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Half-Sandwich Arene Ruthenium(II) and Osmium(II) Thiosemicarbazone Complexes: Solution Behavior and Antiproliferative Activity.
We
report the synthesis, characterization, and antiproliferative
activity of organo-osmium(II) and organo-ruthenium(II) half-sandwich
complexes [(η 6 - p -cym)Os(L)Cl]Cl
( 1 and 2 ) and [(η 6 - p -cym)Ru(L)Cl]Cl ( 3 and 4 ), where
L = N -(2-hydroxy)-3-methoxybenzylidenethiosemicarbazide
( L1 ) or N -(2,3-dihydroxybenzylidene)-3-phenylthiosemicarbazide
( L2 ), respectively. X-ray crystallography showed that
all four complexes possess half-sandwich pseudo-octahedral “three-legged
piano-stool” structures, with a neutral N,S-chelating thiosemicarbazone
ligand and a terminal chloride occupying three coordination positions.
In methanol, E / Z isomerization of
the coordinated thiosemicarbazone ligand was observed, while in an
aprotic solvent like acetone, partial dissociation of the ligand occurs,
reaching complete displacement in a more coordinating solvent like
DMSO. In general, the complexes exhibited good activity toward A2780
ovarian, A2780Cis cisplatin-resistant ovarian, A549 lung, HCT116 colon,
and PC3 prostate cancer cells. In particular, ruthenium complex 3 does not present cross-resistance with the clinical drug
cisplatin in the A2780 human ovarian cancer cell line. The complexes
were more active than the free thiosemicarbazone ligands, especially
in A549 and HCT116 cells with potency improvements of up to 20-fold
between organic ligand L1 and ruthenium complex 1 .
## Introduction
Introduction The discovery of highly
efficient anticancer drugs with increased
selectivity and less toxic side effects is an area of intense research
in bioinorganic chemistry. 1 Thiosemicarbazones
(TSCs) and their metal complexes display a wide spectrum of biological
activities, 2 − 5 in particular they possess anticancer, antibacterial, and antiviral
properties. 6 − 8 A variety of cellular mechanisms of action appears
to be involved in the activity of this class of ligands, 9 including the inhibition of cellular iron uptake
by transferrin, 10 − 12 the mobilization of iron from cells, 6 − 8 the inhibition of ribonucleotide reductase activity, 13 − 15 the up-regulation of the metastasis suppressor protein, N -myc downstream regulated gene I, 16 , 17 and the formation of redox active metal complexes that produce reactive
oxygen species. 11 , 18 − 20 Moreover, various
studies 21 have demonstrated that the biological
properties of TSC ligands can be modified and improved upon binding
to transition metal ions. 6 , 22 Metal coordination
presents an opportunity to improve synergistically the efficacy of
a biologically active organic scaffold 23 such as lipophilicity, which influences cell permeability. 24 Diversity arises from not only the choice of
the metal itself and its oxidation state but also from the type and
number of coordinated ligands, as well as the coordination geometry
of the complex. 23 Metal complexes
of TSCs are playing a promising role in anticancer
research, as is evident from the number of recent publications. 8 , 25 − 27 Platinum drugs are still widely used to treat cancer, 5 , 28 but their therapeutic use can be limited by intrinsic or acquired
resistance and by the occurrence of numerous deleterious side effects. 29 , 30 It is imperative, therefore, to develop new and more effective drugs.
Ruthenium, a second row transition metal, continues to attract much
attention, 31 , 32 as its complexes have long been
known to be well-suited for biological applications. 33 , 34 Organometallic Ru(II) complexes with half-sandwich structure have
demonstrated antiproliferative potential, 35 and there are numerous possibilities to modulate their biological
and pharmacological properties by the appropriate choice of the ligands. 11 , 36 In particular, the presence of a chelating ligand offers structural
stability and the opportunity to tune the electronic and steric features
of the complex. 37 Additional features to
be considered include water solubility and air stability. 37 , 38 The biological activity of osmium compounds has been much less explored,
perhaps because of the reputation of osmium (as osmium tetroxide)
as being highly toxic. 39 Nevertheless,
several half-sandwich piano-stool osmium(II) complexes have exhibited
promising in vitro activity and no cisplatin cross-resistance. 40 − 42 Investigations of osmium complexes as alternatives to ruthenium-based
anticancer agents have resulted in structurally diverse libraries
of osmium complexes with different oxidation states and nuclearity. 43 − 46 Organometallic chemistry offers a potentially rich field for
biological
and medicinal application; 47 however, lack
of understanding of the aqueous chemistry of the organometallic complexes
has emerged as a major obstacle for further developments. This is
particularly true for osmium(II) arene complexes. 48 Third row transition metals are more inert than those of
the first and second row. For example, aquation of Pt(II) chlorido
complexes often occurs up to 10 4 times more slowly compared
to the lighter congener Pd(II), and similarly, organo-Os(II) complexes
react typically 100 times more slowly than Ru(II). 49 − 51 However, reports
on ruthenium arene complexes have shown that their aqueous reactivity
is highly dependent on the nature of the coordinated ligands, as well
as the arene, rather than on the metal and its oxidation state alone. 52 , 53 The aim of the present study is to investigate the reactivity
in
solution and the antiproliferative activity toward cancer cells of
two Os(II) complexes [(η 6 - p -cym)Os(L)Cl]Cl
( 1 and 2 ) and two analogous Ru(II) complexes
[(η 6 - p -cym)Ru(L)Cl]Cl ( 3 and 4 ), where L = N -(2-hydroxy)-3-methoxybenzylidenethiosemicarbazide
( L1 ) or N -(2,3-dihydroxybenzylidene)-3-phenylthiosemicarbazide
( L2 ), respectively ( Figure 1 ). This type of ligands, which could in principle
be tridentate, can confer solution stability on their metal complexes;
moreover they have shown interesting cytotoxic properties 54 and could offer synergic antitumor activity.
Different substituents were considered for ligands L1 and L2 on both the phenyl ring and at the N(3) nitrogen,
since this can modulate lipophilicity and/or complex–substrate
interactions. The solution behavior of complexes 1 – 4 was studied both in a protic solvents such as methanol or
water/DMSO mixture and in coordinating aprotic solvents like acetone,
DMSO and DMF. The antiproliferative activity of 1 – 4 was evaluated for A2780 human ovarian carcinoma and its
cisplatin resistant variant A2780Cis, A549 lung, HCT116 colon, and
PC3 prostate tumor cell lines. Figure 1 Schematic representation of ligands L1 and L2 and corresponding osmium(II) and ruthenium(II)
complexes 1 – 4 .
## Results and Discussion
Results and Discussion Synthesis and Characterization of the Complexes Ligands N -(2-hydroxy)-3-methoxybenzylidenethiosemicarbazide
( L1 ) and N -(2,3-dihydroxybenzylidene)-3-phenylthiosemicarbazide
( L2 ) were synthesized according to previously reported
procedures. 54 , 55 The reactions between [(η 6 - p -cym)MCl 2 ] 2 (M =
Os and Ru) and the corresponding thiosemicarbazone ligands were carried
out in a mixture of dry CH 3 OH and CH 2 Cl 2 at ambient temperature and led to the isolation of pseudo-octahedral
complexes 1 – 4 of general formula
[(η 6 - p -cym)M(L)Cl]Cl in good yields.
The identity of the complexes was verified by 1 H NMR spectroscopy
and ESI-MS spectrometry, and their structures were confirmed by single
crystal X-ray crystallography. In all cases, the metal coordinates
to a chloride ion, a η 6 - p -cymene
ring and a NS -bidentate thiosemicarbazone chelating
ligand. One chloride is present as the counterion ( Figure 1 ). X-ray Crystallographic
Studies Crystals suitable for
X-ray diffraction analysis were obtained by slow evaporation of saturated
solutions in methanol for compounds 1 and 3 and in acetone for compounds 2 and 4 .
The crystal structures and atomic numbering schemes for [(η 6 - p -cym)Os( L1 )Cl]Cl ( 1 ), [(η 6 - p -cym)Os( L2 )Cl]Cl·(CH 3 ) 2 CO ( 2· (CH 3 ) 2 CO), [(η 6 - p -cym)Ru( L1 )Cl]Cl ( 3 ), and [(η 6 - p -cym)Ru( L2 )Cl]Cl · (CH 3 ) 2 CO ( 4· (CH 3 ) 2 CO) are shown in Figure 2 . Selected bond lengths and angles are listed in Table 1 , other crystallographic
data are reported in Table S1 . Complexes 1 and 3 crystallize in the orthorhombic system
with the chiral space group P 2 1 2 1 2 1 , while complexes 2 and 4 crystallize
in triclinic system with centrosymmetric space group P 1̅. Both 2 and 4 crystallize with
an acetone solvent molecule. The complexes adopt the expected half-sandwich
pseudo-octahedral “three-legged piano-stool” geometry
with η 6 - p -cymene as the seat and
the neutral N,S-chelating TSC ligand and a terminal chloride as the
three legs. The positive charge of the complex is balanced by a chloride
counterion. It is notable that in all the complexes, the ligand is
present as the E isomer. Figure 2 X-ray crystal structures
of complexes 1 – 4 with thermal ellipsoids
drawn at 50% probability. Hydrogens
are drawn as fixed-size spheres of 0.11 Å radius and solvent
molecules have been omitted for clarity. The edge-to-face stacking
between one of the hydrogens of the p -cymene ring
and an aromatic ring of the thiosemicarbazone ligands is indicated. Table 1 Selected Bond Lengths
(Å) and
Angles (deg) for Complexes 1 – 4 bond
distance (Å) bond angle (deg) 1 Os1–Cl1 2.4113(12) S1–Os1–Cl1 86.52(4) Os1–S1 2.3551(13) N4–Os1–Cl1 81.63(10) Os1–N4 2.118(4) N4–Os1–S1 81.63(11) S1–C2 1.695(5) H14–CE1 2.563 2· (CH 3 ) 2 CO Os1–Cl1 2.4030(5) S8–Os1–Cl1 87.81(2) Os1–S8 2.3527(5) N4–Os1–Cl1 83.44(5) Os1–N10 2.1227(17) N10–Os1–S8 81.79(5) S8–C8 1.693(2) H21–CE1 2.500 3 Ru1–Cl1 2.4046(11) S1–Ru1–Cl1 86.90(4) Ru1–S1 2.3501(10) N4–Ru1–Cl1 83.06(9) Ru1–N4 2.125(3) N4–Ru1–S1 81.95(10) S1–C2 1.695(4) H14–CE1 2.548 4· (CH 3 ) 2 CO Ru1–Cl1 2.3993(3) S8–Ru1–Cl1 88.338(11) Ru1–S8 2.3508(3) N10–Ru1–Cl1 84.77(3) Ru1–N10 2.1256(9) N10–Ru1–S8 81.94(3) S8–C8 1.6923(12) H21–CE1 2.486 In 1 and 3 , the uncoordinated chloride
anion forms a NH···Cl hydrogen bond of 3.034(4) Å
and 175.1° for 1 , and 3.031(3) and 176.7° for 3 . In 2 and 4 , a similar H-bond
occurs between the uncoordinated chloride and the 3-OH group of the
aromatic ring with a bond distance OH···Cl of 3.0651(16)
Å and 169.0° for 2 , and 3.0605(9) Å and
168.7° for 4 . The thiosemicarbazone ligands bind
to the metal center through the imine nitrogen and the thione sulfur
forming a five member chelate ring with an angle of 82° for N–Ru–S,
indicating a distortion from a regular octahedron, in analogy with
similar Ru–arene thiosemicarbazone complexes. 56 The length of the S–C bond (∼1.69 Å)
is in accord with a double bond nature; in the free ligands, it is
∼1.69–1.70 Å. 57 − 59 It is worth noting
that in some osmium(II) and ruthenium(II) arene
complexes the potentially NNO tridentate hydrazone ligands behave
as NN bidentate ligands. It has been highlighted that the ligands
are not flexible enough to occupy a facial arrangement in the complex
and are therefore bidentate. 60 An analogous
situation could occur with L1 and L2 that
can span the three facial coordination sites of the metal only with
difficulty. Interestingly, these hydrazone ligands were found in both E and Z configuration upon complexation
with Ru(II) and Os(II). The dihedral angles between the aromatic ring
plane and the thiosemicarbazones are around 70° in complexes 1 and 3 and about 78° in 2 and 4 . Usually, this type of ligand adopts a flat conformation: 58 , 59 , 61 In our structures, the lack of
coplanarity is related to metal coordination. In the crystal structures
of 1 – 4 , the same T-shaped edge-to-face
stacking π-interactions, between one of the hydrogens of the p -cymene ring and the π electron density of the aromatic
ring of the thiosemicarbazone ligands, are observed (distances from
2.50 to 2.86 Å, Figure 2 ). Solution Studies 1 H NMR
studies were used
to investigate the stability of the four complexes in various solvents. 1 H NMR spectra of 1 – 4 were
first recorded in MeOD- d 4 , due to their
low solubility in chlorinated solvents such as chloroform or dichloromethane.
For all the metal complexes, the spectra displayed just one set of
signals, corresponding to the E isomer of the bidentate
ligand coordinated to the metal center, the isomer in the crystallized
complexes. The aromatic protons of the thiosemicarbazone ligands displayed
peaks between 6.5 and 8.2 ppm, and the iminic protons displayed peaks
between 8.7 and 8.9 ppm, as expected for the ligand in the E form. 55 , 62 The complexes contain chiral
metal centers and in the 1 H NMR spectra recorded at 298
K a doublet is present for each p -cymene proton in
the range 4.90–5.90 ppm; the isopropyl methyl groups appear
as two doublets at 1.1 and 1.2 ppm. The resonance of one proton of
the p -cymene ring displays a marked high-field shift
in comparison with the other p -cymene protons, in
particular up to 4.90 ppm for osmium compounds 1 and 2 and 4.87 for ruthenium 3 and 4 ( Figure 3 ). This
is likely due to edge-to-face π-interaction between the C–H
hydrogen and the aromatic ring of the TSC ligand in the E form, as observed previously in analogous systems. 21 , 63 Figure 3 Aromatic
region of the time-dependent 1 H NMR spectrum
of 1 in MeOD- d 4 at T = 298 K followed over 30 days. E and Z isomers are labeled as a and b sets, respectively. The percentage of the Z isomer ( b set) increases with time. The time dependence of the 1 H NMR spectra
of 1 – 4 (5 mM) in MeOD- d 4 was monitored over 30 days at 298 K, and is illustrated
for complex 1 in Figure 3 . As shown in Figure 3 , a second set of peaks started to appear after 24
h (set b ) and increased in intensity until a 1:1
ratio for the
two species was reached over a period of 21 days. Variable-temperature 1 H NMR spectra were recorded from 298 to 323 K over a period
of 2 h. The 1:1 ratio of the a / b peak areas for the two species recorded at t =
30 days did not change over this temperature range (data not shown).
NOESY experiments carried out for 1 at t = 30 days, gave evidence that in the b set of peaks
there is an interaction between the iminic hydrogen of the ligand
and one of the aromatic protons of the p -cymene ( Figure S1 ); this interaction is absent in the a set. A possible explanation for the presence, in solution,
of two species (corresponding to set a and set b ) is the establishment of an E / Z equilibrium for coordinated ligand L1 ( Figure 4 ). The presence of
both the E and the Z isomers of
the ligand coordinated to the metal center would explain the interaction
of the iminic proton with the p -cymene moiety, observed
for set b in the NOESY experiment. This interaction
is possible only for a Z conformation of the ligand
and not with the E conformation. TSCs are known to
undergo E / Z interconversion not
only as free ligands but also upon coordination (for a mechanistic
insight see ref ( 64 ) and references therein). Figure 4 (A) E / Z interconversion
for L1 and L2 . (B) Chemical structures of
the E and Z isomers of ligand L1 in the complex 1 ; the interaction between
the iminic
proton and one proton of the p -cymene is depicted
by circles. The increase in the percentage
of Z isomer suggests that the presence
of a protic solvent could lead to the formation of a negative charge
on the iminic nitrogen and to the rotation around the single bond,
resulting in the isomerization and the formation of the Z isomer, as proposed in Scheme 1 . This mechanism is supported by the 1 H
NMR spectrum of the crystals of the complexes in methanol. In the
X-ray crystal structures of 1 and 3 , obtained
from a methanol solution, the ligand is in the E conformation,
but the 1 H NMR spectra of the same crystals recorded in
MeOD- d 4 showed the presence of both isomers
of the ligands after 24 h, suggesting that the solvent plays a crucial
role in the isomerization process. Recently, examples of pentamethylcyclopentadienyl
iridium(III) complexes with TSCs ligands that crystallize with the
coordinated ligand either with E or Z conformation have been reported, confirming the possibility of having
both isomers in organometallic complexes. 65 Scheme 1 Proposed Mechanism for the E / Z Interconversion
Process of the Coordinated Ligand L1 for Metal Complexes 1 and 3 in Methanol Analysis of the data provides evidence that the interconversion
is slightly faster for the ruthenium compound: at 298 K the Z isomer takes 2 weeks to reach the equilibrium with the E isomer (1:1 ratio), whereas 3 weeks are required for the
osmium complex. The situation is slightly different for complexes 2 and 4 . For these complexes a second set of
signals arises over time (1:1 ratio at t = 7 days
and 298 K, Figure S2 ). However, the 1 H NMR spectra of these complexes show broad signals in the
aromatic region for the Z isomer ( Figure S2 ). For complex 4 , for example, at t = 7 days only very broad overlapping signals can be seen
( Figure S3 ). The presence of two hydroxyl
groups on the aromatic ring of the coordinated ligand perhaps gives
rise to exchange processes or paramagnetic species which broaden signals
in the 1 H NMR spectra. Due to the long-time scale
of the NMR experiments and the catecholic
nature of ligand L2 , complexes 2 and 4 can be subjected to oxidation. UV–visible spectroscopy
was performed in order to verify whether the catechol moiety of 2 is involved in oxidation processes in methanol solution.
The development of a stable and strong absorption band of a methanol
solution of 2 around 337 nm, related to π–π*
transition of the catechol aromatic ring, was followed over 3 days
in air ( Figure S4 ). No changes in the UV–vis
spectra were detected, indicating that the catechol moiety is not
involved in redox processes. 1 H NMR spectra of complexes 1 – 4 were also recorded in an aprotic solvent,
acetone. In this case, two different sets of signals were observed
immediately after dissolution in acetone- d 6 at 298 K for all the complexes ( Figure 5 ). Comparison with the 1 H NMR
obtained in MeOD at t = 0 indicates that one set
of signals is related to the parent organometallic compound, as shown
in Figure 5 for compound 2 . The presence of free ligand was excluded by comparison
with the 1 H NMR spectrum of L2 recorded in
acetone- d 6 . It is notable that the 1 H NMR spectra change with time at 298 K. As shown in Figure 5 , both a shift and
a modification of the pattern of the signals is observed over 2 days.
After this time the two sets of signals did not change their ratio
(ca. 1:1.2). Probably, the second set of signals is due to a species
containing a coordinated solvent molecule ( Figure 5 ). Figure 5 Aromatic region of the 1 H NMR spectrum
of 2 recorded in acetone- d 6 at 298 K and
followed over 7 days. Red circles indicate proton resonances related
to the species with a coordinated solvent molecule. Due to the limited aqueous solubility of the metal
complexes, antiproliferative
cell assays were performed using stock solutions prepared by dissolution
of the compound in DMSO followed by dilution with water (final concentration
of DMSO 0.5%). The hydrolysis processes are of interest as indicators
of the stability of the pro-drug under such biological testing conditions;
therefore, the solution behavior of 1 – 4 was investigated also in DMSO- d 6 . In
the 1 H NMR spectra of 1 and 3 in DMSO- d 6 recorded at 298 K, three
different sets of signals were observed. A comparison with the 1 H NMR spectrum of L1 obtained in the same solvent
confirmed the presence of free ligand in a 1:1 ratio versus the metal
complex ( Figure 6 ).
The two doublets observed at 6.08 and 6.00 ppm can be assigned to
a complex of the type [Os(η 6 - p -cym)(DMSO) 2 Cl]Cl, in a 1:1 ratio with parent organometallic complex 1 and free ligand L1 . As recently pointed out
in the literature, such a pattern of signals frequently arises after
displacement of the organic ligand in [Ru(η 6 - p -cym)( L )Cl 2 ] complexes. 66 Ligand dissociation was apparent visually; addition
of DMSO to the orange powder of 1 leads to an orange
solution that became green as dissociation proceeded. Figure 6 Comparison of the aromatic
region of the 1 H NMR of complex 1 (upper spectrum)
and that of the corresponding free ligand L1 (lower spectrum)
in DMSO- d 6 at t = 0 and
298 K. Complexes 2 and 4 in DMSO- d 6 gave a complex pattern
of 1 H NMR signals.
Comparison with the spectrum of 2 in MeOD at t = 0 indicates that the major set of signals is related
to parent compound 2 . However, other sets of signals
of lower intensity were observed ( Figure S5 ). Both sets of signals for free ligand L2 and the [Os(η 6 - p -cym)(DMSO) 2 Cl]Cl species, each
accounted for about 10% of the major set. In case of 2 and 4 in DMSO, however, a further set of signals, corresponding
to about 25% of the major set, arises in the 1 H NMR spectrum.
A possible explanation for this set of signals is the presence of
a monosolvated species of the type [Os(η 6 - p -cym)(DMSO)( L )Cl]Cl ( Figure S5 ). To determine whether the degradation process correlates
with the
concentration of DMSO, the analysis was performed using a solution
of D 2 O–(10%)DMSO, monitored for 24 h to mimic the
biological test conditions. The 1 H NMR spectra of the solutions
of 1 – 4 displayed in all cases broad
signals, with complicated splitting patterns, indicating the presence
of several dissociation equilibria in solution. This behavior prevented
the use of DMSO in biological tests; therefore, the possibility of
preparing stock solutions of the compounds in DMF was investigated.
In this case, all complexes 1 – 4 presented
a unique set of signals, stable over 7 days at 298 K ( Figure S6 ). Anticancer Activity The antiproliferative activity
of ligands L1 and L2 and of the related
osmium and ruthenium complexes 1 – 4 toward A549 lung, A2780 ovarian, HCT116 colon, and PC3 prostate
human cancer cells lines was investigated. All experiments included
untreated negative controls and cells treated with the clinical drug
cisplatin (CDDP) as positive control. The anticancer activity of the
organometallic complexes was investigated by performing dose–response
studies in the various cell lines ( Figure S7 ). A stock solution of each compound was prepared in cell culture
medium with DMF to aid solubilization. IC 50 values (concentrations
which caused 50% of cell growth inhibition) were determined as duplicates
of triplicates in two independent sets of experiments and are reported
in Table 2 . Importantly,
all experiments designed to determine the antiproliferative activity
of the complexes included three set of controls (negative, vehicle,
and positive). The cell survival in the negative controls and the
vehicle controls were compared, and in all cases, the differences
were not statistically significant to 99%. This indicates that the
DMF in the sample solutions of complexes 1 – 4 is not toxic and does not interfere with the measurements.
Hence, the effects on cell survival observed arise only from the activity
of the ligands or the metal-based complexes. Table 2 IC 50 Values (μM)
for L1 and L2 and Related Metal Complexes 1 – 4 towards Human Ovarian (A2780), Cisplatin-Resistant
Ovarian (A2780Cis), Lung (A549), Colon (HCT116), and Prostate (PC3)
Cancer Cell Lines a cell
lines IC 50 (μM) resistance factors compound A2780 A2780Cis A549 HCT116 PC3 A2780Cis/A2780 L1 0.85 ± 0.03 0.12 ± 0.02 42 ± 2 30.6 ± 0.5 6.1 ± 0.1 0.14 L2 0.27 ± 0.02 1.23 ± 0.08 23 ± 1 33 ± 5 4.6 ± 0.2 4.55 1 1.60 ± 0.02 6.6 ± 0.9 2.4 ± 0.2 24 ± 2 21 ± 1 4.12 2 0.75 ± 0.08 7.2 ± 0.1 17 ± 1 2.7 ± 0.2 1.60 ± 0.08 9.60 3 4.2 ± 0.3 5.6 ± 0.8 10.5 ± 0.3 19 ± 1 1.33 4 0.36 ± 0.03 1.25 ± 0.06 1.64 ± 0.08 1.38 ± 0.04 3.47 CDDP 1.2 ± 0.2 13.5 ± 0.3 3.1 ± 0.2 5.2 ± 0.1 9.8 ± 0.4 11.25 a Clinical drug cisplatin (CDDP)
is used as positive control. Both thiosemicarbazones L1 and L2 are
highly potent toward ovarian cell lines A2780 and A2780Cis. L1 in particular exhibits IC 50 values of 0.85 and
0.12 μM, respectively. Ligand L2 shows submicromolar
activity in A2780 cells (0.27 μM) and low micromolar potency
in A2780Cis (1.23 μM). Although the metal complexes are less
active than their corresponding ligands, they show IC 50 values of the same order of magnitude as that of CDDP in the parental
cell line and improved resistant factors. Resistance factors, calculated
as the ratio between the antiproliferative activity in the parental
cell line and its resistant derivative, give an indication of whether
the cellular mechanisms of resistance to CDDP are involved in the
mechanism of action of the novel metal complexes. It has been proposed
that the underlying resistance associated with A2780Cis involves a
2-fold more efficient efflux of the platinum drug and a consequent
reduction in cellular accumulation as compared to the parental A2780,
as well as an increase in DNA repair mechanisms. 67 The corresponding resistance factor for CDDP is 11.25.
Complexes 3 and 4 are particularly promising
for overcoming CDDP resistance as they have the lowest factors of
1.33 and 3.4, respectively, highlighting the importance of the substituents
in the chelating ligands and in particular the incorporation of a
phenyl ring at the N(3) of the thiosemicarbazone, when compared to
−NH 2 . For the A549 lung and HCT116 colon cancer
cells, there is an improvement in the activity of metal complexes
compared to their corresponding ligands, with thiosemicarbazones L1 and L2 exhibiting an order of magnitude higher
IC 50 concentrations than the clinical drug CDDP. It is
important to highlight the 17-fold improvement in potency between L1 and its osmium complex 1 increasing from 42
to 2.4 μM in A549 cells, as well as the 12-fold increase in
potency between L2 (33 μM) and osmium complex 2 (2.7 μM) and 20-fold compared to ruthenium complex 4 (1.64 μM) in the HCT116 colon cell line. The prostate
cancer cell line PC3 shows mixed results with increments in potency
for complexes 2 and 4 derived from L2 but reduction in anticancer activity for complexes 1 and 3 derived from L1 . The former
are more active than CDDP in this cell line. The observed trends in
the anticancer activity, across all cell lines and all compounds,
point toward complexes with ligand L2 being more potent
than those which bear ligand L1 , and within this, ruthenium
complex 4 has a more potent activity compared to the
osmium analogue. This highlights that the anticancer activity of the
complexes is not only the result of the metal center per se , but also of the nature of the substituents on the thiosemicarbazone
ligands.
## Synthesis and Characterization of the Complexes
Synthesis and Characterization of the Complexes Ligands N -(2-hydroxy)-3-methoxybenzylidenethiosemicarbazide
( L1 ) and N -(2,3-dihydroxybenzylidene)-3-phenylthiosemicarbazide
( L2 ) were synthesized according to previously reported
procedures. 54 , 55 The reactions between [(η 6 - p -cym)MCl 2 ] 2 (M =
Os and Ru) and the corresponding thiosemicarbazone ligands were carried
out in a mixture of dry CH 3 OH and CH 2 Cl 2 at ambient temperature and led to the isolation of pseudo-octahedral
complexes 1 – 4 of general formula
[(η 6 - p -cym)M(L)Cl]Cl in good yields.
The identity of the complexes was verified by 1 H NMR spectroscopy
and ESI-MS spectrometry, and their structures were confirmed by single
crystal X-ray crystallography. In all cases, the metal coordinates
to a chloride ion, a η 6 - p -cymene
ring and a NS -bidentate thiosemicarbazone chelating
ligand. One chloride is present as the counterion ( Figure 1 ).
## X-ray Crystallographic
Studies
X-ray Crystallographic
Studies Crystals suitable for
X-ray diffraction analysis were obtained by slow evaporation of saturated
solutions in methanol for compounds 1 and 3 and in acetone for compounds 2 and 4 .
The crystal structures and atomic numbering schemes for [(η 6 - p -cym)Os( L1 )Cl]Cl ( 1 ), [(η 6 - p -cym)Os( L2 )Cl]Cl·(CH 3 ) 2 CO ( 2· (CH 3 ) 2 CO), [(η 6 - p -cym)Ru( L1 )Cl]Cl ( 3 ), and [(η 6 - p -cym)Ru( L2 )Cl]Cl · (CH 3 ) 2 CO ( 4· (CH 3 ) 2 CO) are shown in Figure 2 . Selected bond lengths and angles are listed in Table 1 , other crystallographic
data are reported in Table S1 . Complexes 1 and 3 crystallize in the orthorhombic system
with the chiral space group P 2 1 2 1 2 1 , while complexes 2 and 4 crystallize
in triclinic system with centrosymmetric space group P 1̅. Both 2 and 4 crystallize with
an acetone solvent molecule. The complexes adopt the expected half-sandwich
pseudo-octahedral “three-legged piano-stool” geometry
with η 6 - p -cymene as the seat and
the neutral N,S-chelating TSC ligand and a terminal chloride as the
three legs. The positive charge of the complex is balanced by a chloride
counterion. It is notable that in all the complexes, the ligand is
present as the E isomer. Figure 2 X-ray crystal structures
of complexes 1 – 4 with thermal ellipsoids
drawn at 50% probability. Hydrogens
are drawn as fixed-size spheres of 0.11 Å radius and solvent
molecules have been omitted for clarity. The edge-to-face stacking
between one of the hydrogens of the p -cymene ring
and an aromatic ring of the thiosemicarbazone ligands is indicated. Table 1 Selected Bond Lengths
(Å) and
Angles (deg) for Complexes 1 – 4 bond
distance (Å) bond angle (deg) 1 Os1–Cl1 2.4113(12) S1–Os1–Cl1 86.52(4) Os1–S1 2.3551(13) N4–Os1–Cl1 81.63(10) Os1–N4 2.118(4) N4–Os1–S1 81.63(11) S1–C2 1.695(5) H14–CE1 2.563 2· (CH 3 ) 2 CO Os1–Cl1 2.4030(5) S8–Os1–Cl1 87.81(2) Os1–S8 2.3527(5) N4–Os1–Cl1 83.44(5) Os1–N10 2.1227(17) N10–Os1–S8 81.79(5) S8–C8 1.693(2) H21–CE1 2.500 3 Ru1–Cl1 2.4046(11) S1–Ru1–Cl1 86.90(4) Ru1–S1 2.3501(10) N4–Ru1–Cl1 83.06(9) Ru1–N4 2.125(3) N4–Ru1–S1 81.95(10) S1–C2 1.695(4) H14–CE1 2.548 4· (CH 3 ) 2 CO Ru1–Cl1 2.3993(3) S8–Ru1–Cl1 88.338(11) Ru1–S8 2.3508(3) N10–Ru1–Cl1 84.77(3) Ru1–N10 2.1256(9) N10–Ru1–S8 81.94(3) S8–C8 1.6923(12) H21–CE1 2.486 In 1 and 3 , the uncoordinated chloride
anion forms a NH···Cl hydrogen bond of 3.034(4) Å
and 175.1° for 1 , and 3.031(3) and 176.7° for 3 . In 2 and 4 , a similar H-bond
occurs between the uncoordinated chloride and the 3-OH group of the
aromatic ring with a bond distance OH···Cl of 3.0651(16)
Å and 169.0° for 2 , and 3.0605(9) Å and
168.7° for 4 . The thiosemicarbazone ligands bind
to the metal center through the imine nitrogen and the thione sulfur
forming a five member chelate ring with an angle of 82° for N–Ru–S,
indicating a distortion from a regular octahedron, in analogy with
similar Ru–arene thiosemicarbazone complexes. 56 The length of the S–C bond (∼1.69 Å)
is in accord with a double bond nature; in the free ligands, it is
∼1.69–1.70 Å. 57 − 59 It is worth noting
that in some osmium(II) and ruthenium(II) arene
complexes the potentially NNO tridentate hydrazone ligands behave
as NN bidentate ligands. It has been highlighted that the ligands
are not flexible enough to occupy a facial arrangement in the complex
and are therefore bidentate. 60 An analogous
situation could occur with L1 and L2 that
can span the three facial coordination sites of the metal only with
difficulty. Interestingly, these hydrazone ligands were found in both E and Z configuration upon complexation
with Ru(II) and Os(II). The dihedral angles between the aromatic ring
plane and the thiosemicarbazones are around 70° in complexes 1 and 3 and about 78° in 2 and 4 . Usually, this type of ligand adopts a flat conformation: 58 , 59 , 61 In our structures, the lack of
coplanarity is related to metal coordination. In the crystal structures
of 1 – 4 , the same T-shaped edge-to-face
stacking π-interactions, between one of the hydrogens of the p -cymene ring and the π electron density of the aromatic
ring of the thiosemicarbazone ligands, are observed (distances from
2.50 to 2.86 Å, Figure 2 ).
## Solution Studies
Solution Studies 1 H NMR
studies were used
to investigate the stability of the four complexes in various solvents. 1 H NMR spectra of 1 – 4 were
first recorded in MeOD- d 4 , due to their
low solubility in chlorinated solvents such as chloroform or dichloromethane.
For all the metal complexes, the spectra displayed just one set of
signals, corresponding to the E isomer of the bidentate
ligand coordinated to the metal center, the isomer in the crystallized
complexes. The aromatic protons of the thiosemicarbazone ligands displayed
peaks between 6.5 and 8.2 ppm, and the iminic protons displayed peaks
between 8.7 and 8.9 ppm, as expected for the ligand in the E form. 55 , 62 The complexes contain chiral
metal centers and in the 1 H NMR spectra recorded at 298
K a doublet is present for each p -cymene proton in
the range 4.90–5.90 ppm; the isopropyl methyl groups appear
as two doublets at 1.1 and 1.2 ppm. The resonance of one proton of
the p -cymene ring displays a marked high-field shift
in comparison with the other p -cymene protons, in
particular up to 4.90 ppm for osmium compounds 1 and 2 and 4.87 for ruthenium 3 and 4 ( Figure 3 ). This
is likely due to edge-to-face π-interaction between the C–H
hydrogen and the aromatic ring of the TSC ligand in the E form, as observed previously in analogous systems. 21 , 63 Figure 3 Aromatic
region of the time-dependent 1 H NMR spectrum
of 1 in MeOD- d 4 at T = 298 K followed over 30 days. E and Z isomers are labeled as a and b sets, respectively. The percentage of the Z isomer ( b set) increases with time. The time dependence of the 1 H NMR spectra
of 1 – 4 (5 mM) in MeOD- d 4 was monitored over 30 days at 298 K, and is illustrated
for complex 1 in Figure 3 . As shown in Figure 3 , a second set of peaks started to appear after 24
h (set b ) and increased in intensity until a 1:1
ratio for the
two species was reached over a period of 21 days. Variable-temperature 1 H NMR spectra were recorded from 298 to 323 K over a period
of 2 h. The 1:1 ratio of the a / b peak areas for the two species recorded at t =
30 days did not change over this temperature range (data not shown).
NOESY experiments carried out for 1 at t = 30 days, gave evidence that in the b set of peaks
there is an interaction between the iminic hydrogen of the ligand
and one of the aromatic protons of the p -cymene ( Figure S1 ); this interaction is absent in the a set. A possible explanation for the presence, in solution,
of two species (corresponding to set a and set b ) is the establishment of an E / Z equilibrium for coordinated ligand L1 ( Figure 4 ). The presence of
both the E and the Z isomers of
the ligand coordinated to the metal center would explain the interaction
of the iminic proton with the p -cymene moiety, observed
for set b in the NOESY experiment. This interaction
is possible only for a Z conformation of the ligand
and not with the E conformation. TSCs are known to
undergo E / Z interconversion not
only as free ligands but also upon coordination (for a mechanistic
insight see ref ( 64 ) and references therein). Figure 4 (A) E / Z interconversion
for L1 and L2 . (B) Chemical structures of
the E and Z isomers of ligand L1 in the complex 1 ; the interaction between
the iminic
proton and one proton of the p -cymene is depicted
by circles. The increase in the percentage
of Z isomer suggests that the presence
of a protic solvent could lead to the formation of a negative charge
on the iminic nitrogen and to the rotation around the single bond,
resulting in the isomerization and the formation of the Z isomer, as proposed in Scheme 1 . This mechanism is supported by the 1 H
NMR spectrum of the crystals of the complexes in methanol. In the
X-ray crystal structures of 1 and 3 , obtained
from a methanol solution, the ligand is in the E conformation,
but the 1 H NMR spectra of the same crystals recorded in
MeOD- d 4 showed the presence of both isomers
of the ligands after 24 h, suggesting that the solvent plays a crucial
role in the isomerization process. Recently, examples of pentamethylcyclopentadienyl
iridium(III) complexes with TSCs ligands that crystallize with the
coordinated ligand either with E or Z conformation have been reported, confirming the possibility of having
both isomers in organometallic complexes. 65 Scheme 1 Proposed Mechanism for the E / Z Interconversion
Process of the Coordinated Ligand L1 for Metal Complexes 1 and 3 in Methanol Analysis of the data provides evidence that the interconversion
is slightly faster for the ruthenium compound: at 298 K the Z isomer takes 2 weeks to reach the equilibrium with the E isomer (1:1 ratio), whereas 3 weeks are required for the
osmium complex. The situation is slightly different for complexes 2 and 4 . For these complexes a second set of
signals arises over time (1:1 ratio at t = 7 days
and 298 K, Figure S2 ). However, the 1 H NMR spectra of these complexes show broad signals in the
aromatic region for the Z isomer ( Figure S2 ). For complex 4 , for example, at t = 7 days only very broad overlapping signals can be seen
( Figure S3 ). The presence of two hydroxyl
groups on the aromatic ring of the coordinated ligand perhaps gives
rise to exchange processes or paramagnetic species which broaden signals
in the 1 H NMR spectra. Due to the long-time scale
of the NMR experiments and the catecholic
nature of ligand L2 , complexes 2 and 4 can be subjected to oxidation. UV–visible spectroscopy
was performed in order to verify whether the catechol moiety of 2 is involved in oxidation processes in methanol solution.
The development of a stable and strong absorption band of a methanol
solution of 2 around 337 nm, related to π–π*
transition of the catechol aromatic ring, was followed over 3 days
in air ( Figure S4 ). No changes in the UV–vis
spectra were detected, indicating that the catechol moiety is not
involved in redox processes. 1 H NMR spectra of complexes 1 – 4 were also recorded in an aprotic solvent,
acetone. In this case, two different sets of signals were observed
immediately after dissolution in acetone- d 6 at 298 K for all the complexes ( Figure 5 ). Comparison with the 1 H NMR
obtained in MeOD at t = 0 indicates that one set
of signals is related to the parent organometallic compound, as shown
in Figure 5 for compound 2 . The presence of free ligand was excluded by comparison
with the 1 H NMR spectrum of L2 recorded in
acetone- d 6 . It is notable that the 1 H NMR spectra change with time at 298 K. As shown in Figure 5 , both a shift and
a modification of the pattern of the signals is observed over 2 days.
After this time the two sets of signals did not change their ratio
(ca. 1:1.2). Probably, the second set of signals is due to a species
containing a coordinated solvent molecule ( Figure 5 ). Figure 5 Aromatic region of the 1 H NMR spectrum
of 2 recorded in acetone- d 6 at 298 K and
followed over 7 days. Red circles indicate proton resonances related
to the species with a coordinated solvent molecule. Due to the limited aqueous solubility of the metal
complexes, antiproliferative
cell assays were performed using stock solutions prepared by dissolution
of the compound in DMSO followed by dilution with water (final concentration
of DMSO 0.5%). The hydrolysis processes are of interest as indicators
of the stability of the pro-drug under such biological testing conditions;
therefore, the solution behavior of 1 – 4 was investigated also in DMSO- d 6 . In
the 1 H NMR spectra of 1 and 3 in DMSO- d 6 recorded at 298 K, three
different sets of signals were observed. A comparison with the 1 H NMR spectrum of L1 obtained in the same solvent
confirmed the presence of free ligand in a 1:1 ratio versus the metal
complex ( Figure 6 ).
The two doublets observed at 6.08 and 6.00 ppm can be assigned to
a complex of the type [Os(η 6 - p -cym)(DMSO) 2 Cl]Cl, in a 1:1 ratio with parent organometallic complex 1 and free ligand L1 . As recently pointed out
in the literature, such a pattern of signals frequently arises after
displacement of the organic ligand in [Ru(η 6 - p -cym)( L )Cl 2 ] complexes. 66 Ligand dissociation was apparent visually; addition
of DMSO to the orange powder of 1 leads to an orange
solution that became green as dissociation proceeded. Figure 6 Comparison of the aromatic
region of the 1 H NMR of complex 1 (upper spectrum)
and that of the corresponding free ligand L1 (lower spectrum)
in DMSO- d 6 at t = 0 and
298 K. Complexes 2 and 4 in DMSO- d 6 gave a complex pattern
of 1 H NMR signals.
Comparison with the spectrum of 2 in MeOD at t = 0 indicates that the major set of signals is related
to parent compound 2 . However, other sets of signals
of lower intensity were observed ( Figure S5 ). Both sets of signals for free ligand L2 and the [Os(η 6 - p -cym)(DMSO) 2 Cl]Cl species, each
accounted for about 10% of the major set. In case of 2 and 4 in DMSO, however, a further set of signals, corresponding
to about 25% of the major set, arises in the 1 H NMR spectrum.
A possible explanation for this set of signals is the presence of
a monosolvated species of the type [Os(η 6 - p -cym)(DMSO)( L )Cl]Cl ( Figure S5 ). To determine whether the degradation process correlates
with the
concentration of DMSO, the analysis was performed using a solution
of D 2 O–(10%)DMSO, monitored for 24 h to mimic the
biological test conditions. The 1 H NMR spectra of the solutions
of 1 – 4 displayed in all cases broad
signals, with complicated splitting patterns, indicating the presence
of several dissociation equilibria in solution. This behavior prevented
the use of DMSO in biological tests; therefore, the possibility of
preparing stock solutions of the compounds in DMF was investigated.
In this case, all complexes 1 – 4 presented
a unique set of signals, stable over 7 days at 298 K ( Figure S6 ).
## Anticancer Activity
Anticancer Activity The antiproliferative activity
of ligands L1 and L2 and of the related
osmium and ruthenium complexes 1 – 4 toward A549 lung, A2780 ovarian, HCT116 colon, and PC3 prostate
human cancer cells lines was investigated. All experiments included
untreated negative controls and cells treated with the clinical drug
cisplatin (CDDP) as positive control. The anticancer activity of the
organometallic complexes was investigated by performing dose–response
studies in the various cell lines ( Figure S7 ). A stock solution of each compound was prepared in cell culture
medium with DMF to aid solubilization. IC 50 values (concentrations
which caused 50% of cell growth inhibition) were determined as duplicates
of triplicates in two independent sets of experiments and are reported
in Table 2 . Importantly,
all experiments designed to determine the antiproliferative activity
of the complexes included three set of controls (negative, vehicle,
and positive). The cell survival in the negative controls and the
vehicle controls were compared, and in all cases, the differences
were not statistically significant to 99%. This indicates that the
DMF in the sample solutions of complexes 1 – 4 is not toxic and does not interfere with the measurements.
Hence, the effects on cell survival observed arise only from the activity
of the ligands or the metal-based complexes. Table 2 IC 50 Values (μM)
for L1 and L2 and Related Metal Complexes 1 – 4 towards Human Ovarian (A2780), Cisplatin-Resistant
Ovarian (A2780Cis), Lung (A549), Colon (HCT116), and Prostate (PC3)
Cancer Cell Lines a cell
lines IC 50 (μM) resistance factors compound A2780 A2780Cis A549 HCT116 PC3 A2780Cis/A2780 L1 0.85 ± 0.03 0.12 ± 0.02 42 ± 2 30.6 ± 0.5 6.1 ± 0.1 0.14 L2 0.27 ± 0.02 1.23 ± 0.08 23 ± 1 33 ± 5 4.6 ± 0.2 4.55 1 1.60 ± 0.02 6.6 ± 0.9 2.4 ± 0.2 24 ± 2 21 ± 1 4.12 2 0.75 ± 0.08 7.2 ± 0.1 17 ± 1 2.7 ± 0.2 1.60 ± 0.08 9.60 3 4.2 ± 0.3 5.6 ± 0.8 10.5 ± 0.3 19 ± 1 1.33 4 0.36 ± 0.03 1.25 ± 0.06 1.64 ± 0.08 1.38 ± 0.04 3.47 CDDP 1.2 ± 0.2 13.5 ± 0.3 3.1 ± 0.2 5.2 ± 0.1 9.8 ± 0.4 11.25 a Clinical drug cisplatin (CDDP)
is used as positive control. Both thiosemicarbazones L1 and L2 are
highly potent toward ovarian cell lines A2780 and A2780Cis. L1 in particular exhibits IC 50 values of 0.85 and
0.12 μM, respectively. Ligand L2 shows submicromolar
activity in A2780 cells (0.27 μM) and low micromolar potency
in A2780Cis (1.23 μM). Although the metal complexes are less
active than their corresponding ligands, they show IC 50 values of the same order of magnitude as that of CDDP in the parental
cell line and improved resistant factors. Resistance factors, calculated
as the ratio between the antiproliferative activity in the parental
cell line and its resistant derivative, give an indication of whether
the cellular mechanisms of resistance to CDDP are involved in the
mechanism of action of the novel metal complexes. It has been proposed
that the underlying resistance associated with A2780Cis involves a
2-fold more efficient efflux of the platinum drug and a consequent
reduction in cellular accumulation as compared to the parental A2780,
as well as an increase in DNA repair mechanisms. 67 The corresponding resistance factor for CDDP is 11.25.
Complexes 3 and 4 are particularly promising
for overcoming CDDP resistance as they have the lowest factors of
1.33 and 3.4, respectively, highlighting the importance of the substituents
in the chelating ligands and in particular the incorporation of a
phenyl ring at the N(3) of the thiosemicarbazone, when compared to
−NH 2 . For the A549 lung and HCT116 colon cancer
cells, there is an improvement in the activity of metal complexes
compared to their corresponding ligands, with thiosemicarbazones L1 and L2 exhibiting an order of magnitude higher
IC 50 concentrations than the clinical drug CDDP. It is
important to highlight the 17-fold improvement in potency between L1 and its osmium complex 1 increasing from 42
to 2.4 μM in A549 cells, as well as the 12-fold increase in
potency between L2 (33 μM) and osmium complex 2 (2.7 μM) and 20-fold compared to ruthenium complex 4 (1.64 μM) in the HCT116 colon cell line. The prostate
cancer cell line PC3 shows mixed results with increments in potency
for complexes 2 and 4 derived from L2 but reduction in anticancer activity for complexes 1 and 3 derived from L1 . The former
are more active than CDDP in this cell line. The observed trends in
the anticancer activity, across all cell lines and all compounds,
point toward complexes with ligand L2 being more potent
than those which bear ligand L1 , and within this, ruthenium
complex 4 has a more potent activity compared to the
osmium analogue. This highlights that the anticancer activity of the
complexes is not only the result of the metal center per se , but also of the nature of the substituents on the thiosemicarbazone
ligands.
## Conclusions
Conclusions Two new osmium(II) and
two ruthenium(II) half-sandwich complexes
[(η 6 - p -cym)M(L)Cl]Cl containing
a thiosemicarbazone ligand (L) were synthesized and characterized
by 1 H NMR, ESI-MS spectrometry and single crystal X-ray
crystallography. Complexes 1 – 4 are
structurally very similar and characterized by a distorted octahedral
geometry. In the crystal structures, the E configuration
of the thiosemicarbazone ligand was evident. In a protic solvent,
such as methanol, an interconversion takes
place and peaks for both E and Z isomers of the ligand appear in the 1 H NMR spectrum.
The conformational change in the ligand is probably promoted by the
interaction of the solvent with the acidic proton of the aromatic
ring. When the complexes were dissolved in the nonprotic, coordinating
acetone or in DMSO, solvation reactions prevailed. On the contrary,
in DMF solution, the complexes remained stable. Hence, DMF (5%) and
not DMSO was used to aid solubility for cancer cell screening. Promising
results were obtained, particularly toward HCT116 colon cancer cells,
in which the metal complexes are up to 20-fold more potent than corresponding
free ligand L2 . Ruthenium complex 3 shows
promising anticancer activity, and the possibility to overcome CDDP
resistance as demonstrated by the data for A2780 ovarian cancer cells
and its derived CDDP-resistant cell line A2780Cis. In fact all complexes
showed lower resistance factors than the clinical drug cisplatin.
Future work will aim at optimizing the pharmacological profiles of
these complexes, especially to increase stability under biological
testing conditions.
## Experimental Section
Experimental Section Materials All commercial reagents were used as received.
2-Hydroxy-3-methoxybenzaldehyde, 2,3-dihydroxybenzaldehyde, thiosemicarbazide,
and 4-phenylthiosemicarbazide were purchased from Sigma-Aldrich; OsCl 3 · n H 2 O and RuCl 3 · n H 2 O were from Alfa Aesar. All
reactions were performed under an inert atmosphere of nitrogen using
standard Schlenk line techniques, and all glassware was oven-dried
(120 °C) overnight. Dry solvents were purchased from Sigma-Aldrich
and stored under nitrogen. [(η 6 - p -cym)OsCl 2 ] 2 and [(η 6 - p -cym)RuCl 2 ] 2 were synthesized according
to literature procedures. 49 , 68 Cell Culture Cell
lines used in this work included
A2780 human ovarian carcinoma and its cisplatin-resistant variant
A2780Cis, A549 human caucasian lung carcinoma, HCT116 human colon
carcinoma, and PC3 human prostate carcinoma. They were all obtained
from the European Collection of Cell Cultures (ECACC), used between
passages 5 and 18 and were grown in Roswell Park Memorial Institute
medium (RPMI-1640) supplemented with 10% (v/v) of fetal calf serum,
1% (v/v) of 2 mM glutamine and 1% (v/v) penicillin/streptomycin. They
were grown as adherent monolayers at 310 K in a 5% CO 2 humidified
atmosphere and passaged at ca. 70–80% confluence. In
Vitro Growth Inhibition Assay Briefly,
5000 cells were seeded per well in 96-well plates. The cells were
preincubated in drug-free media at 310 K for 48 h before adding different
concentrations of the compounds to be tested. A stock solution of
the metal complex was first prepared in 5% DMF (v/v) and a mixture
0.9% saline and medium (1:1) (v/v) following serial dilutions in RPMI-1640.
The drug exposure period was 24 h. After this, supernatants were removed
by suction, and each well was washed with PBS. A further 72 h was
allowed for the cells to recover in drug-free medium at 310 K. The
SRB assay was used to determine cell viability. Absorbance measurements
of the solubilized dye allowed the determination of viable treated
cells compared to untreated controls. IC 50 values (concentrations
which caused 50% of cell growth inhibition) were determined as duplicates
of triplicates in two independent sets of experiments and their standard
deviations were calculated. All experiments included three sets of
controls: (a) negative controls, in which cells were kept untreated,
(b) vehicle controls, in which cells were exposed to medium with vehicle
only (in this case DMF, at the highest concentration used for the
complexes), and (c) positive controls, in which cells were exposed
to different concentrations of the anticancer drug cisplatin. Syntheses General
Procedure for the Synthesis of Thiosemicarbazone Ligands
( L1 and L2 ) The synthesis of ligands L1 and L2 was performed using the following adapted
literature procedure. 54 , 55 The appropriate aldehyde (1 mol
equiv) was dissolved in a hot toluene solution (20 mL) containing
few drops of glacial acetic acid. An equimolar amount of the corresponding
thiosemicarbazide (1 mol equiv) was added to the solution, and the
reaction mixture was heated under reflux for 8 h. The solution was
cooled to ambient temperature, and the TSC ligands were obtained as
precipitate. After filtration the solid was washed several times with
toluene and ether and dried under vacuum. N -(2-Hydroxy)-3-methoxybenzylidenethiosemicarbazide
( L1 ) White powder, yield: 87%. 1 H
NMR (DMSO- d 6 ): δ 11.39 (s, 1H, NH),
9.17 (s, 1H, OH), 8.40 (s, 1H, CH=N), 8.10–7.88 (2s,
1H+1H, NH 2 ), 7.52 (d, 1H, J = 7.5 Hz,
CH Ar ), 6.95 (d, 1H, J = 7.5 Hz, CH Ar ), 6.75 (t, 1H, J = 7.5 Hz, CH Ar ), 3.81 (s, 3H, OCH 3 ). ESI-MS (C 9 H 11 N 3 SO 2 , MeOH): m / z = 225 [M + H] + . N -(2,3-Dihydroxybenzylidene)-3-phenylthiosemicarbazide
( L2 ) White powder, yield: 81%. 1 H
NMR (DMSO- d 6 ): δ 11.76 (s, 1H, NH),
10.01–9.54 (2s, 1H+1H, OH), 9.01 (s, 1H, NH), 8.49 (s, 1H,
CH=N), 7.56 (d, 2H, J = 7.5 Hz, CH Ar ), 7.49 (d, 2H, J = 8 Hz, CH Ar ), 7.34
(t, 2H, J = 7.5 Hz, CH Ar ), 7.17 (t, 1H, J = 7.5 Hz, CH Ar ), 6.81 (d, 1H, J = 8 Hz, CH Ar ), 6.64 (t, J = 8 Hz, CH Ar ). ESI-MS (C 14 H 13 N 3 SO 2 , MeOH): m / z = 287 [M + H] + . General Procedure for the Metal Complexes
Synthesis ( 1 – 4 ) The TSC
ligand (2 mol equiv) was
dissolved in dry methanol (20 mL), and the solution was acidified
with the addition of 1 drop of HCl 37%. [(η 6 - p -cym)MCl 2 ] 2 (1 mol equiv) was dissolved
in 10 mL of dry dichloromethane, and the solution was added to the
previous one. The reaction mixture was maintained under stirring at
ambient temperature under nitrogen for 24 h. The volume was then reduced
to half on the rotary evaporator, and diethyl ether was added until
the precipitation of a solid occurred. The product was then collected
by filtration and dried under vacuum. [Os(η 6 -p-cym)Cl(L1)]Cl ( 1 ) Orange powder, yield:
98%. Anal. Calcd for C 19 H 25 Cl 2 N 3 O 2 OsS: C, 36.77; H, 4.06; N,
6.77. Found: C, 36.51; H, 4.56; N, 6.70. 1 H NMR (MeOD- d 4 ): δ 8.76 (s, 1H, CH=N), 7.86
(d, 1H, J = 8 Hz, CH Ar ), 7.25 (d, 1H, J = 8 Hz, CH Ar ), 7.01 (t, 1H, J = 8 Hz, CH Ar ), 5.87 (d, 1H, J = 5.5
Hz, CH p-cym ), 5.44 (d, 1H, J =
5.5 Hz, CH p -cym ), 5.31 (d, 1H, J = 5.5 Hz, CH p -cym ),
4.90 (d, 1H, J = 5.5 Hz, CH p -cym ), 3.99 (s, 3H, OCH 3 ), 2.54 (m, 1H, J = 7 Hz, CH i -prop ),
2.16 (s, 3H, CH 3 ), 1.20–1.11 (2d, 3H+3H, J = 7 Hz, CH 3 i -prop ).
ESI-MS (positive ions, MeOH): m / z = 585 [M – Cl] + . Crystals suitable for X-ray analysis
were obtained by vapor diffusion of ether into a saturated methanol
solution of the compound. [Os(η 6 -p-cym)Cl(L2)]Cl ( 2 ) Orange powder, yield: 72%. Anal. Calcd for C 24 H 27 Cl 2 N 3 O 2 OsS·H 2 O:
C, 41.14; H, 4.17; N, 6.00. Found: C, 40.81; H, 4.16; N, 6.23. 1 H NMR (MeOD- d 4 ): δ 8.87
(s, 1H, CH=N), 7.75 (d, 1H, J = 7 Hz, CH Ar ), 7.48 (t, 2H, J = 7 Hz, CH Ar ), 7.43 (d, 2H, J = 7 Hz, CH Ar ), 7.35
(t, 1H, J = 7 Hz, CH Ar ), 7.08 (dd, 1H, J = 8 Hz, CH Ar ), 6.88 (t, 1H, J = 8 Hz, CH Ar ), 5.86 (d, 1H, J = 5.5
Hz, CH p -cym ), 5.49 (d, 1H, J = 5.5 Hz, CH p -cym ),
5.31 (d, 1H, J = 5.5 Hz, CH p -cym ), 4.93 (d, 1H, J = 5.5 Hz, CH p -cym ), 2.55 (m, 1H, J = 7 Hz, CH i -prop ), 1.21–1.13
(2d, 3H+3H, J = 7 Hz, CH 3 i -prop ). ESI-MS (positive ions, CH 3 OH): m / z = 648 [M – Cl] + . Crystals suitable for
X-ray analysis were obtained by slow evaporation of a saturated acetone
solution. [Ru(η 6 -p-cym)Cl(L1)]Cl ( 3 ) Red powder, yield: 78%. Anal. Calcd for C 19 H 25 Cl 2 N 3 O 2 RuS·CH 3 OH:
C, 42.63; H, 5.19; N, 7.46. Found: C, 41.92; H, 5.21; N, 7.34. 1 H NMR (MeOD- d 4 ): δ 8.79
(s, 1H, CH=N), 8.06 (d, 1H, J = 8 Hz, CH Ar ), 7.28 (d, 1H, J = 8 Hz, CH Ar ), 7.07 (t, 1H, J = 8 Hz, CH Ar ), 5.71
(d, 1H, J = 6 Hz, CH p -cym ), 5.17 (d, 1H, J = 6 Hz, CH p -cym ), 5.04 (d, 1H, J = 6 Hz, CH p -cym ), 4.00 (s, 3H, OCH 3 ), 2.64 (m, 1H, J = 7 Hz, CH i -prop ), 2.10 (s, 3H, CH 3 ), 1.20–1.14
(2d, 3H+3H, J = 7 Hz, CH 3 i -prop ). ESI-MS (positive ions, CH 3 OH): m / z = 496 [M – Cl] + . Crystals suitable for
X-ray analysis were obtained by vapor diffusion of ether into a saturated
methanol solution of the compound. [Ru(η 6 -p-cym)Cl(L2)]Cl ( 4 ) Red powder, yield: 87%.
Anal. Calcd for C 24 H 27 Cl 2 N 3 O 2 RuS·CH 3 COCH 3 : C, 49.77; H,
5.10; N, 6.45. Found: C, 49.54; H, 5.23; N,
7.01. 1 H NMR (MeOD- d 4 ): δ
8.90 (s, 1H, CH=N), 7.95 (dd, 1H, J = 8 Hz, J ′ = 1 Hz, CH Ar ), 7.48 (t, 2H, J = 7.5 Hz, CH Ar ), 7.41 (d, 2H, J = 7.5 Hz, CH Ar ), 7.37 (d, 1H, J = 7
Hz, CH Ar ), 7.09 (td, 1H, J = 8 Hz, J ′ =1 Hz, CH Ar ), 6.94 (t, 1H, J = 7.5 Hz, CH Ar ), 5.86 (d, 1H, J = 5.5 Hz, CH p -cym ), 5.49 (d,
1H, J = 5.5 Hz, CH p -cym ), 5.31 (d, 1H, J = 5.5 Hz, CH p -cym ), 4.93 (d, 1H, J = 5.5 Hz, CH p -cym ), 2.55 (m, 1H, J = 7 Hz, CH i -prop ), 1.21–1.13
(2d, 3H+3H, J = 7 Hz, CH 3 i -prop ). ESI-MS (positive ions, CH 3 OH): m / z = 558 [M – Cl] + . Crystals suitable for
X-ray analysis were obtained by slow evaporation of a saturated acetone
solution of the compound. X-ray Crystallography Diffraction data were obtained
on an Xcalibur Gemini diffractometer four-circle system with a Ruby
CCD area detector using Mo Kα radiation. Absorption corrections
were applied using ABSPACK. 69 The crystals
were mounted on a glass fiber with Fromblin oil and kept at 150(2)
K during data collection. Using Olex2, 70 the structure was solved with the ShelXT 71 structure solution program using Direct Methods and refined with
the ShelXL refinement package using least-squares minimization. NMR Spectroscopy 1 H NMR spectra were obtained
in 5 mm NMR precision tubes at 298 K on either Bruker DPX-300 or DPX-400
NMR spectrometers. 1 H NMR chemical shifts were internally
referenced to residual protiated solvent for DMSO- d 6 (2.50 ppm), CD 3 OD (3.31 ppm), D 2 O (4.79 ppm), (CD 3 ) 2 CO (2.05 ppm). 1 H NMR spectra at variable temperature were obtained in 5 mm NMR precision
tube on a Bruker AV-III 400 NMR spectrometer. NOESY spectra were obtained
in 5 mm NMR precision tubes at 298 K on a Bruker DPX-500 NMR spectrometer. 1 H NMR peaks were internally referenced to CHD 2 OD
(3.31 ppm) for methanol- d 4 or 1,4-dioxane
(3.66 ppm). All data processing was carried out using MestReNova 9.0.1. Mass Spectrometry Electrospray ionization mass spectra
(ESI-MS) were obtained by preparing the sample in methanol using a
Bruker Esquire 2000 ion trap spectrometer. Samples were prepared in
methanol. The mass spectra were recorded with a scan range of m / z 50–500 for positive ions for L 1 -L 2 and m / z 400–1000 for positive ions for the complexes 1 – 4 . UV–Vis Spectroscopy UV–vis absorption
spectra were recorded on a Cary 300 spectrometer using quartz cuvettes
of 1 cm path-length (600 μL). The sample temperature was adjusted
to 298 K by PTP1 Peltier temperature controller. Samples were prepared
in methanol. Spectra were recorded from 200 to 600 nm. Data were processed
with Microsoft Excel 14.3.6 Mac version.
## Materials
Materials All commercial reagents were used as received.
2-Hydroxy-3-methoxybenzaldehyde, 2,3-dihydroxybenzaldehyde, thiosemicarbazide,
and 4-phenylthiosemicarbazide were purchased from Sigma-Aldrich; OsCl 3 · n H 2 O and RuCl 3 · n H 2 O were from Alfa Aesar. All
reactions were performed under an inert atmosphere of nitrogen using
standard Schlenk line techniques, and all glassware was oven-dried
(120 °C) overnight. Dry solvents were purchased from Sigma-Aldrich
and stored under nitrogen. [(η 6 - p -cym)OsCl 2 ] 2 and [(η 6 - p -cym)RuCl 2 ] 2 were synthesized according
to literature procedures. 49 , 68
## Cell Culture
Cell Culture Cell
lines used in this work included
A2780 human ovarian carcinoma and its cisplatin-resistant variant
A2780Cis, A549 human caucasian lung carcinoma, HCT116 human colon
carcinoma, and PC3 human prostate carcinoma. They were all obtained
from the European Collection of Cell Cultures (ECACC), used between
passages 5 and 18 and were grown in Roswell Park Memorial Institute
medium (RPMI-1640) supplemented with 10% (v/v) of fetal calf serum,
1% (v/v) of 2 mM glutamine and 1% (v/v) penicillin/streptomycin. They
were grown as adherent monolayers at 310 K in a 5% CO 2 humidified
atmosphere and passaged at ca. 70–80% confluence.
In
Vitro Growth Inhibition Assay Briefly,
5000 cells were seeded per well in 96-well plates. The cells were
preincubated in drug-free media at 310 K for 48 h before adding different
concentrations of the compounds to be tested. A stock solution of
the metal complex was first prepared in 5% DMF (v/v) and a mixture
0.9% saline and medium (1:1) (v/v) following serial dilutions in RPMI-1640.
The drug exposure period was 24 h. After this, supernatants were removed
by suction, and each well was washed with PBS. A further 72 h was
allowed for the cells to recover in drug-free medium at 310 K. The
SRB assay was used to determine cell viability. Absorbance measurements
of the solubilized dye allowed the determination of viable treated
cells compared to untreated controls. IC 50 values (concentrations
which caused 50% of cell growth inhibition) were determined as duplicates
of triplicates in two independent sets of experiments and their standard
deviations were calculated. All experiments included three sets of
controls: (a) negative controls, in which cells were kept untreated,
(b) vehicle controls, in which cells were exposed to medium with vehicle
only (in this case DMF, at the highest concentration used for the
complexes), and (c) positive controls, in which cells were exposed
to different concentrations of the anticancer drug cisplatin.
## Syntheses
Syntheses General
Procedure for the Synthesis of Thiosemicarbazone Ligands
( L1 and L2 ) The synthesis of ligands L1 and L2 was performed using the following adapted
literature procedure. 54 , 55 The appropriate aldehyde (1 mol
equiv) was dissolved in a hot toluene solution (20 mL) containing
few drops of glacial acetic acid. An equimolar amount of the corresponding
thiosemicarbazide (1 mol equiv) was added to the solution, and the
reaction mixture was heated under reflux for 8 h. The solution was
cooled to ambient temperature, and the TSC ligands were obtained as
precipitate. After filtration the solid was washed several times with
toluene and ether and dried under vacuum. N -(2-Hydroxy)-3-methoxybenzylidenethiosemicarbazide
( L1 ) White powder, yield: 87%. 1 H
NMR (DMSO- d 6 ): δ 11.39 (s, 1H, NH),
9.17 (s, 1H, OH), 8.40 (s, 1H, CH=N), 8.10–7.88 (2s,
1H+1H, NH 2 ), 7.52 (d, 1H, J = 7.5 Hz,
CH Ar ), 6.95 (d, 1H, J = 7.5 Hz, CH Ar ), 6.75 (t, 1H, J = 7.5 Hz, CH Ar ), 3.81 (s, 3H, OCH 3 ). ESI-MS (C 9 H 11 N 3 SO 2 , MeOH): m / z = 225 [M + H] + . N -(2,3-Dihydroxybenzylidene)-3-phenylthiosemicarbazide
( L2 ) White powder, yield: 81%. 1 H
NMR (DMSO- d 6 ): δ 11.76 (s, 1H, NH),
10.01–9.54 (2s, 1H+1H, OH), 9.01 (s, 1H, NH), 8.49 (s, 1H,
CH=N), 7.56 (d, 2H, J = 7.5 Hz, CH Ar ), 7.49 (d, 2H, J = 8 Hz, CH Ar ), 7.34
(t, 2H, J = 7.5 Hz, CH Ar ), 7.17 (t, 1H, J = 7.5 Hz, CH Ar ), 6.81 (d, 1H, J = 8 Hz, CH Ar ), 6.64 (t, J = 8 Hz, CH Ar ). ESI-MS (C 14 H 13 N 3 SO 2 , MeOH): m / z = 287 [M + H] + . General Procedure for the Metal Complexes
Synthesis ( 1 – 4 ) The TSC
ligand (2 mol equiv) was
dissolved in dry methanol (20 mL), and the solution was acidified
with the addition of 1 drop of HCl 37%. [(η 6 - p -cym)MCl 2 ] 2 (1 mol equiv) was dissolved
in 10 mL of dry dichloromethane, and the solution was added to the
previous one. The reaction mixture was maintained under stirring at
ambient temperature under nitrogen for 24 h. The volume was then reduced
to half on the rotary evaporator, and diethyl ether was added until
the precipitation of a solid occurred. The product was then collected
by filtration and dried under vacuum. [Os(η 6 -p-cym)Cl(L1)]Cl ( 1 ) Orange powder, yield:
98%. Anal. Calcd for C 19 H 25 Cl 2 N 3 O 2 OsS: C, 36.77; H, 4.06; N,
6.77. Found: C, 36.51; H, 4.56; N, 6.70. 1 H NMR (MeOD- d 4 ): δ 8.76 (s, 1H, CH=N), 7.86
(d, 1H, J = 8 Hz, CH Ar ), 7.25 (d, 1H, J = 8 Hz, CH Ar ), 7.01 (t, 1H, J = 8 Hz, CH Ar ), 5.87 (d, 1H, J = 5.5
Hz, CH p-cym ), 5.44 (d, 1H, J =
5.5 Hz, CH p -cym ), 5.31 (d, 1H, J = 5.5 Hz, CH p -cym ),
4.90 (d, 1H, J = 5.5 Hz, CH p -cym ), 3.99 (s, 3H, OCH 3 ), 2.54 (m, 1H, J = 7 Hz, CH i -prop ),
2.16 (s, 3H, CH 3 ), 1.20–1.11 (2d, 3H+3H, J = 7 Hz, CH 3 i -prop ).
ESI-MS (positive ions, MeOH): m / z = 585 [M – Cl] + . Crystals suitable for X-ray analysis
were obtained by vapor diffusion of ether into a saturated methanol
solution of the compound. [Os(η 6 -p-cym)Cl(L2)]Cl ( 2 ) Orange powder, yield: 72%. Anal. Calcd for C 24 H 27 Cl 2 N 3 O 2 OsS·H 2 O:
C, 41.14; H, 4.17; N, 6.00. Found: C, 40.81; H, 4.16; N, 6.23. 1 H NMR (MeOD- d 4 ): δ 8.87
(s, 1H, CH=N), 7.75 (d, 1H, J = 7 Hz, CH Ar ), 7.48 (t, 2H, J = 7 Hz, CH Ar ), 7.43 (d, 2H, J = 7 Hz, CH Ar ), 7.35
(t, 1H, J = 7 Hz, CH Ar ), 7.08 (dd, 1H, J = 8 Hz, CH Ar ), 6.88 (t, 1H, J = 8 Hz, CH Ar ), 5.86 (d, 1H, J = 5.5
Hz, CH p -cym ), 5.49 (d, 1H, J = 5.5 Hz, CH p -cym ),
5.31 (d, 1H, J = 5.5 Hz, CH p -cym ), 4.93 (d, 1H, J = 5.5 Hz, CH p -cym ), 2.55 (m, 1H, J = 7 Hz, CH i -prop ), 1.21–1.13
(2d, 3H+3H, J = 7 Hz, CH 3 i -prop ). ESI-MS (positive ions, CH 3 OH): m / z = 648 [M – Cl] + . Crystals suitable for
X-ray analysis were obtained by slow evaporation of a saturated acetone
solution. [Ru(η 6 -p-cym)Cl(L1)]Cl ( 3 ) Red powder, yield: 78%. Anal. Calcd for C 19 H 25 Cl 2 N 3 O 2 RuS·CH 3 OH:
C, 42.63; H, 5.19; N, 7.46. Found: C, 41.92; H, 5.21; N, 7.34. 1 H NMR (MeOD- d 4 ): δ 8.79
(s, 1H, CH=N), 8.06 (d, 1H, J = 8 Hz, CH Ar ), 7.28 (d, 1H, J = 8 Hz, CH Ar ), 7.07 (t, 1H, J = 8 Hz, CH Ar ), 5.71
(d, 1H, J = 6 Hz, CH p -cym ), 5.17 (d, 1H, J = 6 Hz, CH p -cym ), 5.04 (d, 1H, J = 6 Hz, CH p -cym ), 4.00 (s, 3H, OCH 3 ), 2.64 (m, 1H, J = 7 Hz, CH i -prop ), 2.10 (s, 3H, CH 3 ), 1.20–1.14
(2d, 3H+3H, J = 7 Hz, CH 3 i -prop ). ESI-MS (positive ions, CH 3 OH): m / z = 496 [M – Cl] + . Crystals suitable for
X-ray analysis were obtained by vapor diffusion of ether into a saturated
methanol solution of the compound. [Ru(η 6 -p-cym)Cl(L2)]Cl ( 4 ) Red powder, yield: 87%.
Anal. Calcd for C 24 H 27 Cl 2 N 3 O 2 RuS·CH 3 COCH 3 : C, 49.77; H,
5.10; N, 6.45. Found: C, 49.54; H, 5.23; N,
7.01. 1 H NMR (MeOD- d 4 ): δ
8.90 (s, 1H, CH=N), 7.95 (dd, 1H, J = 8 Hz, J ′ = 1 Hz, CH Ar ), 7.48 (t, 2H, J = 7.5 Hz, CH Ar ), 7.41 (d, 2H, J = 7.5 Hz, CH Ar ), 7.37 (d, 1H, J = 7
Hz, CH Ar ), 7.09 (td, 1H, J = 8 Hz, J ′ =1 Hz, CH Ar ), 6.94 (t, 1H, J = 7.5 Hz, CH Ar ), 5.86 (d, 1H, J = 5.5 Hz, CH p -cym ), 5.49 (d,
1H, J = 5.5 Hz, CH p -cym ), 5.31 (d, 1H, J = 5.5 Hz, CH p -cym ), 4.93 (d, 1H, J = 5.5 Hz, CH p -cym ), 2.55 (m, 1H, J = 7 Hz, CH i -prop ), 1.21–1.13
(2d, 3H+3H, J = 7 Hz, CH 3 i -prop ). ESI-MS (positive ions, CH 3 OH): m / z = 558 [M – Cl] + . Crystals suitable for
X-ray analysis were obtained by slow evaporation of a saturated acetone
solution of the compound.
## General
Procedure for the Synthesis of Thiosemicarbazone Ligands
(
General
Procedure for the Synthesis of Thiosemicarbazone Ligands
( L1 and L2 ) The synthesis of ligands L1 and L2 was performed using the following adapted
literature procedure. 54 , 55 The appropriate aldehyde (1 mol
equiv) was dissolved in a hot toluene solution (20 mL) containing
few drops of glacial acetic acid. An equimolar amount of the corresponding
thiosemicarbazide (1 mol equiv) was added to the solution, and the
reaction mixture was heated under reflux for 8 h. The solution was
cooled to ambient temperature, and the TSC ligands were obtained as
precipitate. After filtration the solid was washed several times with
toluene and ether and dried under vacuum. N -(2-Hydroxy)-3-methoxybenzylidenethiosemicarbazide
( L1 ) White powder, yield: 87%. 1 H
NMR (DMSO- d 6 ): δ 11.39 (s, 1H, NH),
9.17 (s, 1H, OH), 8.40 (s, 1H, CH=N), 8.10–7.88 (2s,
1H+1H, NH 2 ), 7.52 (d, 1H, J = 7.5 Hz,
CH Ar ), 6.95 (d, 1H, J = 7.5 Hz, CH Ar ), 6.75 (t, 1H, J = 7.5 Hz, CH Ar ), 3.81 (s, 3H, OCH 3 ). ESI-MS (C 9 H 11 N 3 SO 2 , MeOH): m / z = 225 [M + H] + . N -(2,3-Dihydroxybenzylidene)-3-phenylthiosemicarbazide
( L2 ) White powder, yield: 81%. 1 H
NMR (DMSO- d 6 ): δ 11.76 (s, 1H, NH),
10.01–9.54 (2s, 1H+1H, OH), 9.01 (s, 1H, NH), 8.49 (s, 1H,
CH=N), 7.56 (d, 2H, J = 7.5 Hz, CH Ar ), 7.49 (d, 2H, J = 8 Hz, CH Ar ), 7.34
(t, 2H, J = 7.5 Hz, CH Ar ), 7.17 (t, 1H, J = 7.5 Hz, CH Ar ), 6.81 (d, 1H, J = 8 Hz, CH Ar ), 6.64 (t, J = 8 Hz, CH Ar ). ESI-MS (C 14 H 13 N 3 SO 2 , MeOH): m / z = 287 [M + H] + .
N -(2-Hydroxy)-3-methoxybenzylidenethiosemicarbazide
( L1 ) White powder, yield: 87%. 1 H
NMR (DMSO- d 6 ): δ 11.39 (s, 1H, NH),
9.17 (s, 1H, OH), 8.40 (s, 1H, CH=N), 8.10–7.88 (2s,
1H+1H, NH 2 ), 7.52 (d, 1H, J = 7.5 Hz,
CH Ar ), 6.95 (d, 1H, J = 7.5 Hz, CH Ar ), 6.75 (t, 1H, J = 7.5 Hz, CH Ar ), 3.81 (s, 3H, OCH 3 ). ESI-MS (C 9 H 11 N 3 SO 2 , MeOH): m / z = 225 [M + H] + .
N -(2,3-Dihydroxybenzylidene)-3-phenylthiosemicarbazide
( L2 ) White powder, yield: 81%. 1 H
NMR (DMSO- d 6 ): δ 11.76 (s, 1H, NH),
10.01–9.54 (2s, 1H+1H, OH), 9.01 (s, 1H, NH), 8.49 (s, 1H,
CH=N), 7.56 (d, 2H, J = 7.5 Hz, CH Ar ), 7.49 (d, 2H, J = 8 Hz, CH Ar ), 7.34
(t, 2H, J = 7.5 Hz, CH Ar ), 7.17 (t, 1H, J = 7.5 Hz, CH Ar ), 6.81 (d, 1H, J = 8 Hz, CH Ar ), 6.64 (t, J = 8 Hz, CH Ar ). ESI-MS (C 14 H 13 N 3 SO 2 , MeOH): m / z = 287 [M + H] + .
## General Procedure for the Metal Complexes
Synthesis (
General Procedure for the Metal Complexes
Synthesis ( 1 – 4 ) The TSC
ligand (2 mol equiv) was
dissolved in dry methanol (20 mL), and the solution was acidified
with the addition of 1 drop of HCl 37%. [(η 6 - p -cym)MCl 2 ] 2 (1 mol equiv) was dissolved
in 10 mL of dry dichloromethane, and the solution was added to the
previous one. The reaction mixture was maintained under stirring at
ambient temperature under nitrogen for 24 h. The volume was then reduced
to half on the rotary evaporator, and diethyl ether was added until
the precipitation of a solid occurred. The product was then collected
by filtration and dried under vacuum. [Os(η 6 -p-cym)Cl(L1)]Cl ( 1 ) Orange powder, yield:
98%. Anal. Calcd for C 19 H 25 Cl 2 N 3 O 2 OsS: C, 36.77; H, 4.06; N,
6.77. Found: C, 36.51; H, 4.56; N, 6.70. 1 H NMR (MeOD- d 4 ): δ 8.76 (s, 1H, CH=N), 7.86
(d, 1H, J = 8 Hz, CH Ar ), 7.25 (d, 1H, J = 8 Hz, CH Ar ), 7.01 (t, 1H, J = 8 Hz, CH Ar ), 5.87 (d, 1H, J = 5.5
Hz, CH p-cym ), 5.44 (d, 1H, J =
5.5 Hz, CH p -cym ), 5.31 (d, 1H, J = 5.5 Hz, CH p -cym ),
4.90 (d, 1H, J = 5.5 Hz, CH p -cym ), 3.99 (s, 3H, OCH 3 ), 2.54 (m, 1H, J = 7 Hz, CH i -prop ),
2.16 (s, 3H, CH 3 ), 1.20–1.11 (2d, 3H+3H, J = 7 Hz, CH 3 i -prop ).
ESI-MS (positive ions, MeOH): m / z = 585 [M – Cl] + . Crystals suitable for X-ray analysis
were obtained by vapor diffusion of ether into a saturated methanol
solution of the compound. [Os(η 6 -p-cym)Cl(L2)]Cl ( 2 ) Orange powder, yield: 72%. Anal. Calcd for C 24 H 27 Cl 2 N 3 O 2 OsS·H 2 O:
C, 41.14; H, 4.17; N, 6.00. Found: C, 40.81; H, 4.16; N, 6.23. 1 H NMR (MeOD- d 4 ): δ 8.87
(s, 1H, CH=N), 7.75 (d, 1H, J = 7 Hz, CH Ar ), 7.48 (t, 2H, J = 7 Hz, CH Ar ), 7.43 (d, 2H, J = 7 Hz, CH Ar ), 7.35
(t, 1H, J = 7 Hz, CH Ar ), 7.08 (dd, 1H, J = 8 Hz, CH Ar ), 6.88 (t, 1H, J = 8 Hz, CH Ar ), 5.86 (d, 1H, J = 5.5
Hz, CH p -cym ), 5.49 (d, 1H, J = 5.5 Hz, CH p -cym ),
5.31 (d, 1H, J = 5.5 Hz, CH p -cym ), 4.93 (d, 1H, J = 5.5 Hz, CH p -cym ), 2.55 (m, 1H, J = 7 Hz, CH i -prop ), 1.21–1.13
(2d, 3H+3H, J = 7 Hz, CH 3 i -prop ). ESI-MS (positive ions, CH 3 OH): m / z = 648 [M – Cl] + . Crystals suitable for
X-ray analysis were obtained by slow evaporation of a saturated acetone
solution. [Ru(η 6 -p-cym)Cl(L1)]Cl ( 3 ) Red powder, yield: 78%. Anal. Calcd for C 19 H 25 Cl 2 N 3 O 2 RuS·CH 3 OH:
C, 42.63; H, 5.19; N, 7.46. Found: C, 41.92; H, 5.21; N, 7.34. 1 H NMR (MeOD- d 4 ): δ 8.79
(s, 1H, CH=N), 8.06 (d, 1H, J = 8 Hz, CH Ar ), 7.28 (d, 1H, J = 8 Hz, CH Ar ), 7.07 (t, 1H, J = 8 Hz, CH Ar ), 5.71
(d, 1H, J = 6 Hz, CH p -cym ), 5.17 (d, 1H, J = 6 Hz, CH p -cym ), 5.04 (d, 1H, J = 6 Hz, CH p -cym ), 4.00 (s, 3H, OCH 3 ), 2.64 (m, 1H, J = 7 Hz, CH i -prop ), 2.10 (s, 3H, CH 3 ), 1.20–1.14
(2d, 3H+3H, J = 7 Hz, CH 3 i -prop ). ESI-MS (positive ions, CH 3 OH): m / z = 496 [M – Cl] + . Crystals suitable for
X-ray analysis were obtained by vapor diffusion of ether into a saturated
methanol solution of the compound. [Ru(η 6 -p-cym)Cl(L2)]Cl ( 4 ) Red powder, yield: 87%.
Anal. Calcd for C 24 H 27 Cl 2 N 3 O 2 RuS·CH 3 COCH 3 : C, 49.77; H,
5.10; N, 6.45. Found: C, 49.54; H, 5.23; N,
7.01. 1 H NMR (MeOD- d 4 ): δ
8.90 (s, 1H, CH=N), 7.95 (dd, 1H, J = 8 Hz, J ′ = 1 Hz, CH Ar ), 7.48 (t, 2H, J = 7.5 Hz, CH Ar ), 7.41 (d, 2H, J = 7.5 Hz, CH Ar ), 7.37 (d, 1H, J = 7
Hz, CH Ar ), 7.09 (td, 1H, J = 8 Hz, J ′ =1 Hz, CH Ar ), 6.94 (t, 1H, J = 7.5 Hz, CH Ar ), 5.86 (d, 1H, J = 5.5 Hz, CH p -cym ), 5.49 (d,
1H, J = 5.5 Hz, CH p -cym ), 5.31 (d, 1H, J = 5.5 Hz, CH p -cym ), 4.93 (d, 1H, J = 5.5 Hz, CH p -cym ), 2.55 (m, 1H, J = 7 Hz, CH i -prop ), 1.21–1.13
(2d, 3H+3H, J = 7 Hz, CH 3 i -prop ). ESI-MS (positive ions, CH 3 OH): m / z = 558 [M – Cl] + . Crystals suitable for
X-ray analysis were obtained by slow evaporation of a saturated acetone
solution of the compound.
## [Os(η
[Os(η 6 -p-cym)Cl(L1)]Cl ( 1 ) Orange powder, yield:
98%. Anal. Calcd for C 19 H 25 Cl 2 N 3 O 2 OsS: C, 36.77; H, 4.06; N,
6.77. Found: C, 36.51; H, 4.56; N, 6.70. 1 H NMR (MeOD- d 4 ): δ 8.76 (s, 1H, CH=N), 7.86
(d, 1H, J = 8 Hz, CH Ar ), 7.25 (d, 1H, J = 8 Hz, CH Ar ), 7.01 (t, 1H, J = 8 Hz, CH Ar ), 5.87 (d, 1H, J = 5.5
Hz, CH p-cym ), 5.44 (d, 1H, J =
5.5 Hz, CH p -cym ), 5.31 (d, 1H, J = 5.5 Hz, CH p -cym ),
4.90 (d, 1H, J = 5.5 Hz, CH p -cym ), 3.99 (s, 3H, OCH 3 ), 2.54 (m, 1H, J = 7 Hz, CH i -prop ),
2.16 (s, 3H, CH 3 ), 1.20–1.11 (2d, 3H+3H, J = 7 Hz, CH 3 i -prop ).
ESI-MS (positive ions, MeOH): m / z = 585 [M – Cl] + . Crystals suitable for X-ray analysis
were obtained by vapor diffusion of ether into a saturated methanol
solution of the compound.
## [Os(η
[Os(η 6 -p-cym)Cl(L2)]Cl ( 2 ) Orange powder, yield: 72%. Anal. Calcd for C 24 H 27 Cl 2 N 3 O 2 OsS·H 2 O:
C, 41.14; H, 4.17; N, 6.00. Found: C, 40.81; H, 4.16; N, 6.23. 1 H NMR (MeOD- d 4 ): δ 8.87
(s, 1H, CH=N), 7.75 (d, 1H, J = 7 Hz, CH Ar ), 7.48 (t, 2H, J = 7 Hz, CH Ar ), 7.43 (d, 2H, J = 7 Hz, CH Ar ), 7.35
(t, 1H, J = 7 Hz, CH Ar ), 7.08 (dd, 1H, J = 8 Hz, CH Ar ), 6.88 (t, 1H, J = 8 Hz, CH Ar ), 5.86 (d, 1H, J = 5.5
Hz, CH p -cym ), 5.49 (d, 1H, J = 5.5 Hz, CH p -cym ),
5.31 (d, 1H, J = 5.5 Hz, CH p -cym ), 4.93 (d, 1H, J = 5.5 Hz, CH p -cym ), 2.55 (m, 1H, J = 7 Hz, CH i -prop ), 1.21–1.13
(2d, 3H+3H, J = 7 Hz, CH 3 i -prop ). ESI-MS (positive ions, CH 3 OH): m / z = 648 [M – Cl] + . Crystals suitable for
X-ray analysis were obtained by slow evaporation of a saturated acetone
solution.
## [Ru(η
[Ru(η 6 -p-cym)Cl(L1)]Cl ( 3 ) Red powder, yield: 78%. Anal. Calcd for C 19 H 25 Cl 2 N 3 O 2 RuS·CH 3 OH:
C, 42.63; H, 5.19; N, 7.46. Found: C, 41.92; H, 5.21; N, 7.34. 1 H NMR (MeOD- d 4 ): δ 8.79
(s, 1H, CH=N), 8.06 (d, 1H, J = 8 Hz, CH Ar ), 7.28 (d, 1H, J = 8 Hz, CH Ar ), 7.07 (t, 1H, J = 8 Hz, CH Ar ), 5.71
(d, 1H, J = 6 Hz, CH p -cym ), 5.17 (d, 1H, J = 6 Hz, CH p -cym ), 5.04 (d, 1H, J = 6 Hz, CH p -cym ), 4.00 (s, 3H, OCH 3 ), 2.64 (m, 1H, J = 7 Hz, CH i -prop ), 2.10 (s, 3H, CH 3 ), 1.20–1.14
(2d, 3H+3H, J = 7 Hz, CH 3 i -prop ). ESI-MS (positive ions, CH 3 OH): m / z = 496 [M – Cl] + . Crystals suitable for
X-ray analysis were obtained by vapor diffusion of ether into a saturated
methanol solution of the compound.
## [Ru(η
[Ru(η 6 -p-cym)Cl(L2)]Cl ( 4 ) Red powder, yield: 87%.
Anal. Calcd for C 24 H 27 Cl 2 N 3 O 2 RuS·CH 3 COCH 3 : C, 49.77; H,
5.10; N, 6.45. Found: C, 49.54; H, 5.23; N,
7.01. 1 H NMR (MeOD- d 4 ): δ
8.90 (s, 1H, CH=N), 7.95 (dd, 1H, J = 8 Hz, J ′ = 1 Hz, CH Ar ), 7.48 (t, 2H, J = 7.5 Hz, CH Ar ), 7.41 (d, 2H, J = 7.5 Hz, CH Ar ), 7.37 (d, 1H, J = 7
Hz, CH Ar ), 7.09 (td, 1H, J = 8 Hz, J ′ =1 Hz, CH Ar ), 6.94 (t, 1H, J = 7.5 Hz, CH Ar ), 5.86 (d, 1H, J = 5.5 Hz, CH p -cym ), 5.49 (d,
1H, J = 5.5 Hz, CH p -cym ), 5.31 (d, 1H, J = 5.5 Hz, CH p -cym ), 4.93 (d, 1H, J = 5.5 Hz, CH p -cym ), 2.55 (m, 1H, J = 7 Hz, CH i -prop ), 1.21–1.13
(2d, 3H+3H, J = 7 Hz, CH 3 i -prop ). ESI-MS (positive ions, CH 3 OH): m / z = 558 [M – Cl] + . Crystals suitable for
X-ray analysis were obtained by slow evaporation of a saturated acetone
solution of the compound.
## X-ray Crystallography
X-ray Crystallography Diffraction data were obtained
on an Xcalibur Gemini diffractometer four-circle system with a Ruby
CCD area detector using Mo Kα radiation. Absorption corrections
were applied using ABSPACK. 69 The crystals
were mounted on a glass fiber with Fromblin oil and kept at 150(2)
K during data collection. Using Olex2, 70 the structure was solved with the ShelXT 71 structure solution program using Direct Methods and refined with
the ShelXL refinement package using least-squares minimization.
## NMR Spectroscopy
NMR Spectroscopy 1 H NMR spectra were obtained
in 5 mm NMR precision tubes at 298 K on either Bruker DPX-300 or DPX-400
NMR spectrometers. 1 H NMR chemical shifts were internally
referenced to residual protiated solvent for DMSO- d 6 (2.50 ppm), CD 3 OD (3.31 ppm), D 2 O (4.79 ppm), (CD 3 ) 2 CO (2.05 ppm). 1 H NMR spectra at variable temperature were obtained in 5 mm NMR precision
tube on a Bruker AV-III 400 NMR spectrometer. NOESY spectra were obtained
in 5 mm NMR precision tubes at 298 K on a Bruker DPX-500 NMR spectrometer. 1 H NMR peaks were internally referenced to CHD 2 OD
(3.31 ppm) for methanol- d 4 or 1,4-dioxane
(3.66 ppm). All data processing was carried out using MestReNova 9.0.1.
## Mass Spectrometry
Mass Spectrometry Electrospray ionization mass spectra
(ESI-MS) were obtained by preparing the sample in methanol using a
Bruker Esquire 2000 ion trap spectrometer. Samples were prepared in
methanol. The mass spectra were recorded with a scan range of m / z 50–500 for positive ions for L 1 -L 2 and m / z 400–1000 for positive ions for the complexes 1 – 4 .
## UV–Vis Spectroscopy
UV–Vis Spectroscopy UV–vis absorption
spectra were recorded on a Cary 300 spectrometer using quartz cuvettes
of 1 cm path-length (600 μL). The sample temperature was adjusted
to 298 K by PTP1 Peltier temperature controller. Samples were prepared
in methanol. Spectra were recorded from 200 to 600 nm. Data were processed
with Microsoft Excel 14.3.6 Mac version.