Exploring the versatility of cycloplatinated thiosemicarbazones as antitumor and antiparasitic agents

Article abstract of DOI:10.1021/om300334z

Tridentate cycloplatinated thiosemicarbazone complexes have been prepared from a biologically significant ligand, 3,4-dichloroacetophenone thiosemicarbazone (1). The tetranuclear complex 2 was prepared by reaction of the ligand with K₂[PtCl₄]. Two mononuclear (3 and 4) and two dinuclear (5 and 6) complexes were isolated upon cleavage of the Pt-S _bridging bonds of the tetranuclear complex 2 with the appropriate phosphane ligand. Each complex was characterized using various analytical and spectroscopic techniques, and the molecular structures of 2-4 were also elucidated. The in vitro antiparasitic activities of these complexes against Plasmodium falciparum strains (D10 (chloroquine sensitive) and Dd2 (chloroquine resistant)) and Trichomonas vaginalis have been determined. Preliminary studies into their potential plasmodial target in the form of β-hematin formation inhibition assays were also completed. Preliminary results suggest that ligand 1 and complex 3 do not hinder formation of β-hematin. The antiproliferative activity of the complexes against the cisplatin-senstive A2780 and cisplatin-resistant A2780cisR human ovarian cancer cell lines has been evaluated. The complexes were found to exhibit moderate to weak inhibitory activities.

Full text of DOI:10.1021/om300334z

Article
pubs.acs.org/Organometallics
Exploring the Versatility of Cycloplatinated Thiosemicarbazones as
Antitumor and Antiparasitic Agents
Prinessa Chellan,† Kirkwood M. Land,‡ Ajit Shokar,‡ Aaron Au,‡ Seung Hwan An,‡ Catherine M. Clavel,§
Paul J. Dyson,§ Carmen de Kock,∥ Peter J. Smith,∥ Kelly Chibale,†,⊥ and Gregory S. Smith*,†
†Department of Chemistry, University of Cape Town, Private Bag, Rondebosch 7701, South Africa
‡Department of Biological Sciences, University of the Pacific, Stockton, California 95211, United States
§Institut des Sciences et Ingen
́ ́ ́
ierie Chimiques, Ecole Polytechnique Federale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland
∥Division of Pharmacology, Department of Medicine, University of Cape Town, K45, OMB, Groote Schuur Hospital, Observatory,
7925, South Africa
⊥Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch 7701, South Africa
S
* Supporting Information
ABSTRACT: Tridentate cycloplatinated thiosemicarbazone complexes
have been prepared from a biologically significant ligand, 3,4-
dichloroacetophenone thiosemicarbazone (1). The tetranuclear complex
2 was prepared by reaction of the ligand with K2[PtCl4]. Two mononuclear
(3 and 4) and two dinuclear (5 and 6) complexes were isolated upon
cleavage of the Pt−Sbridging bonds of the tetranuclear complex 2 with the
appropriate phosphane ligand. Each complex was characterized using
various analytical and spectroscopic techniques, and the molecular
structures of 2−4 were also elucidated. The in vitro antiparasitic activities
of these complexes against Plasmodium falciparum strains (D10
(chloroquine sensitive) and Dd2 (chloroquine resistant)) and Trichomonas
vaginalis have been determined. Preliminary studies into their potential
plasmodial target in the form of β-hematin formation inhibition assays were
also completed. Preliminary results suggest that ligand 1 and complex 3 do
not hinder formation of β-hematin. The antiproliferative activity of the complexes against the cisplatin-senstive A2780 and
cisplatin-resistant A2780cisR human ovarian cancer cell lines has been evaluated. The complexes were found to exhibit moderate
to weak inhibitory activities.
inhibitory mechanisms would compromise the parasite’s ability
to degrade host hemoglobin required for (parasite) protein
synthesis.18−21
INTRODUCTION
■
Thiosemicarbazones are a class of Schiff base compounds that
contain a thiourea moiety. They are well-known for their
biological activity as antiparasitics,1−6 antibacterial7−9 and
antitumoral agents.10−14 As chemotherapeutics their mode of
action is believed to be through the inhibition of ribonucleotide
reductase, thus arresting DNA synthesis.15 As antiparasitics,
thiosemicarbazones may affect processes, some associated with
hemoglobin (Hb) digestion in the food vacuole of the parasite
through several possible inhibitory mechanisms of action. Their
ability to chelate endogenous metals such as Fe(III) can inhibit
parasite growth by withholding it from metal-dependent
enzymes such as ribonucleotide reductase and enzymes in the
heme biosynthetic pathway.4,16 Alternatively, the resulting
chelate complexes could inhibit cysteine proteases, effectively
enhancing the natural inhibitory effect of endogenous metals on
the protease catalytic site.17 Another possible mechanism of
cysteine protease inhibition could be through covalent
modification of the cysteine thiol groups within the parasite
via electrophilic centers (the thione carbon and/or imine
carbon) of the thiosemicarbazone moiety. All of the above
The ability of thiosemicarbazones to form stable complexes
with a variety of metals is also believed to be of great
importance to their biological activities. The lipophilic nature of
the complex might be enhanced in comparison to the free
ligand, and certain long-term side effects may decrease or be
altogether avoided, since the coordinated ligand of the metal
complexes can dissociate and the metal ion can then interact
with the organism.22−24 In addition, the metal complex could
exhibit bioactivities not shown by the free thiosemicarbazone
ligand and coordination could yield a significant reduction in
drug resistance.25 Research into the synthesis and application of
thiosemicarbazone metal complexes as biological agents is thus
Special Issue: Organometallics in Biology and Medicine
Received: April 23, 2012
Published: July 26, 2012
© 2012 American Chemical Society
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Scheme 1
Reaction conditions: (i) K2[PtCl4], EtOH−H2O, 50 °C, 5 days; (ii) L or P∧P, acetone, room temperature, 3 h.
an area of great promise, and there have been several reports on
this subject published by us26−29 as well as others.30−37
The discovery of cisplatin and its analogues as antitumoral
agents had a profound influence on establishing the field of
metal complexes in medicine,38 and the amount of research
already accomplished within this area is substantial.39−44
Despite the considerable quantity of work completed, there
are still many little-researched avenues such as the preparation
of mono- and polynuclear complexes from biologically active
hybrid ligands. Hybrid ligands are defined as bis- or polydentate
molecules which contain at least two different functionalities
containing donor atoms capable of binding to a metal.45
Thiosemicarbazones are prime hybrid candidates, as they fulfill
this basic criterion. Recently, we have explored the chemistry
and application of biologically significant thiosemicarbazones
and their platinum group metal (PGM) complexes. Our studies
have included the synthesis of ruthenium-, palladium-, and
platinum-containing thiosemicarbazones.26−29 To demonstrate
the versatility of these complexes, we have extended our studies
into their biological and catalytic applications and the results so
far have been promising.27−29,46,47 Our studies of their
inhibitory effects against malarial and T. vaginalis parasitic
strains are among the first to be reported in the literature for
palladium(II) thiosemicarbazone complexes.28,29,48 In light of
these results, together with recent research carried out in other
laboratories,33,49−54 we have been encouraged to expand our
studies into the biological relevance of thiosemicarbazone PGM
complexes.
RESULTS AND DISCUSSION
■
Synthesis and Characterization. The tetranuclear com-
plex 2 was prepared by stirring ligand 1 and K2[PtCl4] in an
ethanol−water mixture at 50 °C for 5 days (Scheme 1). The
mono- and diplatinum complexes were synthesized by cleavage
of the bridging Pt−S bonds of 2 with either a mono- or
diphosphane in acetone at room temperature.
On examination of the 1H NMR spectral data for complex 2,
the absence of proton resonances for the thioamide and ortho
protons (observed at 10.20 and 8.20 ppm, respectively, for 1)
presents evidence of the coordination of the ortho carbon to
platinum and the formation of a second imine bond, thus
suggesting that sulfur bonds to the metal in the thiolato form.
In comparison to the tetranuclear complex 2, a slight upfield
shift is observed for the thiosemicarbazone aromatic protons of
complexes 3, 5, and 6. In complexes 3, 5, and 6, platinum
satellites are observed for the proton of the carbon adjacent to
the ortho-metalated carbon. Figure 1 shows the proton NMR
spectrum for complex 3, where these satellite peaks are clearly
As part of our continuing search for improved metal-
lotherapeutics, we have now turned our attention to cyclo-
platinated thiosemicarbazone complexes. In this paper, we
report the synthesis and structural characterization of five new
cycloplatinated thiosemicarbazone complexes. The preliminary
biological screening of these complexes for inhibitory effects
against chloroquine-resistant and chloroquine-sensitive Plasmo-
dium falciparum strains, Trichomonas vaginalis, and cisplatin-
resistant (A2780cisR) and cisplatin-sensitive ovarian (A2780)
cancer cell lines is also reported.
1
Figure 1. Expansion of the H NMR aromatic region of complex 3.
The proton resonance with Pt satellites is circled.
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visible. A coupling constant of 20−25 Hz is observed, and this
Table 1. Selected Bond Lengths (Å) and Angles (deg) for 2−
4
55,56
3
corresponds to a J coupling of 195Pt.
A downfield shift of the azomethyl protons by approximately
0.25 ppm is observed for 3−6 in comparison to 2. This is
consistent with deshielding of these protons upon coordination
of the imine nitrogen to the metal. For complex 4, two sets of
resonances are observed for the PTA ancillary ligand. A singlet
at 4.20 ppm is assigned to the CH2 protons adjacent to
phosphorus. The CH2 protons of the N−CH2−N moiety
2·3DMSO
A
B
3·2DMSO
4
Pt1−C1
Pt1−N1
Pt1−S1
Pt1−P1
N1−C7
N2−C9
C1−Pt1−S1
C1−Pt1−P1
N1−Pt1−C1
N1−Pt1−S1
N1−Pt1−P1
P1−Pt1−S1
2.004(3)
1.996(3)
2.3169(8)
1.999(4)
1.995(3)
2.3513(8
2.039(3)
2.038(2)
2.2359(7)
2.2359(7)
1.298(3)
1.316(4)
163.57(8)
97.00(8)
80.78(10)
82.80(7)
177.17(7)
99.43(3)
2.027(3)
2.035(2)
2.3322(11)
2.2101(7)
1.301(3)
1.313(3)
163.74(7)
98.56(8)
80.73(9)
83.07(6)
168.56(6)
97.59(3)
1.307(4)
1.293(5)
164.12(11)
1.302(5)
1.300(5)
100.70(3)
usually exhibit two doublets due to geminal coupling between
57,58
Hequatorial and Haxial
.
In complex 4, these protons are
observed as a quartet, possibly due to the influence of platinum.
In the 13C{1H} NMR spectra, the thiolato carbon resonance
shifts from 179.1 ppm in the free ligand 1 to 166.6 ppm in
complex 2, further confirming deprotonation and coordination
of sulfur in the thiolato form. The ortho carbon resonance shifts
downfield from 128.0 to 153.8 ppm, displaying evidence of
cyclometalation, and the imine carbon also shifts downfield as a
result of coordination of the imine nitrogen to platinum. For
complexes 3−6, the thioamide (C−S) carbon resonates at
approximately 177.0 ppm, similar to that of the tetranuclear
complex 2. The ortho carbon is observed between 154.0 and
155.0 ppm. The chloro-substituted carbons of the thiosemi-
carbazone ligand appear to be equivalent and are observed at
ca. 131.0 ppm for all complexes except complex 4. In complex
4, two distinct resonances are seen at 135.3 and 133.15 ppm.
The 31P{1H} NMR analysis for complexes 3−5 revealed one
singlet for the phosphorus nuclei with platinum satellites due to
platinum−phosphorus coupling. A 1J coupling constant of
between 1680 and 1888 Hz was observed, and this is consistent
with the literature values for analogous cycloplatinated
compounds.59 In complex 6, where the bridging ligand is
trans-bis(diphenylphosphino)ethylene, the phosphorus nucleus
resonates as a triplet with platinum satellites and is a
consequence of phosphorus−phosphorus and phosphorus−
platinum coupling. For complex 2, infrared spectral analysis
revealed two strong absorption bands (three observed for the
ligand) in the N−H region at 3321 and 3149 cm−1, agreeing
with the presence of two N−H bonds for the primary amine in
the complex after cyclometalation. Absorption bands at 1598
and 1561 cm−1 correspond to two CN bonds, the high-
frequency band being assigned to the newly formed imine and
the lower energy band to the metal-coordinated imine (via
nitrogen). For the mononuclear (3 and 4) and dinuclear (5 and
6) complexes, similar absorption bands are observed.
X-ray Structure Analysis. Single crystals of complexes 2−
4 suitable for single-crystal X-ray diffraction were obtained by
exposing dimethyl sulfoxide solutions of each complex to air.
Crystal data for complexes 2·3DMSO,60 3·2DMSO,61 and 462
are summarized in the reference section, and Table 1 gives
selected bond angles and lengths.
Complex 2 (Figure 2) crystallizes with three solvent
molecules of DMSO with a monoclinic system. Each platinum
center is bridged by a sulfur of the adjacent Pt−
thiosemicarbazone unit to form an eight-membered Pt4S4
core, around which the four thiosemicarbazone ligands reside.
Each metal center is coordinated to the imine nitrogen, ortho
carbon, and sulfur of one thiosemicarbazone ligand, and the
fourth coordination site is occupied by the sulfur of the
adjacent thiosemicarbazone ligand. A slightly distorted square-
planar geometry is observed around each metal center.
80.61(13)
83.97(9)
81.42(15)
84.08(9)
tetranuclear platinum thiosemicarbazone complexes.63
A
lengthening of the C−S bond for S(1A)−C(9A) and S(1B)−
C(9B) is observed with values for the bond distances in close
agreement with the bond length expected for a C−S single
bond (1.82 Å) rather than a normal CS double bond (1.56
Å),64 corroborating that sulfur coordinates to palladium in the
thiolato form.65 The bond angles N(1A)−Pt(1A)−S(1B)#2,
N(1B)−Pt(1B)−S(1A), C(1A)−Pt(1A)−S(1A), and C(1B)−
Pt(1B)−S(1B) are all less than 180°, confirming the slight
deviation from the expected square-planar geometry around
platinum.
The mononuclear complexes 3 and 4 (parts a and b,
respectively, of Figure 3) crystallize in the orthorhombic and
monoclinic systems, respectively. The molecular structure of
each complex confirms that the platinum−sulfur bridging
bonds in complex 2 are cleaved and the tridentate coordination
of the thiosemicarbazone remains intact. As with 2, there is a
distorted square-planar coordination sphere around the metal
and this is confirmed by the slight deviation of the bond angles
observed around platinum for complexes 3 and 4.
The bond distances observed between the metal and the
coordinated carbon, nitrogen, and sulfur atoms in 3 and 4 are
similar to those in the tetranuclear complex 2, indicating that
the electron density distribution between platinum and the
thiosemicarbazone ligand does not change after cleavage. These
bond distances are also in close agreement with similar
mononuclear platinum thiosemicarbazone complexes.59,66
In Vitro Antitumor Activity. The in vitro antiproliferative
effects of compounds 1−6 were evaluated against the cisplatin-
sensitive (A2780) and cisplatin-resistant (A2780cisR) ovarian
cancer cell lines. Table 2 summarizes the cytotoxic values
obtained.
Ligand 1 exhibited the highest activity out of all the
compounds tested against both cell lines. Complexes 2−5 are
moderately cytotoxic against both cell lines, with complex 4
showing the lowest IC50 values. Complexes 2, 4, and 5 are less
active against the cisplatin-resistant (A2780cisR) cell line, while
complex 3 is more active in this cell line in comparison to
A2780. While none of the compounds tested showed activities
comparable to cisplatin against the A2780 cell line, ligand 1
shows a markedly better activity than cisplatin against the
A2780cisR line. Metalation of the thiosemicarbazone ligand
seems to have an adverse effect on cytotoxicity, and the number
of platinum thiosemicarbazone moieties does not appear to
influence the biological activity. The tetraplatinum complex 2 is
The bond lengths observed between platinum and each
coordinated atom agree with those reported for similar
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Figure 2. (a) Molecular structure of 2·3DMSO with ellipsoids at the 25% probability level and another half-molecule generated via the symmetry
code 1 − x, y, 0.5 − z. Solvent molecules and hydrogen atoms have been omitted for clarity. (b) View of complex 2 down the 010 plane, showing the
ring formed by the Pt4S4 core. Atom labeling, solvent molecules, and hydrogen atoms are omitted for clarity.
Figure 3. (a) Structure of complex 3·2DMSO with ellipsoids at the 30% probability level. Solvent molecules and hydrogen atoms have been omitted
for clarity. (b) Structure of complex 4 with ellipsoids at the 40% probability level. Hydrogen atoms are omitted for clarity.
In Vitro Antiparasitic Activity. The antiparasitic activity of
complexes 2−6 in vitro against two Plasmodium falciparum
strains, D10 (chloroquine-sensitive) and Dd2 (chloroquine-
resistant), and the parasite Trichomonas vaginalis was also
determined. For the in vitro assays, the complexes were tested
as suspensions if not completely dissolved in the assay medium.
Table 3 gives the antiplasmodial data ascertained for complexes
2−6.
Complexes 2−4 shows only moderate activity compared to
chloroquine, while 5 and 6 show no activity at the highest
compound concentration tested. Complex 3, which contains
the triphenylphosphane ancillary ligand, shows better activity
compared to 4 (containing a PTA ancillary ligand) against both
Table 2. IC50 Determinations for 1−6 against A2780 and
A2780cisR Cancer Cell Lines
a
IC50 (μM)
compd
A2780
A2780cisR
1
2
3
4
5
6
11.3 2.7
51.5 1.0
129 31
21.2 1.2
36.5 3.6
>200
13.8 2.6
67 10
99.0 4.7
32
7
>200
175 1.7
b
cisplatin
1.5
25
a
b
IC50: minimum concentration for 50% cell death. IC50 values for
cisplatin were determined using the same experimental assay and
protocol as for 1−6 but were not determined at the same time as
compounds 1−6.
Table 3. Antiplasmodial Data Obtained for Complexes 2−6
a
IC50 (μM)
complex
D10
Dd2
more active than complex 6 (containing the bis-
(diphenylphosphino)ethylene spacer) but is less active than
the dppf-containing complex 5. The type of ancillary ligand
used in the design of platinum thiosemicarbazone chemo-
therapeutics may play a key role in enhancing activity. The
mononuclear complex 4, containing the most hydrophilic
phosphane ligand, showed the best activity, followed by 2,
which has no phosphane ligands. Complex 6 shows moderate
activity against the A2780 line yet was not active in the
cisplatin-resistant cell line.
2
3
4
5
6
32.29 2.28
19.93 3.74
21.42 1.22
not tested
14.47 1.98
24.90 3.24
not tested
b
not active
b
not active
not tested
chloroquine
0.012 0.001
0.146 2.08
a
The minimum compound concentration required for 50% inhibition
in vitro. Up to the highest compound concentration tested (100 μg/
b
cm3).
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P. falciparum strains. The PTA ligand is widely used in the
design and synthesis of metallotherapeutics, as it is a water-
soluble compound that is believed to enhance the hydro-
philicity of the metal complex.67,68 Against the Dd2 strain,
complex 3 shows enhanced activity (IC50 = 14.47 μM) in
comparison to the D10 strain. None of the compounds tested
show activities comparable to chloroquine against both strains.
In contrast to the inhibitory studies for P. falciparum,
complex 4 is the only compound that showed a significant
percentage inhibition (92.5%) of the Trichomonas vaginalis
parasite (Table 4). Metronidazole, the current FDA-approved
potential drug to inhibit formation of hemozoin can be
measured using a β-hematin (synthetic hemozoin) inhibition
assay. Complex 3 exhibits the best antiplasmodial activity
against the two malarial strains tested and was thus chosen,
along with ligand 1, for β-hematin inhibition studies.
Compounds 1 and 3 were tested using a modified NP-40
detergent mediated hematin formation screen.74 Figure 4 shows
Table 4. Preliminary Inhibition Data of Compounds 1−6
against T. vaginalis
compd
inhibition at 100 μM (%)
IC50 T1 strain (μM)
1
2
3
4
5
6
51.3
68.7
42.3
92.5
24.5
42.3
100.0
21.1
0.72
a
metronidazole
Figure 4. Concentration dose−response curves for NP-40 detergent
mediated β-hematin assays of 1, 3, amodiaquine, and chloroquine.
a
Current FDA-approved treatment for T. vaginalis infections.
treatment, exhibited a 100% inhibition at this concentration
(100 μM), comparable to that for 4. Since 4 was deemed to be
the most potent inhibitor out of all compounds tested, its IC50
value was determined and it was found to be 21.1 μM.
Although this compound is not as potent as metronidazole, its
inhibitory effect suggests that their targets are necessary for
parasite viability.
the dose−response curves obtained for compounds 1 and 3
along with amodiaquine and chloroquine. Neither ligand 1 nor
complex 3 is found to hinder β-hematin formation at the
concentrations tested, in comparison to amodiaquine and
chloroquine. This result suggests that arylthiosemicarbazones
and their cycloplatinated complexes target other processes
within the Plasmodium falciparum parasite, possibly cysteine
protease function.
Cysteine proteases are integral to the parasitic life cycle.
They are involved in several parasite functions, including host
invasion, nutrition, and protein processing.69 Thiosemicarba-
zones are believed to inhibit parasite growth through reversible
interactions with cysteine proteases in the parasite, and a recent
study of the interactions of Pt(II) complexes and cysteine
proteases revealed that Pt(II) complexes can bind to the active
site cysteine of different cysteine proteases.70 Furthermore, it
was found that the presence of an aromatic ring enhanced the
inhibitory activity of these complexes in comparison to the
clinical drugs cisplatin and carboplatin. However, the type and
rate of active site cysteine binding were unique for each
cysteine protease. Different cysteine proteases have structurally
different folds, and these can sterically influence how the
complex binds to the cysteine protease targets within each
parasite.70 This could account for the contrasting activities
observed for complexes 3 and 4 against the P. falciparum and T.
vaginalis parasite strains. Moreover, it has been shown that the
PTA ligand forms specific interactions within the active site of a
cysteine protease when bound to a different metal center.71
β-Hematin Inhibition Assay. In the life cycle of the
Plasmodium falciparum parasite, degradation of the infected
host’s hemoglobin to provide essential amino acids for its
growth and nutrition is a vital step.72 A side product of this
hemoglobin digestion is free heme, which is toxic to the
parasite. The parasite removes this threat by conversion of the
free heme into a crystalline solid known as hemozoin, which is
nontoxic to the parasite. Thus, this process is one potential
target in the design of possible antiplasmodial therapeutics. The
clinically used drugs amodiaquine and chloroquine are believed
to inhibit the formation of hemozoin.73 The ability of a
CONCLUSIONS
■
Five new cycloplatinated thiosemicarbazone complexes (2−6)
have been prepared using the ligand 3,4-dichloroacetopheno-
nethiosemicarbazone (1). The molecular structures of 2−4
show that the ligand coordinates to platinum in a tridentate
manner via the ortho carbon, imine nitrogen, and thiolato
sulfur. All of the compounds prepared were screened for
antiparasitic activity against Plasmodium falciparum and
Trichomonas vaginalis strains and antitumor activity against
cisplatin-sensitive and -resistant human ovarian cancer cell lines.
They were found to exhibit moderate to low parasite inhibition
and antiproliferative activities. When ligand 1 and complex 3
were evaluated for their ability to inhibit β-hematin formation,
it was found that the compounds do not target this process.
Thiosemicarbazones and their metal complexes may act upon
cysteine protease functions within the parasite, and a study of
their interaction with different cysteine protease enzymes must
be carried out to validate this hypothesis.
EXPERIMENTAL SECTION
■
General Methods. Thiosemicarbazide, 3,4-dichloroacetophenone,
triphenylphosphane, 1,3,5-triaza-7-phosphaadamantane, bis-
(diphenylphosphino)ferrocene, and trans-bis(diphenylphosphino)-
ethylene were purchased from Sigma-Aldrich and used without further
purification. Potassium tetrachloroplatinate was kindly donated by
AngloAmerican Platinum Ltd. Ligand 1, 3,4-dichloroacetophenone-
thiosemicarbazone, was prepared using a previously reported literature
method.6 All solvents used were analytical grade and were dried over
molecular sieves. Nuclear magnetic resonance (NMR) spectra were
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recorded on a Varian Unity XR400 (1H at 399.95 MHz, 13C at 100.58
MHz, 31P at 161.90 MHz), Varian Mercury XR300 (1H at 300.08
MHz, 13C at 75.46 MHz, 31P at 121.47 MHz), or Bruker Biospin
GmbH (1H at 400.22 MHz, 13C at 100.65 MHz, 31P at 162.00 MHz)
spectrometer at ambient temperature. Chemical shifts for 1H and
13C{1H} NMR are reported using tetramethylsilane (TMS) as the
internal standard, and 31P{1H} NMR spectra were measured relative to
H3PO4 as the external standard. NMR spectra were recorded in
deuterated dimethyl sulfoxide (DMSO-d6) unless otherwise stated.
Infrared absorptions (IR) were measured on a Perkin-Elmer Spectrum
100 FT-IR spectrometer as KBr pellets. Microanalyses for C, H, and N
were carried out using a Thermo Flash 1112 Series CHNS-O analyzer,
The product (5) was isolated as an orange amorphous solid (0.120 g,
70%). Mp: 225−227 °C. 1H NMR: δ/ppm 7.42−7.54 (m, 20H, PPh2
H), 7.17 (s, 2H, Ar H), 7.15 (s, 4H, NH2), 6.13(s, 2H, Ar H, 3J(Pt−H)
= 20.0 Hz), 5.04 (br s, 4H, Cp H), 4.23 (br s, 4H, Cp H), 2.36 (s, 6H,
CH3). 13C NMR: δ/ppm 177.1 (C−S), 166.5 (CN), 154.1 (Ar C),
152.0 (Ar C), 132.9 (PPh2 C), 131.0 (2C, Ar C), 128.0 (PPh2 C),
126.7 (Ar C), 124.8 (Ar C), 73.5−74.9 (Cp C), 13.1 (CH3). 31P
NMR: δ/ppm 11.62 (PPh2, 1J(Pt−P) = 1881.8 Hz). IR: ν/cm−1 3475
(N−H stretch), 3383 (N−H stretch), 1595 (CN stretch), 1573
(CN stretch). Anal. Found: C, 42.36; H, 2.71; N, 5.53. Calcd for
C52H42Cl4FeN6P2Pt2S2: C, 42.64; H, 2.89; N, 5.74. ESI-MS: m/z
1465.0 [M + H]+, 100%.
Synthesis of Complex 6. trans-Bis(diphenylphosphino)ethylene
(0.0468 g, 0.1180 mmol) was reacted with complex 2 (0.102 g, 0.0557
mmol). The product (6) was isolated as an orange amorphous solid
and melting points were determined using a Buchi B-540 melting
̈
point apparatus. Mass spectrometry determinations were carried out
on all new compounds using either electron impact (EI) on a JEOL
GC Matell instrument or electrospray ionization (ESI) on a Waters
API Quattro Micro instrument in either the positive or negative mode.
Synthesis of [Pt(3,4-dichloroacetophenone thiosemicarba-
zone)]4 (2). K2[PtCl4] (0.862 g, 2.08 mmol) was dissolved in
deionized water (10 cm3), and ethanol (150 cm3) was added. The
ligand 1 (0.520 g, 2.08 mmol) was then added to the resulting milky
orange suspension. The reaction mixture was heated to 50 °C for 5
days before being cooled to room temperature. The orange-yellow
product (2) was isolated by vacuum filtration, washed with ethanol (3
× 10 cm3), and dried. Yield: 0.880 g, 23%. Mp: 226−228 °C dec
without melting. 1H NMR: δ/ppm 7.47 (s, 4H, Ar H), 7.25 (br s, 8H,
NH2), 6.74 (s, 4H, Ar H), 2.08 (s, 12H, CH3). 13C NMR: δ/ppm
166.6 (C−S), 165.0 (CN), 153.8 (Ar C), 149.2 (Ar C), 130.88 (2C,
Ar C), 126.4 (Ar C), 124.5 (Ar C), 13.2 (CH3). IR: ν/cm−1 3321 (N−
H stretch), 3149 (N−H stretch), 1598 (CN stretch), 1561 (CN
stretch). Anal. Found: C, 25.42; H, 2.36; N, 8.34. Calcd for
C36H28Cl8N12Pt4S4·C4H18O: C, 25.35; H, 2.44; N, 8.87. ESI-MS: m/
z 1821.8 ([C36H28Cl8N12Pt4S4 + H]+, 20%).
1
(0.0976 g, 67%). Mp: 312−314 °C. H NMR: δ/ppm 7.31−7.52 (m,
22H, Hα, PPh2 H), 7.13(s, 2H, Ar H), 7.12 (s, 4H, NH2), 6.30 (s, 2H,
3
Ar H, J(Pt−H) = 24.1 Hz), 2.33 (s, 6H, CH3). 13C NMR: δ/ppm
177.3 (C−S), 167.4 (CN), 154.6 (Ar C), 152.4 (Ar C), 135.0
(Ph2P−C), 134.0 (PPh2 C), 132.11 (2C, Ar C), 129.3 (PPh2 C),
127.5 (Ar C), 125.7 (Ar C), 13.7 (CH3). 31P NMR: δ/ppm 16.25 (t,
3
1
PPh2, J(P−P) = 22.80 Hz, J(Pt−P) = 1870.0 Hz). IR: ν/cm−1 3456
(N−H stretch), 3324 (N−H stretch), 1627 (CN stretch), 1606
(CN stretch). Anal. Found: C, 40.67; H, 2.95; N, 6.65. Calcd for
C44H36Cl4N6P2Pt2S2: C, 40.44; H, 2.78; N, 6.43. ESI-MS: m/z 1307.0
[M + H]+, 100%.
X-ray Structure Analysis. Crystals suitable for single-crystal X-ray
diffraction of complexes 2·3DMSO, 3·2DMSO, and 4 were grown
from the DMSO-d6 NMR samples by exposure of the solutions to air.
X-ray single-crystal intensity data were collected on a Nonius Kappa-
CCD diffractometer using graphite-monochromated Mo Kα radiation
(λ = 0.710 73 Å). The temperature was controlled by an Oxford
Cryostream cooling system (Oxford Cryostat). The strategy for the
data collections was evaluated using the Bruker Nonius “Collect”
program. Data were scaled and reduced using DENZO-SMN
software.75 Absorption corrections were performed using SADABS.76
The structure was solved by direct methods and refined employing
full-matrix least squares with the program SHELXL-9777 refining on
F2. Packing diagrams were produced using the program PovRay and
graphic interface X-seed.78 For 2·3DMSO, all non-hydrogen atoms,
except the carbon and oxygen atoms on one of the DMSO molecules,
were refined anisotropically. All hydrogen atoms were positioned
geometrically with an N−H distance of 0.88 Å and C−H distances
ranging from 0.95 to 0.98 Å and refined as riding on their parent
atoms, with Uiso(H) = 1.2−1.5[Ueq(C or N)]. In the asymmetric unit,
one DMSO molecule is on general positions. Another molecule is on a
2-fold rotation axis, and it is disordered and shows high thermal
motions; therefore the carbon and oxygen atoms were refined
isotropically and the hydrogen atoms were not placed. The complex
2 main molecule is located at special positions on a 2-fold rotation axis.
The structure was refined successfully to an R factor of 0.0204. For
3·2DMSO, all non-hydrogen atoms were refined anisotropically. All
the hydrogen atoms, except those on the N3 atom, were placed in
idealized positions in a riding model with Uiso set at 1.2 or 1.5 times
those of their parent atoms and fixed C−H bond lengths ranging from
0.95 to 0.98 Å. The hydrogen atoms H3A and H3B on N3 were
located in the difference electron density maps and refined with simple
bond length constraints. One DMSO molecule was disordered over
two positions with a site occupancy of 0.5 each. For complex 4, all
non-hydrogen atoms were refined anisotropically. All the hydrogen
atoms were placed in idealized positions in a riding model with Uiso set
at 1.2 or 1.5 times those of their parent atoms and restrained with fixed
bond lengths ranging from 0.95 to 0.99 Å for C−H and 0.88 Å for N−
H.
General Method for Synthesis of Mono- and Bis-Phosphane
Pt(II) Thiosemicarbazone Complexes. The tetrameric complex 2
(1 mol equiv) was suspended in acetone (15 cm3). For complexes 3
and 4, the monophosphane (4 mol equiv) was added, and for
complexes 5−7, the bis(diphenylphoshane) (2 mol equiv) was added
and the reaction mixture was stirred at room temperature for 3 h. The
product was collected as an orange solid by filtration, washed with
acetone (2 × 5 cm3), and dried under vacuum.
Synthesis of Complex 3. Triphenylphosphane (0.145 g, 0.552
mmol) was reacted with complex 2 (0.255 g, 0.140 mmol). The
product (3) was isolated as an orange amorphous solid (0.198 g, 50%).
1
Mp: 299−301 °C dec without melting. H NMR: δ/ppm 7.46−7.59
(m, 15H, PPh3 H), 7.16 (s, 1H, Ar H), 7.02 (s, 2H, NH2), 6.19 (s, 1H,
3J(Pt−H) = 25.5 Hz, Ar H), 2.35 (s, 3H, CH3). 13C NMR: δ/ppm
177.5 (C−S), 167.1 (CN), 155.2 (Ar C), 152.7 (Ar C), 134.4−
134.3 (PPh3 C), 131.8 (2C, Ar C), 129.05 (PPh3 C), 130.3 (Ar C),
125.5 (Ar C), 13.6 (CH3). 31P NMR: δ/ppm 25.70 (PPh3, 1J(Pt−P) =
1888 Hz). IR: ν/cm−1 3472 (N−H stretch), 3340 (N−H stretch),
1598 (CN stretch), 1579 (CN stretch). Anal. Found: C, 45.47;
H, 3.15; N, 5.63. Calcd for C27H22Cl2N3PPtS: C, 45.20; H, 3.09; N,
5.86. EI-MS: m/z 716.85 [M − H]+, 100%.
Synthesis of Complex 4. 1,3,5-Triaza-7-phosphaadamantane
(0.0383 g, 0.244 mmol) was reacted with complex 2 (0.102 g,
0.0558 mmol). The product (4) was isolated as an orange amorphous
solid (0.102 g, 74%.). Mp: 303−304 °C dec without melting. 1H
NMR: δ/ppm 7.19−7.20 (m, 4H, Ar H, NH2), 4.51 (q, 6H,
3J(Hβ(axial)Hβ(equatorial)) = 3.10, 13.48 Hz, PTA H), 4.20 (s, 6H, PTA
H), 2.28 (s, 3H, CH3). 13C NMR: δ/ppm 177.7 (C−S), 166.7 (C
N), 154.8 (Ar C), 152.6 (Ar C), 135.3 (Ar C), 133.15 (Ar C), 127.9
(Ar C), 125.7 (Ar C), 72.43 (PTA C), 51.0 (PTA C), 13.6 (CH3). 31
P
1
NMR: δ/ppm −65.70 (PTA, J(Pt−P) = 1680 Hz). IR: ν/cm−1 3413
(N−H stretch), 3257 (N−H stretch), 1621 (CN stretch), 1563
(CN stretch). Anal. Found: C, 29.63; H, 3.23; N, 13.91. Calcd for
C15H19Cl2N6PPtS: C, 29.42; H, 3.13; N, 13.72. ESI-MS: m/z 613.0 [M
+ H]+, 100%.
P. falciparum in Vitro Assay. The test samples were tested in
triplicate on one occasion against chloroquine-sensitive (CQS) D10
strain and chloroquine-resistant (CQR) Dd2 strain of Plasmodium
falciparum. Continuous in vitro cultures of asexual erythrocyte stages of
P. falciparum were maintained using a modified method.79 Quantitative
assessment of antiplasmodial activity in vitro was determined via the
Synthesis of Complex 5. Bis(diphenylphosphino)ferrocene (0.0657
g, 0.118 mmol) was reacted with complex 2 (0.106 g, 0.0580 mmol).
5796
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Organometallics
Article
parasite lactate dehydrogenase assay using a modified method.80 The
test samples were prepared to a 20 mg/cm3 stock solution in 100%
DMSO and sonicated to enhance solubility. Samples were tested as a
suspension if not completely dissolved. Stock solutions were stored at
−20 °C. Further dilutions were prepared on the day of the experiment.
Chloroquine diphosphate (Sigma) was used as the reference drug in all
experiments. A full dose−response was performed for all compounds
to determine the concentration inhibiting 50% of parasite growth
(IC50 value). Test samples were tested at a starting concentration of
100 μg/cm3, which was then serially diluted 2-fold in complete
medium to give 10 concentrations, with the lowest concentration
being 0.2 μg/cm3. The same dilution technique was used for all
samples. CQ was tested at a starting concentration of 100 ng/cm3
against the CQR strain and 1000 ng/cm3 against the CQS strain. The
highest concentration of solvent (0.5%) to which the parasite was
exposed had no measurable effect on the parasite viability (data not
shown). The IC50 values were obtained using a nonlinear dose−
response curve fitting analysis via Graph Pad Prism v.4.0 software.
T. vaginalis in Vitro Assay. Cultures of T. vaginalis T1 strain were
grown in 5 cm3 completed TYM Diamond’s media in a 37 °C
incubator for 24 h. One hundred millimolar stocks of the compounds
were made by dissolving in DMSO, were screened against T1 stain of
T. vaginalis. Samples were tested as a suspension if not completely
dissolved. Cells untreated and inoculated with 5 μL of DMSO were
used as controls. A 5 μL portion of 100 mM stock of the compound
library was inoculated for a final concentration of 100 μM. Results
were calculated on the basis of counts utilizing a hemocytometer after
24 h. For IC50 values, increasing concentrations of the compound (0−
100 μM) were tested for inhibitory activity, and concentrations that
inhibited at approximately 50% were then obtained by linear
regression analysis. These predicted IC50 values were then confirmed
by direct testing on T. vaginalis strain T1 as described above.
A2780 and A2780cisR Cancer in Vitro Assay. The human
A2780 and A2780cisR ovarian carcinoma cells were obtained from the
European Collection of Cell Cultures (Salisbury, U.K.). Cells were
grown routinely in RPMI-1640 medium with 10% fetal calf serum
(FCS) and antibiotics at 37 °C and 5% CO2. Cytotoxicity was
determined using the MTT assay (MTT = 3(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide). Cells were seeded
in 96-well plates as monolayers with 100 cm3 of cell solution
(approximately 20 000 cells) per well and preincubated for 24 h in
medium supplemented with 10% FCS. Compounds were prepared as a
DMSO solution and then dissolved in the culture medium and serially
diluted to the appropriate concentration, to give a final DMSO
concentration of 1%. A 100 cm3 portion of the drug solution was
added to each well and the plates were incubated for another 72 h.
Subsequently, MTT (5 mg/cm3 solution) was added to the cells, and
the plates were incubated for a further 2 h. The culture medium was
aspirated, and the purple formazan crystals formed by the
mitochondrial dehydrogenase activity of vital cells were dissolved in
DMSO. The optical density, directly proportional to the number of
surviving cells, was quantified at 540 nm using a multiwell plate reader,
and the fraction of surviving cells was calculated from the absorbance
of untreated control cells. The evaluation is based on means from
three independent experiments, each comprising three microcultures
per concentration level.
the assay was carried out using the pyridine−ferrichrome method
developed by Ncokazi and Egan.81 A solution of 50% (v/v) pyridine,
30% (v/v) H2O, 20% (v/v) acetone, and 0.2 M HEPES buffer (pH
7.4) was prepared and 32 μL added to each well to give a final pyridine
concentration of 5% (v/v). Acetone (60 μL) was then added to assist
with hematin dispersion. The UV−vis absorbance of the plate wells
was read on a SpectraMax plate reader. Sigmoidal dose−response
curves were fitted to the absorbance data using GraphPad Prism v3.02.
ASSOCIATED CONTENT
■
S
* Supporting Information
CIF files giving crystallographic data for complexes 2·3DMSO,
3·2DMSO, and 4. This material is available free of charge via
the Internet at http://pubs.acs.org. CCDC 866790, 866791,
and 866792 also contain supplementary crystallographic data
for this paper. These data can be obtained free of charge at
www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cam-
bridge Crystallographic Data Centre, 12, Union Road, Cam-
bridge CB2 1EZ, U.K.; fax (internat.) +44-1223/336-033; e-
mail deposit@ccdc.cam.ac.uk).
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +27-21-6505279. Fax: +27-21-6505195. E-mail: gregory.
smith@uct.ac.za.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Professor Timothy J. Egan and Miss Kathryn Wicht of the
University of Cape Town are gratefully acknowledged for their
assistance with β-hematin inhibition assays. Financial support
from the University of Cape Town, the National Research
Foundation (NRF) of South Africa, and the Medical Research
Council (MRC) of South Africa is gratefully acknowledged.
Anglo American Platinum Ltd. is kindly acknowledged for the
donation of metal salts. Even though the work is supported by
the MRC, the views and opinions expressed are not those of
the MRC but of the authors of the material produced or
publicized.
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c; a (Å), 14.7265(6); b (Å), 30.6788(14); c (Å), 14.2547(6); β (deg),
111.9020(10); V (Å3), 5975.3(4); Z, 4; Dc (g cm−3), 2.285; μ (mm−1),
9.987; θ range for data collection (deg), 1.63−28.33; limiting indices,
−19 < h < 18, −40 < k < 40, −19 < l < 19; measured reflections, 67
775; reflections used (Rint), 7432 (0.0358); parameters, 342; R1,
0.0204; wR2, 0.0454; goodness of fit on F2, 1.068.
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formula weight, 873.75; crystal system, orthorhombic; space group,
Pca21; a (Å), 15.9511(4); b (Å), 9.2259(2); c (Å), 23.0990(6); β
(deg), 90; V (Å3), 3399.32(14); Z, 4; Dc (g cm−3), 1.707; μ (mm−1),
4.550; θ range for data collection (deg), 3.49−28.28; limiting indices,
−21 < h < 21, −12 < k < 12, −30 < l < 30; measured reflections, 136
324; reflections used (Rint), 8405 (0.0498); parameters, 414; R1,
0.0214; wR2, 0.0382; goodness of fit on F2, 1.145.
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612.38; crystal system, monoclinic; space group, P21/c; a (Å),
19.176(4); b (Å), 6.0493(12); c (Å), 17.977(4); β (deg), 114.13(3);
V (Å3), 1903.2(7); Z, 4; Dc (g cm−3), 2.137; μ (mm−1), 7.860; θ range
for data collection (deg), 3.36−28.28; limiting indices, −25 < h < 23,
−8 < k < 0, 0 < l < 23; measured reflections, 4703; reflections used
(Rint), 4703 (0.0000); parameters, 237; R1, 0.0182; wR2, 0.0389;
goodness of fit on F2, 1.126.
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