New palladium(II) and platinum(II) complexes with 9-aminoacridine: Structures, luminiscence, theoretical calculations, and antitumor activity

Article abstract of DOI:10.1021/ic800589m

The new complexes [Pd(dmba)(N10-9AA)(PPh₃)]ClO₄ (1), [Pt(dmba)(N9-9AA)(PPh₃)]ClO₄ (2), [Pd(dmba)(N10-9AA)Cl] (3), and [Pd(C₆F₅)(N10-9AA)(PPh₃)Cl] (4) (9-AA = 9-aminoacridine; dmb

Full text of DOI:10.1021/ic800589m

Inorg. Chem. 2008, 47, 6990-7001
New Palladium(II) and Platinum(II) Complexes with 9-Aminoacridine:
Structures, Luminiscence, Theoretical Calculations, and Antitumor
Activity
Jose´ Ruiz,*,† Julia Lorenzo,‡ Consuelo Vicente,† Gregorio Lo´pez,† Jose´ Mar´ıa Lo´pez-de-Luzuriaga,§
Miguel Monge,§ Francesc X. Avile´s,‡ Delia Bautista,| Virtudes Moreno,⊥ and Antonio Laguna*,#
Departamento de Qu´ımica Inorga´nica, UniVersidad de Murcia, E-30071 Murcia, Spain, Institut de
Biotecnolog´ıa i Biomedicina, UniVersitat Auto`noma de Barcelona, E-08193 Barcelona, Spain,
Departamento de Qu´ımica, UniVersidad de La Rioja, E-26004 Logron˜o, Spain, SAI, UniVersidad
de Murcia, E-30100 Murcia, Spain, Departament de Qu´ımica Inorga`nica, UniVersitat de
Barcelona, E-08028 Barcelona, Spain, and Departamento de Qu´ımica Inorga´nica, Instituto de
Ciencia de Materiales de Arago´n UniVersidad de Zaragoza-CSIC, E-50009 Zaragoza, Spain
Received April 3, 2008
The new complexes [Pd(dmba)(N10-9AA)(PPh3)]ClO4 (1), [Pt(dmba)(N9-9AA)(PPh3)]ClO4 (2), [Pd(dmba)(N10-9AA)Cl]
(3), and [Pd(C6F5)(N10-9AA)(PPh3)Cl] (4) (9-AA ) 9-aminoacridine; dmba ) N,C-chelating 2-(dimethylaminom-
ethyl)phenyl) have been prepared. The crystal structures have been established by X-ray diffraction. In complex 2,
an anagostic C-H · · · Pt interaction is observed. All complexes are luminescent in the solid state at room temperature,
showing important differences between the palladium and platinum complexes. Complex 2 shows two structured
emission bands at high and low energies in the solid state, and the lifetimes are in agreement with excited states
of triplet parentage. Density functional theory and time-dependent density functional theory calculations for complex
2 have been done. Values of IC50 were also calculated for the new complexes 1-4 against the tumor cell line
HL-60. All of the new complexes were more active than cisplatin (up to 30-fold in some cases). The 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.
Scheme 1. Schematic Representation of the Two Tautomeric Forms of
Introduction
the 9-Aminoacridine
Acridine is an aza-aromatic compound, and its derivatives
have been widely used in pharmacology.1,2 Its polyaromatic
nature facilitates the intercalation between DNA base pairs.1
The 9-aminoacridine (9-AA) molecule has two symmetrically
distributed nitrogen atoms with nucleophilic properties: the
* Authors to whom correspondence should be addressed. E-mail:
jruiz@um.es (J.R.); alaguna@unizar.es (A.L.).
†
Departamento de Qu´ımica Inorga´nica, Universidad de Murcia.
Institut de Biotecnolog´ıa i Biomedicina, Universitat Auto`noma de
exocyclic N(9) and the endocyclic N(10). Two tautomeric
forms of the free 9-aminoacridine moiety can be adopted in
‡
Barcelona.
§
solution (see Scheme 1).
Departamento de Qu´ımica, Universidad de La Rioja.
SAI, Universidad de Murcia.
Departament de Qu´ımica Inorga`nica, Universitat de Barcelona.
|
To the best of our knowledge,3 there are no examples of
9-aminoacridine metal complexes where the ligand is
coordinated in its amine form, bound to the metal atom in a
monodentate fashion by the endocyclic nitrogen atom, and
⊥
# Instituto de Ciencia de Materiales de Arago´n Universidad de Zaragoza.
(1) Farrell, N. Transition Metal Complexes as Drugs and Chemothera-
peutic Agents; Kluwer Academic Publishers: Dordrecht, The Nether-
lands, 1989.
(2) Korth, C.; May, B. C. H.; Cohen, F. E.; Prusiner, S. B. Proc. Natl.
Acad. Sci. U. S. A. 2001, 98, 9836–9841.
(3) CCDC CSD, version 5.28, November 2006, update May 2007.
6990 Inorganic Chemistry, Vol. 47, No. 15, 2008
10.1021/ic800589m CCC: $40.75  2008 American Chemical Society
Published on Web 07/01/2008

Pd(II) and Pt(II) 9-Aminoacridine Complexes
Scheme 2
only two examples, reported by Lippard et al.,4 where the
ligand is coordinated in its imine form, which binds to the
metal atom in a monodentate fashion by the exocyclic
nitrogen atom: cis-[Pt(NH3)2(N9-9-AA)Cl](NO3) and cis-
[Pt(NH3)2(N9-9-AA)2](NO3)2. Metalations of 9-aminoacridine
and nitro-9-[(2-aminoethyl)amino]acridines have been also
reported.5–7 The interaction of 9-aminoacridinecarboxamide
platinum complexes with DNA has been investigated.8
Moreover, platinum-acridinylthiourea conjugates are a new
class of cytotoxic DNA-targeted agents that act through a
hybrid mechanism involving monofunctional platination of
nucleobase nitrogen and classical intercalation.9–11 On the
other hand, the excitation spectra for 9-aminoacridine and
some platinum and ruthenium derivatives have been
reported.12,13
In this work, we describe the syntheses, structures, and
luminescent properties of new 9-aminoacridine palladium and
platinum derivatives from the N,C-chelating 2-(dimethy-
laminomethyl)phenyl (dmba) and pentafluorophenyl C6F5
groups. Density functional theory (DFT) and time-dependent
density functional theory (TD-DFT) calculations are reported.
To our knowledge, the structures of complexes [Pd(dm-
ba)(N10-9AA)(PPh3)]ClO4, [Pd(dmba)(N10-9AA)Cl], and
[Pd(C6F5)(N10-9AA)(PPh3)Cl] represent the first examples
of metal complexes where the ligand 9-AA is coordinated
in a monodentate fashion by the endocyclic nitrogen atom.
The in vitro antiproliferative activity of the new complexes
Figure 1. ORTEP representation (50% probability) of 1. Selected bond
lengths (Å) and angles (deg): Pd(1)-C(21) ) 2.011(3), Pd(1)-N(10) )
2.121(6), Pd(1)-N(1) ) 2.139(2), Pd(1)-P(1) ) 2.2579(7), C(21)-Pd(1)-
N(10) ) 173.51(19), C(21)-Pd(1)-N(1) ) 81.14(10), N(10)-Pd(1)-N(1)
) 94.03(18), C(21)-Pd(1)-P(1) ) 93.85(8), N(10)-Pd(1)-P(1) )
91.40(16), N(1)-Pd(1)-P(1) ) 172.10(7).
in the HL-60 cell line has also been studied. All of the new
complexes were more active than cisplatin (up to 30-fold in
some cases).
Results and Discussion
Syntheses and Structures of [M(dmba)(PPh3)(9-AA)]
ClO4. The dmba complexes [Pd(dmba)(PPh3)(N10-9-AA)]-
ClO4 (1) and [Pt(dmba)(PPh3)(N9-9-AA)]ClO4 · acetone (2)
have been prepared from the corresponding chlorometal
complex [M(dmba)(PPh3)Cl] (M ) Pd or Pt). After precipi-
tation of AgCl by the addition of AgClO4 in a 1:1 molar
ratio in acetone, the solvent complexes [M(dmba)(PPh3)(Me2-
CO)]ClO4 (M ) Pd or Pt), generated in situ, react with 1
equiv of 9-AA to give the cationic complexes [M(dm-
ba)(PPh3)(9-AA)]ClO4 (1 and 2) (Scheme 2).
Complexes 1 and 2 · acetone are yellow, air-stable solids
that decompose upon heating above 180 °C in a dynamic
N2 atmosphere. An IR band is observed at ca. 1095, which
is assigned to the ν3 mode of free perchlorate (Td symmetry).
The observation of an additional band at ca. 623 cm-1 for
the ν4 mode confirms the presence of free perchlorate.14
Complexes 1 and 2·acetone were isolated as single crystals
suitable for X-ray diffraction studies following procedures
described in the Experimental Section. The crystal structure
of compound 1 consists of mononuclear [Pd(dmba)(PPh3)(N10-
9-AA)]+ cations (Figure 1) and perchlorate anions. The
acridine ligand 9-AA is coordinated in a monodentate fashion
by the endocyclic nitrogen atom N(10), no examples of this
type of coordination mode being previously reported.3 The
palladium coordination geometry is approximately square-
planar, although the angles around palladium deviate from
90° due to the bite of the cyclometallated ligand. The acridine
ring is nearly perpendicular to the Pd square plane. The
(4) Sundquist, W. I.; Bancroft, D. P.; Lippard, S. J. J. Am. Chem. Soc.
1990, 112, 1590–1596.
(5) Ceci, E.; Cini, R.; Konopa, J.; Maresca, L.; Natile, G. Inorg. Chem.
1996, 35, 876–882.
(6) Riera, X.; Moreno, V.; Ciudad, C. J.; Noe, V.; Font-Bard´ıa, M.; Solans,
X. Bioinorg. Chem. Appl. 2007, 2007; Article ID 98732, 15 pages,
DOI:, 10.1155/2007/98732.
(7) Maresca, L.; Pacifico, C.; Pappadopoli, M. C.; Natile, G. Inorg. Chim.
Acta 2000, 304, 274–282.
(8) Temple, M. D.; McFadyen, W. D.; Holmes, R. J.; Denny, W. A.;
Murray, V. Biochemistry 2000, 39, 5593–5599.
(9) Guddneppanavar, R.; Choudhury, J. R.; Kheradi, A. R.; Steen, B. D.;
Saluta, G.; Kucera, G. L.; Day, C. S.; Bierbach, U. J. Med. Chem.
2007, 50, 2259–2263.
(10) Baruah, H.; Rector, C. L.; Monnier, S. M.; Bierbach, U. Biochem.
Pharmacol. 2002, 64, 191–200.
(11) Baruah, H.; Barry, C. G.; Bierbach, U. Curr. Top. Med. Chem. 2004,
4, 1537–1549.
(12) de Carvalho, I. M. M.; Moreira, I. D.; Gehlen, M. H. Inorg. Chem.
2003, 42, 1525–1531.
(14) Nakamoto, K. Infrared and Raman Spectra of Inorganic and Coor-
dination Compounds, 5th ed.; Wiley-Interscience: New York, 1997;
p 199.
(13) Scolaro, L. M.; Alibrandi, G.; Romeo, R.; Ricevuto, V.; Campagna,
S. Inorg. Chem. 1992, 31, 2074–2078.
Inorganic Chemistry, Vol. 47, No. 15, 2008 6991

Ruiz et al.
Scheme 3
hydrogen-bonding interactions with the perchlorate anion.
Intermolecular aromatic CH/π interactions15 are also found
[distance H36 · · ·centroid (C5,C6,C7,C8,C11,C12): 2.583 Å].
The 1H and 13C NMR spectra in acetone-d6 of complexes
1 and 2·acetone at room temperature are quite different (see
the Experimental Section), indicating that in solution the
amine tautomeric form of 9-AA is present in complex 1,
whereas the imine tautomeric form is observed in complex
2 · acetone, as it also occurs in the solid state (Figures 1 and
2). Thus, for complex 1, a unique set of four resonances is
observed for the aromatic resonances of AA in a 2:2:2:2 ratio.
The observation at 25 °C of the NCH2 and NMe2 proton
signals as doublets (due to phosphorus coupling) suggests
that the inversion of the configuration at the PdCCCN chelate
ring is faster than the NMR time scale.18,26 Broad resonances
are observed at room temperature for the NH2 group and
the PPh3 protons, the PPh3 proton resonances becoming sharp
at -60 °C and +50 °C. No other significant changes are
Figure 2. ORTEP representation (50% probability) of 2·acetone. Selected
bond lengths (Å) and angles (deg): Pt(1)-C(21) ) 2.015(3), Pt(1)-N(9)
) 2.096(3), Pt(1)-N(1) ) 2.137(3), Pt(1)-P(1) ) 2.2315(8), C(21)-Pt(1)-
N(9) ) 167.97(12), C(21)-Pt(1)-N(1) ) 81.61(12), N(9)-Pt(1)-N(1) )
89.37(11), C(21)-Pt(1)-P(1) ) 96.07(9), N(9)-Pt(1)-P(1) ) 93.35(8),
N(1)-Pt(1)-P(1) ) 176.15(8).
complex shows extensive intermolecular N-H · · · O and
C-H · · · O bond links between the palladium ligands and
perchlorate counterions. Intermolecular aromatic CH/π in-
teractions15 are also found.
The structure of 2 · acetone consists of mononuclear [Pt-
(dmba)(PPh3)(N9-9-AA)]+ cations and perchlorate anions.
The cation of 2 · acetone is depicted in Figure 2. In contrast
to complex 1, here, the acridine ligand adopts an imino-type
configuration with the proton of the exocyclic 9-amino group
shifted on N(10), being N(9) and N(10) sp2-hybridized, as
was previously observed in the related complex cis-[Pt(NH3)2-
(N9-9-AA)Cl](NO3).4
The C(9)-N(9) bond distance [1.305(4) Å] is quite short,
and the value is very close to the mean value reported for
free acridines in the imino tautomeric configuration.5 The
asymmetric coordination of 9-aminoacridine in this complex
renders the acridine ring protons inequivalent and brings the
H(8) atoms close to the platinum atom, with a Pt· · · H(8)
distance of 2.539 Å and a Pt · · ·H(8)-C(8) angle of 143(12)°,
which clearly indicates the presence of an anagostic
interaction.16,17
The acridine moiety is nearly perpendicular to the Pt
square plane (average dihedral angle, 75.73°) and is folded
about the C(9)-N(10) vector with an average angle between
outer rings of 8.99°. The C(21)-Pt-N(1) angle of 81.61(12)°
is within the normal range for such dmba metal com-
plexes.18–22 The PPh3 ligand is trans to the nitrogen donor,
displaying no tendency to occupy the position trans to the
σ-bound orthoplatinated aryl group.23 Furthermore, there is
an intramolecular interaction by phenyl-AA π-stacking
(centroid-centroid distance: 3.654 Å).24,25 The complex also
shows extensive intermolecular N-H · · · O and C-H · · · O
bond links between the platinum ligands, perchlorate coun-
terions, and lattice acetone molecules. In particular, the imino
proton on the acridine N(10) position is stabilized by
1
observed in the variable-temperature H NMR spectra of
complex 1. The assignments given in the Experimental
Section for complex 1 were supported by the pertinent
heteronuclear (1H-13C) correlation spectroscopy (COSY)
technique.
On the other hand, for complex 2 · acetone, NMR spec-
troscopic studies demonstrated the persistence of the asym-
metric imino coordination of 9-AA in acetone-d6 solutions
of the complex 2·acetone at 25 °C (Scheme 3). No significant
changes are observed in the 1H NMR spectra over the -60°
to +50 °C range. The 1H spectra for 2·acetone were assigned
as follows (for the numbering scheme, see Scheme 3): the
proton resonance at very low field was assigned as H8 on
the basis of the crystal structure of 2·acetone, which reveals
the unique location of this proton above the platinum square
plane, the short Pt-H8 distances resulting in paramagnetic
deshielding of the H8 protons by approximately 2.64 ppm
versus aminoacridine free base. Protons H7, H6, and H5
could then be assigned from the connectivity observed in a
COSY spectrum. As shown by 1H NMR, protons on H9 and
(18) Ruiz, J.; Cutillas, N.; Rodr´ıguez, V.; Sampedro, J.; Lo´pez, G.;
Chaloner, P. A.; Hitchcock, P. B. J. Chem. Soc., Dalton Trans. 1999,
2939–2946.
(19) Bedford, R. B.; Cazin, C. S. J.; Coles, S. J.; Gelbrich, T.; Horton,
P. N.; Hursthouse, M. B.; Light, M. E. Organometallics 2003, 22,
987–999.
(20) Ruiz, J.; Cutillas, N.; Lo´pez, F.; Lo´pez, G.; Bautista, D. Organome-
tallics 2006, 25, 5768–5773.
(21) Dupont, J.; Pfeffer, M.; Spencer, J. Eur. J. Inorg. Chem. 2001, 1917–
1927.
(15) Nishio, M. CrystEngComm 2004, 6, 130–158.
(16) Braga, D.; Grepioni, F.; Tedesco, E.; Biradha, K.; Desiraju, G. R.
Organometallics 1997, 16, 1846–1856.
(22) Ruiz, J.; Villa, M. D.; Cutillas, N.; Lo´pez, G.; de Haro, C.; Bautista,
D.; Moreno, V.; Valencia, L. Inorg. Chem. 2008, 47, 4490–4505.
(23) Otto, S.; Samuleev, P. V.; Polyakov, V. A.; Ryabov, A. D.; Elding,
L. I. Dalton Trans. 2004, 3662–3668.
(17) Brookhart, M.; Green, M. L. H.; Parkin, G. PNAS 2007, 104, 6908–
6914.
6992 Inorganic Chemistry, Vol. 47, No. 15, 2008

Pd(II) and Pt(II) 9-Aminoacridine Complexes
Scheme 4
H10 of the imino 9-AA of complex 2·acetone were observed
directly in acetone-d6 solution at 9.16 and 10.6 ppm,
respectively. Selective irradiation of the H9 resonance
resulted in a strong nuclear Overhauser enhancement (NOE)
of the H8 (16%), but not the H1 resonance (Scheme 3).
Selective irradiation of the H10 resonance resulted in a NOE
(4%) of both the H4 and H5 resonances, as expected. The
1H NMR spectrum shows also that both the NMe2 and the
CH2N group of the dmba are diastereotopic, two separate
signals being observed for the former (coupled to 31P) and
an AB quartet for the latter (the resonance at δ 4.22 appears
as a doublet of doublets, due also to 31P coupling). Therefore,
there is no plane of symmetry in the palladium coordination
plane.
Figure 3. ORTEP representation (50% probability) of 3·DMF. Selected
bond lengths (Å) and angles (deg): Pd(1)-C(21) ) 1.983(2), Pd(1)-N(10)
) 2.0541(17), Pt(1)-N(1) ) 2.0913(18), Pd(1)-Cl(1) ) 2.4297(5),
C(21)-Pd(1)-N(10) ) 91.92(8), C(21)-Pd(1)-N(1) ) 82.97(8), N(10)-
Pd(1)-N(1))174.30(7),C(21)-Pd(1)-Cl(1))173.57(6),N(10)-Pd(1)-Cl(1)
) 90.89(5), N(1)-Pd(1)-Cl(1) ) 93.93(5).
Table 1. Hydrogen Bonds [Å and deg] for Complex 3·DMF
D-H· · ·A
d(D-H) d(H· · ·A) d(D· · ·A) <(DHA)
C(91)-H(91)· · ·Cl(1)
N(9)-H(09B)· · ·O(1)#1
N(9)-H(09A)· · ·Cl(1)#1
C(1)-H(1)· · ·Cl(1)#1
C(8)-H(8)· · ·O(1)#1
C(6)-H(6)· · ·Cl(1)#2
0.96(3)
0.89(3)
0.83(3)
0.95
0.95
0.95
2.90(3)
1.99(3)
2.46(3)
2.86
2.51
2.86
3.786(2) 155(2)
2.839(3) 161(3)
3.254(2) 161(2)
3.777(2) 162.5
3.439(3) 166.1
3.761(2) 158.4
3.623(3) 135.4
C(93)-H(93B)· · ·Cl(1)#3 0.98
2.86
fashion by the endocyclic nitrogen atom N(10) and is planar
(average angle between outer rings of 1.47°). The Cl ligand
is trans to the carbon donor, as found in related complexes
of the type [Pd(dmba)Cl(L)] (L ) py,28,29 PTA,22 or PPh329),
the Pd-Cl bond length being rather long, consistent with
the large ground-state trans influence of the σ-bound carbon
atom.23 The acridine ring is nearly perpendicular to the Pd
square plane (average dihedral angle, 85.32°). The complex
also shows extensive intermolecular N-H · · · O, C-H · · ·O,
N-H· · ·Cl, and C-H· · ·Cl bond links between the palladium
ligands and lattice DMF molecules (Table 1). Furthermore,
there is an intermolecular interaction by DMF-AA π-stacking
(distance N(4) · · ·centroid (C5,C6,C7,C8,C11,C12): 3.592 Å
and O(1) · · · centroid (C9-C14): 3.337 Å).
The structure of complex 4 · acetone · toluene is shown in
Figure 4. The palladium atom is in a slightly distorted square-
planar geometry. The acridine ligand 9-AA is coordinated
by the endocyclic nitrogen atom N(10) and is planar, the
Pd(1)-N(10) (2.1201 Å) being longer than that found in
complex 3 · DMF (2.0541 Å). The Pd-C6F5 bond length is
in the range found in the literature for pentafluorophenyl
complexes.30–34
Syntheses and Structures of [Pd(dmba)(9-AA)(Cl)]
and [Pd(C6F5)(9-AA)(Cl)]. The ready reaction of [Pd-
(dmba)(µ-Cl)]2 and [Pd(C6F5)(PPh3)(µ-Cl)]2 in CHCl3 with
9-AA in a 1:2 molar ratio gives mononuclear complexes 3
and 4, respectively (Scheme 4).
The complexes are air-stable, both in the solid state and
in solution, and they are yellow. In the far-IR spectra of
complexes 3 and 4 is exhibited a band assigned to ν(Pd-Cl)
at 358 cm-1 (for 3) and 298 cm-1 (for 4). In the zone between
1670 and 1550 cm-1, the stretching ν(>CdN-) and bending
δ(NH2) bands can be assigned. The δ(NH2) band of the free
ligand appears at 1670 cm-1. The IR spectrum of complex
4 shows also an absorption at 783 cm-1 that is observed as
a single band for the so-called X-sensitive mode,27 as
expected from the presence of only one C6F5 group in the
coordination sphere of the palladium atom. In the IR spectra
of compound 4, the form of the bands between 550 and 520
cm-1, corresponding to the triphenylphosphine molecules,
confirms the coordination of the palladium atom to only one
molecule of PPh3.
The structure of complex 3 · DMF is shown in Figure 3.
The acridine ligand 9-AA is coordinated in a monodentate
(24) Janiak, C. J. Chem. Soc., Dalton Trans. 2000, 3885–3896.
(25) Ruiz, J.; Lorenzo, J.; Sanglas, L.; Cutillas, N.; Vicente, C.; Villa, M. D.;
Avile´s, F. X.; Lo´pez, G.; Moreno, V.; Pe´rez, J.; Bautista, D. Inorg.
Chem. 2006, 45, 6347–6360.
(28) Lu, Z. L.; Neverov, A. A.; Brown, R. S. Org. Biomol. Chem. 2005, 3,
3379–3387.
(29) Mentes, A.; Kemmitt, R. D. W.; Fawcett, J.; Russell, D. R. J. Mol.
Struct. 2004, 693, 241–246.
(26) Ruiz, J.; Rodr´ıguez, V.; Cutillas, N.; Lo´pez, G.; Pe´rez, J. Organome-
tallics 2002, 21, 4912–4918.
(30) Althoff, G.; Ruiz, J.; Rodr´ıguez, V.; Lo´pez, G.; Pe´rez, J.; Janiak, C.
CrystEngComm 2006, 8, 662–665.
(27) Maslowski, E. Vibrational Spectra of Organometallic Compounds;
Wiley: New York, 1977; p 437.
(31) Fornie´s, J.; Mart´ınez, F.; Navarro, R.; Urriolabeitia, E. P. J. Organomet.
Chem. 1995, 495, 185–194.
Inorganic Chemistry, Vol. 47, No. 15, 2008 6993

Ruiz et al.
centroid-centroid contact (Ct · · ·Ct < 3.8 Å),30 although the
ring planes are not coplanar. The C6F5 ring is surrounded
by C-H moieties from phenyl rings, and intermolecular
interaction contacts C-H · · · F-C are observed.30,35
The 1H and 13C NMR spectra in DMSO-d6 of complexes
3 · CHCl3 and 4 ·1/2CHCl3 indicate that in solution the amine
tautomeric form of 9-AA is present in both complexes. The
19F NMR spectrum of 4 · 1/2CHCl3 in solution, at room
temperatue, shows a unique set of sharp resonances, indicat-
ing the presence of only one type of C6F5 group.
Luminescence Studies on Complexes 1-4. All com-
plexes are luminescent in the solid state at room temperature,
showing important differences between the palladium and
platinum complexes. As we have seen, from a structural
viewpoint, the main difference is that, in the platinum
complex 2 · acetone, the 9-AA ligand is bonded to the metal
center in a zwitterionic (acridinium) form, whereas in the
palladium complexes, the amino form of the 9-AA ligand is
present.4 Thus, complexes 1, 3 · CHCl3, and 4 ·1/2CHCl3
display high-energy structured emissions at 490 nm (exc.
400 nm), 500 nm (exc. 400 nm), and 505 nm (exc. 400 nm),
respectively. This high-energy structured emission at ca. 500
nm observed for all the palladium complexes is similar to
that displayed by the free 9-AA ligand.36 By contrast, the
platinum complex 2 · acetone shows, under the same condi-
tions, two structured emission bands at high energy (492 nm,
exc. 450 nm) and low energy (602 nm, exc. 350 nm).
All complexes are also luminescent in 5 × 10-4 M CH2Cl2
solutions at room temperature, showing only high-energy
emissions at ca. 500 nm. In the case of the low-energy
emission for complex 2 · acetone, it appears when the
temperature is decreased to 77 K (Figure 7).
Figure 4. ORTEP representation (50% probability) of 4·acetone·toluene.
Selected bond lengths (Å) and angles (deg): Pd(1)-C(21) ) 1.974(7),
Pd(1)-C(21′)) 2.050(8), Pd(1)-N(10) ) 2.1201(18), Pd(1)-P(1) )
2.2518(6), Pd(1)-Cl(1) ) 2.3629(6), C(21)-Pd(1)-C(21′) ) 6.5(3),
C(21)-Pd(1)-N(10) ) 88.1(2), C(21′)-Pd(1)-N(10) ) 89.3(2), C(21)-
Pd(1)-P(1) ) 91.29(19), C(21′)-Pd(1)-P(1) ) 90.2(2), N(10)-Pd(1)-P(1)
) 178.64(5), C(21)-Pd(1)-Cl(1) ) 178.0(2), C(21′)-Pd(1)-Cl(1) )
175.4(2), N(10)-Pd(1)-Cl(1) ) 92.16(5), Cl(1)-Pd(1)-P(1) ) 88.38(2).
We have studied in detail the photoluminescent properties
of platinum complex 2. The lifetimes of both emissions
observed for complex 2 · acetone are quite different, that of
the high-energy emission being ca. 10 µs, whereas that of
the low-energy emission is ca. 1.25 ms. These lifetimes are
in agreement with excited states of triplet parentage.
The excitation spectrum of this complex shows that a high-
energy excitation favors the low-energy emission and that a
low-energy excitation favors the high-energy emission (see
Figure 6). Since both emissions are obtained upon excitation
at high or low energy, it can be stated that both excited states
have a common energetic zone. In view of the experimental
data (see below), it seems plausible that the emissions arise
from intercrossed excited states of triplet parentage, since
the high-energy excitation produces the low-energy emission
and Vice Versa (see inset in Figure 6).
Taking into account that the structured high-energy band
is similar for all complexes and also is similar to that of the
9-AA ligand, the assignment of the excited state is likely to
arise from a triplet ligand (acridine)-centered (3LC) excited
state.
By contrast, the assignment of the low-energy emission
for complex 2 · acetone is more complicated because there
Figure 5. The dimeric structure of complex 4·acetone·toluene, showing
the hydrogen bonds.
An interesting feature of the crystal structure is the
presence of dimeric units of [Pd(C6F5)(N10-9AA)(PPh3)Cl]2
due mainly to the intermolecular NH · · · Cl hydrogen bond
(Figure 5). A stacking interaction between AA pairs is also
present, which may result in synergism with the observed
N-H· · ·Cl H-bonding interaction to favor the supramolecular
association. It is noteworthy that the intramolecular π-in-
teraction appears between the electron-poor C6F5 ring and a
phenyl ring of the phosphine, showing a rather short
(32) 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,
3650–3661.
(33) 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–
932.
(34) 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,
7365–7376.
(35) Ruiz, J.; Villa, M. D.; Rodr´ıguez, V.; Cutillas, N.; Vicente, C.; Lo´pez,
G.; Bautista, D. Inorg. Chem. 2007, 46, 5448–5449.
(36) Sandeep, P.; Bisht, P. B. Chem. Phys. 2006, 326, 521–526.
6994 Inorganic Chemistry, Vol. 47, No. 15, 2008

Pd(II) and Pt(II) 9-Aminoacridine Complexes
Figure 6. Emission spectra for complex 2·acetone in the solid state at room temperature upon excitation at 450 nm (red) and 350 nm (black). (Inset)
Mechanism of excitation from the ground state (S0) to the lowest triplet excited states (T1 and T2) (left). Emission spectra for complex 2·acetone in solid
state at 77K (right).
Figure 7. Excitation and emission spectra for complex 2·acetone in 5 × 10-4 M CH2Cl2 solution at room temperature (left) and at 77 K (right).
metal origin are usually unstructured and broad, this suggests
3
that this emission is not a pure MC transition and that the
dmba ligand is, to some extent, involved.
Second, since palladium complex 1 bears the same ligands
as complex 2 · acetone and does not display the low-energy
emission, a possible 3L′C origin of the low-energy emission
can be ruled out.
Therefore, it is highly likely that the high-energy band
arises from a metal-perturbed 3LC excited state, and the low-
energy emission arises from a ligand-metal-to-ligand charge
transfer. In the case of the palladium complexes, the absence
of the low-energy emission is likely to be due to a higher
energy difference between the metal and ligand orbitals.
Finally, the fact that the low-energy emission is missed
in solution is probably due to its very long lifetime, since
the collisions between complex 2 and solvent molecules in
the excited state are more effective and, therefore, the
Figure 8. Theoretical model [Pt(Acridinium)(PPh3)(C6H4-CH2NMe2)]+.
nonradiative deactivation is favored. Moreover, when the
spectrum is registered in CH2Cl2 at 77 K, the low-energy
emission is recovered because the nonradiative deactivation
processes are reduced when the temperature is decreased
(Figure 7).
DFT and TD-DFT Calculations. In view of the results
reported in the photophysical studies section and in order to
demonstrate these assumptions, single-point DFT and time-
dependent DFT calculations have been carried out on a model
are several possibilities, including triplet ligand (dmba)-
centered (3L′C), triplet metal-centered (3MC), or triplet
ligand-to-metal charge transfer (3LMCT).
First, the structured profile of the low-energy emission
suggests that the difference between the vibrational levels
in the ground state is large. Since the emissions having a
Inorganic Chemistry, Vol. 47, No. 15, 2008 6995

Ruiz et al.
Scheme 5. Frontier Orbitals 164a (HOMO-1), 165a (HOMO), and
166a (LUMO)
(LUMO) (HOMO ) highest occupied molecular orbital;
LUMO ) lowest unoccupied molecular orbital). The shape
of these orbitals is given in Scheme 5. Thus, while orbital
164a is mainly located at the acridinium ligand with a
small contribution from the platinum center, orbital 165a
is mostly centered at the arylic ligand (C6H4-CH2NMe2)
and the platinum center. The lower-energy empty orbital
166a (LUMO) is placed at the acridinium ligand with some
contribution from the platinum center. This preliminary
DFT calculation indicates that, regarding the shape of the
frontier orbitals, both ligand (dmba)-metal-to-ligand (acri-
dinium) charge transfer (L′MLCT) and ligand (acri-
dinium)-centered transitions would be favored.
In the experimental results, the two emissions observed
for complex 2 display lifetimes in the microsecond range.
As we have mentioned above, these transitions can be related
to triplet excited states. The use of TD-DFT calculations
permits the estimation of the energies of the two lowest triplet
excited states and which are the orbitals involved in each
transition. The TD-DFT results confirm the anticipated
tendency observed in the DFT calculations. Scheme 6 shows
the orbitals involved in the two first triplet transitions. The
lowest triplet transition (T1) occurs at a theoretical value of
497 nm (exp 450 nm) between orbitals 164a and 166a. As
we have commented upon before, both orbitals are mainly
located at the acridinium ligand, which agrees with an
internal transition in this ligand (3LC). The second triplet
transition (T2) appears at a theoretical value of 424 nm (exp
350 nm) between orbitals 165a and 166a. The previous
analyses of the orbitals have shown that they are located at
the (C6H4-CH2NMe2)Pt fragment (165a) and the acridinium
ligand (166a), which suggests a triplet ligand (dmba)-metal-
to-ligand (acridinium) charge transfer (3L′MLCT) transition.
The prediction of T1 and T2 excited states using TD-DFT
system consisting of the complete structure of the cation
[Pt(Acridinium)(PPh3)(C6H4-CH2NMe2)]+ of complex 2
(Figure 8).
We have analyzed first the electronic structure of this
model system obtained through single-point DFT calcula-
tions. Hence, we can check the contribution of each part
of the molecule to the frontier orbitals involved in the
transitions responsible for the photoluminescent properties.
The most important frontier orbitals are the occupied 164a
(HOMO-1) and 165a (HOMO) and the empty 166a
Scheme 6. Lowest Triplet Excitations T1 (left) and T2 (right)
6996 Inorganic Chemistry, Vol. 47, No. 15, 2008

Pd(II) and Pt(II) 9-Aminoacridine Complexes
calculations is in good agreement with those obtained in the
experimental results.
Biological Assays. Circular Dichroism Spectroscopy.
The circular dichroism (CD) spectra of calf thymus DNA
alone and incubated with compounds 1-4 and free 9-AA at
37 °C for 24 h with several molar ratios were recorded
(Figure 9). The new metal complexes induce modifications
in the secondary structure of DNA. In principle, they can
interact with the DNA by intercalation of the ligand
aminoacridine. Looking at the spectrum recorded for the ct-
DNA incubated with the complex 3, an increasing of the
ellipticity of the positive and negative bands with increasing
values of ri ([DNA]/[complex]) can be observed.
For the complexes 1, 2, and 4, an increase of the ellipticity
of the bands when ri ) 0.1 and 0.3 is observed, and a
decrease is observed when ri ) 0.5. All of the new complexes
show a hypsochromic shift of the bands, as happens for the
free classical intercalator 9-aminoacridine (9AA) (Figure 9).
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 platinum
complexes and the free ligand 9-AA were incubated at the
molar ratio ri ) 0.5 with pBR322 plasmid DNA at 37 °C
for 24 h. Representative gels obtained for complexes 1-4
are shown in Figure 10. The behavior of the gel electro-
phoretic mobility of both forms, ccc and oc, of pBR322
plasmid and DNA/cisplatin adducts is consistent with previ-
ous reports.37 When the pBR322 was incubated with
complexes 1, 3, and 4 and the ligand 9-AA, the rate of
migration of the ccc form decreased, indicating that these
complexes modify the tertiary structure of pBR322. These
electrophoretic studies indicate that the strength of the
interaction of 2 to DNA (lane 3) is much larger than that of
ethydium bromide (the dye). In the case of 1, this situation
is not so drastic, but nevertheless, lane 2 in Figure 10 is not
as brilliant as lanes 4-6.
Figure 9. CD spectra of calf thymus DNA incubated with complexes 1-4
and free 9-AA at different ri.
Atomic Force Microscopy (AFM) Study of Com-
pound-pBR322 Complexes. AFM images of the plasmid
pBR322 DNA incubated in the appropriate new metal
complexes in concentrations corresponding to a molar ratio
of ri ) 0.005 are shown in Figure 11. In all of the images,
toroidal supercoiled forms of the plasmid DNA can be
observed. These modifications are likely due to the strong
effect of intercalation of the 9-aminoacridine ligand. Super-
coiling in the plasmid DNA tertiary structure has been
observed before for other classic intercalators such as
ethydium bromide and planar heterocycles.6,25,38 The back-
grounds of the Figure 11c and d indicate 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. In complexes 1 and 4, a greater interaction on the
DNA is observed with some formation of agglomerates.
Figure 10. Electrophoretic mobility pattern of pBR322 plasmid DNA
incubated with 9-AA and their metal complexes: lane 1, pBR322; lane 2,
[Pd(dmba)(N10-9AA)(PPh3)]ClO4; lane 3, [Pt(dmba)(N9-9AA)(PPh3)]ClO4;
lane 4, [Pd(dmba)(N10-9-AA)Cl]; lane 5, [Pd(C6F5)(N10-9-AA)Cl(PPh3)];
lane 6, 9-AA; lane 7, CDDP.
When the pBR322 was incubated with a higher concentration
of complexes 2 and 3, a greater interaction on the DNA is
observed with the formation also of aggregates in balls.
Consequently, all of the new complexes modify the tertiary
structure of pBR322, which was also observed in electro-
phoretic pattern.
Cytotoxicity Studies. The in vitro growth inhibitory effect
of the new complexes 1-4 and cisplatin was evaluated in
the HL-60 human tumor cell line. As listed in Table 2, both
at 24 h and 72 h incubation times, all of the new complexes
were more active than cisplatin. Complexes 1 and 2 are
significantly more active than 3 and 4. A possible reason
might be the cationic nature of 1 and 2 that may enhance
(37) Ushay, H. M.; Tullius, T. D.; Lippard, S. J. Biochemistry 1981, 20,
3744–3748.
(38) Pope, L. H.; Davies, M. C.; Laughton, C. A.; Roberts, C. J.; Tendler,
S. J. B.; Williams, P. M. J. Microscopy 2000, 199, 68–78.
Inorganic Chemistry, Vol. 47, No. 15, 2008 6997

Ruiz et al.
Figure 11. TMAFM image corresponding to (a) pBR322, (b) pBR-complex 1, (c) pBR-complex 2, (d) pBR-complex 3, and (e) pBR-complex 4.
Table 2. IC50(µM) for Cisplatin and Complexes 1-4 for the Tumor
Table 3. Percentages of Vital Cells, Apoptotic Cells, Dead Cells, and
Damaged Cells after Treatment of a HL-60 Cell Population with
Cisplatin and Complexes 1-4 for 24 h
Cell Line HL-60
complex
24 h
72 h
treatment
(IC50 24 h, µM) cells (R1)
% vital
% apoptotic
cells (R2)
% dead
cells (R3)
% damaged
cells (R4)
cisplatin
15.6
0.52
0.91
5.92
6.04
2.1
1
2
3
4
0.45
0.85
4.64
4.68
control
91.02
60.93
75.42
66.73
77.13
70.47
4.81
33.06
19.32
29.34
14.06
20.30
3.96
4.94
4.89
3.50
7.63
8.77
0.21
1.06
0.37
0.43
1.17
0.45
CDDP (15.6)
1 (0.52)
2 (0.91)
3 (5.92)
4 (6.04)
their interaction with DNA (as shown in the electrophoretic
studies). In fact, at a short incubation time (24 h), complex
1 was about 30-fold more active than cisplatin toward HL-
60 cells.
For palladium complexes, the ligand is coordinated in its
amine form, bound to the metal atom in a monodentate
fashion by the endocyclic nitrogen atom, whereas for
platinum the acridine ligand adopts an imino-type configu-
ration with the proton of the exocyclic 9-amino group shifted
on N(10). In the platinum complex, an anagostic CH· · ·Pt
interaction is observed. All complexes are luminescent in
the solid state at room temperature, showing important
differences between the palladium and platinum complexes.
The platinum complex shows two structured emission bands
at high and low energies in the solid state, and the lifetimes
are in agreement with excited states of triplet parentage. DFT
and TD-DFT calculations for complex 2 have been done.
The prediction of T1 and T2 excited states using TD-DFT
calculations is in good agreement with those obtained in the
experimental results. Values of IC50 were also calculated for
the new complexes, 1-4, against the tumor cell line HL-60.
All of the new complexes were more active than cisplatin
(up to 30-fold in some cases). The DNA adduct formation
of the new complexes synthesized was followed by circular
dichroism and electrophoretic mobility. Atomic force mi-
croscopy images of the modifications caused by the com-
plexes on plasmid DNA pBR322 were also obtained.
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.39 The type of cell death
induced by the new complexes was investigated through an
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 3.
Conclusions
Four organometallic palladium(II) and platinum(II) com-
plexes with 9-aminoacridine have been prepared, and their
crystal structures have been established by X-ray diffraction.
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-
(39) Martin, S. J.; Green, D. R. Curr. Opin. Oncol. 1994, 6, 616–621.
6998 Inorganic Chemistry, Vol. 47, No. 15, 2008

Pd(II) and Pt(II) 9-Aminoacridine Complexes
50 thermobalance at a heating rate of 5 °C min-1 and the solid
CH(7) of 9-AA + C6H4 of dmba), 72.69 (CH2NMe2), 51.02 (NMe2).
31P{1H} NMR (acetone-d6) δ (H3PO4) 44.04 (s). ESI+ mass spectra
(DMSO): m/z +502.0 ([Pd(dmba)(PPh3)]+).
1
samples under nitrogen flow (100 mL min-1). The H, 13C, 31P,
19F, and 195Pt NMR spectra were recorded on a Bruker AC 300E
or a Bruker AV 400 spectrometer. Chemical shifts are cited relative
to SiMe4 (1H and 13C, external), CFCl3 (19F, external), 85% H3PO4
(31P, external), and Na2[PtCl6] (195Pt, external). Infrared spectra were
recorded on a Perkin-Elmer 1430 spectrophotometer using Nujol
mulls between polyethylene sheets. Electrospray ionization (ESI)
mass (+mode) analyses were performed on a LC-MS Agilent VL
system. Excitation and emission spectra were recorded with a Jobin-
Yvon Horiba Fluorolog 3-22 Tau-3 spectrofluorimeter. Phospho-
rescence lifetime was recorded with a Fluoromax phosphorimeter
accessory containing an UV xenon flash tube with a flash rate
between 0.05 and 25 Hz. The lifetime data were fitted using the
Jobin-Yvon software package and the Origin 6.1 program. Solvents
were dried by the usual methods.
Preparation of [Pt(dmba)(N9-9AA)(PPh3)]ClO4 ·acetone (2). To
a solution of [Pt(dmba)Cl(PPh3)] (100 mg, 0.16 mmol) in acetone
(20 mL) was added AgClO4 (33.4 mg, 0.16 mmol). AgCl im-
mediately formed. The resulting suspension was stirred for 12 h
and then filtered through a short pad of celite. To the filtrate was
then added 9-AA (31 mg, 0.16 mmol). The solution was stirred
for 24 h, and then hexane was added to precipitate a yellow solid,
which was collected by filtration and air-dried.
Data for Complex 2. Yield: 64%. Anal. calcd for C43H43-
N3ClO5PPt: C, 54.8; H, 4.6; N, 4.5. Found: C, 54.6; H, 4.7; N, 4.4.
Mp: 188 °C (dec). IR (Nujol, cm-1): 1710 [ν(>CdO), acetone]
1628, 1599 [ν(>CdN-)], 1094, 620 (ClO4), 547-500 (PPh3). 1H
NMR (acetone-d6): δ(SiMe4) 10.97 (d, 1H, H(1) of 9-AA, JH1H2
)
8.4 Hz), 10.64 (br, 1H, NH, H10), 9.16 (br, 1H, NH, H9), 7.99 (d,
1H, H(8) of 9-AA, JH8H7 ) 8.4 Hz), 7.70 (m, 1H, H(2) of 9-AA),
7.64 (m, 6H of PPh3), 7.41(m, 3H, H(3) + H(4) + H(5) of 9-AA),
7.31 (m, 3H of PPh3), 7.21 (m, 8H, H(7) of 9-AA + C6H4 of dmba
+ 6H of PPh3), 6.91 (false t, 1H, C6H4, JHH ≈ JHH ) 7.3 Hz), 6.59
(dd, 1H, C6H4, JHH ) 7.3 Hz, JHP ) 2.7 Hz, JHPt ) 43 Hz), 6.40
(false t, 1H, C6H4, JHH ≈ JHH ) 7.3 Hz), 4.47 (d, 1H, NCH2 of
dmba, JHH ) 13.6 Hz), 4.22 (dd, 1H, NCH2 of dmba, JHH ) 13.6
Hz, JHP ) 3 Hz), 3.08 (d, 3H, NMe2, JHP ) 2.4 Hz, JHPt ) 29 Hz),
2.91 (d, 3H, NMe2, JHP ) 3.3 Hz, JHPt ) 25 Hz), 2.08 (s, 6H,
Me2CO). 13C{1H} NMR (acetone-d6): δ(SiMe4) 139.04 (CH(1′) of
dmba), 135.32 (aromatic CH of PPh3), 133.91 (CH(2) of 9-AA),
133.16 (CH(6) of 9-AA), 131.42 (aromatic CH of PPh3), 128.40
(aromatic CH of PPh3), 127.0 (CH of 9-AA), 125.3 (CH(8) of
9-AA), 124.37 (CH of dmba), 122.19 (CH of dmba), 121.59 (CH(3)
of 9-AA), 117.62, 117.44 (CH(4) + CH(5) of 9-AA), 73.67
(CH2NMe2 of dmba), 51.41 (NMe2), 50.84 (NMe2), 29.5 (Me2CO).
31P NMR (acetone-d6): δ (H3PO4) 20.49 (s, JPPt ) 4214 Hz). 195Pt
NMR (acetone-d6): δ(Na2[PtCl6]) -3930.7 (d, JPtP ) 4214 Hz).
ESI+ mass spectra (DMSO): m/z +784.7 ([Pt(dmba)(9AA)-
(PPh3)]+); 590.0 ([Pt(dmba)(PPh3)]+ -1).
Materials
The starting complexes [M(dmba)Cl(PPh3)] (M ) Pd and Pt),40,41
[{Pd(dmba)(µ-Cl)}2],42 and [{Pd(C6F5)(PPh3)(µ-Cl)}2]43 were pre-
pared by procedures described elsewhere. The 9-AA was purchased
from Aldrich. The sodium salt of calf thymus DNA, EDTA
(ethylenediaminetetraacetic acid), and Tris-HCl (tris-(hydroxym-
ethyl)aminomethane hydrochloride) used in the CD study were
obtained from Sigma (Madrid, Spain), and pBR322 plasmid DNA
was obtained from Boehringer-Mannheim (Mannheim, Germany).
Caution! 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 [Pd(dmba)(N10-9AA)(PPh3)]ClO4 (1). To a
solution of [Pd(dmba)Cl(PPh3)] (100 mg, 0.19 mmol) in acetone
(20 mL) was added AgClO4 (39 mg, 0.19 mmol). AgCl immediately
formed. The resulting suspension was stirred for 12 h and then
filtered through a short pad of celite. To the filtrate was then added
9-AA (36 mg, 0.19 mmol). The solution was stirred for 24 h, and
then the solvent was partially evaporated under a vacuum and
hexane added to precipitate a yellow solid, which was collected
by filtration and air-dried.
Preparation of [Pd(dmba)(N10-9-AA)Cl] ·CHCl3 (3). To a solu-
tion of [Pd(dmba)(µ-Cl)]2 (100 mg, 0.18 mmol) in CHCl3 (20 mL)
was added 9-AA (70.34 mg, 0.36 mmol). The solution was stirred
for 24 h. The resulting suspension was filtered. A yellow solid was
collected by filtration and air-dried.
Data for Complex 1. Yield: 65%. Anal. calcd for C40H37-
N3ClO4PPd: C, 60.3; H, 4.7; N, 5.3. Found: C, 60.1; H, 4.9; N,
5.2. Mp: 234 °C (dec). IR (Nujol, cm-1): 1642, 1618, 1574
[ν(>CdN-) and bending δ(NH2)], 1102, 624 (ClO4), 539-492
(PPh3). 1H NMR (acetone-d6): δ(SiMe4) 9.41 (d, 2H, H(4) + H(5)
of 9-AA, JHH ) 8 Hz), 8.29 (d, 2H, H(1) + H(8) of 9-AA, JHH
8 Hz), 7.92 (br, 2H, NH2), 7.87 (m, 2H, H(3) + H(6) of 9-AA),
7.46 (br, 6H aromatic of PPh3), 7.38 (m, 2H, H(2) + H(7) of 9-AA),
Data for Complex 3. Yield: 90%. Anal. calcd for C23H23-
N3Cl4Pd: C, 46.9; H, 3.9; N, 7.1. Found: C, 47.6; H, 4.2; N, 7.2.
Mp: 194 °C (dec). IR (Nujol, cm-1): 3393, 3322, 3209 [ν(N-H)],
1637, 1609, 1566 [ν(>CdN-) and bending δ(NH2)], 358
)
1
[ν(Pd-Cl)]. H NMR (DMSO-d6): δ(SiMe4) 9.47 (d, 2H, H(4) +
7.34 (br, 3H aromatic of PPh3), 7.19 (d, 1H, C6H4 of dmba, JHH
)
H(5) of 9-AA, JHH ) 9 Hz), 8.55 (br, 2H, NH2),), 8.46 (d, 2H,
H(1) + H(8) of 9-AA, JHH ) 9 Hz), 8.29 (s, 1H, CHCl3), 7.81
(false t, 2H, H(3) + H(6) of 9-AA, JHH ≈ JHH ) 7.6 Hz), 7.41
(false t, 2H, H(2) + H(7) of 9-AA, JHH ≈ JHH ) 7.6 Hz), 6.91 (d,
7.2 Hz), 7.14 (br, 6H aromatic of PPh3), 6.94 (m, 1H, C6H4), 6.58
(false t, 1H, C6H4, JHH ≈ JHP ) 7.2 Hz), 6.46 (m, 1H, C6H4,), 4.42
(d, 2H, NCH2 of dmba, JHP ) 2.4 Hz), 2.60 (d, 6H, NMe2 of dmba,
JHP ) 2.4 Hz). 13C{1H} NMR (acetone-d6): δ(SiMe4) 139.35 (C6H4
of dmba), 132.94 (CH(3) + CH(6) of 9-AA), 131.40 (aromatic CH
of PPh3), 128.37 (d, aromatic CH of PPh3, JCP ) 10.6 Hz), 126.55
(CH(4) + CH(5) of 9-AA), 125.37 (C6H4 of dmba), 125.13 (C6H4
of dmba), 123.84 (CH(1) + CH(8) of 9-AA), 123.34 (CH(2) +
1H, C6H4 of dmba, JHH ) 7.2 Hz), 6.74 (false t, 1H, C6H4, JHH
≈
JHH ) 7.2 Hz), 6.30 (false t, 1H, C6H4, JHH ≈ JHH ) 7.2 Hz), 5.31
(d, 1H, C6H4 of dmba, JHH ) 7.2 Hz), 4.09 (s, 2H, NCH2 of dmba),
2.95 (s, 6H, NMe2 of dmba). 13C{1H} NMR (DMSO-d6): δ(SiMe4)
131.9 (CH of dmba), 131.4 (CH(3) + CH(6) of 9-AA), 130.4
(CH(4) + CH(5) of 9-AA), 124.5 (CH of dmba), 123.5 (CH of
dmba), 123.2 (CH(1) + CH(8) of 9-AA), 122.4 (CH(2) + CH(7)
of 9-AA), 121.2 (CH of dmba), 73.1 (CH2NMe2), 52.0 (NMe2 of
dmba). ESI+ mass spectra (DMSO): m/z +433.8 ([Pd(dm-
ba)(AA)]+); 710.6 ([Pd2(dmba)2(AA)Cl]+).
(40) Deeming, A. J.; Rothwell, I. P.; Hursthouse, M. B.; New, L. J. Chem.
Soc., Dalton Trans. 1978, 1490–1496.
(41) 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,
264–271.
(42) Cope, A. C.; Friedrich, E. C. J. Am. Chem. Soc. 1968, 90, 909–913.
(43) Uso´n, R.; Fornie´s, J.; Navarro, R.; Garc´ıa, M. P. Inorg. Chim. Acta
1979, 33, 69–75.
Preparation of [Pd(C6F5)(N10-9-AA)Cl(PPh3)] ·1/2CHCl3 (4). To
a solution of [Pd(C6F5)(PPh3)(µ-Cl)]2 (100 mg, 0.09 mmol) in
Inorganic Chemistry, Vol. 47, No. 15, 2008 6999

Ruiz et al.
Table 4. Crystal Structure Determination Details
param
1
2·acetone
3·DMF
4·acetone·toluene
empirical formula
fw
C40H37ClN3O4PPd
796.55
C43H43ClN3O5PPt
943.31
C25H29ClN4OPd
543.37
C47H39ClF5N2OPPd
915.62
cryst syst
space group
a (Å)
b (Å)
c (Å)
R (deg)
ꢀ (deg)
γ (deg)
V (Å3)
monoclinic
P21/c
18.3063(11)
9.5252(5)
19.8347(11)
90
94.184(2)
90
3449.4(3)
100(2)
monoclinic
P21/c
13.8181(6)
14.6014(6)
20.0898(8)
90
105.367(2)
90
3908.5(3)
100(2)
orthorhombic
Pbca
11.7887(6)
11.5472(6)
35.4910(19)
90
90
90
4831.3(4)
100(2)
8
triclinic
P1
j
11.6276(6)
12.4619(6)
14.5469(8)
79.679(2)
80.245(2)
83.291(2)
2035.64(18)
100(2)
temp (K)
Z
4
4
2
µ (mm-1
)
0.710
38866
3.749
42435
0.903
49656
0.623
22610
reflns collcd
ind reflns
8031
7984
4942
8270
data/restraints/params
R (int)
8031/44/519
0.0293
0.0410
7984/2/494
0.0285
0.0277
0.0615
0.771 and -0.528
4942/0/305
0.0270
0.0271
0.0638
0.569 and -0.500
8270/17/521
0.0191
0.0327
0.0815
0.727 and -0.638
2
R1 [I > 2σ(I)]a
wR2 (all data)b
largest diff. peak and hole (e·Å-3
0.0983
)
2.301 and -0.735
a R1 ) ∑|Fo| - |Fc|/∑|Fo|, wR2 ) [∑[w(Fo - Fc )2]/∑ w(Fo )2]0.5
constants set by the program.
.
w ) 1/[σ2 - (Fo ) + (aP)2 + bP], where P ) (2Fc + Fo )/3 and a and b are
b
2
2
2
2
2
CHCl3 (20 mL) was added 9-AA (34.96 mg, 0.18 mmol). The
resulting solution was stirred for 24 h. The resulting suspension
was filtered to precipitate a yellow solid, which was collected by
filtration and air-dried.
groups were refined using rigid groups, and the other hydrogens
were refined using a riding mode.
Special Features. For compound 1, the oxygen atoms of the
perchlorate anions are disordered over two positions, 64:36%. The
acridine ligand is disordered over two positions, 71:29%. The
hydrogen atoms of NH2 from the minor positions of the acridine
were not included in the refinement.
For compound 2·acetone, the acetone of solvation is disordered
over two positions, ca. 53:47%. Its hydrogen atoms were not
included in the refinement.
For compound 4·acetone ·toluene, the C6F5 ligand is disordered
over two positions, ca. 53:47%. The acetone of solvation is
disordered over two positions, ca. 66:34%, and its hydrogen atoms
were not included in the refinement.
Data for Complex 4. Yield: 88%. Anal. calcd for C37H25N2-
ClF5PPd ·1/2CHCl3: C, 54.6; H, 3.1; N, 3.4. Found: C, 54.3; H, 3.3;
N, 3.4. Mp: 160 °C (dec). IR(Nujol, cm-1): 3479, 3323, 3242
[ν(N-H)], 1646, 1571 [ν(>CdN-) and bending δ(NH2)], 1059,
958 (C6F5), 783 (Pd-C6F5 str), 535-500 (PPh3), 298 ν(Pd-Cl).
1H NMR (DMSO-d6): δ(SiMe4) 9.42 (dd, 2H, H(4) + H(5) of
9-AA, JHH ) 8.4 Hz, JHP ) 2.7 Hz), 8.53 (brs, 2H, NH2), 8.41 (d,
2H, H(1) + H(8) of 9-AA, JHH ) 8.4 Hz), 8.30 (s, 0.5H, CHCl3),
7.96 (false t, 2H, H(3) +H(6), JHH ≈ JHH ) 7.6 Hz), 7.74 (m, 6H
of PPh3), 7.4 (m, 9H of PPh3), 7.43 (false t, 2H, H(2)+H(7) of
9-AA, JHH ≈ JHH ) 7.6 Hz). 19F NMR (DMSO-d6): δ(CFCl3)
-113.8 (m, 2Fo), -161.5 (t, 1Fp, Jmp ) 20 Hz), -162.6 (m, 2Fm).
31P NMR (DMSO-d6): δ(H3PO4) 28.86 (t, JPF ) 8 Hz). 13C{1H}
NMR (DMSO-d6): δ(SiMe4) 133.9 (d, aromatic CH of PPh3, JPF
) 10.3 Hz), 131.3 (CH(3) + CH(6) of 9-AA), 131.0 (aromatic CH
of PPh3), 128.4 (aromatic CH of PPh3), 128.3 (CH(4) + CH(5) of
9-AA), 123.3 (CH(1) + CH(8) of 9-AA), 122.4 (CH(2) + CH(7)
of 9-AA). ESI+ mass spectra (DMSO): m/z +729.0
Computational Details for DFT and TD-DFT Calculations.
The model system used in the theoretical studies of cation
[Pt(Acridinium)(PPh3)(C6H4-CH2NMe2)]+ 2 was taken from the
X-ray diffraction data for complex 2·acetone. Keeping all distances,
angles, and dihedral angles frozen, single-point DFT calculations
were performed on both model systems. In both the ground-state
calculations and the subsequent calculations of the electronic
excitation spectra, the B3LYP functional45 as implemented in
TURBOMOLE46 was used. The excitation energies were obtained
at the density functional level by using the time-dependent
perturbation theory approach (TD-DFT),47–51 which is a DFT
generalization of the Hartree-Fock linear response or random-phase
([Pd(C6F5)(9AA)(PPh3)]+); 1005.6 ([Pd2(C6F5)2(PPh3)(9AA)]+
1).
+
X-Ray Crystal Structure Analysis. Suitable crystals of 1 and
2·acetone were grown from acetone/hexane. Crystals from 3·DMF
were grown from DMF evaporation. Crystals from 4·acetone ·toluene
were grown from acetone/toluene. The crystal and molecular structures
of the compounds 1, 2·acetone, 3·DMF, and 4·toluene ·acetone have
been determined by X-ray diffraction studies (Table 4).
The crystals were mounted onto the tip of a glass fiber, and the
data collection was performed with a Bruker Smart APEX diffrac-
tometer. Data were collected using monochromated Mo KR
radiation in the ꢀ scan mode. The structures of 1 and 3·DMF were
solved by direct methods and 2·acetone and 4·toluene ·acetone by
the heavy atom method. All were refined anisotropically on F2.44
Restraints to local aromatic ring symmetry or light atom displace-
ment factor components were applied in some cases. The NH’s
were refined as free and the NH2 as free with SADI; the methyl
(44) Sheldrick, G. M. SHELXL-97, A program for Crystal Structure
Refinement; University of Go¨ttingen: Go¨ttingen, Germany, 1997.
(45) (a) Becke, A. D. J. Chem. Phys. 1992, 96, 2155–2160. (b) Becke,
A. D. J. Chem. Phys. 1993, 98, 5648–5652. (c) Lee, C.; Yang, W.;
Parr, R. G. Phys. ReV. Lett. 1998, 37, 785.
(46) Ahlrichs, R.; Ba¨r, M.; Ha¨ser, M.; Horn, H.; Ko¨lmel, C. Chem. Phys.
Lett. 1989, 162, 165–169.
(47) Bauernschmitt, R.; Ahlrichs, R. Chem. Phys. Lett. 1996, 256, 454–
464.
(48) Bauernschmitt, R.; Ahlrichs, R. J. Chem. Phys. 1996, 104, 9047–9052.
(49) Bauernschmitt, R.; Ha¨ser, M.; Ahlrichs, R. Chem. Phys. Lett. 1997,
264, 573, and refs therein.
(50) Gross, E. K. U.; Kohn, W. AdV. Quantum Chem. 1990, 21, 255.
(51) Casida, M. E. Recent adVances in density functional methods; Chong,
D. P., Ed.; World Scientific: Singapore, 1995; Vol. 1.
7000 Inorganic Chemistry, Vol. 47, No. 15, 2008

Pd(II) and Pt(II) 9-Aminoacridine Complexes
approximation method52 In all calculations, the Karlsruhe split-
valence-quality basis sets53 augmented with polarization functions54
were used. The Stuttgart effective core potential in TURBOMOLE
was used for Pt.55
frequency 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 a high aspect ratio. In general,
the images were obtained at room temperature (T ) 23 ( 2 °C),
and the relative humidity 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). Cells were routinely maintained
in a RPMI-1640 medium supplemented with 10% (v/v) heat
inactivated fetal bovine serum, 2 mmol/L of glutamine, 100 U/mL
of penicillin, and 100 µg/mL of streptomycin (Life Technologies,
Inc.) in a highly humidified atmosphere of 95% air with 5% CO2
at 37 °C.
Cytotoxicity Assay. The growth inhibitory effect of metal
complexes on the leukemia HL-60 cell line was measured by the
microculture tetrazolium [3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide, MTT] assay.56
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 cells/well.
Biological Assays
Circular Dichroism Study. Spectra were recorded at room
temperature on an Applied Photophysics Π*-180 spectrometer with
a 75 W xenon lamp using a computer for spectral substraction and
smooth reduction. The palladium and platinum samples (ri ) 0.1,
0.3, 0.5) were prepared by the addition of aliquots of each
compound, from stock solutions (1 mg/mL, 4% in DMSO), to a
solution of calf thymus DNA (Sigma) in a TE buffer (20 µg/mL)
and incubated for 24 h at 37 °C. As a blank was used a solution of
each compound in a TE buffer (50 mM NaCl, 10 mM Tris-HCl,
0.1 mM EDTA, pH 7.4). 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 ellipticity values are
given in millidegrees (mdeg).
Electrophoretic Mobility Study. pBR322 plasmid DNA of 0.25
µg/µL concentration was used for the experiments. A total of 4 µL
of charge maker was added to aliquot parts of 20 µL of the complex
DNA compound containing 0.7 µg DNA previously incubated at
37 °C for 24 h. The mixtures were electrophoretised in 1% agarose
gel in 1× TBE buffer (45 mM Tris-borate, 1 mM EDTA, pH 8.0)
for 5 h at 30 V. The gel was subsequently stained in the same
buffer containing ethidium bromide (1 µg/mL) for 20 min. The
DNA bands were visualized with a Typhoon 9410 Variable Mode
Imager (Amersham Biosciences).
Atomic Force Microscopy. Preparation of adducts DNA-
metal complexes. pBR322 DNA (25 µg/µL), previously heated at
60 °C, was incubated in an appropriate volume with the required
metal 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 (Schle-
icher & 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-Schoon-
maker 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) 5 times and
plunged 3 times each in ethanol (100%). The samples were blow-
dried with compressed argon over silica gel and then imaged in
the AFM.
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 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; the percent cell viability
versus 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.
In Vitro Apoptosis Assay. The induction of apoptosis in Vitro
by the metal complexes 1-4 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 six-well plates (5 × 105 cells/well) were exposed
to IC50 concentrations of cisplatin or complexes 1-4 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. This work was supported by the
Direccio´n General de Investigacio´n del Ministerio de Ciencia
y Tecnolog´ıa (Project CTQ2005-09231-C02-01/BQU and
BIO2007-6846-C02-01), Spain, and the Fundacio´n Se´neca
de la Comunidad Auto´noma de la Regio´n de Murcia (Project
00448/PI/04), Spain. We thank Dr. M. J. Prieto for AFM
facilities (Universitat de Barcelona) and Dr. Francisca Garc´ıa
(Cell Culture Facility) and Manuela Costa (Cytometry
Facility) for technical assistance.
Imaging by Atomic Force Microscopy. The samples were
imaged in a Nanoscope III Multimode AFM (Digital Instrumentals
Inc., Santa Barbara, CA) operating in the tapping mode in air at a
scan rate of 1-3 Hz. The AFM probe was a 125-mm-long
monocrystalline silicon cantilever with integrated conical-shaped
Si tips (Nanosensors GmbH Germany) with an average resonance
Supporting Information Available: X-ray crystallography data
in CIF format for complexes 1, 2 · acetone, 3 · DMF, and
4·acetone ·toluene. This material is available free of charge via the
Internet at http://pubs.acs.org.
(52) Olsen, J.; Jørgensen, P. Modern electronic structure theory; Yarkony,
D. R., Ed.; World Scientific: Singapore, 1995; Vol. 2.
IC800589M
(53) Scha¨fer, A.; Horn, H.; Ahlrichs, R. J. Chem. Phys. 1992, 97, 2571.
(54) Dunning, T. H., Jr J. Chem. Phys. 1994, 100, 5829.
(55) Andrae, D.; Haeussermann, U.; Dolg, M.; Stoll, H.; Preuss, H. Theor.
Chim. Acta 1990, 77, 123–141.
(56) Mosmann, T. J. Immunol. Methods 1983, 65, 55–63.
(57) Vermes, I.; Haanen, C.; Steffensnakken, H.; Reutelingsperger, C.
J. Immunol. Methods 1995, 184, 39–51.
Inorganic Chemistry, Vol. 47, No. 15, 2008 7001