Novel bis(thiosemicarbazones) of the 3,5-diacetyl-1,2,4-triazol series and their platinum(ii) complexes: chemistry, antiproliferative activity and preliminary nephrotoxicity studies

Article abstract of DOI:10.1039/c1dt10212e

The preparation and characterization of three novel ⁴N- monosubstituted bis(thiosemicarbazone) ligands of 3,5-diacetyl-1,2,4-triazol series and their dinuclear platinum complexes are described. The crystal and molecular structure of the [Pt(μ-H

Full text of DOI:10.1039/c1dt10212e

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PAPER
Novel bis(thiosemicarbazones) of the 3,5-diacetyl-1,2,4-triazol series and their
platinum(II) complexes: chemistry, antiproliferative activity and preliminary
nephrotoxicity studies†
Ana I. Matesanz,a Carolina Herna´ndez,a,b Ana Rodr´ıguezc and Pilar Souza*a
Received 8th February 2011, Accepted 18th March 2011
DOI: 10.1039/c1dt10212e
The preparation and characterization of three novel 4N-monosubstituted bis(thiosemicarbazone)
ligands of 3,5-diacetyl-1,2,4-triazol series and their dinuclear platinum complexes are described. The
crystal and molecular structure of the [Pt(m-H3L3)]2 complex derived of 3,5-diacetyl-1,2,4-triazol
bis(4N-p-tolylthiosemicarbazone), H5L3, has been resolved by single crystal X-ray diffraction. The
ligands coordinate, in an asymmetric dideprotonate form, to the platinum ions in a tridentate fashion
(NNS) and S-bridging bonding modes. Thus the molecular units of the platinum complexes are stacked
as dimers. The new compounds synthesized have been evaluated for antiproliferative activity in vitro
against NCI-H460, A2780 and A2780cisR human cancer cell lines. The cytotoxicity data suggest that
these compounds may be endowed with important antitumour properties since are capable of not only
circumventing cisplatin resistance in A2780cisR cells but also exhibit high antiproliferative activity in
human non-small cell lung cancer NCI-H460 cells. The interactions of these compounds with calf
thymus DNA was investigated by UV-vis absorption and a nephrotoxic study, in LLC-PK1 cells, has
also been carried out.
structure and mode of action differ from that of cisplatin.6–9 In
Introduction
this regard, and taking into account that one of the mechanisms
Although platinum metallo-drugs are among the most effective
inducing the nephrotoxicity is due to the inactivation of some
agents for the treatment of cancer its clinical utility is restricted
enzymes because of the reaction between platinum ions and sulfur
due to the frequent development of drug resistance, the limited
containing proteins, an active area of research focuses on the
spectrum of tumours against which these drugs are active and
synthesis of chelate platinum(II) complexes bearing nitrogen and
severe normal tissue toxicity, including nephrotoxicity, being
sulfur mixed donor atoms, which should prevent the adverse
reaction described.10,11
an important side effect which interferes with their therapeutic
efficiency.1–5
a-(N)-Heterocyclic thiosemicarbazones, (N)-TSCs, are typical
tridentate NNS chelate ligands and moreover some of them
have shown antineoplastic activity by themselves.12 It has been
demonstrated that the biochemical mechanism of action involves,
among others, ribonucleotide reductase (RR) inhibition and DNA
interaction by intercalation. Particularly, the 3-aminopyridine-2-
carboxaldehyde thiosemicarbazone (Triapine, Vion Pharmaceuti-
cals, New Haven, CT) is currently being screened for antitumour
effects using the National Cancer Institute panel of 60 tumour cell
lines and selected for Phase I and II clinical trials.13–15
These disadvantages have driven the development of improved
platinum-based anticancer drugs but, the great majority of
antitumour metal complexes synthesized are structural analogs
of cisplatin and it has been demonstrated that these compounds
having similar DNA-binding modes also exhibit similar biological
profiles.
Therefore, there is a considerable interest in the synthesis and
characterization of new structural types of compounds whose
aDepartamento de Qu´ımica Inorga´nica (Mo´dulo 07), Facultad de Ciencias,
c/Francisco Toma´s y Valiente n◦ 7, Universidad Auto´noma de Madrid,
28049, Madrid, Spain. E-mail: pilar.souza@uam.es; Fax: (+34)914974833;
Tel: (+34)914975156
For thiosemicarbazone/metal compounds, several mechanisms
of antitumour action have also been proposed. For example,
they a) could interact with DNA by formation of a metal-DNA
covalent bond, b) could interfere in the DNA synthesis by RR
inhibition, and c) could interact with DNA through non-covalent
interactions including H-bonding and intercalation.16
Recently, investigations from our laboratory have led to
the development of a series of 3,5-diacetyl-3,5-triazol bis(4N-
monosubstituted thiosemicarbazones).17 The in vitro antitu-
mour studies have shown that the 3,5-diacetyl-3,5-triazol
bDepartamento de Qu´ımica Inorga´nica, Orga´nica y Bioqu´ımica, Facultad de
Ciencias del Medio Ambiente, Avd. Carlos III s/n, Universidad de Castilla-
La Mancha, 45071, Toledo, Spain
cDepartamento de Qu´ımica Inorga´nica, Orga´nica y Bioqu´ımica, ETS
Ingenieros Industriales, Avd. Camilo Jose´ Cela n◦ 3, Universidad de Castilla-
La Mancha, 13071, Ciudad Real, Spain
† CCDC reference number 805583. For crystallographic data in CIF or
other electronic format see DOI: 10.1039/c1dt10212e
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bis(4N-phenylthiosemicarbazone) ligand exhibits important an-
tiproliferative activity in A2780 and A2780cisR human cancer
cell lines. These observations prompted us to synthesize more
analogous compounds by introducing a methyl substituent on
the phenyl ring in ortho, meta and para positions to modify
steric hindance and electronic effects of the 4N-substituent. Thus,
this work is aimed at describing the synthesis and chemical
characterization of the new 3,5-diacetyl-1,2,4-triazol bis(4N-
tolylthiosemicarbazone) ligands, namely H5L1 (4N-ortho-tolyl),
H5L2 (4N-meta-tolyl) and H5L3 (4N-para-tolyl) and their dinuclear
platinum(II) complexes [Pt(m-H3L1)]2, [Pt(m-H3L2)]2 and [Pt(m-
H3L3)]2, respectively (Scheme 1).
[Pt(m-H3L3)]2. Analytical and spectroscopic results are consistent
with the formation of the neutral non-conducting species.
The significant IR vibrational bands and the 1H chemical
shift values of the free ligands and their platinum(II) complexes
are listed in the experimental section and Scheme 1 shows the
numbered structure of the free ligands. Comparison of the IR
spectra of the complexes with that of the free ligands show the
expected differences given the dideprotonation and coordination
shown by the X-ray study of [Pt(m-H3L3)]2, thus, the n(NH-triazol)
disappears in the spectra of platinum complexes as consequence of
the deprotonation of the triazole ring. Regarding the typical bands
from the thioamide group,18 –NH–C(S)–, which are expected to
be affected differently by different modes of coordination after
complexation with metal ions, the band corresponding to n(C S)
disappears upon complexation indicating coordination via the
thioamide sulfur atom. On the other hand, coordination only
induces minor changes in n(C N).
4
In order to investigate the influence of N-substitution on the
antitumour activity, the new compounds synthesized have been
evaluated for antiproliferative activity in vitro against three human
cancer cell lines: NCI-H460 (non-small cell lung cancer), A2780
and A2780cisR (epithelian ovarian cancer). The interaction of
these compounds with calf thymus DNA (CT-DNA) was also
investigated. In addition nephrotoxicity studies, on normal human
renal LLC-PK1 cells, have been carried out as an attempt to
provide an insight into the pharmacological properties of these
compounds.
1
In the H NMR spectra of the ligands, the triazolic proton
was observed as a singlet between 15.09 and 15.28 ppm and two
independent singlets at ~12.9 and ~11.3 ppm were observed for
2N-hydrazinic hydrogens suggesting the presence of the Z (sulfur
atom cis to the azomethine nitrogen atom) and E (sulfur atom
trans to the azomethine nitrogen atom) structural isomers in
solution in accordance with data reported in the literature for other
thiosemicarbazones.19 It may be surprising that these three signals
remain in the spectra of the dimeric dideprotonated complexes,
however this fact can be explained taking in account that the
complexes are dimeric and contain four thiosemicarbazone arms
which can exist, in solution, in various tautomeric forms (asym-
metric dideprotonation, or symmetric deprotonation) forming
Results and discussion
Synthesis and spectroscopic characterization
4
Three new N-monosubstituted bis(thiosemicarbazone) ligands
have been synthesized with high purities and acceptable yields.
The compounds, obtained as ethanol solvates, are yellow powders,
stable to air and moisture and were characterized by elemental
analysis, FAB+ spectrometry and IR and 1H NMR spectroscopy.
3,5-diacetyl-1,2,4-triazol bis(4N-tolylthiosemicarbazone) lig-
ands react with K2PtCl4 to yield, stable to air and moisture,
orange platinum(II) complexes [Pt(m-H3L1)]2, [Pt(m-H3L2)]2 and
1
both intra- and intermolecular hydrogen bonds. The H NMR
integration values of these signals in the complexes (0.5 : 0.5 : 1)
are in agreement with the loss of two protons upon complexation.
The rest of the proton signals appear, in the complexes, at nearly
identical positions if each one is compared with its corresponding
parent ligand.
Scheme 1 Structure of bis(thiosemicarbazone) ligands and their dinuclear platinum complexes.
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Table 1 Crystal data and structure refinement for [Pt(l-H3L3)]2 complex
Molecular formula
Formula weight
T/K
C44H46N18Pt2S4
1345.43
230(2)
˚
Wavelength (A)
0.71073
Crystal system
Triclinic
P1
14.654(4)
16.423(8)
17.538(8)
117.584(11)
97.032(17)
¯
Space group
˚
a/A
˚
b/A
˚
c/A
a/◦
b/◦
g /◦
105.311(18)
3457(2)
3
˚
Volume/A
Z
2
Density (calculated) (g cm-3)
Absorption coefficient (mm-1)
F(000)
1.292
4.201
1312
Crystal size/mm
0.20 ¥ 0.06 ¥ 0.05
-18 £ h £ 17, -16 £ k £ 20, -21 £ l £ 10
21429
13682 [R(int) = 0.0391]
13682/432/621
Index ranges
Reflections collected
Independent reflections
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2s(I)]
R indices (all data)
0.968
R1 = 0.0518, wR2 = 0.1398
R1 = 0.0775, wR2 = 0.1508
-3
˚
Largest diff. peak and hole, e A 0.924 and -0.677
Fig. 1 Molecular structure of [Pt(l-H3L3)]2 (hydrogen atoms are omitted
for clarity).
◦
3
˚
Table 2 Selected bond distances (A) and angles ( ) for [Pt(l-H3L )]2
complex
and sulfur atoms belonging to the deprotonated arm of one ligand
molecule, and the fourth position being occupied by a sulfur atom
of the non-deprotonated arm from the other ligand. Thus, the
deprotonated thiosemicarbazone arm behaves as a bidentate and
the neutral one behaves as a monodentate acting as a bridge.
The bond angle data indicates that the stereochemistry around
each platinum(II) ion is almost planar. The angles deviate slightly
from that expected for a regular square-planar geometry, this
distortion may be attributed to the restricted bite angle of the
tridentate moieties. The coordination results in the formation of
two five membered (PtSCNN and PtNCCN) chelate rings for
each platinum(II) ion, which are coplanar with the deprotonated
triazole ring.
S(1)–C(1)
1.792(10)
1.733(8)
1.807(10)
1.745(9)
1.315(11)
1.385(10)
1.433(11)
1.32(1)
1.387(8)
1.431(9)
2.282(3)
2.327(2)
2.323(2)
2.298(2)
2.034(7)
2.046(7)
2.023(7)
2.054(7)
S(1)–Pt(1)–S(4)
S(1)– Pt(1)–N(1)
S(1)–Pt(1)–N(4)
S(4)–Pt(1)–N(1)
S(4)–Pt(1)–N(4)
N(1)–Pt(1)–N(4)
S(2)–Pt(2)–S(3)
S(3)–Pt(2)–N(10)
S(3)–Pt(2)–N(13)
S(2)–Pt(2)–N(10)
S(2)–Pt(2)–N(13)
N(10)–Pt(2)–N(13)
95.60(9)
84.2(2)
164.0(2)
179.6(2)
100.4(2)
79.9(3)
96.30(9)
85.1(2)
164.6(2)
179.9(2)
99.0(2)
79.6(3)
S(2)–C(15)
S(3)–C(23)
S(4)–C(37)
C(1)–N(2)
C(1)–N(3)
C(2)–N(3)
C(23)–N(11)
N(1)–N(2)
N(10)–N(11)
Pt(1)–S(1)
Pt(1)–S(4)
Pt(2)–S(2)
Pt(2)–S(3)
Pt(1)–N(1)
Pt(1)–N(4)
Pt(2)–N(10)
Pt(2)–N(13)
˚
˚
The Pt–N [2.024–2.054 A] and Pt–S [2.282–2.327 A] bond
distances are comparable with those reported for Pt(II) thiosemi-
carbazone complexes.20–22 It is important to note that upon co-
ordination, the deprotonated arms undergo significant evolution
from the thione to the thiol form [C(1)–S(1) 1.792(10) and
These spectroscopic similarities suggest the same dimeric struc-
ture for the three platinum(II) complexes synthesized.
˚
C(23)–S(3) 1.807(10) A], while the neutral thiosemicarbazone
arms present shorter C–S bond lengths [C(37)–S(4) 1.755(9) and
Description of the crystal structure
˚
C(15)–S(2) 1.733(8) A]. The C–N and N–N bond distances are
Good quality crystals suitable for single crystal X-ray diffraction
analysis were obtained for [Pt(m-H3L3)]2 by recrystallization
in dimethylsulfoxide (DMSO). The molecular structure of the
platinum complex together with the atomic numbering scheme
is shown in Fig. 1. Crystallographic data is shown in Table 1 with
selected bond lengths and angles shown in Table 2.
intermediate between formal single and double bonds, pointing to
extensive delocalization over the entire 3,5-diacetyl-1,2,4-triazole
bis(thiosemicarbazone) skeleton.
Interestingly, the flexibility of the ligand originating from the
free rotation of the two thiosemicarbazone arms around the
C(9)–C(11), C(12)–C(13), and C(31)–C(34), C(33)–C(35) single
bonds, allows each ligand to ligate to two metal ions in a
twist conformation generating two parallel coordination planes.23
Particularly, between the two triazole moieties, the interplane sep-
This neutral platinum(II) dinuclear compound crystallizes in
¯
the triclinic P1 space group with Z = 2 and its crystallographic
analysis reveals unambiguously a dimeric structure which results
from the pairing of two mononuclear subunits through two
thiosemicarbazone moieties bridges.
Each Pt(II) center is four coordinated with a [NNSS] donor
environment, via: one triazolic nitrogen atom, the iminic nitrogen
˚
aration being 3.43 A is considered optimal for p–p interactions (in-
tramolecular stacking). This arrangement is reinforced by double
intramolecular hydrogen bonds between the 2NH of the bridging
thiosemicarbazone moieties and uncoordinated triazole nitrogen
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Table 3 In vitro antiproliferative activity of the bis(thiosemicarbazone) compounds and cisplatin, evaluated in human NCI-H460 (non-small lung cancer),
A2780 and A2780cisR (epithelian ovarian cancer) cell lines
A2780
A2780cisR
NCI-H460
%Inhibition(100 mM)
IC50(mM)
%Inhibition(100 mM)
IC50(mM)
15
5.0 0.05
3.8 0.4
RFa
%Inhibition(100 mM)
IC50(mM)
H5L1·EtOH
91
94
90
86
93
88
94
1
1
2
1
1
1
1
8.2 0.19
3.0 0.02
3.7 0.02
7.4 0.08
93
92
93
91
90
92
90
1
1
1
1
1
1
3
1
(1.8)
(1.7)
(1.0)
(2.0)
(2.2)
(1.9)
(5.9)
65
87
85
60
45
60
90
1
2
1
2
4
2
2
17
1
H5L2·EtOH
7.4 0.41
H5L3·EtOH
13
17
2
1
[Pt(l-H3L1)]2
[Pt(l-H3L2)]2
[Pt(l-H3L3)]2·2DMSO
Cisplatin
15
35
1
1
16
1
> 100
6.3 0.26
5.9 0.2
2.6 0.04
0.85 0.02
4.9 0.06
5 0.5
a RF, resistance factor (n-fold) in parentheses. The fold resistance equals the IC50 of the resistant cell divided by the IC50 of the parental cells.
Fig. 2 View of [Pt(l-H3L3)]2, along the bc plane, showing the parallel disposition of the molecules as a result of p–p stacking interactions.
atoms. The crystal lattice is further stabilized by intermolecular
A2780cisR, cisplatin resistant cell lines. Of particular relevance
are the values of the resistance factor, RF (defined as IC50 in
A2780cisR/IC50 in A2780) which indicate that the three ligands
are able to circumvent cisplatin resistance. The ligands also exhibit
antiproliferative activity in NCI-H460 cell line having IC50 values
in the low micromolar range.
From the chemical point of view, the position of the methyl
group on the tolyl ring does not appear to influence cytotoxicity
since the antiproliferative activity of the ligands [H5L1 (4N-
ortho-tolyl), H5L2 (4N-meta-tolyl) and H5L3 (4N-para-tolyl)] is
comparable in the three lines tested.
Upon complexation, the three resulting complexes [Pt(m-
H3L1)]2, [Pt(m-H3L2)]2 and [Pt(m-H3L3)]2 also show antiprolifera-
tive activity, in both A2780 and A2780cisR, in the low micromolar
range. The RF values of 2.0, 2.2 and 1.9, lower than that of
cisplatin (5.9), indicate that the three complexes are able to
circumvent cisplatin resistance. In contrast, NCI-H460 cell line
p–p stacking interactions between successive thiosemicarbazone
˚
moieties (3.78 A) that give rise to the environment for the structure
that is represented in Fig. 2.
Antiproliferative activity in human NCI-H460, A2780 and
A2780cisR cell lines
To analyze the potential of the compounds as antitumour agents,
the new compounds synthesized were tested (in powder solid
form) for their antiproliferative activity in vitro against the
human cancer cell lines: NCI-H460 (non-small cell lung cancer),
A2780 and A2780cisR (ephileian ovarian cancer). For comparison
purposes the cytotoxicity of cisplatin was evaluated under the same
experimental conditions.
Table 3 shows that the three new ligands present important
antiproliferative activity in both A2780, cisplatin sensitive, and
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show distinctive sensitivity to the platinum complexes, thus [Pt(m-
H3L1)]2 and [Pt(m-H3L3)]2, with the methyl group in the ortho and
para positions of the phenyl ring respectively, were comparable
in activity to cisplatin while the meta-tolyl substituted, complex
[Pt(m-H3L2)]2, show minimal activity (inhibition of 45% at the
maximum concentration of 100 mM).
DNA Binding Studies
To study the binding affinity between DNA and the new ligands
and platinum(II) complexes, which have significant effect against
the tested cell lines, the 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 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.24
Fig. 3 UV absorption spectra of CT-DNA in absence (bottom) and
presence of H5L1·EtOH ligand. The data were collected for [CT-DNA] =
1 ¥ 10-4 M and mixing ratio R of 1, 2, 3, 4, 5, 7 and 10 (from top to bottom).
Table 5 In vitro antiproliferative activity of the bis(thiosemicarbazone)
compounds and cisplatin, evaluated in normal human LLC-PK1 renal
cells
In UV experiments, the spectra of CT-DNA in the presence
of each compound have been recorded, by keeping constant CT-
DNA concentrations in diverse [CT-DNA]/[compound] mixing
ratios (R = 1–10) 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 ligands and complexes
displayed intense absorptions bands in the UV region (lmax 295–
323 nm) assigned to intraligand p → p* transitions of aromatic
chromophores which are also sensitive to the interaction of the
compound with CT-DNA.
LLC-PK1
%Inhibition (100 mM)
IC50 (mM)
H5L1·EtOH
54
80
79
47
55
62
85
1
3
2
2
2
2
1
43
9.5 0.62
14
> 100
44
4
H5L2·EtOH
H5L3·EtOH
2
[Pt(l-H3L1)]2
[Pt(l-H3L2)]2
[Pt(l-H3L3)]2·2DMSO
Cisplatin
3
8.7 0.14
7.9 0.09
For all compounds, the absorption spectra displayed an increase
in the intensity of the characteristic CT-DNA absorbance while
increasing the compound concentration, and the percentage of
hyperchromism observed is given in Table 4. Only in the spectrum
of H5L1 ligand, the hyperchromism is accompanied by a red-shift
up to 275 nm. In addition, the absorption spectra of the three
ligands show an increase of the intensity of the ligand centered
transitions, and these changes are also more pronounced in the
case of H5L1 ligand for which the band at lmax 322 nm is split into
three bands at lmax 327, 241 and 357 nm (Fig. 3).
These characteristics suggest noncovalent interactions such as
electrostatic and surface (major or minor groove) binding along
the outside of the DNA helix. Intercalative DNA-binding has been
excluded on the basis of the absence of hypochromism; in the other
hand both ligands and platinum(II) complexes show similar UV
absorption profiles therefore a covalent interaction between Pt(II)
and guanine N7 is unlikely.
Preliminary nephrotoxicity studies
In order to investigate possible adverse side effects that may occur,
such as nephrotoxicity, the compounds were subsequently tested
(in powder solid form) in vitro on normal human renal LLC-
PK1 cells. For comparison purposes the toxicity of cisplatin was
evaluated under the same experimental conditions.
As is shown in Table 5, the [Pt(m-H3L1)]2 complex exhibited
very low cellular growth inhibition (<50%), at the maximum
concentration of 100 mM, and therefore did not have evaluable
cytotoxicity (IC50 > 100 mM). In addition, compounds H5L1 and
[Pt(m-H3L2)]2 were demonstrated to be much less toxic (over 5 fold)
to the LLC-PK1 cells than cisplatin by comparing their IC50.
The three remaining compounds H5L2, H5L3 and [Pt(m-H3L3)]2
havenephrotoxicprofilessimilartothatofcisplatinbutalsosimilar
cytotoxic profiles with the advantage of circumventing cisplatin
resistance in the A2780cisR cell line.
Table 4 Absorption spectral properties of bis(thiosemicarbazone) lig-
ands and their platinum(II) complexes bound to CT-DNA
Conclusion
The compounds investigated here are capable not only of circum-
venting cisplatin resistance in A2780cisR cells but also exhibit high
antiproliferative activity in human non-small cell lung cancer NCI-
H460 cells with the only exception of [Pt(m-H3L2)]2. Taking into
account that these compounds are active in A2780cisR cisplatin
resistant cells and moreover, both ligands and platinum(II) com-
plexes do efficiently interact with CT-DNA by inducing structural
changes on the DNA secondary structure different from those
induced by cisplatin, it is most likely that part of the biochemical
Increase in absorbance (%)a
H5L1·EtOH
65
55
60
50
70
60
H5L2·EtOH
H5L3·EtOH
[Pt(l-H3L1)]2
[Pt(l-H3L2)]2
[Pt(l-H3L3)]2·2DMSO
a % hyperchromism = (ADNA bound - ADNA free)/ADNA bound
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mechanism of action of these bis(thiosemicarbazone) compounds
involves groove binding. The compounds synthesized here are
neutral, aromatic and hydrophobic molecules and therefore should
tend to accumulate within the lypophilic DNA grooves.
3,5-diacetyl-1,2,4-triazol◦
bis(4N-o-tolylthiosemicarbazone),
H5L1: yield (70%), mp 220 C. Elemental analysis found, C, 54.95;
H, 5.41; N, 23.75; S, 12.80; C22H25N9S2·EtOH requires C, 54.85;
H, 5.95, N, 23.95; S 12.15%. MS (FAB+ with mNBA matrix) m/z
480.2 (43%) for [C22H25N9S2+H]+. IR (KBr pellet): n/cm-1 3521
In conclusion, this study indicates that the newly synthesized
bis(thiosemicarbazone) ligands and platinum(II) complexes may
be endowed with important antitumour properties. Moreover
these compounds show less nephrotoxity than cisplatin on LLC-
PK1 cells, and the platinum complex [Pt(m-H3L1)]2 being the most
promising compound of this new series since it is active in the
low micromolar range but not nephrotoxic at 100 mM. Based on
these results, these compounds represent a valuable lead in the
development of new anticancer chemotherapeutic agents capable
of improving antitumour activity and reducing nephrotoxicity.
2
(s, OH-EtOH), 3340 (s, NH-triazol), 3224 and 3172 (s, NH and
4NH), 1586 (s, CN), 857 (w, CS-thioamide IV band). H NMR
1
(300 MHz, DMSO-d6): d (ppm) 15.09 (s, NH-triazol, 1H), 12.92
2
4
and 11.35 (s, NH, 1H), 10.23 and 10.09 (s, NH, 1H), 7.27-7.20
(m, aromatic protons, 8H), 3.30 (s, CH3-thiosemicarbazide, 6H),
2.23 and 2.22 (s, CH3-triazol, 3H). UV–vis (DMSO): l max/nm
295, 322.
3,5-Diacetyl-1,2,4-triazo◦l
bis(4N-m-tolylthiosemicarbazone)
H5L2: yield (60%), mp 194 C. Elemental analysis found, C, 55.00;
H, 5.50, N, 24.35; S 12.50; C22H25N9S2·EtOH requires C, 54.85;
H, 5.95, N, 23.95; S 12.15%. MS (FAB+ with mNBA matrix) m/z
480.1 (100%) for [C22H25N9S2+H]+. IR (KBr pellet): n/cm-1 3584
Experimental
2
(s, OH-EtOH), 3379 (s, NH-triazol), 3286 and 3197 (s, NH and
4NH), 1606 (s, CN), 756 (w, CS-thioamide IV band). H NMR
Measurements
1
(300 MHz, DMSO-d6): d (ppm) 15.28 (s, NH-triazol, 1H), 12.91
Elemental analyses were performed on a LECO CHNS-932
microanalyzer. Fast atom bombardment (FAB) mass spectra
were performed on a VG AutoSpec spectrometer (nitrobenzyl
2
4
and 11.27 (s, NH, 1H), 10.30 and 10.19 (s, NH, 1H), 7.47-7.00
(m, aromatic protons, 8H), 3.32 (s, CH3-thiosemicarbazide, 6H),
2.43 and 2.31 (s, CH3-triazol, 3H). UV–vis (DMSO): l max/nm
323.
1
alcohol matrix). H NMR spectra (DMSO-d6) were recorded on
a BRUKER AMX-300 spectrometer. All cited physical measure-
ments were obtained out by the Servicio Interdepartamental de
Investigacio´n (SIDI) of the Universidad Auto´noma de Madrid.
Melting points were determined with a Stuart Scientific SMP3
apparatus. Conductivity measurements were made on a Crison
Basic 30+ conductimeter. Infrared spectra (KBr pellets) were
recorded on a Bomen–Michelson spectrophotometer (4000–400
cm-1). Electronic spectra were recorded on an Unicam UV/vis
UV4 spectrophotometer.
3,5-Diacetyl-1,2,4-triazo◦l
bis(4N-p-tolylthiosemicarbazone)
H5L3: yield (70%), mp 189 C. Elemental analysis found, C, 55.00;
H, 5.45, N, 24.10; S 12.50; C22H25N9S2·EtOH requires C, 54.85;
H, 5.95, N, 23.95; S 12.15%. MS (FAB+ with mNBA matrix) m/z
480.0 (35%) for [C22H25N9S2+H]+. IR (KBr pellet): n/cm-1 3592
(s, OH-EtOH), 3360 (s, NH-triazol), 3237 and 3129 (w, 2NH and
4NH), 1588 (s, CN), 879 (w, CS-thioamide IV band). H NMR
1
(300 MHz, DMSO-d6): d (ppm) 15.27 (s, NH-triazol, 1H), 12.89
and 11.25 (s, 2NH, 1H), 10.28 and 10.19 (s, 4NH, 1H), 7.49–7.14
(m, aromatic protons, 8H), 3.22 (s, CH3-thiosemicarbazide, 6H),
2.43 and 2.33 (s, CH3-triazol, 3H). UV–vis (DMSO): l max/nm
323.
Materials
Solvents were purified and dried according to standard procedures.
Hydrazine hydrate, L-lactic acid, ortho-tolyl isothiocyanate, meta-
tolyl isothiocyanate, para-tolyl isothiocyanate and potassium
tetrachloridoplatinate(II) were commercially available. Calf thy-
mus DNA (CT-DNA), Tris-HCl and NaCl were purchased from
Aldrich.
Platinum(II) complexes. They were obtained by reacting a
methanol (20 mL) suspension of the desired ligand with a
water solution (5 mL) of potassium tetrachloridoplatinate(II). The
reaction mixture was stirred at room temperature in the dark for
5 h. The resulting orange solid obtained was filtered, washed with
methanol and diethyl ether and dried in vacuo. Finally it was
crystallized by slow evaporation from a DMSO solution.
Synthesis of compounds
3,5-Diacetyl-1,2,4-triazol bis(4N-tolylthiosemicarbazone) lig-
ands. The thiosemicarbazides were prepared by reacting an
ethanolic solution of the desired (ortho, meta or para) tolylisoth-
iocyanate with an ethanolic solution of hydrazine hydrate, in a
1 : 1 molar ratio. The reaction mixture was stirred for one more
hour and then the white product formed was filtered, washed with
ethanol and diethyl ether, dried in vacuo and recrystallised from
ethanol.
The thiosemicarbazones were obtained by refluxing an ethano-
lic solution (20 mL) of the desired 4N-tolylthiosemicarbazide
(2 mmol) with 3,5-diacetyl-1,2,4-triazol (1 mmol), which was
prepared as described in reference,25 for five hours 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.
[Pt(l-H3L1)]2: yield (45%), mp 300 ◦C (decomposes). Elemental
analysis found, C, 39.65; H, 4.00, N, 19.25; S 9.55; C44H50N18S4Pt2
requires C, 39.15; H, 3.75, N, 18.65; S 9.50%. Molar conductivity
(DMF: 10-3 M, mS cm-1): 13. IR (KBr pellet): n/cm-1 3176 (w,
1
NH), 1585 (s, CN). H NMR (300 MHz, DMSO-d6): d (ppm)
15.11 (s, NH-triazol, 0.5H), 12.92 (s, 2NH, 0.5H), 11.34 (s, 2NH,
1H), 10.23 and 10.09 (s, 4NH, 2H), 7.27–7.21 (m, aromatic protons,
16H), 3.60 (s, CH3-thiosemicarbazide, 12H), 2.41 (s, CH3-triazol,
12H). UV–vis (DMSO): l max/nm 323.
[Pt(l-H3L2)]2: yield (55%), mp 259 ◦C (decomposes). Elemental
analysis found, C, 38.90; H, 3.85, N, 19.25; S 9.50; C44H50N18S4Pt2
requires C, 39.15; H, 3.75, N, 18.65; S 9.50%. Molar conductivity
(DMF: 10-3 M, mS cm-1): 21. IR (KBr pellet): n/cm-1 3197 (w,
1
NH), 1607 (s, CN). H NMR (300 MHz, DMSO-d6): d (ppm)
15.30 (s, NH-triazol, 0.5H), 12.91 (s, 2NH, 0.5H), 11.27 (s, 2NH,
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1H), 10.30 and 10.20 (s, 4NH, 2H), 7.47–7.00 (m, aromatic protons,
16H), 3.45 (s, CH3-thiosemicarbazide, 12H), 2.35 and 2.31 (s, CH3-
triazol, 12H). UV–vis (DMSO, l max/nm) 320.
Tris base for determination of optical density (at 515 nm) in a
Tecan Ultra Evolution spectrophotometer.
The normal human cells (LLC-PK1) were grown in 199 medium
supplemented with 3% foetal bovine serum (FBS) and 1.5 g L-1
of sodium bicarbonate 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 10,000
cells per well with 100 mL of medium and were then incubated
for 24 h. After attachment to the culture surface the cells were
incubated with various concentrations of the compounds tested
freshly dissolved in DMSO (1 mg mL-1) and diluted in the culture
[Pt(l-H3L3)]2: yield (55%), mp 292 ◦C (decomposes). Ele-
mental analysis found, C, 38.50; H, 4.55, N, 16.50; S 12.70;
C44H50N18S4Pt2·2DMSO requires C, 38.30; H, 4.15, N, 16.75; S
12.75%. Molar conductivity (DMF: 10-3 M, mS cm-1): 09. IR (KBr
pellet): n/cm-1 3234 (w, NH), 1592 (s, CN). 1H NMR (300 MHz,
2
DMSO-d6): d (ppm) 15.30 (s, NH-triazol, 0.5H), 12.90 (s, NH,
0.5H), 11.25 (s, 2NH, 1H), 10.30 and 10.19 (s, 4NH, 2H), 7.49–7.15
(m, aromatic protons, 16H), 3.48 (s, CH3-thiosemicarbazide, 12H),
2.33 and 2.30 (s, CH3-triazol, 12H). UV–vis (DMSO, l max/nm)
322.
◦
medium (DMSO final concentration 1%) for 48 h at 37 C. The
cells were fixed by adding 50 mL 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 effects of complexes were expressed as corrected percentage
inhibition values according to the following equation:
Crystallography
A summary of the crystal data collection and refinement param-
eters for complex [Pt(m-H3L3)]2 is given in Table 1. The crystals
were placed in a Bruker–Nonius X8 APEX II CCD area-detector
diffractometer equipped with a graphite-monochromated Mo-
˚
Ka radiation source (l = 0.71073 A). Data were integrated
using SAINT26 which also applied corrections for Lorentz and
polarization effects. An absorption correction was performed
with the program SADABS.27 The software package SHELXTL28
was used for space group determination, structure solution, and
refinement. The structure was solved by direct methods, completed
with different Fourier syntheses, and refined with anisotropic
displacement parameters. The position of the hydrogen atoms
were calculated geometrically and were allowed to ride on their
parent carbon atoms with fixed isotropic U.
% Inhibition = [1 - (T/C)] ¥ 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:
E = Emax/[1 + (IC50/C)n]
Refinement included application of the squeeze procedure29 to
model diffuse electron density in one substantial void per unit
cell presumably occupied by disordered DMSO solvent molecules.
Chemical formula and derivate quantities are given without
solvent.
CCDC-805583 contains the supplementary crystallographic
data for [Pt(l-H3L3)]2 compound described in this paper (please
see ESI†).
where E is the percentage inhibition observed, Emax is the maximal
effects, 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
software.30
For comparison purposes, the antiproliferative activity of
cisplatin was evaluated under the same experimental conditions.
All compounds were tested in two independent studies with
triplicate points. These experiments were carried out at the Unidad
de Evaluacio´n de Actividades Farmacolo´gicas de Compuestos
Qu´ımicos (USEF), Universidad de Santiago de Compostela.
In vitro antiproliferative activity
The human cancer cells (A2780, A2780cisR and NCI-H460) were
grown in RPMI-1640 medium supplemented with 10% foetal
bovine serum (FBS) and 2 mM L-glutamine 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 15 ¥ 103 (for NCI-H460) or 4000 (for A2780 and
A2780cisR) cells per well with 100 mL of medium and were then
incubated for 24 h. After attachment to the culture surface the cells
were incubated with various concentrations 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) or 96 h (for A2780 and A2780cisR). The cells were
fixed by adding 50 mL of 30% trichloroacetic acid (TCA) per well.
DNA-Binding experiments
CT-DNA stock solution was prepared by dissolving the
lyophilized 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
spectrophotometrically31 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, of ca. 1.8, indicating that the DNA
was sufficiently free of protein. Stock solutions were kept frozen
until the day of the experiment.
◦
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
Concentrated stock solutions (5 ¥ 10-3 M) of both ligands
and platinum(II) complexes were prepared dissolving each one
of the compounds in DMSO. From these stock solutions, for all
experiments the desired concentration of compound was achieved
5744 | Dalton Trans., 2011, 40, 5738–5745
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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 a DMSO content 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 ¥ 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 the their UV-vis absorption spectra were recorded, at
room temperature, in the wavelength interval of 240–400 nm.
11 J. S. Casas, E. E. Castellano, J. Ellena, M. S. Garc´ıa-Tasende, M.
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Acknowledgements
We are grateful to Ministerio de Ciencia e Innovacio´n, Instituto de
Salud Carlos III (PI080525), Universidad Auto´noma de Madrid
and Comunidad de Madrid (CCG08-UAM/SAL-4000) of Spain
for financial support.
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©