Cyclometalated Complexes of Platinum and Palladium with N-(4-Chlorophenyl)-α-benzoylbenzylideneamine. in Vitro Cytostatic Activity, DNA Modification, and Interstrand Cross-Link Studies

Article abstract of DOI:10.1021/ic960050y

In the present paper we report the synthesis and structural IR and ¹H and ¹³C NMR characterization of four platinum(II) and palladium(II) cyclometalated complexes of the formula [Pd(4-ClC₆H₄N=C(COC₆H₅)C ₆H₄)X]₂, where X = OAc (2) or Cl (3), and [Pt(4-ClC₆H₄N=C(COC₆H₅)C ₆H₄)X]₂, where X = Cl (4) or OAc (5). The acetate-bridged compounds 2 and 5 have an open-book structure. The chloro-bridged compounds 3 and 4 have an unfolded structure. Complex 2 crystallizes in the centrosymmetric triclinic space group P1?, with Z = 2. Unit cell parameters are as follows: a = 11.765(3) ?,b = 12.862(4) ?, c = 15.022(5) ?, α = 84.08(2)°, β= 78.56(2)°, γ = 83.13(2)°. All these compounds were screened against MDA-MB 468 (breast carcinoma) and HL-60 (leukemic) tumor cells. The low IC₅₀ values of compound 5 (1.05 μM) and of compound 4 (1.58 μM) against MDA-MB 468 and of 0.82 μM (5) and 1.37 μM (4) against HL-60 relative to cis-DDP (2.67 and 2.07 μM, respectively) and other cyclometalated compounds suggest that the synthesized compounds may be regarded as having potential antitumor properties. The data suggest that the antiproliferative activity of compound 5 may correlate with its covalent binding to DNA and the induction of important modifications on the helix.

Full text of DOI:10.1021/ic960050y

Inorg. Chem. 1996, 35, 5181-5187
5181
Cyclometalated Complexes of Platinum and Palladium with
N-(4-Chlorophenyl)-r-benzoylbenzylideneamine. In Vitro Cytostatic Activity, DNA
Modification, and Interstrand Cross-Link Studies
Carmen Navarro-Ranninger,*,† Isabel Lo´pez-Solera,† V´ıctor M. Gonza´lez,‡ Jose´ M. Pe´rez,‡
Amparo Alvarez-Valde´s,† Avelino Mart´ın,§ Paul R. Raithby,§ Jose´ R. Masaguer,† and
Carlos Alonso*,‡
Departamento de Qu´ımica Inorga´nica and Centro de Biolog´ıa Molecular “Severo Ochoa”,
Facultad de Ciencias, Universidad Auto´noma de Madrid, 28049-Madrid, Spain, and Department of
Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K.
ReceiVed January 17, 1996X
1
In the present paper we report the synthesis and structural IR and H and 13C NMR characterization of four
platinum(II) and palladium(II) cyclometalated complexes of the formula [Pd(4-ClC6H4NdC(COC6H5)C6H4)X]2,
where X ) OAc (2) or Cl (3), and [Pt(4-ClC6H4NdC(COC6H5)C6H4)X]2, where X ) Cl (4) or OAc (5). The
acetate-bridged compounds 2 and 5 have an open-book structure. The chloro-bridged compounds 3 and 4 have
an unfolded structure. Complex 2 crystallizes in the centrosymmetric triclinic space group P1h, with Z ) 2. Unit
cell parameters are as follows: a ) 11.765(3) Å, b ) 12.862(4) Å, c ) 15.022(5) Å, R ) 84.08(2)°, â ) 78.56-
(2)°, γ ) 83.13(2)°. All these compounds were screened against MDA-MB 468 (breast carcinoma) and HL-60
(leukemic) tumor cells. The low IC50 values of compound 5 (1.05 µM) and of compound 4 (1.58 µM) against
MDA-MB 468 and of 0.82 µM (5) and 1.37 µM (4) against HL-60 relative to cis-DDP (2.67 and 2.07 µM,
respectively) and other cyclometalated compounds suggest that the synthesized compounds may be regarded as
having potential antitumor properties. The data suggest that the antiproliferative activity of compound 5 may
correlate with its covalent binding to DNA and the induction of important modifications on the helix.
I. Introduction
high antitumor activity, showing specific activity towards some
forms of cancer.9 (2) Different optical isomers of these
Since the detection of the antitumor activity of cis-dichlo-
rodiammineplatinum (cis-DDP), a wide range of analogous
compounds with the general formula ML2X2 have been syn-
thesized and characterized.1-5 By alteration of the nature of
the metal M, the kinetically inert ligands L, or the kinetically
labile ligands X, the kinetic, structural, and electronic properties
of the molecules can be “fine-tuned” to maximize the activity
of these complexes. Thus, the search for systems with antitumor
activity has been extended to a whole range of coordination
and organometallic compounds and even to compounds which
do not contain metals.6 Our interest in this area has been
focused on complexes analogous to cis-DDP, where the ligands
X and L have specific properties (where L is a derivative of
benzoylbenzylideneamine which allows cyclometalation reac-
tions and where X is either chlorine or acetate).7,8 The present
study is based on the following facts: (1) Pd and Pt cyclo-
metalated complexes with benzoylbenzylideneamines display
compounds show different levels of antitumor activity.10 (3)
The palladium and platinum cyclometalated complexes contain-
ing carboxylate with a folded structure show greater activity
than complexes containing chloride with an unfolded structure.9
(4) Different substituents on the ligand modify the reactivity of
the resulting complexes. (5) The palladium and platinum
complexes produce conformational changes in the double helix
of DNA.11 In order to perform a further analysis of the influence
of some of these factors on the biochemical properties of
cyclometalated complexes, we have synthesized new compounds
which contain activated ligands having a chloro substituent,
N-(4-chlorophenyl)-R-benzoylbenzylideneamine. The activity
of the newly synthesized compounds has been compared with
the activity of related Pd(II) and Pt(II) cyclometalated complexes
previously described,9 showing that the former exhibit higher
antiproliferative activity. Our studies suggest that the biochemi-
cal mode of action of compound 5 may be due to the formation
of monofunctional or bifunctional intrastrand adducts on DNA
and that it does not induce interstrand cross-linking.
* To whom correspondence should be addressed.
† Departamento de Qu´ımica Inorganica, Universidad Auto´noma de
Madrid.
‡ Centro de Biolog´ıa Molecular “Severo Ochoa”, Universidad Autono´ma
de Madrid.
II. Results and Discussion
§ University of Cambridge.
Synthesis and Characterization. The reaction of equimolar
amounts of Pd(OAc)2 with the ligand N-(4-chlorophenyl)-R-
benzoylbenzylideneamine (1) in HOAc resulted in the formation
of the acetate-bridged cyclometalated compound 2. The
X Abstract published in AdVance ACS Abstracts, August 1, 1996.
(1) Reedijk, J. Inorg. Chim. Acta 1992, 198, 873.
(2) Ko¨ft-Maier, P. Chem. ReV. 1987, 87, 1137.
(3) Hollis, S. L.; Amundsen, A. R.; Stern, E. W. J. Med. Chem. 1989,
32, 128.
(4) Farrell, N.; Hacker, M. P. J. Med. Chem. 1990, 33, 2179.
(5) Pasini, A.; Zunino, F. Angew. Chem. 1987, 26, 615.
(6) Keppler, B. K. New J. Chem. 1990, 14, 389.
(7) Garc´ıa-Ruano, J. L.; Lo´pez-Solera, I.; Masaguer, J. R.; Navarro-
Ranninger, C.; Rodr´ıguez, J. H. Organometallics 1992, 11, 3013.
(8) Navarro-Ranninger, C.; Lo´pez-Solera, I.; Alvarez-Valde´s, A.; Rod-
r´ıguez-Ramos, J. H.; Masaguer, J. R.; Garc´ıa-Ruano, J. L. Organo-
metallics 1993, 12, 4104.
(9) Navarro-Ranninger, C.; Lo´pez-Solera, I.; Pe´rez, J. M.; Rodr´ıguez, J.;
Garc´ıa-Ruano, J. L.; Raithby, P. R.; Masaguer, J. R.; Alonso, C. J.
Med. Chem. 1993, 36, 3795.
(10) Navarro-Ranninger, C.; Lo´pez-Solera, I.; Pe´rez, J. M.; Masaguer, J.
R.; Alonso, C. Appl. Organomet. Chem. 1993, 7, 57.
(11) Navarro-Ranninger, C.; Amo-Ochoa, P.; Pe´rez, J. M.; Gonza´lez, V.
M.; Masaguer, J. R.; Alonso, C. J. Inorg. Biochem. 1994, 53, 177.
S0020-1669(96)00050-X CCC: $12.00 © 1996 American Chemical Society

5182 Inorganic Chemistry, Vol. 35, No. 18, 1996
Navarro-Ranninger et al.
Scheme 1
Table 1. 1H NMR Spectral Parameters (δ, ppm)a for Ligand 1 and
posterior metathetical reaction of this compound with NaCl led
to the formation of the chloro-bridged compound 3, which can
also be obtained by reaction of the ligand with PdCl2 in MeOH.
The ortho-platinated compound 4 was obtained from [Pt(µ-Cl)2-
(η3-C4H7)2] using CHCl3 as solvent.12 The chloro-bridged
complex 4 was converted into the acetate-bridged form (com-
pound 5) by treatment with silver acetate in CHCl3 (Scheme
1). All the compounds are air stable. The acetate dimers,
compounds 2 and 5, are soluble in most organic solvents. The
chloro derivatives, compounds 3 and 4, are slightly soluble in
organic solvents such as CHCl3 but soluble in DMSO and DMF.
The microanalytical data of compounds 2, 3, 4, and 5 (see
Experimental Section) are consistent with the empirical formula
[M(4-ClC6H4NdC(COC6H5)C6H4)X] (M ) Pd, X ) OAc, 2;
M ) Pd, X ) Cl, 3; M ) Pt, X ) Cl, 4; M ) Pt, X ) OAc,
5).
Compounds 3 and 4 in DMSO-d6
10
9
11
8
Cl
6
M
5
N
2
4
O
14
15
Cl
1
3 (M ) Pd)
4 (M ) Pt)
7.81, m, 2H
7.52, m, 2H
7.71, m, 1H
H4,4′
H5,5′
H6
7.87, m, 2H 7.81, m, 2H
7.38, m, 2H 7.51, m, 2H
7.42, m, 1H 7.70, m, 1H
H8
7.75, m, 2H 6.90, d (7.3), 1H 7.01, dd (1.2,7.6), 1H
H9
H10
H11
7.28, m, 2H 7.03, t (7.3), 1H
7.43, m, 1H 7.18, m, 1H
7.84, m, 1H
7.35, dt (1.2, 7.6), 1H
7.13, dt (1.2, 7.6), 1H
8.31,b dd (1.2, 7.6), 1H
7.30-7.00, m
The IR analysis showed that the bands assigned to the ν-
(CdO) stretching frequencies remain unchanged in all the
compounds relative to the ligand. The presence of negative
shifts in the ν(CdN) stretching frequency and of a band
assignable to ν(MsN) about 450 cm-1 in all compounds
suggests that the ligand coordinates to the metal through the
nitrogen. The IR spectra of complexes 2 and 5 exhibit the
typical asymmetric and symmetric stretching modes of the
acetate groups with strong absorptions at 1584 and 1421 cm-1
for compound 2 and at 1584 and 1418 cm-1 for compound 5.
The IR spectrum of compound 3 exhibits, on the other hand,
two asymmetric ν(Pd-Cl) stretching absorptions at 319 and 295
cm-1. Also, compound 4 exhibits two ν(PtsCl) stretching
H14,14′ 6.38,c 2H
H15,15′ 7.06,c 2H
7.14,c 2H
7.28,c 2H
7.30-7.00, m
a Abbreviations: d, doublet; t, triplet; dd, double doublet; dt, double
triplet; m, multiplet. The numbers in parentheses correspond to J(1H-
1H) in Hz. b 3J(195Pt-1H) ) 39.1 Hz. c AA′BB′ system.
compounds, as suggested by the X-ray data of similar com-
plexes7 and by the X-ray diffraction data of compound 2 (see
1
below). In contrast, the H NMR spectra of complexes 3 and
4 show well-resolved patterns in the aromatic region, indicating
the unfolded structure of these complexes. The main difference
between the spectra of the isostructural compounds 3 and 4 is
the larger chemical shift observed for H11 (7.84 ppm in 3 and
8.31 ppm in 4). This suggests that the platinum atom has a
stronger deshielding effect than palladium on the ortho position.
The strong shielding effect at H8 must be a consequence of the
change in the spatial arrangement of the COC6H5 group of the
cyclometalated complexes, with respect to that exhibited by the
ligand. The signal corresponding to H11 in Pt compound 4
shows a splitting due to the coupling with the metal (3J(195Pt-
1H) ) 39.1 Hz).
absorptions at 328 and 324 cm-1
.
1
The H NMR data for ligand 1 and their ortho-palladated
derivatives compounds 3 and 4 are shown in Table 1. For
compounds 2 and 5, see the Experimental Section. The spectra
were assigned on the basis of chemical shifts and of spin-spin
coupling information and were confirmed by selective proton
decoupling. The trans arrangement of the ligands in the acetates
2 and 5 is inferred from the fact that the acetate-bridge methyl
groups appear as singlets at 1.80 and 1.95 ppm, respectively.
The presence of a broadened unresolved pattern in the aromatic
region can explain the open-book-shape structure of the
The 13C NMR parameters of ligand 1 and those of the
palladium and platinum compounds are shown in Table 2. The
spectra were assigned by heteronuclear 2D correlation spec-
troscopy,13 and the quaternary carbon atoms were assigned by
the heteronuclear NOE effect.14 The spectra of the palladium
(12) Pregosin, P. S.; Wombacher, F.; Albinati, A.; Linaza, F. J. Organomet.
Chem. 1991, 418, 249.

Cyclometalated Complexes of Pt and Pd
Inorganic Chemistry, Vol. 35, No. 18, 1996 5183
Table 2. 13C NMR Parameters (δ, ppm)a of the Ligand 1 and
Complexes 2-5
10
9
X
11
12
8
Cl
X
6
5
7
1
M
N
3
2
4
13
O14
18 CH3
15
O
O
17
16
Cl
2b
3c
4c
5b
carbon
1
(M ) Pd) (M ) Pd)
(M ) Pt)
(M ) Pt)
C1
C2
C3
C4,4′
C5,5′
C6
C7
C8
166.8
197.0
134.3
128.0
128.7
134.4
134.6
129.1
128.8
131.8
128.8
129.1
147.6
180.4
191.8
133.2
129.2
129.2
135.1
145.5
128.4
124.5
131.5
133.2
156.3
143.0
125.7
127.8
180.7
191.9
n.o. 133.0
186.4 (89.5)
191.5 (48.5)
182.3 (41.6)
192.0 (77.7)
131.9
129.1
129.1
135.0
144.9
127.9
126.2
129.4
129.6
135.9
145.5
129.0
124.8
131.6
136.0
155.1
143.4
126.1
128.1
133.0
129.5
129.4
136.0
Figure 1. Diagram of the compound 2. Thermal ellipsoids are at the
20% probability level.
144.3 (56.3)
130.3 (36.1)
124.7
134.2 (41.6)
133.8 (56.9)
147.2 (1095.9) 140.7 (1183.2)
143.1 (23.6)
126.7
C9
Table 3. Selected Bond Distances and Angles for Compound 2
C10
C11
C12
C13
C14,14′ 121.7
C15,15′ 128.0
C16
C17
C18
132.4
133.2
Distances (Å)
Pd(1)-N(1)
Pd(1)-C(11)
Pd(1)-O(1)
Pd(1)-O(3)
N(1)-C(17)
C(17)-C(16)
C(17)-C(18)
C(16)-C(15)
C(15)-C(14)
C(14)-C(13)
C(13)-C(12)
C(12)-C(11)
C(16)-C(11)
O(1)-C(1)
2.017(2)
1.939(3)
2.034(5)
2.123(5)
1.311(9)
1.44(1)
1.49(1)
1.39(1)
1.38(1)
1.36(1)
1.41(1)
1.40(1)
1.43(1)
1.271(9)
1.233(9)
1.51(1)
1.21(1)
1.47(1)
2.939(1)
Pd(2)-N(2)
Pd(2)-C(21)
Pd(2)-O(4)
Pd(2)-O(2)
N(2)-C(27)
C(27)-C(26)
C(27)-C(28)
C(26)-C(25)
C(25)-C(24)
C(24)-C(23)
C(23)-C(22)
C(22)-C(21)
C(26)-C(21)
O(4)-C(3)
2.021(6)
1.938(7)
2.030(5)
2.131(5)
1.297(8)
1.464(9)
1.52(1)
1.39(1)
1.37(1)
1.39(1)
1.38(1)
1.41(1)
1.40(1)
1.27(1)
1.242(8)
1.52(1)
1.202(9)
1.476(9)
143.1
123.1
129.1
n.o.
183.7
23.5
127.7
131.7
n.o. 132.8
n.o.
23.8
a The numbers in parentheses correspond to J(195Pt-13C) in Hz; n.o.
) not observed. b CDCl3. c DMSO-d6.
and platinum compounds are very similar, suggesting that they
have analogous structures. The most significant differences
between compounds 3 and 4 are observed in the C1 and C12
atoms directly involved in the cyclometalated ring. For the
platinum derivatives, the largest shielding is observed in the
C12 linked directly to the metal. These compounds exhibit a
larger deshielding for C1. Both effects must be attributed to
electronic perturbations due to the metal itself. On the other
hand, the almost identical chemical shifts for C8, C9, and C10
in both types of compounds suggest that the palladium and
platinum atoms have similar electronic back-bonding effects.
Crystal Structure of [Pd(4-ClC6H4NdC(COC6H5)C6H4)-
(µ-OAc)]2. The crystal structure of compound 2 is shown in
Figure 1 together with the atomic numbering scheme. Signifi-
cant bond distances and bond angles are listed in Table 3. The
structure consists of discrete molecules separated by van der
Waals distances without any element of symmetry. Each
palladium atom is bonded in a distorted-square-planar coordina-
tion, approaching tetrahedral geometry, to the nitrogen atom,
to the ortho carbon atom of the phenyl ring supporting the iminic
carbon, and to one of the oxygen atoms from each of the two
bridging acetates. The dihedral angles between planes N-Pd-C
and O-Pd-O for Pd(1) and Pd(2) are 6.9 and 5.4°, respectively.
The Pd-N and Pd-C bonds form the basis for a five-membered
chelate ring. The bond lengths of Pd(1)-C(11) ) 1.939(3) Å
and Pd(2)-C(21) ) 1.938(7) Å suggest the multiple-bond-like
character of the Pd-C linkages due to metal-to-ligand back-
bonding.8 This conclusion is supported by the NMR data. The
bond lengths of Pd(1)-N(1) ) 2.017(2) Å and of Pd(2)-N(2)
) 2.021(6) Å together with the Pd-C distances suggest an
electronic delocalization in the five-membered ring. The Pd-O
(Pd(1)-O(1) ) 2.034(5) Å and Pd(2)-O(4) ) 2.030(5) Å)
C(1)-O(2)
C(3)-O(3)
C(1)-C(112)
C(18)-O(5)
C(18)-C(19)
Pd(1)-Pd(2)
C(3)-C(212)
C(28)-O(6)
C(28)-C(29)
Angles (deg)
O(1)-Pt(1)-O(3)
O(1)-Pd(1)-O(3)
C(11)-Pd(1)-O(1)
N(1)-Pd(1)-O(3)
N(1)-Pd(1)-C(11)
Pd(1)-N(1)-C(17)
N(1)-C(17)-C(16)
87.4(7) O(4)-Pt(2)-O(2)
91.6(2) O(4)-Pd(2)-O(2)
92.3(3) C(21)-Pd(2)-O(4)
94.5(2) N(2)-Pd(2)-O(2)
81.4(3) N(2)-Pd(2)-C(21)
115.6(5) Pd(2)-N(2)-C(27)
114.6(6) N(2)-C(27)-C(26)
89.7(8)
89.7(2)
91.7(3)
97.3(2)
81.1(3)
114.8(4)
116.0(6)
C(17)-C(16)-C(11) 114.4(6) C(27)-C(26)-C(21) 112.5(6)
Pd(1)-C(11)-C(16) 113.7(5) Pd(2)-C(21)-C(26) 117.1(6)
bonds having nitrogen atoms in a trans position are significantly
shorter than those showing a trans relationship with respect to
the aromatic carbon (Pd(1)-O(3) ) 2.123(5) Å and Pd(2)-
O(2) ) 2.131(5) Å), as a consequence of the different trans
effects of both atoms.15 All distances and angles within the
acetate and the aromatic groups are normal. The Pd-Pd
distance of 2.939(1) Å is within the range observed for other
Pd complexes.16 The iminic and carbonyl groups retain an
almost orthogonal arrangement, as exhibited in the N-substituted
R-benzoylbenzylideneamines.17 The torsion angles N(1)-
C(17)-C(18)-O(5) and N(2)-C(27)-C(28)-O(6) are 101.4
and -68.2°, respectively. The angle between the PdOONC
planes is 36.4°. This angle is larger than that found in other
(15) Caygill, G. B.; Steel, P. J. J. Organomet. Chem. 1987, 327, 115.
(16) Vicente, J.; Chicote, M. T.; Mart´ın, J.; Artiago, M.; Solans, X.; Font-
Altaba, M.; Aguilo´, M. J. Chem. Soc., Dalton Trans. 1988, 141.
(17) Fonseca, I.; Mart´ınez-Carrera, S.; Garc´ıa-Blanco, S. Acta Crystallogr.
1979, B35, 2643; 1982, B38, 2735.
(13) Bax, A.; Morris, G. A. J. Magn. Reson. 1981, 42, 501.
(14) Sa´nchez-Farrando, F. Magn. Reson. Chem. 1985, 23, 185.

5184 Inorganic Chemistry, Vol. 35, No. 18, 1996
Navarro-Ranninger et al.
Table 4. IC50 Valuesa of the Cyclometalated Compounds 2-5 and
cis-DDP against MDA-MB 468 and HL-60 Human Cancer Cells
compd
MDA-MB 468
HL-60
cis-DDP
2.67 ( 0.13
6.75 ( 0.57
8.95 ( 0.42
1.58 ( 0.09
1.05 ( 0.08
2.07 ( 0.21
6.35 ( 0.70
8.37 ( 0.63
1.37 ( 0.08
0.82 ( 0.02
2
3
4
5
a
Values are given as (mean) ( (standard deviation).
cyclometalated compounds with acetate bridges (24.46 and
23.96°, where the ligands are benzothiazole and benzoxazole,
respectively).18
Antiproliferative Analysis. Table 4 shows the IC50 values
(µM) of the cyclometalated compounds 2-5 and cis-DDP
against MDA-MB 468 and HL-60 human cancer cells. The
data show that there are not significative differences in the
antiproliferative activity of all compounds against either the HL-
60 or the MDA-MB 468 cell lines. It was observed, however,
that the antiproliferative activity against both types of tumor
cells of compounds 4 and 5, which contain Pt in their molecular
structures, is higher than that of compounds 2 and 3, which
contain Pd. In fact, compounds 2 and 3 exhibit IC50 values
higher than those of cis-DDP. In contrast, the IC50 values
obtained for compound 5 (1.05 and 0.82 µM for MDA-MB 468
and HL-60, respectively) and for compound 4 (1.58 and 1.37
µM for MDA-MB 468 and HL-60, respectively) are lower than
those showed by cis-DDP19 (2.67 and 2.07 µM for MDA-MB
468 and HL-60, respectively). We think that the large anti-
proliferative activity shown by the platinated compounds,
particularly of compound 5, may be due to the fact that this
drug provokes extensive DNA conformational changes (see
below). These changes may lead to blockage of several
biological processes such as DNA replication and transcription,
as has been proposed.20
Figure 2. CD spectra of CT DNA incubated with compound 5: (s)
CT DNA; (‚‚‚) ri ) 0.01; (- - -) ri ) 0.10.
Table 5. CD Spectral Data for CT DNA Incubated with Compound
5
compd
ri
θmax
λmax
θmin
λmin
CT DNAb
5-DNA
5-DNA
9.38
11.31
2.12
276
282
292
-12.20
-8.39
244
244
0.01
0.10
a
b
θ in units of 103 deg cm2 dmol-1; λ in units of nm. DNA control.
Table 6. UV Data and ∆Tm Values of CT DNA Incubated with
Compound 5a
compd
ri
λmax
OD260 nm
H (%)
∆Tm
CT DNAb
5-DNA
5-DNA
258
257
257
0.500
0.516
0.596
0.01
0.10
3.2
19.2
-5.5
-4.0
a
b
λ in units of nm; ∆Tm in units of °C. DNA control.
DNA Metalation. In order to determine the amount of metal
bound to DNA, a total refraction X-ray fluorescence analysis
was performed. We observed that a DNA metalation plateau
was reached at 24 h of incubation with compounds 2-5 and
cis-DDP at ri ) 0.10 (ri ) metal/nucleotide molar ratio). It
was observed, moreover, that at 24 h of incubation the rb value
(metal covalently bound to DNA) of complex 5-DNA was 1
order of magnitude superior to that of complex 4-DNA ((76.58
( 0.28) × 10-3 and (6.58 ( 0.24) × 10-3, respectively), while
the rb value of cis-DDP-DNA complex is close to 0.1 ((99.60
( 7.80) × 10-3). The differences in rb between both complexes
may be a consequence of the differences in lability of the salient
groups (chloride or acetate). It is likely that the high antipro-
liferative activity of compounds 4 and 5 may not be exclusively
dependent on DNA metalation, since DNA metalation due to
compound 4 is significantly lower than that due to compound
5. The total X-ray fluorescence data indicated, on the other
hand, that the Pd compounds 2 and 3 do not interact with the
DNA through covalent interactions, because no Pd was detected
in any case. These data were confirmed by CD spectroscopy,
which showed that the CD spectra of EtOH-precipitated Pd
complexes were identical with the CD spectra of the native
DNA. In contrast, the CD spectra of EtOH-precipitated Pt
complexes were identical with those of the native complexes
(data not shown) as an indication of covalent binding of the
metal to DNA.
Circular Dichroism of the 5-DNA Complex. Since
compound 5 shows high antiproliferative activity and it binds
covalently to DNA, we have performed experiments to analyze
the type of modifications induced upon binding of this com-
pound to DNA. The CD spectra and the wavelengths at which
the maximum and minimum values of ellipticity appear in
control CT DNA and in CT DNA incubated at ri ) 0.01 and
0.10 are shown in Figure 2 and Table 5. It was observed that
the maximum difference in the ellipticity value of the positive
band relative to control CT DNA was obtained at ri ) 0.10
since from a value of 9.38 units in control CT DNA at 276 nm,
a value of 2.12 units was observed in compound 5-DNA at
292 nm. Moreover, the negative band was not observed, as a
further indication of the large distortion of the helix at this ri
value. At lower ri this distortion did not occur. There is an
isodichroic point at 255 nm, although at ri ) 0.10 the distortion
of the helix is quite drastic. The modification in the CD
spectrum induced by compound 5 suggests that a certain opening
and rotation of the bases in the DNA by this compound must
occur, since the CD spectral changes observed indicate relax-
ation of the original B-DNA form.
UV Behavior of the 5-DNA Complex. Table 6 shows that
cyclometalated compound 5 induces alteration of the DNA
secondary structure, since a hyperchromic effect at 260 nm and
variations in the value of λmax were detected. Compound 5
induces a higher hyperchromic effect at ri ) 0.10. Table 6 also
shows the Tm increase induced by compound 5 after 24 h of
complex formation at ri ) 0.01 and 0.10. It can be observed
that the compound induces a destabilizing effect on DNA higher
(18) Churchill, M. R.; Wassermann, M. J.; Young, G. J. Inorg. Chem. 1980,
19, 762.
(19) Farrell, N. J. Med. Chem. 1989, 32, 2240.
(20) Umapathy, P. Coord. Chem. ReV. 1989, 95, 129.

Cyclometalated Complexes of Pt and Pd
Inorganic Chemistry, Vol. 35, No. 18, 1996 5185
Figure 4. Pattern of DNA bands of linear pF18 plasmid DNA
incubated with compound 5 and cis-DDP at ri ) 0.10 for different
periods of time. (lanes 1 and 2) control; (lanes 3-6) cis-DDP; (lanes
7-12) compound 5. Legend: ds ) double strand, ss ) single strand;
Cn ) native DNA, Cd ) denatured DNA.
Figure 3. Changes in the electrophoretic mobility of the ccc (covalently
closed circular) and oc (open circular) forms of native pUC8 plasmid
DNA after incubation with compound 5: (lane 1) control; (lanes 2-5)
compound 5; (lanes 6 and 7) cis-DDP.
incubation with the drug, most of the DNA migrates as the
double-stranded form (lane 6) as an indication of interstrand
cross-link formation. A quantitative analysis of the autorra-
diographs indicated that after 3 and 6 h of incubation of the
DNA with cis-DDP about 20% and 100% of the DNA remained
in the double-stranded form. In contrast, compound 5 did not
induce interstrand cross-linking, since all the DNA remained
in the single-stranded form.
Conclusion. In view of these results we think that the
biochemical activity of compound 5 must be attributed to
covalent binding to DNA, forming mono- or bifunctional Pt-
DNA adducts, which in turn induces important modifications
of the helix. We suggest that compound 5 induces intrastrand
adducts in way similar to that for cis-DDP.
than that induced by cis-DDP.21 In fact, while the Tm value of
native DNA is 62 °C, the Tm value of compound 5-DNA
formed at ri ) 0.01 decreases to 56.5 °C (∆Tm ) -5.5 °C).
The Tm value increases to 58 °C (∆Tm ) -4 °C) at ri ) 0.10.
It is interesting to note that when ri increases from 0.01 to 0.10
in complex 5-DNA, a Tm increment of 1.5 °C is observed, in
contrast with what was detected for cis-DDP. The decrease in
Tm of the DNA due to compound 5 binding probably results
from intrastrand adduct formation in a similar way as for cis-
DDP, since it has been shown that these kinds of adducts are
responsible for DNA destabilization.22
Electrophoretic Behavior of Drug-DNA Complexes. The
analysis of the influence of compound 5 on the tertiary structure
of DNA was determined by its ability to alter the electrophoretic
mobility of the covalently closed circular (ccc) and open circular
(oc) forms of pUC8 plasmid DNA. Figure 3 shows the mobility
of native pUC8 plasmid DNA and of plasmid DNA incubated
with compound 5 at ri ) 0.01, 0.05, 0.10, and 0.25, and with
cis-DDP at ri ) 0.10 and 0.25. It was observed that as the ri
value increases, incubation of the DNA with compound 5
gradually induces a decrease in mobility of the ccc form and
an increase in mobility of the oc forms of pUC8 DNA. It is
likely that the decrease in mobility of the supercoiled DNA must
be attributed to unwinding of the superhelix. The increase in
mobility of the oc form might possibly be associated with a
DNA-shortening effect.23,24 The changes in mobility due to cis-
DDP binding are slightly higher than those due to compound
5. The lower level of DNA uncoiling relative to cis-DDP shown
by compound 5 may be explained either by less bending of the
double helix (less than 42°) due to intrastrand bifunctional
adducts or by a lower level of DNA binding and intrastrand
monofunctional DNA adducts imposed by the bulky cyclo-
metalated residues in compound 5.
III. Experimental Section
General Procedures. The infrared spectra were recordered in Nujol
mulls and KBr pellets in the 4000-200 cm-1 range using a Perkin-
Elmer Model 283 spectrophotometer. NMR spectra were recordered
on a Bruker WP-200-SY (200 MHz) spectrometer in CDCl3 with TMS
as internal standard and in DMSO-d6. The C, H, and N analyses were
carried out in a Perkin-Elmer 240B microanalyzer. All solvents were
purified, prior to use, by standard methods.25 Pd(OAc)2 and K2PtCl4
were purchased from Strem and Johnson Matthey, respectively. Ligand
1 (L; N-(4-chlorophenyl)-R-benzoylbenzylideneamine) was synthesized
as previously described.26
Synthesis of [PdL(µ-OAc)]2 (2). A mixture of equimolar amounts
of Pd(OAc)2 and L in HOAc was heated at 50 °C under nitrogen for
2 h. After removal of the solvent in vacuo the residue was extracted
with CH2Cl2. The elution with CH2Cl2-EtOH (1%) gave the desired
complex 2 (yield 56%). Anal. Calcd for C44H32N2O6Cl2Pd2: C, 54.56;
H, 3.31; N, 2.89. Found: C, 54.51; H, 3.32; N, 2.92. IR (νmax, cm-1):
CdO, 1672; CdN, 1598; CsOacet 1584, 1418; PdsN, 450; PdsC,
610. 1H NMR (CDCl3, δ (ppm)): 7.72-6.80 (m, 9H, aromatic
protons), 6.93 and 6.65 (AA′BB′, 4H), 1.80 (s, 3H, OAc).
Synthesis of [PdL(µ-Cl)]2 (3). Method A. To a solution of the
acetate-bridged dimer (compound 2) in acetone was added an equimolar
amount of NaCl. The solid obtained (compound 3) was filtered and
dried in vacuo (yield 78%).
Method B. A mixture of equimolar amounts of PdCl2 and of L in
MeOH was stirred for 2 days at 25 °C. The solid obtained (compound
3) was filtered and dried in vacuo (yield 40%). Anal. Calcd for C40-
H26N2O2Cl4Pd2: C, 52.14; H, 2.82; N, 3.04. Found: C, 52.09; H, 2.79;
N, 3.03. IR (νmax, cm-1): CdO, 1673; CdN, 1594; PdsN, 451; PdsC,
606; PdsCl, 319, 295.
Interstrand Cross-Link Formation. Since gel electrophore-
sis experiments suggested that compound 5 induces DNA
intrastrand drug-DNA adducts in a way similar to that of cis-
DDP, we further tested whether compound 5 is also able to
induce interstrand cross-links. Figure 4 shows the pattern of
DNA bands of linear pF18 plasmid DNA incubated with
compounds 5 and cis-DDP at ri ) 0.10 for different periods of
time. Figure 4 shows that native linear pF18 melted DNA
migrates as a band which corresponds to the single-stranded
DNA form (lane 2). As expected, upon cis-DDP binding there
is a gradual increase in the double stranded form. After 6 h of
Synthesis of [PtL(µ-Cl)]2 (4). To a solution of bis(µ-chloro)bis-
[(η3-2-methylallyl)platinum] in CHCl3 was added 2 equiv of L. This
mixture was refluxed until a precipitate was formed. The precipitate
was filtered, washed with CHCl3 and ether, and dried in vacuo (yield
62%). Anal. Calcd for C40H26N2O2Cl4Pt2: C, 43.72; H, 2.37; N, 2.55.
(21) Eastman, A. Biochemistry 1983, 22, 3927.
(22) Johnson, N. P.; Lapetoule, P.; Razaka, H.; Villani, G. In Biochemical
Mechanisms of Platinum Antitumor Drugs; McBrien, D. H. C., Slater,
T. F., Eds.; IRL Press: Oxford, U.K., 1986; pp 1-28.
(23) Cohen, G. L.; Lippard, S. J. Science 1979, 203, 1014.
(24) Hermans, T. S.; Teicher, B. A.; Chan, V.; Collins, L. S. Cancer Res.
1988, 48, 2335.
(25) Perrin, D. D.; Armarego, W. L. F.; Perrin, D. R. Purification of
Laboratory Chemicals, 2nd ed.; Pergamon Press: Oxford, U.K., 1980.
(26) Alcaide, B.; Leo´n-Santiago, M. A.; Pe´rez-Ossorio, R.; Plumet, J.;
Sierra, M. A.; De la Torre, M. Synthesis 1982, 989.

5186 Inorganic Chemistry, Vol. 35, No. 18, 1996
Navarro-Ranninger et al.
Table 7. Crystal Analysis Parameters of Compound 2
Biological Assays. Formation of Drug-DNA Complexes. Stock
solutions of each compound (1 mg/mL) were stored in the dark at room
temperature until use. Drug-DNA complex formation was ac-
complished by addition to CT DNA (calf thymus DNA, purchased from
Sigma) of aliquots of each of the compounds at different concentrations
in 0.02 SSPE (SSPE ) 180 mM NaCl, 10 mM NaH2PO4, 1 mM EDTA,
pH 7.0). The amount of drug added to the DNA solution was expressed
as ri (the input molar ratio of Pt or Pd to nucleotides). The mixture
was incubated at 37 °C for various periods of time, as indicated below.
Cytotoxicity Assays. MDA-MB 468 cells were grown in DMEM
medium without sodium pyruvate and with glucose (4.5 g/L), supple-
mented with 10% FCS (fetal calf serum), 10 µg/mL of insulin, and
1% antibiotic-antimycotic solution. The replication period of MDA-
MB 468 cells was 24 h at 37 °C under an atmosphere with 5% CO2,
reaching the logarithmic growth phase at 72 h. HL-60 cells were
cultured in RPMI 1640 medium suplemented with 10% FCS and 1%
antibiotic-antimycotic solution. The replication period of HL-60 cells
was 16 h, reaching the logarithmic growth phase at 48 h under an
atmosphere of 5% CO2. In order to calculate the concentration of the
compounds which produces 50% inhibition of cell growth (IC50), 200
µL aliquots of cell culture (2.5 × 105 cells/mL) were incubated with
the compound at concentrations between 0 and 100 µg/mL for 48 h
(HL60 cell line) or 72 h (MDA-MB 468 cell line). Cell density was
determined in control and drug-treated cultures using a Neubauer
haemocitometer. Data were obtained from survival curves in which
the percentage of cell survival was plotted against the logarithm of
drug concentration. All experiments were carried out in triplicate.
Quantitation of Pt/Pd Binding to DNA. A 20 µg/mL solution of
CT DNA in TE buffer (10 mM Tris‚HCl, pH 8.0, 0.1 mM EDTA) was
incubated at 37 °C with the drugs at ri ) 0.10. After 24 h of incubation,
the DNA was precipitated twice with 2.5 volumes of cold ethanol and
0.1 volume of 3 M NaAc, pH 4.8. The DNA was washed with 70%
ethanol and resuspended in 1 mL of TE buffer. The amount of DNA
in each sample was measured by UV spectrophotometry (Beckman Acta
cIII) at 260 nm. The percentage of metal covalently bound to DNA
was determined by total X-ray fluorescence (TXRF) using a Seifert
EXTRA-II apparatus. As controls, Pt and Pd determinations were also
made in native DNA and in DNA precipitated immediately after
addition of the compounds. The assays were made in triplicate.
Circular Dichroism Spectroscopy. The CD spectra were performed
in a 1 cm rectangular quartz cell in a JASCO J-600 spectropolarimeter
attached to a temperature programmer using a computer for spectral
subtraction and noise reduction. The CD analysis was done at 20 °C.
Each sample was scanned twice in a range of wavelengths between
220 and 310 nm. The generated CD spectra represent the mean of
three independent scans.
Ultraviolet Spectral Data. UV measurements of the drug-DNA
complexes (20 µg/mL of DNA, ri ) 0.01 and 0.10) were carried out at
25 °C by differential spectrophotometry in a Shimadzu PR-1 spectro-
photometer. The Tm values of native DNA and compound 4-DNA
and compound 5-DNA complexes (20 µg/mL) were recorded at 260
nm by differential spectrophotometry at an increase rate of 1 °C/min
from 45 to 95 °C in a Beckman Acta cIII spectrophotometer attached
to a temperature programmer. The maximum value of hyperchromicity
in control DNA at 95 °C was 33%. The data represent the mean values
of three independent determinations.
Gel Electrophoresis of Drug-pUC8 Complexes. pUC8 DNA
aliquots (50 µg/mL) were incubated with the drugs in a buffer solution
containing 50 mM NaCl, 10 mM Tris‚HCl (pH 7.4), and 0.1 mM EDTA
at several ri values. Incubations were performed in the dark at 37 °C.
Aliquots (20 µL) of the drug-DNA complexes containing 1 µg of DNA
were subjected to 1.5% agarose gel electrophoresis for 16 h at 25 V in
40 mM Tris‚acetate and 2 mM EDTA pH 8.0 buffer. The DNA was
stained with ethidium bromide (0.5 µg/mL). The gels were photo-
graphed with a MP-4 Polaroid camera using 665 Polaroid film and an
orange filter.
Crystal Data
formula
sym
unit cell determination
C44H32N2O6Cl2Pd2‚C4H10O
triclinic, P1h
least-squares fit from 25 rflns
unit cell dimens
a
b
c
11.765(3) Å
12.862(4) Å
15.022(5) Å
84.08(2)°
78.56(2)°
83.13(2)°
R
â
γ
packing
V, Z
2204(11) Å-3, 2
1.570 g cm-3, 1052
0.991 mm-1
Dc, F(000)
µ
Experimental Data
Stoe four-circle
Mo KR (λ ) 0.710 73 Å)
180 K
highly oriented graphite cryst
ω/θ
diffractometer used
radiation
temp
monochromator
scan type
no. of rflns
measd
6164
obsd
range of hkl
std rflns
4720 (F > 4.0σ(F))
-12 to +12, -13 to +13, -10 to +16
3 measd every 100 rflns, no variation
Solution and Refinement
Siemens SHELXTL PLUS (PC version)a
direct methods
system used
soln
refinement
full-matrix least squares
H atoms
riding model, fixed isotropic U
weighting scheme
final ∆Fo peaks (e Å-3
final R and Rw
scattering factors
anomalous dispersion
w-1 ) σ2(F) + 0.0050F2
)
1.37
0.063, 0.084
b
b
a SHELXTL PLUS, program version 4.0, Siemens Analytical Instru-
ments, Madison, WI, 1990. b International Tables of X-ray Crystal-
lography; Kynoch Press: Birmingham, U.K., 1974; Vol. IV, pp 99-
100, 149.
Found: C, 43.68; H, 2.33; N, 2.52. IR (νmax, cm-1): CdO, 1672;
CdN, 1595; PdsN, 453; PdsC, 616; PtsCl, 328, 324.
Synthesis of [PtL(µ-OAc)]2 (5). To a suspension of [PtL(µ-Cl)]2
in CHCl3 was added an equimolar amount of AgOAc. The whitish
solution changed to red in 10 min. Afterward, the solution was filtered
and concentrated. A deep red solid product precipitated after addition
of MeOH (yield 78%). Anal. Calcd for C44H32N2O6Cl2Pt2: C, 46.11;
H, 2.79; N, 2.44. Found: C, 46.07; H, 2.80; N, 2.42. IR (νmax, cm-1):
CdO, 1672; CdN, 1595; CsOacet, 1584, 1424; PdsN, 452; PdsC,
615 cm-1 1H NMR (CDCl3, δ (ppm)): 7.64-6.60 (m, 13H, aromatic
.
protons), 1.95 (s, 3H, OAc).
Structural Determination and Refinement of Compound 2.
Orange crystals were obtained after recrystallization of compound 2
by slow evaporation from a CH2Cl2-EtOH (1%) solution. Intensity
data were recorded on a Stoe four-circle diffractometer using graphite-
monochromated Mo KR (λ ) 0.710 73 Å) radiation. The data, the
details of the data collection, and the structural analysis are summarized
in Table 7. Three standard reflections were measured every 100
reflections. There was no evidence of crystal decomposition. The data
were corrected for absorption. They were averaged to give 4720 unique
observed reflections with F > 4σ(F). The structure was solved by
direct methods, and refinement, based on F2, was by full-matrix least-
squares techniques.27 All non-H atoms were refined anisotropically
until R ) 0.063. H atoms were placed in idealized positions and
allowed to ride on the relevant C atom; CsH ) 0.96 Å. A Et2O solvate
molecule was located.
Interstrand Cross-Link Assay. The pF18 plasmid DNA was
linearized by digestion in 150 mM NaCl with 10 units/µg of DNA of
Bam HI at 37 °C for 4 h. pF18 DNA was 3′-end labeled by incubation
with 2.5 µCi/µg of DNA of [R-32P]dCTP and 1.25 units/µg of DNA of
Klenow fragment of Escherichia coli DNA pol I for 30 min at room
temperature. The reaction was stopped by heating to 70 °C for 5 min.
(27) (a) Sheldrick, G. M. SHELXS86: Program for the Solution of Crystal
Structures; University of Go¨ttingen, Go¨ttingen, Germany, 1985. (b)
Sheldrick, G. M. SHELXL93: Program for the Refinement of Crystal
Structures; University of Go¨ttingen, Go¨ttingen, Germany, 1993.

Cyclometalated Complexes of Pt and Pd
Inorganic Chemistry, Vol. 35, No. 18, 1996 5187
Unincorporated radioactivity and proteins were removed by passing
the labeling reaction mixture through a Sephadex G-50 column.
Sonicated CT DNA was added to the eluted solution of labeled pF18
DNA to a final DNA concentration of 180 µg/mL. DNA at a
concentration of 90 µg/µL was incubated with the drug in Bam HI
buffer (150 mM NaCl) at ri ) 0.10. Aliquots (10 µL) were removed
after several periods of incubation (5 min, 15 min, 30 min, 1 h, 3 h,
and 6 h for compound 5 and 15 min, 1 h, 3 h, and 6 h for cis-DDP),
and the reaction was terminated by addition of an equal volume of
loading dye (90% formamide, 10 mM EDTA, 0.1% xylene cyanol,
and 0.1 bromophenol blue). DNA was melted for 10 min at 90 °C,
and samples were chilled on ice prior to loading onto 1% agarose gel.
Electrophoresis was carried out in agarose gel in TAE buffer at 20 V
for 16 h. The gel was dried and autoradiographed. The density of the
bands was determined in a Molecular Dynamics Computing Densito-
meter (Model 300A).
Acknowledgment. We thank the CICYT (Grant Nos. SAF
93-1122, HB-059, SAF 93-0140, and CAM 160/92) for financial
support. We thank Johnson-Matthey Ltd. for their generous
loan of K2PtCl4.
Supporting Information Available: Listings of atomic coordinates,
anisotropic thermal parameters for non-hydrogen atoms, positional and
isotropic thermal parameters for hydrogen atoms, and all bond distances
and angles for 2 (8 pages). Ordering information is given on any
current masthead page.
IC960050Y