 8. Figure 4 shows the X-ray structure
of complex 8 with selected bond lengths and angles listed
in Table 5. The crystal structure of compound 8 shows two
independent molecules in the asymmetric unit with the
palladium atoms in slightly distorted square-planar geom-
etries. The angles between the two C6F5 rings are 85.47(9)°
and 86.24(9)°, and the angles between the two 1-Mecyt
ligands are 88.14(7)° and 90.05(7)°. The two coordinated
Table 5. Selected Bond Lengths and Angles for Complex 8
bond lengths, Å
bond angles, deg
molecule 1 Pd(1)-C(21)
Pd(1)-C(31)
1.998(2)
2.005(2)
2.1111(18) C(31)-Pd(1)-N(4)
2.1187(18) C(21)-Pd(1)-N(1)
C(31)-Pd(1)-N(1)
C(21)-Pd(1)-C(31)
C(21)-Pd(1)-N(4)
85.47(9)
175.97(8)
93.97(8)
92.76(8)
174.68(8)
88.14(7)
86.24(9)
173.39(8)
92.39(8)
91.87(8)
Pd(1)-N(4)
Pd(1)-N(1)
N(4)-Pd(1)-N(1)
molecule 2 Pd(2)-C(51)
Pd(2)-C(41)
2.002(2)
2.003(2)
C(51)-Pd(2)-C(41)
C(51)-Pd(2)-N(7)
Pd(2)-N(7)
2.1169(18) C(41)-Pd(2)-N(7)
2.1171(19) C(51)-Pd(2)-N(10)
Pd(2)-N(10)
C(41)-Pd(2)-N(10) 174.61(8)
N(7)-Pd(2)-N(10) 90.05(7)
1-Mecyt ligands are arranged in a head-to-tail fashion such
the complex possesses approximate C2 molecular symmetry
as observed previously in cis-[Pt(NH3)2(1-Mecyt)2].18 There
is a relatively short intramolecular contact N2‚‚‚O2 (3.071
Å). The other contacts N‚‚‚O are longer (3.162, 3.243, and
3.420 Å). The Pd-N(1-Mecyt) bond distances (Pd(1)-N(4)
(26) Uso´n, R.; Fornie´s, J.; Mart´ınez, F.; Toma´s, M. J. Chem. Soc., Dalton
Trans. 1980, 888.
(27) Uso´n, R.; Fornie´s, J.; Toma´s, M.; Menjo´n, B. Organometallics 1985,
4, 1912.
Inorganic Chemistry, Vol. 44, No. 21, 2005 7369

Ruiz et al.
Table 6. Hydrogen Bonds for Complex 8a
<(DHA),
deg
D-H‚‚‚A
d(D-H), Å d(H‚‚‚A), Å d(D‚‚‚A), Å
N(2)-H(0B)‚‚‚O(2)#1
N(5)-H(50A)‚‚‚O(4)#2
N(8)-H(80A)‚‚‚O(1)#3
N(11)-H(11A)‚‚‚O(3)#4
0.80(3)
0.85(3)
0.82(3)
0.83(3)
2.09(3)
2.09(3)
1.96(3)
2.03(3)
2.861(3)
2.895(3)
2.776(3)
2.821(3)
162(3)
157(3)
172(3)
160(3)
a Symmetry transformations used to generate equivalent atoms: #1 -x
+ 1, -y, -z + 2; #2 x, y - 1, z; #3 x, y + 1, z; #4 -x + 2, -y + 1, -z
+ 1.
Figure 6. ORTEP of complex 11 showing the atom-numbering scheme.
Displacement ellipsoids are drawn at the 50% probability level.
Table 7. Selected Bond Lengths and Angles for Complex 11
bond lengths, Å
bond angles, deg
Pd(1)-C(1)
Pd(1)-C(11)
Pd(1)-N(3)
Pd(1)-N(2)
1.922(5)
2.010(4)
2.064(4)
2.092(3)
C(1)-Pd(1)-C(11)
C(1)-Pd(1)-N(3)
C(11)-Pd(1)-N(3)
C(1)-Pd(1)-N(2)
C(11)-Pd(1)-N(2)
N(3)-Pd(1)-N(2)
85.19(18)
174.61(16)
90.04(16)
92.27(16)
176.37(16)
92.37(13)
(C6F5)(L′)(1-Mecyt)2]ClO4 (10 and 11) (Scheme 3). In
acetone solution complexes 10 and 11 behave as 1:1
electrolytes.9 The IR spectra of these complexes show an
absorption located at 790 cm-1 that is observed as a single
band for the so-called X-sensitive mode,12 as expected from
the presence of only one C6F5 group in the coordination
sphere of the palladium atom. The IR spectrum of complex
11 also shows an absorption assigned to ν(CN) of the t-BuNC
group33-35 at ca. 2235 cm-1. The 19F NMR spectra of
complexes 10 and 11 at room temperature show a unique
set of sharp resonances indicating the presence of only one
Figure 5. π-π Interactions in complex 8.
) 2.1111(18) Å and Pd(1)-N(1) ) 2.1187(18) Å) are longer
than those observed in complexes 1, trans-[Pd(NH3)2(1-
Mecyt)2]2+, and [Pd(1-Mecyt)4]2+,19 although similar to the
values found in cis-[Pd(C6F5)2(NH3)2], cis-[Pd(C6F5)2-
(NHCMe2)2],28 and cis-[Pd(C6F5)2{NHC(OMe)Me}2].29 The
planes of the cytosine rings are roughly at right angles to
the metal coordination plane with dihedral angles of 72.4°
(N1) and 70.8° (N4) in molecule 1 and 72.9° (N7) and 69.1°
(N10) in molecule 2. The two 1-Mecyt rings are nearly
perpendicular with dihedral angles of 69.1° in molecule 1
and 72.9° in molecule 2. In the crystal there is intermolecular
hydrogen bonding (N-H‚‚‚O) forming infinite chains along
the y axis (Table 6). There are also intermolecular π-π
interactions between pyrimidine rings of adjacent chains
(N(12) of a nucleobase ring of molecules of type 2 with
C(71) of another ring of molecules of type 2 of different
chain ) 3.742 Å; N(3) and C(3) ) 3.687 Å and N(1) and
C(2) ) 3.778 Å of molecules of type 1) (Figure 5). This
type of intermolecular stacking interaction has been previ-
ously observed in trans-[Pt(NH3)Cl2(1-Mecyt)]‚1/2H2O.30
Complexes cis-[Pd(C6F5)(L′)(1-Mecyt)2]ClO4. The reac-
tion of [{Pd(C6F5)(L′)(µ-Cl)}2] (L′ ) PPh3,31 t-BuNC32) with
AgClO4 (1:2 molar ratio) in acetone followed by addition
of 2 equiv of 1-Mecyt gives the cationic complexes [Pd-
1
type of C6F5 group. The H NMR spectra of complexes 10
and 11 show 10 resonances for the coordinated 1-Mecyt
ligands with two distinct sets of resonances for the H(5),
H(6), and Me protons, indicating the presence of two types
of 1-Mecyt groups, one trans to C6F5 and the other trans to
1
L′ (L′ ) PPh3 or t-BuNC). The H NMR spectrum of
complex 11 shows also a singlet resonance for the t-BuNC
group at δ 1.43 ppm.
The 31P MNR spectrum of complex 10 shows a unique
resonance for the phosphine ligand at δ 23.8 ppm.
Crystal Structure of 11. Figure 6 shows the X-ray
structure of complex 11 with selected bond lengths and
angles listed in Table 7. The palladium atom exhibits a
distorted square-planar geometry. The angle between the two
1-Mecyt ligands is 92.37(13)°. The two coordinated 1-Mecyt
ligands are arranged in a head-to-tail fashion as shown in
(30) Lippert, B.; Lock, C. J. L.; Speranzini, R. A. Inorg. Chem. 1981, 20,
(28) Ruiz, J.; Rodr´ıguez, V.; Cutillas, N.; Lo´pez, G.; Pe´rez, J. Organome-
tallics 2002, 21, 4912.
808.
(31) Uso´n, R.; Fornie´s, J.; Navarro, R.; Garc´ıa, M. P. Inorg. Chim. Acta
(29) Ruiz, J.; Cutillas, N.; Rodr´ıguez, V.; Sampedro, J.; Lo´pez, G.;
Chaloner, P. A.; Hitchcock, P. B. J. Chem. Soc., Dalton Trans. 1999,
2939.
1979, 33, 69.
(32) Uso´n, R.; Fornie´s, J.; Espinet, P.; Lalinde, E. J. Organomet. Chem.
1983, 254, 371.
7370 Inorganic Chemistry, Vol. 44, No. 21, 2005

New Palladium(II) and Platinum(II) Complexes
Table 8. Hydrogen Bonds for Complex 11
<(DHA),
deg
D-H‚‚‚A
d(D-H), Å d(H‚‚‚A), Å d(D‚‚‚A), Å
N(4)-H(40B)‚‚‚O(6)
N(5)-H(50A)‚‚‚O(5)
0.75(4)
0.84(3)
2.21(4)
2.05(3)
2.957(6)
2.871(5)
174(7)
168(5)
Figure 7. Modification of the gel electrophoretic mobility of pBR322
plasmid incubated with 1-Mecyt and its palladium and platinum compounds.
Lane 1 shows pBR; lane 2, 1-Mecyt; lane 3, complex 1; lane 4, complex 2;
lane 5, complex 3; lane 6, complex 4; lane 7, complex 5; lane 8, complex
6; lane 9, complex 7; lane 10, complex 8; lane 11, complex 9; lane 12,
complex 10; lane 13, complex 11; lane 14, complex cisplatin.
complex 8. Two relatively short intramolecular contacts N5‚
‚‚O5 (2.871(5) Å) and N4‚‚‚O6 (2.957(6) Å) are observed,
which suggests a significant extra stabilization of this
arrangement due to intramolecular H bonds (Table 8). The
Pd-N(1-Mecyt) bond distances (Pd(1)-N(3) ) 2.064(4) Å
and Pd(1)-N(2) ) 2.092(3) Å) are shorter than those
observed in complex 8. The planes of the cytosine rings are
roughly at right angles to the metal coordination plane with
dihedral angles of 93.0° (N2) and 81.5° (N3). The two
1-Mecyt rings are nearly perpendicular with dihedral angles
of 79.7°. There are also intermolecular π-π interactions
between C6F5 rings.36,37 The planes of both rings make an
angle of 2.8° with an interplanar distance of 3.43 Å, a center-
to-center distance of 4.15 Å, and a deviation of the center-
center line of the perpendicular of the plane of 35°.
Table 9. CD Spectral Data for DNA for Different ri (molar ratio)
θmax
θmin
ri
λ
max (nm) (deg‚cm2‚dmol-1
)
λ
min (nm) (deg‚cm2‚dmol-1
)
DNA
1
274.6
279.6
280.8
282.6
279.8
280.8
282.6
280.2
282.6
284.2
274.0
277.8
281.6
279.0
281.8
282.6
279.0
281.0
282.2
278.4
278.8
281.6
278.6
281.0
280.4
279.2
279.2
280.0
279.8
280.8
226.2
279.6
280.6
282.2
5.01 × 103
4.00 × 103
2.33 × 103
1.54 × 103
3.98 × 103
2.34 × 103
1.54 × 103
4.38 × 103
3.65 × 103
2.75 × 103
4.46 × 103
3.44 × 103
1.87 × 103
4.19 × 103
2.87 × 103
1.54 × 103
4.05 × 103
3.13 × 103
2.17 × 103
4.83 × 103
5.15 × 103
3.89 × 103
3.82 × 103
1.45 × 103
1.33 × 103
4.05 × 103
3.83 × 103
3.45 × 103
3.98 × 103
2.33 × 103
1.79 × 103
4.01 × 103
2.86 × 103
1.68 × 103
245.8
245.8
246.4
247.4
245.8
246.6
247.4
246.4
248.4
249.2
245.4
243.6
247.0
247.2
249.0
247.4
245.8
247.0
246.2
246.8
247.8
246.2
244.4
247.4
246.4
245.8
246.0
246.0
245.8
246.4
247.2
246.0
245.6
243.6
-5.86 × 103
-4.82 × 103
-3.29 × 103
-3.82 × 103
-4.82 × 103
-3.29 × 103
-3.83 × 103
-5.40 × 103
-4.63 × 103
-4.56 × 103
-5.06 × 103
-4.31 × 103
-3.73 × 103
-5.16 × 103
-4.60 × 103
-3.83 × 103
-5.36 × 103
-4.50 × 103
-4.15 × 103
-5.65 × 103
-5.33 × 103
-4.29 × 103
-5.28 × 103
-2.19 × 103
-1.84 × 103
-5.22 × 103
-5.15 × 103
-4.88 × 103
-4.82 × 103
-3.29 × 103
-1.97 × 103
-4.93 × 103
-4.00 × 103
-2.17 × 103
0.1
0.3
0.5
0.1
0.3
0.5
0.1
0.3
0.5
0.1
0.3
0.5
0.1
0.3
0.5
0.1
0.3
0.5
0.1
0.3
0.5
0.1
0.3
0.5
0.1
0.3
0.5
0.1
0.3
0.5
0.1
0.3
0.5
2
3
4
Biological Assays. Circular Dichroism Spectroscopy. The
circular dichroism (CD) spectra of Calf thymus DNA alone
and incubated with 1-Mecyt and its palladium(II) and
platinum(II) compounds at 37 °C for 24 h with several molar
ratios were recorded.
5
6
The free ligand 1-Mecyt 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
complexes are significant (Table 9). All complexes, except
7, decreased the ellipticity of the positive and negative band
with increasing values of ri. An upshift in λmax is also
observed. This effect is more important for the palladium
than the platinum complexes. These modifications are
probably due to a BfC transformation with increasing
winding of the DNA helix.38-42 Complex 7 increased the
ellipticity for ri ) 0.1 and 0.3 and decreased for ri ) 0.5.
Gel Electrophoresis of Compound-pBR322 Complexes.
The influence of the compounds on the tertiary structure of
DNA was determined by their ability to modify the elec-
trophoretic mobility of the covalently closed circular (ccc)
and open (oc) forms of pBR322 plasmid DNA. The
complexes and the 1-Mecyt ligand were incubated at the
7
8
9
10
11
molar ratio ri ) 0.50 with pBR322 plasmid DNA at 37 ° C
for 24 h. Representative gels obtained for the Pd and Pt
complexes 1-11 are shown in Figure 7. The behavior of
the gel electrophoretic mobility of both forms, ccc and oc,
of pBR322 plasmid and DNA:cisplatin adducts is consistent
with previous reports.43 No changes were observed in sample
incubated with the free ligand 1-Mecyt. When pBR322 was
incubated with palladium compounds 1 (lane 3) and 10 (lane
12), a retard in the mobility of the ccc form was observed.
When pBR322 was incubated with compounds 2, 3, 5, 6,
and 8, a single footprinting for both forms, ccc and oc,
coalescent form, was observed.
The behavior observed for the electrophoretic mobility for
the palladium and platinum complexes indicates that some
conformational change occurred. This means that the degree
of superhelicity of the DNA molecules has been altered. In
contrast, the free ligand 1-Mecyt does not seem to modify
the tertiary structure of DNA.
(33) Ruiz, J.; Rodr´ıguez, V.; Cutillas, N.; Florenciano, F.; Pe´rez, J.; Lo´pez,
G. Inorg. Chem. Commun. 2001, 4, 23.
(34) Ruiz, J.; Mart´ınez, M. T.; Vicente, C.; Garc´ıa, G.; Lo´pez, G.; Chaloner,
P. A.; Hitchcock, P. B. Organometallics 1993, 12, 1594.
(35) Ruiz, J.; Lo´pez, J. F. J.; Rodr´ıguez, V.; Pe´rez, J.; Ram´ırez de Arellano,
M. C.; Lo´pez, G. J. Chem. Soc., Dalton Trans. 2001, 2683.
(36) Collings, J. C.; Roscoe, K. P.; Thomas, R. L.; Batsanov, A. S.; Stimson,
L. M.; Howard, J. A. K.; Marder, T. B. New J. Chem. 2001, 25, 1410.
(37) Vangala, V. R.; Nangia, A.; Lynch, V. M. Chem. Commun. 2002,
1304.
(38) Macquet, J. P.; Butour, J. L. Biochimie 1978, 60, 901.
(39) Brabec, V.; Kleinwa¨chter, V.; Butour, J.; Johnson, M. P. Biophys.
Chem. 1990, 35, 129.
(40) Marchan, V.; Moreno, V.; Pedroso, E.; Grandas, A. Chem. Eur. J.
2002, 7, 808.
(41) Zaludova´, R.; Zakouska´, A.; Kaspa´rova´, J.; Balcarova´, Z.; Kleinwa¨cht-
er, V.; Vra´na, O.; Farrel, N.; Brabec, V. Eur. J. Biochem. 1997, 246,
508.
(42) Pe´rez-Cabre´, M.; Cervantes, G.; Moreno, V.; Prieto, M. J.; Pe´rez, J.
M.; Font-Bardia, M.; Solans, X. J. Inorg. Biochem. 2004, 98, 510.
(43) Ushay, H. M.; Tullius, T. D.; Lippard, S. J. Biochemistry 1981, 20,
3744.
Inorganic Chemistry, Vol. 44, No. 21, 2005 7371

Ruiz et al.
Figure 8. TMAFM image corresponding to pBR322 (a), pBR-cisplatin (b), pBR-complex 1 (c), pBR-complex 2 (d), pBR-complex 3 (e), and pBR-
complex 4 (f).
Figure 9. TMAFM image corresponding to pBR-complex 5 (g), pBR-complex 6 (h), pBR-complex 7 (i), pBR-complex 8 (j), pBR-complex 9 (k),
pBR-complex 10 (l), and pBR-complex 11 (m).
AFM Study of Compound-pBR322 Complexes. In
Figures 8 and 9 the images corresponding to the modifica-
tions caused by the Pd(II) and Pt(II) complexes on pBR322
plasmid DNA are shown. In all cases the complexes seem
to modify the morphology of the pBR322 DNA in similar
mode as cisplatin does.44-47 In the AFM images the same
7372 Inorganic Chemistry, Vol. 44, No. 21, 2005

New Palladium(II) and Platinum(II) Complexes
Table 10. IC50 (µM) for Cisplatin and Complexes 1-11 for the Tumor
Table 11. Percentages of Vital Cells, Apoptotic Cells, Dead Cells, and
Damaged Cells after Treatment of a HL-60 Cell Population with
Cisplatin and Complexes 1-11 for 24 h
Cell Line HL-60
complex
24 h
72 h
2.15
11.48
104.90
5.27
treatment
(IC50 24 h, mM) cells (R1)
% vital
% apoptotic
cells (R2)
% dead
cells (R3)
% damaged
cells (R4)
cisplatin
1
2
3
15.61
10.80
124.30
6.24
control
89.65
54.89
33.75
44.46
35.18
44.84
54.67
41.25
40.22
44.30
81.85
74.32
34.76
2.81
37.57
40.42
36.34
39.04
30.39
21.09
36.09
38.80
31.20
7.99
6.79
5.17
0.75
2.37
5.16
2.43
4.31
5.47
4.81
6.45
3.56
6.56
1.27
0.52
1.49
cisplatin (15.6)
1 (10.80)
2 (124.3)
3 (6.4)
4 (10.89)
5 (3.02)
6 (3.24)
7 (1.36)
8 (11.32)
9 (1.66)
10 (4.71)
11 (7.05)
4
5
10.89
3.01
10.48
2.06
20.68
16.77
21.47
19.30
19.43
16.21
17.42
17.94
8.99
6
3.24
2.98
7
1.36
1.08
8
9
11.32
1.65
11.67
1.42
10
11
4.71
7.05
5.37
6.13
14.84
46.63
10.32
17.12
type of effect is observed for palladium or platinum
complexes. The metal complexes attached to DNA cause
kinks and cross-linking in the plasmid forms.
Conclusions
Cytotoxicity Studies. The in vitro growth inhibitory effect
of complexes 1-11 and cisplatin was evaluated in HL-60
human tumor cell line. As listed in Table 10, at a short
incubation time (24 h) all of the new complexes (except
complex 2) were more active than cisplatin. In fact, at a 24
h incubation time complexes 7 and 9 were about 10-fold
more active than cisplatin toward HL60 cells. After 72 h of
incubation time complexes 7 and 9 were also 2-fold more
active than cisplatin, while the rest of complexes were less
active than cisplatin.
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
organisms. 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 an understanding of the basic mechanisms
that underlie apoptosis will offer potential new targets for
therapeutic treatment of diseases.48
New palladium and platinum organometallic complexes
derived from the 2-(dimethylaminomethyl)phenyl (dmba) and
pentafluorphenyl groups with the model nucleobase 1-methyl-
cytosine have been prepared. The crystal structures of [Pd-
(bpzm*)(C6F5)(1-Mecyt)]ClO4,[Pt(dmba)(DMSO)(1-Mecyt)]-
ClO4, cis-[Pd(C6F5)2(1-Mecyt)2], and cis-[Pd(t-BuNC)(C6F5)-
(1-Mecyt)2]ClO4 have been established by X-ray diffraction.
There is extensive hydrogen bonding (N-H‚‚‚O, C-H‚‚‚F,
or C-H‚‚‚O) in all compounds. There are also intermolecular
π-π interactions between pyrimidine rings of adjacent chains
in [Pd(C6F5)2(1-Mecyt)2]. DNA adduct formation of the new
complexes synthesized was followed by circular dichroism
and electrophoretic mobility. Atomic force microscopy
images of the modifications caused by the complexes on
plasmid DNA pBR322 were also obtained. In all cases the
complexes seem to modify the morphology of the pBR322
DNA in similar mode to that of cisplatin. Values of IC50
were also calculated for the new complexes against the tumor
cell line HL-60. At a short incubation time (24 h) almost all
the new complexes were more active than cisplatin. It was
demonstrated by flow cytometry that the majority of the cell
death observed in cytotoxic assays is by apoptosis.
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 11. Exposure of cells to
cisplatin and complexes 1-8 and 11 resulted in a significant
induction of apoptosis (30-45% of apoptotic cells) compared
with that produced by complexes 9 and 10 (8-15% of
apoptotic cells). However, an additional population of cells
appeared to be dying by necrosis or late apoptosis when
HL60 cells were exposed to complexes 1-11.
Experimental Section
Instrumental Measurements. The C, H, and N analyses were
performed with a Carlo Erba model EA 1108 microanalyzer.
Decomposition temperatures were determined with a Mettler TG-
50 thermobalance at a heating rate of 5 °C min-1 and the solid
samples under nitrogen flow (100 mL min-1). Molar conductivities
were measured in acetone solution (c ≈ 5 × 10-4 mol L-1) with a
Crison 525 conductimeter. The 1H, 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. Solvents were dried by the usual methods.
Materials. The starting complexes [(N-N)Pd(C6F5)Cl] (N-N
) bpzm*, bpzm,14 tmeda, and bpy),49 [(o-C6H4CH2NMe2)PdCl-
(PPh3)],22 [(o-C6H4CH2NMe2)PtCl(L′)] (L′ ) PPh3 and DMSO),23
(44) Cervantes, G.; Prieto, M. J.; Moreno, V. Met. Based Drugs 1997, 4,
9.
(45) Onoa, G. B.; Cervantes, G.; Moreno, V.; Prieto, M. J. Nucleic Acid
Res. 1998, 26, 1473.
(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) Onoa, G. B.; Moreno, V. Int. J. Pharm. 2002, 245, 55.
(48) Martin, S. J.; Green, D. R. Curr. Opin. Oncol. 1994, 6, 616.
(49) Uso´n, R.; Fornie´s, J.; Mart´ınez, F. J. Organomet. Chem. 1976, 112,
105.
Inorganic Chemistry, Vol. 44, No. 21, 2005 7373

Ruiz et al.
cis-[Pd2(C6F5)4(µ-Cl)2]2- 26
,
cis-[Pt(C6F5)2(THF)2],27 and [{Pd(C6F5)-
2H, Hγ+Hγ′), 8.08 (d, 1H, Hδ′, J(Hδ′Hγ′) 4.2 Hz), 7.83 (d, 1H, HR′,
J(HR′Hâ′) 4.9 Hz), 7.76 (d, 1H, H6 of 1-Mecyt, J 7.2 Hz), 7.74 (m,
1H, Hâ), 7.33 (m, 1H, Hâ′), 5.83 (d, 1H, H5 of 1-Mecyt, J 7.2 Hz),
3.22 (s, 3H, Me of 1-Mecyt). 19F NMR (DMSO-d6): δ(CFCl3)
-115.8 (d, 1Fo, J(FoFm) 31.1 Hz), -117.4 (d, 1Fo, J(FoFm) 31.1
Hz), -159.2 (t, 1Fp, J(FmFp) 22.6 Hz), -161.6 (m, 1Fm), -162.6
(m, 1Fm).
Preparation of Complexes [M(dmba)(PPh3)(1-Mecyt)]ClO4
[M ) Pd (5), Pt (6)]. To a solution of [M(dmba)(PPh3)Cl] (0.185
mmol) (M ) Pd or Pt) in acetone (20 mL) was added AgClO4
(38.4 mg, 0.185 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 1-methylcytosine (23.1
mg, 0.185 mmol). The solution was stirred for 6 h, and then the
solvent was partially evaporated under vacuum and water added
to precipitate a white solid, which was collected by filtration and
air-dried.
(L′)(µ-Cl)}2] (L′ ) PPh3,31 t-BuNC32) were prepared by procedures
described elsewhere. 1-Methylcytosine (1-Mecyt) was purchased
from Chemogen (Konstanz, Germany). The sodium salt of Calf
thymus DNA, EDTA (ethylenediaminetetraacetic acid), and Tris-
HCl (tris-(hydroxymethyl)aminomethane hydrochloride) used in the
circular dichroism (CD) study were obtained from Sigma (Madrid,
Spain), HEPES (N-2-hydroxyethyl piperazyne-N′-2-ethanesulfonic
acid) was obtained from ICN (Madrid), and pBR322 plasmid DNA
was obtained from Boehringer-Mannheim (Mannheim, Germany).
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(N-N)(C6F5)(1-Mecyt)]ClO4
[N-N ) bpzm* (1), bpzm (2), tmeda (3), bpy (4)]. To a solution
of [PdCl(C6F5)(N-N)] (0.194 mmol) in acetone (20 mL) was added
AgClO4 (40.4 mg, 0.194 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 1-methylcy-
tosine (24.3 mg, 0.194 mmol). The solution was stirred for 24 h,
and then the solvent was partially evaporated under vacuum and
water (for 1 and 2) or hexane (for 3 and 4) added to precipitate a
white solid, which was collected by filtration and air-dried.
Data for Complex 1. Yield: 75 mg, 55%. Anal. Calcd for
C22H23ClF5N7O5Pd: C, 37.6; H, 3.3; N, 14.0. Found: C, 37.9; H,
3.1; N, 13.7. Mp: 260 °C dec. ΛM: 152 S cm2 mol-1. IR (Nujol,
Data for Complex 5. Yield: 81 mg, 60%. Anal. Calcd for
C32H34ClN4O5PPd: C, 52.8; H, 4.7; N, 7.7. Found: C, 52.5; H,
4.7; N, 7.5. Mp: 239 °C dec. ΛM: 165 S cm2 mol-1. IR (Nujol,
1
cm-1): ν(NH), 3432, 3330. H NMR (CDCl3): δ(SiMe4) 7.66-
7.30 (m, 17 H, PPh3 + NH2), 6.97 (d, 1H, C6H4, JHH ) 7.0 Hz),
6.83 (m, 2H, H6 of 1-Mecyt + C6H4), 6.34 (false t, 1 H, C6H4, JHH
= JHH ) 7.0 Hz), 6.26 (false t, 1 H, C6H4, JHH = JHH ) 7.0 Hz),
5.76 (d, 1H, H5 of 1-Mecyt, J 7.2 Hz), 4.30 (br, 1 H, NCH2), 3.83
(br, 1 H, NCH2), 3.03 (s, 3H, Me of 1-Mecyt), 2.70 (br, 3H, NMe2),
2.63 (br, 3H, NMe2). 31P NMR (CDCl3): δ(H3PO4) 43.5 (s).
Data for Complex 6. Yield: 100 mg, 77%. Anal. Calcd for
C32H34ClN4O5PPt: C, 47.1; H, 4.2; N, 6.9. Found: C, 46.9; H,
4.3; N, 6.7; S, 5.6. Mp: 281 °C dec. ΛM: 110 S cm2 mol-1. IR
cm-1): ν(NH), 3327, 3230; ν(Pd-C6F5), 778. H NMR (acetone-
1
d6): δ(SiMe4) 8.37 (br, 1H, NH2), 8.19 (br, 1H, NH2), 7.78 (d,
1H, CH2, J 15 Hz), 7.74 (d, 1H, H6 of 1-Mecyt, J 7.2 Hz), 6.91 (d,
1H, CH2, J 15 Hz), 6.09 (s, 1H, H4′), 6.07 (s, 1 H, H4), 6.01 (d,
1H, H5 of 1-Mecyt, J 7.2 Hz), 3.33 (s, 3H, Me of 1-Mecyt), 2.60
(s, 3H, Me3′), 2.58 (s, 3H, Me5), 2.16 (s, 3H, Me5′), 1.99 (s, 3 H,
Me3). 13C{1H} NMR (CDCl3): δ(SiMe4) 150.4 (C6 of 1-Mecyt),
109.9, 109.5 (C4 or C4′ of bpzm*), 94.5 (C5 of 1-Mecyt), 59.8
(CH2 of bpzm*), 39.0 (Me of 1-Mecyt), 14.7, 14.0, 12.2, 11.9 (Me
of bpzm*). 19F NMR (acetone-d6): δ(CFCl3) -117.1 (d, 2Fo,
J(FoFm) 22.5 Hz), -161.2 (t, 1Fp, J(FmFp) 19.7 Hz), -164.9 (m,
2Fm).
Data for Complex 2. Yield: 80 mg, 57%. Anal. Calcd for
C18H15ClF5N7O5Pd: C, 33.5; H, 2.3; N, 15.2. Found: C, 33.5; H,
2.2; N, 15.0. Mp: 248 °C dec. ΛM: 140 S cm2 mol-1. IR (Nujol,
cm-1): ν(NH), 3425; ν(Pd-C6F5), 773. 1H NMR (acetone-d6): δ-
(SiMe4) 8.29 (m, 2H, H5 + H5′), 8.04 (br, 1H, NH2), 7.93 (br,
1H, NH2), 7.78 (d, 1H, H6 of 1-Mecyt, J 7.2 Hz), 7.77 (d, 1H, H3
or H3′, J 1.8 Hz), 7.40 (d, 1H, H3 or H3′, J 2.0 Hz), 7.37 (d, 1H,
CH2, J 15.2 Hz), 7.25 (d, 1H, CH2, J 15.2 Hz), 6.52 (m, 2H, H4 +
H4′), 5.96 (d, 1H, H5 of 1-Mecyt, J 7.2 Hz), 3.46 (s, 3H, Me of
1-Mecyt). 19F NMR (acetone-d6): δ(CFCl3) -118.6 (br, 2Fo),
-161.4 (t, 1Fp, J (FmFp) 18.8 Hz), -164.8 (br, 2Fm).
1
(Nujol, cm-1): ν(NH), 3423, 3309. H NMR (CDCl3): δ(SiMe4)
7.71-7.37 (m, 17 H, PPh3 + NH2), 7.09 (d, 1H, C6H4, JHH ) 7.1
Hz), 6.90 (m, 2 H, H6 of 1-Mecyt + C6H4), 6.43 (m, 2 H, C6H4),
5.94 (d, 1H, H5 of 1-Mecyt, J 7.2 Hz), 4.35 (d, 1 H, NCH2, JHH
13.5 Hz), 3.94 (dd, 1 H, NCH2, JHH ) 13.5 Hz, JHP ) 3.8 Hz),
3.10 (s, 3H, Me of 1-Mecyt), 2.83 (d, 3H, NMe of dmba, JHP
)
)
2.4 Hz), 2.77 (d, 3H, NMe of dmba, JHP ) 2.7 Hz). 13C{1H} NMR
(CDCl3): δ(SiMe4) 148.3 (C6 of 1-Mecyt), 96.8 (C5 of 1-Mecyt),
71.2 (CH2NMe2), 51.0 (NMe2), 50.4 (NMe2), 39.8 (Me of 1-Mecyt).
31P NMR (CDCl3): δ(H3PO4) 20.7 (JPt-P ) 4104 Hz). 195Pt NMR
(CDCl3): δ(Na2[PtCl6]) - 3761 (d, JPt-P ) 4104 Hz).
Preparation of Complex [Pt(dmba)(DMSO)(1-Mecyt)]ClO4
(7). To a solution of [Pt(dmba)(DMSO)Cl] (100 mg, 0.236 mmol)
in acetone (20 mL) was added AgClO4 (53.2 mg, 0.236 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 1-methylcytosine (29.5 mg, 0.236 mmol).
The solution was stirred for 6 h, and then the solvent was partially
evaporated under vacuum and methanol added to precipitate a
brown solid, which was collected by filtration and air-dried.
Data for Complex 7. Yield: 84 mg, 56%. Anal. Calcd for
C16H25ClN4O6SPt: C, 30.4; H, 4.0; N, 8.9. Found: C, 30.7; H,
4.2; N, 8.5. Mp: 241 °C dec. ΛM: 130 S cm2 mol-1. IR (Nujol,
Data for Complex 3. Yield: 101 mg, 70%. Anal. Calcd for
C17H23ClF5N5O5Pd: C, 33.2; H, 3.8; N, 11.4. Found: C, 33.5; H,
4.1; N, 11.6. Mp: 237 °C dec. ΛM: 135 S cm2 mol-1. IR (Nujol,
cm-1): ν(NH), 3416, 3321; ν(Pd-C6F5), 773. H NMR (acetone-
1
1
d6): δ(SiMe4) 8.55 (br, 1H, NH2), 7.86 (br, 1H, NH2), 7.65 (d,
1H, H6 of 1-Mecyt, J 7.2 Hz), 5.98 (d, 1H, H5 of 1-Mecyt, J 7.2
Hz), 3.37 (s, 3H, Me of 1-Mecyt), 3.13 (br, 4H, CH2), 2.08 (s,
12H, Me).19F NMR (acetone-d6): δ(CFCl3) -117.2 (d, 2Fo, J(FoFm)
18.8 Hz), -162.1 (t, 1Fp, J(FmFp) 26.4 Hz), -164.7 (m, 2Fm).
Data for Complex 4. Yield: 115 mg, 85%. Anal. Calcd for
C21H15ClF5N5O5Pd: C, 38.6; H, 2.3; N, 10.7. Found: C, 38.4; H,
2.3; N, 9.8. Mp: 282 °C dec. ΛM: 132 S cm2 mol-1. IR (Nujol,
cm-1): ν(NH), 3415, 3303. H NMR (CDCl3): δ(SiMe4) 7.67 (s,
1H, NH2), 7.65 (s, 1H, NH2), 7.56 (d, 1H, H6 of 1-Mecyt, J 7.2
Hz), 7.11-6.98 (m, 4 H, C6H4), 6.34 (d, 1H, H5 of 1-Mecyt, J 7.2
Hz), 4.17 (br, 1 H, NCH2 dmba), 4.02 (br, 1 H, NCH2 dmba), 3.49
(s, 3H, Me of 1-Mecyt), 3.39 (brs, 3H, NMe2), 3.25 (brs, 3H, NMe2),
2.73 (s, 6H, Me of DMSO). 195Pt NMR (CDCl3): δ(Na2[PtCl6]) -
3761 (s).
Preparation of Complex cis-[Pd(C6F5)2(1-Mecyt)2] (8). To a
solution of cis-[NBu4]2[Pd2(C6F5)4(µ-Cl)2] (100 mg, 0.070 mmol)
in acetone (20 mL) was added AgClO4 (28.9 mg, 0.140 mmol).
AgCl immediately formed. The resulting suspension was stirred
cm-1): ν(NH), 3328, 3462; ν(Pd-C6F5), 767. H NMR (DMSO-
d6): δ(SiMe4) 8.70 (d, 1H, HR, J(HRHâ) 3.9 Hz), 8.67 (d, 1H, Hδ,
J(HδHγ) 4.2 Hz), 8.60 (br, 1H, NH2), 8.49 (br, 1H, NH2), 8.36 (m,
1
7374 Inorganic Chemistry, Vol. 44, No. 21, 2005

New Palladium(II) and Platinum(II) Complexes
Table 12. Crystal Structure Determination Details
1
7
8
11
formula
fw
C22H23ClF5N7O5Pd
702.32
C19H31ClN4O7PtS
690.08
C25H20F10N6O3Pd
748.87
C21H23ClF5N7O6Pd
706.31
cryst syst
monoclinic
monoclinic
triclinic
orthorhombic
unit cell dimensions
<ind>a (Å)
<ind>b (Å)
<ind>c (Å)
<ind>a (deg)
<ind>b (deg)
<ind>g (deg)
unit cell vol (Å3)
temp
15.1602(17)
22.5242(17)
16.9832(13)
90
110.724(8)
90
5424.0(8)
173(2)
P2(1)/n
8
0.865
10 667
9514
0.0233
0.0333
0.0766
13.6323(6)
13.9239(6)
14.2497(6)
90
116.498(2)
90
2420.66(18)
100(2)
P2(1)/c
4
6.040
26 390
4932
0.0284
0.0239
0.0525
9.7297(11)
16.7595(19)
18.023(2)
96.245(2)
96.146(2)
104.737(2)
2797.5(5)
100(2)
P1h
4
0.771
32 634
33.700(2)
12.1319(11)
13.9242(11)
90
90
90
5692.8(8)
173(2)
Pbcn
space group
Z
8
µ(mm-1
)
0.827
8453
5008
0.0328
0.0401
0.0997
reflns collected
independent reflns
R(int)
12 514
0.0217
0.0330
0.0758
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.
for 30 min and then filtered through a short pad of Celite. To the
filtrate was then added 1-methylcytosine (38.5 mg, 0.308 mmol).
The solution was stirred for 24 h, and then the solvent was partially
evaporated under vacuum and hexane added to precipitate a white
solid, which was collected by filtration and air-dried.
3.2; N, 9.3. Mp: 264 °C dec. ΛM: 140 S cm2 mol-1. IR (Nujol,
cm-1): ν(NH), 3344; ν(Pd-C6F5), 786. 1H NMR (acetone-d6): δ-
(SiMe4) 8.60 (br, 1H, NH2), 8.17 (br, 1H, NH2), 7.86 (br, 1H, NH2),
7.72-7.40 (m, 16 H, PPh3 + NH), 5.95 (d, 1H, H6 of 1-Mecyt, J
7.2 Hz), 5.93 (d, 1H, H6 of 1-Mecyt, J 7.2 Hz), 5.79 (d, 1H, H5 of
1-Mecyt, J 7.2 Hz), 5.77 (d, 1H, H5 of 1-Mecyt, J 7.2 Hz), 3.40
(s, 3H, Me of 1-Mecyt), 3.08 (s, 3H, Me of 1-Mecyt). 19F NMR
(acetone-d6): δ(CFCl3) -115.0 (d, 1Fo, J(FoFm) 24.5 Hz), -116.3
(d, 1Fo, J(FoFm) 28.2 Hz), -162.4 (t, 1Fp, J(FmFp) 20.7 Hz), -164.7
(m, 2Fm). 31P NMR (acetone-d6): δ(H3PO4) 23.8 (s).
Data for Complex 8. Yield: 70 mg, 65%. Anal. Calcd for
C22H14F10N6O2Pd: C, 38.3; H, 2.0; N, 12.2. Found: C, 38.4; H,
2.2; N, 12.3. Mp: 232 °C dec. IR (Nujol, cm-1): ν(NH), 3416;
1
ν(Pd-C6F5), 784. H NMR (acetone-d6): δ(SiMe4) 8.64 (br, 2H,
NH2), 7.65 (br, 2H, NH2), 7.58 (d, 2H, H6 of 1-Mecyt, J 7.2 Hz),
5.88 (d, 2H, H5 of 1-Mecyt, J 7.2 Hz), 3.27 (s, 6H, Me of 1-Mecyt).
19F NMR (acetone-d6): δ(CFCl3) -114.4 (br, 4Fo), -165.6 (t, 2Fp,
J(FmFp) 19.8 Hz), -167.1 (m, 4Fm).
Preparation of Complex cis-[Pt(C6F5)2(1-Mecyt)2] (9). To a
solution of cis-[Pt(C6F5)2(THF)2] (145 mg, 0.215 mmol) in acetone
(20 mL) was added 1-methylcytosine (53.8 mg, 0.43 mmol). The
solution was stirred for 24 h, and then the solvent was partially
evaporated under vacuum and hexane added to precipitate a white
solid, which was collected by filtration and air-dried.
Data for Complex 9. Yield: 129 mg, 79%. Anal. Calcd for
C22H14F10N6O2Pt: C, 33.9; H, 1.8; N, 10.8. Found: C, 34.2; H,
1.9; N, 10.6. Mp: 306 °C dec. IR (Nujol, cm-1): ν(NH), 3375,
3197; ν(Pt-C6F5), 801. 1H NMR (acetone-d6): δ(SiMe4) 8.99 (br,
2H, NH2), 7.66 (br, 2H, NH2), 7.58 (d, 2H, H6 of 1-Mecyt, J 7.2
Hz), 5.92 (d, 2H, H5 of 1-Mecyt, J 7.2 Hz), 3.28 (s, 6H, Me of
1-Mecyt). 13C{1H} NMR (CDCl3): δ(SiMe4) 148.8 (C6 of 1-Mecyt),
94.9 (C5 of 1-Mecyt), 38.9 (Me of 1-Mecyt). 19F NMR (acetone-
d6): δ(CFCl3) -118.2 (br, 4Fo), -167.0 (t, 2Fp, J(FmFp) 19.8 Hz),
-168.0 (m, 4Fm). 195Pt NMR (CDCl3) at 45 °C: δ(Na2[PtCl6]) -
3636 (t, JPt-Fo ) 480 Hz).
Preparation of Complexes cis-[Pd(C6F5)(L′)(1-Mecyt)2]ClO4
[L′ ) PPh3 (10), t-BuNC (11)]. To a solution of [{Pd(C6F5)(L′)-
(µ-Cl)}2] (L′ ) PPh3, t-BuNC) (0.128 mmol) in acetone (20 mL)
was added AgClO4 (52.9 mg, 0.256 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 added
1-methylcytosine (63.8 mg, 0.51 mmol). The solution was stirred
for 24 h, and then the solvent was partially evaporated under
vacuum and hexane added to precipitate a white solid, which was
collected by filtration and air-dried.
Data for Complex 11. Yield: 108 mg, 60%. Anal. Calcd for
C21H23ClF5N6O6Pd: C, 35.7; H, 3.3; N, 13.9. Found: C, 35.6; H,
3.3; N, 13.7. Mp: 293 °C dec. ΛM: 135 S cm2 mol-1. IR (Nujol,
1
cm-1): ν(NH), 3366; ν(CN), 2236; ν(Pd-C6F5), 776. H NMR
(acetone-d6): δ(SiMe4) 8.29 (br, 1H, NH2), 8.292 (br, 1H, NH2),
8.97 (br, 1H, NH2), 7.90 (br, 1H, NH2), 7.89 (d, 1H, H6 of 1-Mecyt,
J 7.2 Hz), 7.71 (d, 1H, H6 of 1-Mecyt, J 7.2 Hz), 6.15 (d, 1H, H5
of 1-Mecyt, J 7.5 Hz), 5.99 (d, 1H, H5 of 1-Mecyt, J 7.2 Hz), 3.51
(s, 3H, Me of 1-Mecyt), 3.35 (s, 3H, Me of 1-Mecyt), 1.43 (s, 9 H,
t-BuNC). 19F NMR (acetone-d6): δ(CFCl3) -118.3 (d, 2Fo, J(FoFm)
20.7 Hz), -161.4 (t, 1Fp, J(FmFp) 20.7 Hz), -165.1 (m, 2Fm).
X-ray Crystal Structure Analysis. Suitable crystals of 1, 7, 8,
and 11 were grown from acetone/hexane. The crystal and molecular
structures of the compounds 1, 7, 8, and 11 have been determined
by X-ray diffraction studies (Table 12). Crystals were mounted on
glass fibers and transferred to the cold gas stream of the diffrac-
tometer (1 and 11 Siemens P4 and the others Bruker Smart APEX).
Data were recorded with Mo KR radiation (λ ) 0.71073Å) in
ω-scan mode. Absorption correction for compounds 1 and 11 was
based on Ψ-scans and for compounds 7 and 8 on multiscans.
Structures 1, 8, and 11 were solved by the heavy atom method;
structure 7 was solved by direct methods and refined anisotropically
on F2.50 The hydrogens at N were located in the Fourier difference
maps and refined freely. Methyl groups were refined using rigid
groups, and other hydrogens were refined using a riding method.
Special Features. For compound 1 the perchlorate anion is
disordered over two sites, ca. 52:48.
For compound 8 the two acetone molecules are disordered over
two sites.
Data for Complex 10. Yield: 90.4 mg, 70%. Anal. Calcd for
C34H29ClF5N6O6PPd: C, 46.1; H, 3.3; N, 9.5. Found: C, 45.8; H,
(50) Sheldrick, G. M. SHELXL-97; University of Go¨ttingen: Go¨ttingen
Germany, 1997.
Inorganic Chemistry, Vol. 44, No. 21, 2005 7375

Ruiz et al.
For compound 11 the tert-butyl group is disordered over two
sites, ca. 58:42, and the perchlorate is also disordered over two
sites, ca. 78:22.
rate of 1-3 Hz. The AFM probe was a 125-mm-long monocrys-
talline 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, 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.
Cytotoxicity Assay. Growth inhibitory effect of palladium and
platinum complexes on the leukemia HL-60 cell line was measured
by the microculture tetrazolium [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide, MTT] assay.51 Briefly, cells growing
in the logarithmic phase were seeded into 96-well microtiter plates
in 100 µL of the appropriate culture medium at a plating density
of 1 × 104/well. Following the addition of different complex
concentrations 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 a wavelength of 490 nm. 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 three trials.
Biological Assays
Formation of Compound-DNA Complexes. All compounds
were dissolved in DMSO prior to dilution with saline. The final
maximum DMSO concentration 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 concentra-
tions in TE buffer (50 mM NaCl, 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, Pd, 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 JASCO J720
spectropolarimeter with a 450 W xenon lamp using a computer for
spectral subtraction and noise 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 of DNA underwent
1% agarose gel electrophoresis for 4 h at 1.5 V/cm in 0.5XTBE
(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 viewed with a TC-312A
transilluminator spectroline and the image captured by a Cohu High
Performance CCD camera.
Atomic Force Microscopy. Preparation of adducts DNA-metal
complexes. pBR322 DNA (25 µg/µL) was incubated in an ap-
propriate volume with the required palladium and platinum
concentration 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 New, 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 ethanol-water mixture (1:1) five times and plunged
three times each in ethanol (100%). 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
In Vitro Apoptosis Assay. Induction of apoptosis in vitro by
complexes 1-11 and cisplatin was determined by a flow cytometric
assay with Annexin V-FITC52 using an Annexin V-FITC Apoptosis
Detection Kit (Roche). Exponentially growing HL-60 cells in six-
well plates (5 × 105 cells/well) were exposed to IC50 concentrations
of cisplatin or complexes 1-11 for 24 h. Afterward 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. Financial support of this work by the
Direccio´n General de Investigacio´n del Ministerio de Ciencia
y Tecnolog´ıa (Project Nos. BQU2001-0979-C02-01 and
Bio2001-2046), Spain, and the Fundacio´n Se´neca de la
Comunidad Auto´noma de la Regio´n de Murcia (Project No.
PI-72-00773-FS-01) is acknowledged. The authors thank Dra.
Mar´ıa Jose´ Prieto (EF and AFM), Dra. Francisca Garc´ıa (Cell
Culture Facility), and Manuela Costa (Cytometry Facility)
for technical assistance.
Supporting Information Available: X-ray crystallography data
for complexes 1, 7, 8, and 11. This material is available free of
charge via the Internet at http://pubs.acs.org.
IC0502372
(51) Mosmann, T. J. Immunol. Methods 1983, 65, 55.
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7376 Inorganic Chemistry, Vol. 44, No. 21, 2005