New palladium and platinum complexes with bioactive 3,5-diacetyl-1,2,4- triazol bis(4-cyclohexyl thiosemicarbazone) ligand: Chemistry, antiproliferative activity and preliminary toxicity studies

Article abstract of DOI:10.1039/c2dt31199b

The preparation and characterization of the new 3,5-diacetyl-1,2,4-triazol bis(4-cyclohexyl thiosemicarbazone) ligand, H₅L¹, is described. Treatment of H₅L¹ with K₂PtCl ₄ gave the dinuclear complex [Pt(H₃L¹)] ₂, 1, but using MCl₂(PPh₃)₂ where M = Pd or Pt, mononuclear complexes 2 and 3, of general formula [M(H ₃L¹)PPh₃], were obtained. Subsequent reaction of the [Pd(H₃L¹)PPh₃] complex with PdCl ₂(PPh₃)₂ yielded a new dinuclear complex [(PPh₃)Pd(H₂L¹)PdCl], 4. All compounds have been characterized by elemental analysis and FAB⁺ spectrometry and by IR and NMR spectroscopy. The molecular structures of mononuclear complexes 2 and 3 and dinuclear complex 4 have been determined by X-ray crystallography. The new compounds synthesized have been evaluated for antiproliferative activity in vitro against NCI-H460, HepG2, MCF-7, A2780 and A2780cisR human cancer cell lines. The cytotoxicity data suggest that the H₅L¹ ligand and [Pt(H₃L¹)]₂, complex 1, may be endowed with important cytotoxic properties since they are capable of not only circumventing cisplatin resistance in A2780cisR but also exhibit antiproliferative activity in NCI-H460. The interactions of these compounds with calf thymus DNA were investigated by UV-vis absorption and a nephrotoxic study, in LLC-PK1 cells, has also been carried out.

Full text of DOI:10.1039/c2dt31199b

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PAPER
New palladium and platinum complexes with bioactive 3,5-diacetyl-1,2,4-
triazol bis(4-cyclohexyl thiosemicarbazone) ligand: chemistry,
antiproliferative activity and preliminary toxicity studies†
Ana I. Matesanz,a Josefina Perlesb and Pilar Souza*a
Received 4th June 2012, Accepted 21st August 2012
DOI: 10.1039/c2dt31199b
The preparation and characterization of the new 3,5-diacetyl-1,2,4-triazol bis(4-cyclohexyl
thiosemicarbazone) ligand, H5L1, is described. Treatment of H5L1 with K2PtCl4 gave the dinuclear
complex [Pt(H3L1)]2, 1, but using MCl2(PPh3)2 where M = Pd or Pt, mononuclear complexes 2 and 3, of
general formula [M(H3L1)PPh3], were obtained. Subsequent reaction of the [Pd(H3L1)PPh3] complex
with PdCl2(PPh3)2 yielded a new dinuclear complex [(PPh3)Pd(H2L1)PdCl], 4. All compounds have been
characterized by elemental analysis and FAB+ spectrometry and by IR and NMR spectroscopy. The
molecular structures of mononuclear complexes 2 and 3 and dinuclear complex 4 have been determined
by X-ray crystallography. The new compounds synthesized have been evaluated for antiproliferative
activity in vitro against NCI-H460, HepG2, MCF-7, A2780 and A2780cisR human cancer cell lines. The
cytotoxicity data suggest that the H5L1 ligand and [Pt(H3L1)]2, complex 1, may be endowed with
important cytotoxic properties since they are capable of not only circumventing cisplatin resistance in
A2780cisR but also exhibit antiproliferative activity in NCI-H460. The interactions of these compounds
with calf thymus DNAwere investigated by UV-vis absorption and a nephrotoxic study, in LLC-PK1
cells, has also been carried out.
Such polydentate ligands quite often are Schiff bases (or the
related reduced derivatives), obtained by condensation of appro-
Introduction
The synthesis of preorganized dinuclear transition metal com-
plexes is a field of research that has been rapidly growing for the
past few years. Scaffolds to support and control a bimetallic
arrangement may be provided by compartmental binucleating
ligands that host two metal ions in suitable proximity.1–5
Among the ligands that are capable of positioning two metal
centers in close proximity, aromatic five-membered heterocycles
containing two or three nitrogen atoms appear to be particularly
suitable candidates. 1,2,4-Triazole-based ligands with chelating
side arms in the 3- and 5-positions of the heterocycle are
especially valuable for this purpose since the 1,2,4-triazole has a
high tendency to span two metal ions, and the individual coordi-
nation spheres, as well as the metal–metal separations, can be
tuned by appropriate alterations of the appended chelate
substituents.6,7
priately designed and prepared formyl- (or keto-) and primary
amine precursors.8–11 Thiosemicarbazones (>CvN–NH–C(S)–
N<) are an important class of Schiff base ligands that are structu-
rally interesting, since both sulfur and nitrogen atoms may be
involved in the coordination, thus invoking the a or b character
of the metallic centre.12–15
In this line, we have been employing the diketone heterocycle
3,5-diacetyl-1,2,4-triazole in condensation reactions with a series
of thiosemicarbazides to obtain ligands containing two thiosemi-
carbazone moieties separated by the five-membered heterocycle
ring 1,2,4-triazol which have been shown to be versatile poly-
dentate ligands towards metal ions with square planar coordi-
nation geometry. Depending on the participation of the central
ring in the coordination (monodentate N1/N2 versus N1N2 brid-
ging mode) we could synthesize and characterize both dimers
[2 + 2] helicates and tetranuclear compounds probing the di-
nucleating tendency of these triazol/bis(thiosemicarbazone)
ligands (Fig. 1).16–19
aDepartamento de Química Inorgánica (Módulo 07), Facultad de
Ciencias, c/Francisco Tomás y Valiente, 7, Universidad Autónoma de
Madrid, 28049 Madrid, Spain. E-mail: pilar.souza@uam.es;
Fax: (+34) 914974833; Tel: (+34) 914975146
A factor of importance in the synthesis of compounds with
1,2,4-triazole is the ease of formation of the triazolate ion by
deprotonation. The 1,2,4-triazolate anion can bind metal ions
through different coordination modes, as is depicted in
Scheme 1.
bServicio Interdepartamental de Apoyo a la Investigación, Facultad de
Ciencias, c/Francisco Tomás y Valiente, 7, Universidad Autónoma de
Madrid, 28049 Madrid, Spain
†CCDC 879640, 879641 and 879642 for complexes 2, 3 and 4. For
crystallographic data in CIF or other electronic format see DOI:
10.1039/c2dt31199b
N1N4 Bridging is relatively rare compared with N1N2 brid-
ging, this is probably because when both hydrazine-type
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Fig. 1 Capped sticks representation of both dimer [2 + 2] helicate
(left) and tetranuclear (right) Pd(II) complexes derived from 3,5-diacetyl-
1,2,4-triazol bis(4-ethyl thiosemicarbazone).
Scheme 2 Structure of the 3,5-diacetyl-1,2,4-triazol bis(4-cyclohexyl
thiosemicarbazone) ligand and its metal complexes synthesized.
complexes based on 3,5-diacetyl-1,2,4-triazol bis(4-monosubsti-
tuted thiosemicarbazone) triphenylphosphine palladium(II) pre-
cursors. Herein, we report on the synthesis and characterization
of the new 3,5-diacetyl-1,2,4-triazol bis(4-cyclohexyl thiosemi-
carbazone) ligand together with the study of its complexation
potential for Pd(II) and Pt(II) ions (Scheme 2).
Scheme 1 Schematic representation of the 1,2,4-triazole geometry and
three types of coordination modes of the 1,2,4-triazolate anion.
nitrogen atoms are bound to metal ions, the N4 easily acts as a
hydrogen acceptor providing additional stability. In fact, in
addition to the ligand–metal interactions, hydrogen bonding
plays a highly important role in the geometry of the coordination
compounds of these ligands.20
On the other hand many α-N-heterocyclic thiosemicarbazones
have been reported to exert tumor-inhibiting effects and one of
the most promising 3-aminopyridine-2-carboxaldehyde thiosemi-
carbazones (Triapine®) has entered several phase I and II clini-
cal trials.21–23 Also, phosphines and phosphine metal containing
complexes are of current interest due to their potential use as
antitumor agents. Particularly, 1,2-bis(diphenylphosphino)ethane
and some of its analogues have been shown to have antitumor
activity against a wide range of tumors, and moreover, their
activity is enhanced upon coordination to metal ions, such as
gold(I).24
The cytotoxic activity of the new compounds synthesized and
cisplatin (assumed as the reference antitumor drug) against five
human cancer cell lines, NCI-H460 (non-small cell lung cancer),
HepG2 (hepatocellular carcinoma), MCF-7 (breast cancer),
A2780 and A2780cisR (epithelian ovarian cancer), has been
studied. The interaction of these compounds with calf thymus
DNA (CT-DNA) was also investigated. In addition toxicity
studies, on normal renal LLC-PK1 cells, have been carried out
as an attempt to provide an insight into the pharmacological
properties of these compounds.
The present work aims to contribute to the understanding of
the structural and cytotoxic characteristics of these compounds.
Results and discussion
Recently, investigations from our laboratory have led to the
development of the first mononuclear Pd(II) complexes derived
from 3,5-diacetyl-1,2,4-triazol bis(4-monosubstituted thiosemi-
carbazones) bearing triphenylphosphine as a coligand. Their
excellent antiproliferative activity was demonstrated by in vitro
experiments in various human cell lines derived from different
types of solid tumours.25
An X-ray structural study demonstrated that 3,5-diacetyl-
1,2,4-triazol bis(4-monosubstituted thiosemicarbazone) ligands
act as tridentate dianion binding Pd(II) ions via one Ntriazolic and
Niminic and S donor atoms from one thiosemicarbazone arm.
Therefore some of the ligand donor atoms are still free and thus
the presence of uncoordinated donor atoms and their relative dis-
positions in space suggested that such complexes might provide
a possibility to bind to a second metal ion.
Synthesis and spectroscopic characterization
A new multidentate ligand, H5L1, which possesses two NNS
coordination sites was synthesized as a platform for metal
arrangement. Complexation with [MCl4]2− ions (M = Pd or Pt)
resulted in the formation of dimers [2 + 2] helicates, in which
each triazol-bis(thiosemicarbazone) behaves as a dianionic
ligand with deprotonation of one hydrazinic (2NH) and the
triazol ring protons and [NNSS] donor set.
Since the cleavage of binuclear Pd(II) and Pt(II) complexes
with tertiary phosphines is well known26 we decided to examine
the behavior of the dimers [2 + 2] helicates synthesized. So, sub-
sequent treatment of both palladium and platinum dimers with
PPh3, in 1 : 2 molar ratios, yielded the expected mononuclear
complexes in which each metal atom is coordinated to a phos-
phine ligand. Interestingly, reaction of the H5L1 ligand with
MCl2(PPh3)2 salts (M = Pd or Pt) in toluene, in the presence of
Keeping in view the above observations, it was planned to
construct a series of dinuclear N1N4triazol-bridged metal
Dalton Trans.
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triethylamine base, in 1 : 1 molar ratios afforded the same mono-
nuclear complexes of stoichiometry [M(H3L1)PPh3].
3, the absence of any signals above 15 ppm, indicative of depro-
tonation of the triazole ring, together with the presence of only
one signal assigned to 2N hydrazinic hydrogens is consistent
with the asymmetric di-deprotonation typical of 3,5-diacetyl-
1,2,4-triazol bis(thiosemicarbazone) ligands. However both the
signals assigned to the triazolic proton and the hydrazinic
protons were absent in the spectrum of complex 4, thus confi-
rming the symmetric tri-deprotonation of the ligand.
Once the mononuclear complexes were obtained, the presence
of several potential uncoordinated donor atoms and their relative
dispositions in space suggested that such complexes might
provide a possibility to bind to a second metal ion. So, upon the
addition of MCl2(PPh3)2 to the [M(H3L1)PPh3] precursors, we
have achieved the synthesis of a new dinuclear complex, in
which the triazol/bis(thiosemicarbazone) ligands have been
found to coordinate two metal ions as a symmetric trianionic
hexadentate ligand with deprotonation of the two hydrazinic
(2NH) and the triazol ring protons. It may be pointed out here
that the two metal ions contain different coordination spheres
[NNSP] and [NNSCl].
31P NMR spectroscopy was used to further confirm complexa-
tion of the ligand. Palladium complexes 2 and 4 display a singlet
at 32.75 and 29.00 ppm respectively for the phosphorus atom of
the triphenylphosphine ligand, while the platinum derivative 3
displays typical resonance at 11.20 ppm.
The compounds described in this paper were characterized by
elemental analysis and FAB+ spectrometry and by IR, H NMR
1
Description of mononuclear crystal structures 2 and 3
and 31P NMR spectroscopy (the significant IR vibrational bands
1
and the H chemical shift values are listed in the Experimental
[Pd(H3L1)PPh3]·EtOH and [Pt(H3L1)PPh3]·EtOH were iso-
lated as neutral compounds. The most significant parameters for
these complexes are shown in Table 1.
The structures together with the atom labelling schemes are
shown in Fig. 2 and 3. Both compounds are isostructural, hence
displaying nearly identical cell parameters, crystallize in the tri-
section). The infrared spectral bands most useful for determining
the mode of coordination of the ligands are the ν(CvN) iminic
and ν(CvS) thioamide IV vibrations. These bands shift to lower
wavenumbers in the spectra of the complexes suggesting coordi-
nation of the imine nitrogen and sulfur atoms. In complexes 2, 3
and 4, the presence of the triphenylphosphine ligand is
confirmed by the existence of the characteristic bands around
3050 and 1097 cm−1 for ν(CH) and ν(P–C), with no significant
change when compared to the precursor PdCl2(PPh3)2.
ˉ
clinic P1 space group with Z = 2 and the asymmetric units
contain one molecule of the neutral complex and one ethanol
molecule (for compound 2 the H atoms of the EtOH molecule
were not located in the Fourier difference map). The metal ion
presents a square planar geometry the bis(thiosemicarbazone)
ligand being attached through the Ntriazolic, and the Niminic and S
atoms from one thiosemicarbazone arm. The fourth coordination
position is occupied by a P atom from the PPh3 coligand which
In the 1H NMR spectrum, the free ligand shows various
peaks at low fields: δ ∼ 15 ppm assigned to the triazolic proton,
δ ∼ 12 ppm assigned to the hydrazinic protons (2NH) and
δ ∼ 10 ppm assigned to the thioamidic protons (4NH). In the
dimeric complex 1 as well as in mononuclear complexes 2 and
is coordinated to Pd or Pt trans to Niminic
.
Table 1 Crystal data and structure refinement for 2, 3 and 4 complexes
2
3
4
Molecular formula
Formula weight
C40H46N9OPPdS2
870.35
C40H52N9OPPtS2
963.07
C45H62ClN9OPPd2S3
1120.44
Temperature (K)
Wavelength (Å)
100(2)
0.71073
100(2)
0.71073
100(2)
1.54178
Crystal system
Space group
Triclinic
P1
Triclinic
P1
Monoclinic
P2(1)/n
ˉ
ˉ
a (Å)
b (Å)
10.7402(7)
11.8911(7)
10.769(5)
12.054(5)
12.4247(4)
16.3391(5)
c (Å)
α (°)
17.4118(12)
81.039(4)
17.499(8)
81.09(2)
24.2489(8)
90
β (°)
89.542(4)
70.946(3)
2074.0(2)
89.09(2)
70.974(19)
2120.1(16)
92.702(2)
90
4917.3(3)
γ (°)
Volume (Å3)
Z
2
2
4
Density (calculated) (g cm−3
)
1.394
0.630
900
1.509
3.488
972
1.513
8.247
2300
Absorption coefficient (mm−1
F(000)
)
Crystal size (mm3)
Index ranges
0.15 × 0.10 × 0.05
−13 ≤ h ≤ 13, −14 ≤ k ≤ 14,
−21 ≤ l ≤ 21
49 659
8437 [R(int) = 0.0644]
8437/0/499
0.30 × 0.34 × 0.38
−13 ≤ h ≤ 13, −15 ≤ k ≤ 13,
−22 ≤ l ≤ 21
51 305
8787 [R(int) = 0.0555]
8787/0/485
0.20 × 0.15 × 0.13
−14 ≤ h≤ 14, −19 ≤ k ≤ 19,
−29 ≤ l ≤ 29
30 211
9122 [R(int) = 0.0340]
9122/11/569
Reflections collected
Independent reflections
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2σ(I)]
R indices (all data)
1.129
1.115
1.039
R1 = 0.0432, wR2 = 0.1129
R1 = 0.0652, wR2 = 0.1373
1.555 and −0.585
R1 = 0.0340, wR2 = 0.1123
R1 = 0.0462, wR2 = 0.1584
2.609 and −3.968
R1 = 0.0412, wR2 = 0.1105
R1 = 0.0459, wR2 = 0.1148
2.164 and −0.727
Largest diff. peak and hole (e Å−3
)
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Table 2 Selected bond distances (Å) and angles (°) for Pd(II)
complexes 2 and 4
2
4
S(1)–C(1)
S(2)–C(6)
C(1)–N(1)
C(1)–N(2)
C(2)–N(3)
C(3)–N(4)
C(3)–N(6)
C(4)–N(5)
C(4)–N(6)
C(5)–N(7)
C(6)–N(8)
C(6)–N(9)
C(7)–N(1)
N(2)–N(3)
N(4)–N(5)
N(7)–N(8)
Pd(1)–S(1)
Pd(1)–N(3)
Pd(1)–N(4)
Pd(1)–P(1)
1.774(4)
1.681(4)
1.338(5)
1.307(5)
1.280(5)
1.338(5)
1.333(5)
1.339(5)
1.354(5)
1.288(4)
1.354(5)
1.315(5)
1.456(5)
1.372(4)
1.349(4)
1.377(4)
2.2434(10)
2.026(3)
2.031(3)
2.2436(10)
1.781(4)
1.763(4)
1.329(6)
1.314(5)
1.302(5)
1.349(5)
1.341(5)
1.331(5)
1.373(5)
1.300(5)
1.312(5)
1.347(5)
1.475(6)
1.368(5)
1.344(5)
1.375(5)
2.2578(9)
2.032(3)
2.037(3)
2.2689(10)
Fig. 2 Molecular structure of complex 2, hydrogen atoms are omitted
for clarity.
N(3)–Pd(1)–N(4)
N(3)–Pd(1)–S(1)
N(3)–Pd(1)–P(1)
N(4)–Pd(1)–S(1)
N(4)–Pd(1)–P(1)
S(1)–Pd(1)–P(1)
N(6)–Pd(2)–N(7)
N(6)–Pd(2)–S(2)
N(6)–Pd(2)–Cl(1)
N(7)–Pd(2)–S(2)
N(7)–Pd(2)–Cl(1)
S(2)–Pd(2)–Cl(1)
79.72(12)
84.00(9)
178.09(10)
163.71(9)
101.19(9)
95.07(4)
79.29(13)
84.00(9)
171.40(9)
163.27(10)
97.56(10)
98.90(4)
81.16(12)
165.05(9)
104.92(9)
84.05(10)
172.98(10)
98.90(4)
Fig. 3 Molecular structure of complex 3, hydrogen atoms are omitted
for clarity.
The bis(thiosemicarbazone) ligand is in dianionic form
showing Z, E configuration, that is the coordinated thiosemicar-
bazone arm, involved in two five-membered (PdSCNN and
PdNCCN or PtSCNN and PtNCCN) chelate rings, with the
sulfur atom cis to the azomethine nitrogen atom, and the un-
coordinated thiosemicarbazone arm with the S atom trans to
the azomethine nitrogen atom. This arrangement is reinforced
by intramolecular hydrogen bonds between the 2NH of the
uncoordinated thiosemicarbazone arm and one triazole nitrogen
atom.
It is important to note that upon coordination, the deproto-
nated arm undergoes significant evolution from the thione to the
thiol form which is reflected in a C–S distance of 1.786(6) Å for
2 and 1.776(5) Å for 3 while the neutral thiosemicarbazone arm
presents a shorter C–S bond length [1.668(6) for 2 and 1.693(6) Å
for 3]. The C–N and N–N bond distances (Tables 2 and 3) are
intermediate between formal single and double bonds, pointing
to extensive delocalization over the entire 3,5-diacetyl-1,2,4-tri-
azole bis(thiosemicarbazone) skeleton, however metal coordi-
nation provokes an important shortening of the C–Nhydrazinic
distance in the deprotonated arm as compared to the undeproto-
nated arm.
Table 3 Selected bond distances (Å) and angles (°) for Pt(II) complex
3
S(1)–C(1)
S(2)–C(6)
C(1)–N(1)
C(1)–N(2)
C(2)–N(3)
C(3)–N(4)
C(3)–N(6)
C(4)–N(5)
C(4)–N(6)
C(5)–N(7)
C(6)–N(8)
C(6)–N(9)
C(7)–N(1)
N(2)–N(3)
N(4)–N(5)
N(7)–N(8)
Pt(1)–S(1)
Pt(1)–N(3)
Pt(1)–N(4)
Pt(1)–P(1)
1.775(6)
1.692(7)
1.352(6)
1.311(8)
1.288(8)
1.340(7)
1.330(7)
1.346(7)
1.370(7)
1.286(7)
1.367(7)
1.331(6)
1.453(8)
1.397(6)
1.355(5)
1.386(8)
2.259(2)
2.027(6)
2.037(4)
2.245(2)
N(3)–Pd(1)–N(4)
N(3)–Pd(1)–S(1)
N(3)–Pd(1)–P(1)
N(4)–Pd(1)–S(1)
N(4)–Pd(1)–P(1)
S(1)–Pd(1)–P(1)
79.3(1)
84.0(1)
171.4(1)
163.3(1)
97.6(1)
98.90(4)
Inspection of the angles formed between the metal ion (M =
Pd2+, Pt2+) and the coordinated atoms shows that the metal is
contained within a slightly distorted square-planar environment.
The distortion is caused by the restricted bite angle of the triden-
tate ligand as reflected in the N(3)–M(1)–N(4) and N(3)–M(1)–
S(1) angles (less than 90°). The angles S(1)–M(1)–P(1) and
P(1)–Pd(1)–N(4) are therefore greater than 90°.
Within each molecule, the bis(thiosemicarbazone)–metal
moiety is close to planar, this disposition appears to be favored
by the presence, at the not coordinated thiosemicarbazone arm,
of an intramolecular hydrogen bond between the hydrazinic
hydrogen and one triazolic nitrogen atom. So the supramolecular
Dalton Trans.
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association involves π–π stacking interactions between parallel
layers of molecules. It is suggested that ethanol solvent mo-
lecules link adjacent units of the complex via intermolecular
hydrogen bonds.
In compound 4 intramolecular hydrogen bonding interactions
are not observed and intermolecular hydrogen bonding is limited
to N(1)–H(1)⋯O(1) with a contact distance of 2.85 Å and <
(NHO) angle 62.1°.
Description of dinuclear crystal structure 4
Antiproliferative activity against tumor and normal cells
The molecular structure of the [(PPh3)Pd(H2L1)PdCl] complex,
which crystallized with one DMSO molecule and one pentane
solvent molecule in the monoclinic P2(1)/n space group,
together with the atom labelling scheme is shown in Fig. 4. The
most characteristic feature is the linear arrangement of the two
Pd(II) ions, which are bridged by the triply deprotonated ligand.
Crystallographic data are shown in Table 1 with selected bond
lengths and angles shown in Table 2.
Cisplatin, cis-diamminedichloridoplatinum(II), and its analogs
are among the most effective agents for the treatment of cancer.
However its restricted clinical utility, due to the frequent
development of drug resistance, the limited spectrum of tumors
against which these drugs are active and severe normal tissue
toxicity, has generated new areas of research, which mainly
focused on the search for new compounds with low toxicity and
improved therapeutic properties.28–32
The 3,5-diacetyl-1,2,4-triazol bis(4-cyclohexyl thiosemicarba-
zone) ligand behaves as a trianion bridging the two metal ions
through the two not adjacent nitrogen atoms of the central tri-
azole ring. So, the ligand coordinates to the Pd(1) center via:
triazolic nitrogen atom N(4), iminic nitrogen N(3) and sulfur
atom S(1); the coordination sphere is completed by the phos-
phorus atom from the PPh3 coligand. The other palladium atom,
Pd(2), is coordinated to the remaining triazolic nitrogen N(6),
iminic nitrogen N(7) and sulfur S(2) atoms of the ligand being
the fourth position occupied by a terminal chlorido ligand.
The geometry around the palladium atoms is square-planar
with slight distortions from ideal values according to the
observed trans bond angles N–Pd–S and N–Pd–P or N–Pd–Cl.
This may be attributed to the restricted bite angle of the triden-
tate moieties.
The Pd–N and Pd–S bond distances in the range 1.9947(3)–
2.151(3) Å and 2.291(10)–2.2578(9) Å, respectively, are com-
parable with those reported for Pd(II) thiosemicarbazone com-
plexes.27 The C–S bond lengths of 1.681(4) and 1.781(4) Å are
within the normal range of a C–S single bond, indicating that the
thiosemicarbazone moieties adopt the thiol tautomeric form.
The C–N and N–N bond distances are intermediate between
formal single and double bonds, pointing to extensive delocali-
zation over the entire 3,5-diacetyl-1,2,4-triazole bis(thiosemicar-
bazone) skeleton.
To assess the antitumor potential of the newly synthesized
compounds, its antiproliferative activity (in powder solid form)
was tested in vitro against a panel of human cancer cell lines
containing examples of lung (NCI-H460), breast (MCF-7), liver
(Hep-G2) and ovarian (A2780 and A2780cisR) cancers. For
comparison purposes the cytotoxicity of cisplatin was always
evaluated under the same experimental conditions (Table 4). Out
of an initial screening of the five compounds synthesized, two of
them presented relevant antiproliferative actions, with IC50
values in the low μM range. The remaining compounds show, at
100 μM concentration, very low cellular growth inhibition
(<50%) and therefore had no evaluable cytotoxicity (IC50
>
100 μM).
In particular, this study identified both the free ligand
H5L1 and the [Pt(H3L1)]2 dimer complex, 1, as having anti-
proliferative activity in the low-micromolar range, against
ovarian (A2780, cisplatin sensitive, and A2780cisR, cisplatin
resistant) and lung (NCI-H460) cancer cells. The ligand also
exhibits antiproliferative activity against breast (MCF-7) cancer
cells.
Of particular relevance are the values of the resistance factor,
RF (defined as IC50 in A2780cisR/IC50 in A2780), since both
compounds tested have a much better RF than cisplatin. This
observation may suggest that these compounds are able to cir-
cumvent cisplatin resistance and this is probably due to a differ-
ent mechanism of action. In fact, it has been demonstrated that
the main molecular target of α-(N)-heterocyclic thiosemicarba-
zones is the ribonucleotide reductase (RR), one of the most
complex enzymes in the cell, from a biological, structural and
regulatory point of view.33,34
Moreover, since one of the mechanisms of cisplatin induced
nephrotoxicity is the inactivation of some enzymes because of
the reaction between platinum ions and sulfur containing pro-
teins, the compounds reported here, bearing nitrogen and sulfur
atoms in their structure, could prevent the adverse reaction
described.35,36
In order to investigate this possible side effect, the compounds
investigated and cisplatin were subsequently tested, in vitro, in
the same μM range (1–100), on normal renal LLC-PK1 cells. As
is shown in Table 5, the H5L1 ligand exhibits very low cellular
growth inhibition (<50%), at the maximum concentration of
100 μM, and therefore had no evaluable cytotoxicity (IC50
>
Fig. 4 Molecular structure of complex 4, hydrogen atoms are omitted
for clarity.
100 μM). In addition, complex 1 was demonstrated to be much
This journal is © The Royal Society of Chemistry 2012
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Table 4 In vitro antiproliferative activity of H5L1, 1, and cisplatin, evaluated in several human cancer cell lines
IC50 a (μM)
RF
Compound
A2780
32
6.7 0.04
0.54 0.02
A2780cisR
NCI-H460
MCF-7
Hep-G2
IC50(A2780cisR)/IC50(A2780)
H5L1
1
Cisplatin
1
47
1
49
19
3
2
54
1
>100
>100
3.23 0.06
1.46
1.34
6.85
8.99 0.02
3.7 0.1
>100
8.75 0.35
3.98 0.2
a 50% inhibitory concentrations in the sulforhodamine B assay (48 h exposure for NCI-H460 and Hep-G2 and 96 h exposure for A2780, A2780cisR
and MCF-7). The compounds were tested in 1 : 99 DMSO–PBS solutions. Values are the means standard deviations obtained from at least three
independent experiments.
Table 5 In vitro antiproliferative activity of H5L1, 1, and cisplatin,
evaluated in normal LLC-PK1 renal cells
IC50
LLC-PK1
Compound
H5L1
1
Cisplatin
>100
33
7.03 0.08
1
less toxic (about 5-fold) to the LLC-PK1 cells than cisplatin by
comparing their IC50.
DNA binding studies
To study the binding affinity between DNA and the new com-
pounds H5L1 and 1, which have a significant effect against the
tested cell lines, UV absorption spectroscopy has been a very
useful technique because the typical B-form DNA gives rise to a
characteristic band at 260 nm in the UV region. Hyperchromism
(increase in absorption of the DNA band) and hypochromism
(decrease in absorption of the DNA band) are the spectral
changes typical of a compound interaction with the DNA helix.
Hypochromism results from contraction of the DNA helix as
well as changes in its conformation, while hyperchromism
results from damage to the DNA double helix structure.37
In UV experiments, the spectra of CT-DNA in the presence of
each compound have been recorded, by keeping constant
CT-DNA concentration in diverse [CT-DNA]/[compound] mixing
ratios (R = 0.5–1.5) and monitoring the change in the absorption
intensity of the typical CT-DNA spectral band at 260 nm.
In the absence of CT-DNA, both compounds displayed intense
absorptions bands in the UV region (λmax 320–350 nm) assigned
to the intraligand π → π* transition of aromatic chromophores
which are also sensitive to their interaction with CT-DNA.
For both compounds, the absorption spectra display an
increase in the intensity of the characteristic CT-DNA absor-
bance while increasing the compound concentration (Fig. 5)
where the percentage of hyperchromism observed [% hyperchro-
mism = (ADNA bound − ADNA free)/ ADNA bound] is about 38% for
the H5L1 ligand and 43% for complex 1.
These characteristics suggest noncovalent interactions such as
electrostatic and surface (major or minor groove) binding along
outside of the DNA helix. Intercalative DNA-binding has been
excluded on the basis of the absence of hypochromism; on the
other hand both the ligand and the platinum(II) complex show
Fig. 5 UV absorption spectra of CT-DNA in the absence and presence
of compounds H5L1 (top) and 1 (bottom). The data were collected for
[CT-DNA] = 1.25 × 10−4 M and mixing ratio R = 0.5–1.5 (from top to
bottom).
similar UV absorption profiles, therefore a covalent interaction
between Pt(II) and guanine N7 is unlikely.
Experimental
Measurements
Elemental analyses were performed on a LECO CHNS-932
microanalyzer. Fast atom bombardment (FAB) mass spectra
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were performed on a VG AutoSpec spectrometer (nitrobenzyl
alcohol matrix). Nuclear Magnetic Resonance (NMR) spectra
were recorded on a BRUKER AMX-300 spectrometer. All cited
physical measurements were obtained by the Servicio Interdepar-
tamental de Investigación (SIDI) of the Universidad Autónoma
de Madrid.
Infrared spectra (KBr pellets) were recorded on a Bomen–
Michelson spectrophotometer (4000–400 cm−1). Electronic
spectra were recorded on a Thermo Scientific Evolution 260 Bio
UV-visible spectrophotometer.
Yield (32%), mp 228 °C (decomposes). Elemental analysis
found, C, 29.70; H, 5.70; N, 15.90; S, 8.20; C40H62N18-
S4Pt2·16H2O requires C, 30.00; H, 5.90; N, 15.70; S, 8.00%.
MS (FAB+ with mNBA matrix) m/z 1313.4 for [{Pt(H3L1)}2 +
H]+. IR (KBr pellets): n/cm−1 3245 (w, 4NH), 2929, 2853
(s, CH), 1549 (s, CN). 1H NMR (300.14 MHz, DMSO-d6):
d (ppm) 12.30 (s, 2NH, 1H), 9.28–8.80, 8.50–7.90 (m, 4NH,
2H), 3.95 (s, CH-cyclohexyl, 2H), 2.37 (s, CH3-triazol, 6H),
1.90–1.75 (m, CH2 equatorial-cyclohexyl, 10H), 1.40–1.20 (m,
CH2 axial-cyclohexyl, 10H).
Attempts to grow crystals of [Pt(H3L1)]2 for X-ray analysis
were unsuccessful.
Materials
[Pd(H3L1)PPh3], 2. This complex was obtained by reaction of
PdCl2(PPh3)2 metallic salt (0.351 g, 0.5 mmol), prepared by a
previously described procedure,39 with the 3,5-diacetyl-1,2,4-
triazol bis(4-cyclohexylthiosemicarbazone) ligand (0.232 g,
0.5 mmol) in toluene, in the presence of Et3N, in 1 : 1 molar
ratios. The reaction mixture was stirred for 2 h at room tempera-
ture. The resulting orange solution was filtered and left to stand
at ambient temperature for two days. The yellow-orange micro-
crystalline solid formed was filtered, washed several times with
hot water, recrystallized from ethanol and finally dried in vacuo.
Yield (73%), mp 176 °C. Elemental analysis found, C, 55.30;
H, 5.60; N, 14.90; S, 7.55; C38H46N9S2PPd requires C, 55.00;
H, 5.55; N, 15.20; S, 7.70%. MS (FAB+ with mNBA matrix)
m/z 830.4 for [Pd(H3L1)PPh3 + H]+. IR (KBr pellets): n/cm−1
3213 (s, NH); 2927, 2851 (s, CH), 1523 (s, CN), 859 (w, CS-
thioamide IV band). 1H NMR (300.14 MHz, DMSO-d6): δ
Solvents were purified and dried according to standard pro-
cedures. Hydrazine hydrate, L-lactic acid, cyclohexyl isothiocya-
nate, palladium(II) chloride, potassium tetrachloridoplatinate(II)
and triphenylphosphine were commercially available.
Synthesis of compounds
3,5-Diacetyl-1,2,4-triazol bis(4-cyclohexylthiosemicarbazone)
ligand, H5L1. An ethanolic solution of hydrazine hydrate
(0.250 g, 5 mmol) was added dropwise with constant stirring to
an ethanolic solution of cyclohexyl isothiocyanate (0.706 g,
5 mmol). The reaction mixture was stirred for one more hour and
then the white product 4-cyclohexylthiosemicarbazide formed
was filtered, washed with cold ethanol and diethyl ether, dried
in vacuo and recrystallized from ethanol. A methanolic solution
of the 4-cyclohexylthiosemicarbazide (0.346 g, 2 mmol) was
then refluxed with 3,5-diacetyl-1,2,4-triazol (0.153 g, 1 mmol),
which was prepared as described in the bibliography,38 for 5 h
and then was left to stand to ambient temperature. The solution
was reduced to half volume and the pale yellow solid formed
was filtered, washed with cold ethanol and diethyl ether and
dried in vacuo.
2
4
(ppm) 12.70 (s, NH, 1H); 8.30–8.25 (d, NH, 2H), 8.05–7.80
(m, aromatic protons, 15H); 4.45 and 4.40 (s, CH-cyclohexyl,
1H), 2.90 and 2.75 (s, CH3-triazol, 3H), 1.90 (m, CH2 equator-
ial-cyclohexyl, 10H), 1.50 (m, CH2 axial-cyclohexyl, 10H). 31P
NMR (121.50 MHz, CDCl3): δ (ppm) 32.75 (PPh3).
Crystallization in ethanol allowed us to isolate single orange
crystals, which were studied by X-ray diffraction techniques.
Yield (55%), mp 205 °C. Elemental analysis found, C, 51.70;
H, 7.20; N, 26.95; S, 13.50; C20H33N9S2 requires C, 51.85;
H, 7.15; N, 27.20; S, 13.80%. MS (FAB+ with mNBA matrix)
m/z 464.3 (100%) for [C20H33N9S2 + H]+. IR (KBr pellets):
[Pt(H3L1)PPh3], 3. This complex was obtained by two differ-
ent routes.
Method 1: using the same procedure as for compound 2,
namely by reaction of PtCl2(PPh3)2 metallic salt (0.395 g,
0.5 mmol), prepared by a previously described procedure,40 with
the 3,5-diacetyl-1,2,4-triazol bis(4-cyclohexylthiosemicarba-
zone) ligand (0.232 g, 0.5 mmol) in toluene, in the presence of
Et3N, in 1 : 1 molar ratios. The reaction mixture was stirred for
2 h at room temperature. The resulting orange solution was
filtered and left to stand at ambient temperature for two days.
The yellow-orange microcrystalline solid formed was filtered,
washed several times with hot water, recrystallized from ethanol
and finally dried in vacuo.
4
n/cm−1 3323 (s, NH-triazol), 3226 and 3099 (w, 2NH and NH),
1547 (s, CN), 853 (w, CS-thioamide IV band). 1H NMR
(300.14 MHz, DMSO-d6): d (ppm) 15.29 (s, NH-triazol, 1H),
12.57 and 10.75 (s, 2NH, 1H), 8.37, 8.34 and 8.20, 8.17 (d,
4NH, 1H), 4.30 (s, CH-cyclohexyl, 2H), 2.38 and 2.37 (s,
CH3-triazol, 3H), 1.91 (m, CH2 equatorial-cyclohexyl, 10H),
1.35 (m, CH2 axial-cyclohexyl, 10H).
[M(H3L1)]2. Dinuclear [2
+ 2] dimer complexes were
obtained by reacting a methanol suspension of the 3,5-diacetyl-
1,2,4-triazol bis(4-cyclohexylthiosemicarbazone) ligand with a
methanolic solution of lithium tetrachloridopalladate(II) (0.140 g,
0.5 mmol) or a water solution of potassium tetrachloridoplatinate
(II) (0.207 g, 0.5 mmol). The reaction mixture was stirred for 5 h
at room temperature and the resulting orange solid obtained
filtered, washed with methanol and diethyl ether, and dried in
vacuo. The difficulty experienced in crystallizing the palladium
complex because of its lower solubility resulted in less satisfac-
tory analyses. For M = Pt the crude mass was crystallized from
ca. 20 mL of DMSO to give pure [Pt(H3L1)]2, 1.
Yield (70%), mp 187 °C. Elemental analysis found, C, 49.90;
H, 5.50; N, 13.60; S, 6.50; C38H46N9S2PPt requires C, 49.70; H,
5.00; N, 13.70; S, 7.00%. MS (FAB+ with mNBA matrix) m/z
918.5 for [Pt(H3L1)PPh3]+. IR (KBr pellets): n/cm−1 3248, 3181
(s, NH); 2924, 2848 (s, CH), 1513 (s, CN), 856 (w, CS-thio-
1
amide IV band). H NMR (300.14 MHz, DMSO-d6): δ (ppm)
2
4
12.20 (s, NH, 1H); 7.92–7.89 (d, NH, 2H), 7.60–7.45 (m, aro-
matic protons, 15H); 4.33–4.29, 4.10 (m, CH-cyclohexyl, 2H),
3.30 and 2.5 (s, CH3-triazol, 3H), 1.85, 1.70 (m, CH2 equatorial-
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Dalton Trans.

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cyclohexyl, 10H), 1.40–1.20 (m, CH2 axial-cyclohexyl, 10H).
31P NMR (121.50 MHz, CDCl3): δ (ppm) 11.20 (PPh3).
Method 2. Triphenylphosphine (0.007 g, 0.04 mmol) and tri-
ethylamine base (1 mL) were added to a suspension of com-
pound 1 (0.026 g, 0.02 mmol) in toluene (20 mL) to give a clear
solution, which was stirred at room temperature for 12 h. The
orange solid that precipitated was filtered, washed with hot water
and Et2O and dried in vacuo.
CO2 at 37 °C. The human cancer cells Hep-G2 were grown in an
EMEM medium supplemented with 10% FBS in an atmosphere
of 5% CO2 at 37 °C.
Cell proliferation was evaluated by the sulforhodamine B
assay. Cells were plated in 96-well sterile plates at a density of
1.5 × 104 (for NCI-H460), 1 × 104 (for MCF-7 and Hep-G2) or
4 × 103 (for A2780 and A2780cisR) cells per well with 100 μL
of medium and were then incubated for 24 h. After attachment to
the culture surface the cells were incubated with various concen-
trations of the compounds tested freshly dissolved in DMSO
(1 mg mL−1) and diluted in the culture medium (DMSO final
concentration 1%) for 48 h (for NCI-H460 and Hep-G2) or 96 h
(for A2780, A2780cisR and MCF-7). The cells were fixed by
adding 50 μL of 30% trichloroacetic acid (TCA) per well. The
plates were incubated at 4 °C for 1 h and then washed five times
with distilled water. The cellular material fixed with TCA was
stained with 0.4% sulforhodamine B dissolved in 1% acetic acid
for 10 min. Unbound dye was removed by rinsing with 0.1%
acetic acid. The protein-bound dye was extracted with 10 mM
unbuffered Tris base for determination of optical density (at
515 nm) in a Tecan Ultra Evolution spectrophotometer.
The normal cells (LLC-PK1) were grown in 199 medium sup-
plemented with 3% FBS and 1.5 g L−1 of sodium bicarbonate in
an atmosphere of 5% CO2 at 37 °C. Cell proliferation was evalu-
ated by the sulforhodamine B assay. Cells were plated in 96-well
sterile plates at a density of 1 × 104 cells per well with 100 μL of
medium and were then incubated for 24 h. After attachment to
the culture surface the cells were incubated with various concen-
trations of the compounds tested freshly dissolved in DMSO
(1 mg mL−1) and diluted in the culture medium (DMSO final
concentration 1%) for 48 h at 37 °C. The cells were fixed by
adding 50 μL of 30% TCA per well. The plates were incubated
at 4 °C for 1 h and then washed five times with distilled water.
The cellular material fixed with TCA was stained with 0.4%
sulforhodamine B dissolved in 1% acetic acid for 10 min.
Unbound dye was removed by rinsing with 0.1% acetic acid.
The protein-bound dye was extracted with 10 mM unbuffered
Tris base for determination of optical density (at 515 nm) in a
Tecan Ultra Evolution spectrophotometer.
Crystallization in ethanol allowed us to isolate single red crys-
tals, which were studied by X-ray diffraction techniques.
[(PPh3)Pd(H2L1)PdCl], 4. To a solution of complex 2 (0.15 g,
0.2 mmol) in toluene was added PdCl2(PPh3)2 metallic salt
(0.09 g, 0.02 mmol), in the presence of Et3N. The reaction
mixture was stirred for 2 h at room temperature. The resulting
orange solution was filtered and left to stand at ambient tempera-
ture for two days. The yellow-orange microcrystalline solid
formed was filtered, washed several times with hot water, recrys-
tallized from ethanol and finally dried in vacuo.
Yield (32%), mp 128 °C. Elemental analysis found, C, 59.10;
H, 5.30; N, 8.05; S, 4.10; C38H44ClN9S2PPd2·2PPh3 requires C,
59.40; H, 4.95; N, 8.45; S, 4.30%. MS (FAB+ with mNBA
matrix) m/z 936.3 for [(M − Cl) + H]+. IR (KBr pellets): n/cm−1
3213 (s, NH); 2927, 2851 (s, CH), 1523 (s, CN), 859 (w,
CS-thioamide IV band). 1H NMR (300.14l MHz, DMSO-d6):
δ (ppm) 12.70 (s, 2NH); 8.30–8.25 (d, 4NH), 8.05–7.80 (m, aro-
matic protons); 4.45, 4.40 (s, CH-cyclohexyl), 2.90 and 2.75 (s,
CH3-triazol), 1.90 (m, CH2 equatorial-cyclohexyl), 1.50 (m,
CH2 axial-cyclohexyl). 31P NMR (121.50 MHz, CDCl3): δ
(ppm) 29.00 (PPh3).
Single orange crystals, suitable for single crystal X-ray diffrac-
tion analysis, were grown by slow evaporation from a dimethyl-
sulfoxide–pentane (3 : 1) solution.
Crystallography
Data were collected on a Bruker X8 APEX II CCD (compounds
2, 3 and 4). Crystallographic data and selected interatomic dis-
tances and angles are listed in Table 1. For all compounds, the
software package SHELXTL was used for space group determi-
nation, structure solution, and refinement.41 The structures were
solved by direct methods, completed with difference Fourier
syntheses, and refined with anisotropic displacement parameters.
All of the compounds here presented crystallize with solvent
molecules: compound 2 contains a disordered ethanol molecule
occupying two alternative positions. Compound 3 contains an
ethanol molecule, and compound 4 crystallizes with one partially
disordered DMSO molecule and one n-pentane molecule.
The effects of complexes were expressed as corrected percen-
tage inhibition values according to the following equation:
% inhibition ¼ ½1 ꢀ ðT=CÞꢁ ꢂ 100
where T is the mean absorbance of the treated cells and C the
mean absorbance in the controls.
The inhibitory potential of compounds was measured by cal-
culating concentration–percentage inhibition curves, these curves
were adjusted to the following equation:
n
E ¼ Emax=½1 þ ðIC50=CÞ ꢁ
In vitro antiproliferative activity
The human cancer cells A2780, A2780cisR and NCI-H460 were
grown in an RPMI-1640 medium supplemented with 10% foetal
bovine serum (FBS) and 2 mM L-glutamine in an atmosphere of
5% CO2 at 37 °C. The human cancer cells MCF-7 were grown
in an EMEM medium with 2 mM L-glutamine and Earle’s BBS
(adjusted with 1.5 g L−1 NaHCO3, 0.1 mM nonessential amino
acids and 1 mM sodium pyruvate) and supplemented with 10%
FBS and 0.01 mg mL−1 bovine insulin in an atmosphere of 5%
where E is the percentage inhibition observed, Emax is the
maximal effect, IC50 is the concentration that inhibits 50% of
maximal growth, C is the concentration of compounds tested and
n is the slope of the semi-logarithmic dose–response sigmoid
curves. This non-linear fitting was performed using GraphPad
Prism 2.01, 1996 software.42
For comparison purposes, the antiproliferative activity of cis-
platin was evaluated under the same experimental conditions.
Dalton Trans.
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All compounds were tested in two independent studies with tri-
plicate points. These experiments were carried out at the Unidad
de Evaluación de Actividades Farmacológicas de Compuestos
Químicos (USEF), Universidad de Santiago de Compostela.
The studies reported here constitute a good example of how
small changes in molecular structure lead to profound differ-
ences in biological activity. In our opinion, the conjunction of
serendipity and rational design (probability) plays a significant
role in cancer drug discovery due to the existence of several very
specific factors to be considered.
Based on these results the H5L1 ligand and the platinum
dimer complex 1, reported here, represent a valuable lead in the
development of new anticancer chemotherapeutic agents capable
of improving antitumor activity and reducing nephrotoxicity.
DNA-binding experiments
CT-DNA stock solution was prepared by dissolving the lyophi-
lized sodium salt in Tris-buffer (NaCl 50 mM, Tris-HCl 5 mM,
pH was adjusted to 7.3 with NaOH 0.5 M) by stirring at 4 °C for
2 days. The CT-DNA solution was standardized spectrophoto-
metrically43 by using its known molar absorption coefficient at
260 nm (6600 M−1 cm−1). The ratio of UV absorbance at 260
and 280 nm, A260/A280, was ca. 1.9, indicating that the DNA was
sufficiently free of protein. Stock solutions were kept frozen
until the day of the experiment.
Acknowledgements
We are grateful to Ministerio de Economía y Competitividad,
Instituto de Salud Carlos III (PI080525 and PI1100659), of
Spain for financial support.
Concentrated stock solutions (5 × 10−3 M) of both the ligand
and complex 1 were prepared by dissolving each one of the com-
pounds in DMSO. From these stock solutions, for all experi-
ments the desired concentration of the compound was achieved
by dilution with Tris-buffer (NaCl 50 mM, Tris-HCl 5 mM, pH
was adjusted to 7.3 with NaOH 0.5 M) to give homogeneous
solutions with DMSO contents of less than 2.5%.
In order to prepare the adducts with double-stranded
CT-DNA, to a mixture of a fixed amount of CT-DNA (1.25 ×
10−4 M) in Tris buffer were added predetermined quantities of
the desired compound solution to achieve different R values (R =
[CT-DNA]/[compound]). The mixtures were incubated for
10 min at 37 °C, and their UV-vis absorption spectra were
recorded, at room temperature, in the wavelength interval of
240–400 nm.
Notes and references
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Conclusions
In summary, a new family of Pt(II) and Pd(II) compounds, 1–4,
containing the 3,5-diacetyl-3,5-triazol bis(4-cyclohexyl thiosemi-
carbazone) ligand, H5L1, as a platform for metal arrangement,
has been successfully prepared and characterized.
This study has identified both the free ligand H5L1 and the
Pt(II) dimer complex 1 as having promising antiproliferative
activity since they are capable of not only circumventing cispla-
tin resistance in A2780cisR cells but they also exhibit antiproli-
ferative activity against lung NCI-H460 cancer cells. The ligand
also exhibits antiproliferative activity against breast (MCF-7)
cancer cells. Moreover, it is important to note that these two
compounds exhibit, in vitro, very low renal toxicity with respect
to cisplatin.
Taking into account that these compounds are active in
A2780cisR cisplatin resistant cells and, moreover, both ligands
and platinum(II) complexes do efficiently interact with CT-DNA
by inducing structural changes on DNA secondary structure
different from those induced by cisplatin, it is most likely that
part of the biochemical mechanism of action of these bis(thiose-
micarbazone) compounds involves groove binding. The com-
pounds synthesized here are neutral, aromatic and hydrophobic
molecules and therefore should tend to accumulate within the
lypophilic DNA grooves.
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