Half-Sandwich Arene Ruthenium(II) and Osmium(II) Thiosemicarbazone Complexes: Solution Behavior and Antiproliferative Activity

Article abstract of DOI:10.1021/acs.organomet.7b00875

We report the synthesis, characterization, and antiproliferative activity of organo-osmium(II) and organo-ruthenium(II) half-sandwich complexes [(η⁶-p-cym)Os(L)Cl]Cl (1 and 2) and [(η⁶-p-cym)Ru(L)Cl]Cl (3 and 4), where L = N-(2-hydro

Full text of DOI:10.1021/acs.organomet.7b00875

Article
Cite This: Organometallics XXXX, XXX, XXX−XXX
Half-Sandwich Arene Ruthenium(II) and Osmium(II)
Thiosemicarbazone Complexes: Solution Behavior and
Antiproliferative Activity
Anna Gatti,†,‡ Abraha Habtemariam,‡ Isolda Romero-Canelon,‡,§ Ji-Inn Song,‡ Bindy Heer,‡
́
Guy J. Clarkson,‡ Dominga Rogolino,† Peter J. Sadler,*,‡ and Mauro Carcelli*,†
†Dipartimento di Scienze Chimiche, della Vita e della Sostenibilita
̀
Ambientale and CIRCMSB (Consorzio Interuniversitario di
Ricerca in Chimica dei Metalli nei Sistemi Biologici), University of Parma, Parco Area delle Scienze 11/A, 43124 Parma, Italy
‡Department of Chemistry, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, United Kingdom
§School of Pharmacy, University of Birmingham, Birmingham B15 2TT, United Kingdom
S
* Supporting Information
ABSTRACT: We report the synthesis, characterization, and
antiproliferative activity of organo-osmium(II) and organo-
ruthenium(II) half-sandwich complexes [(η6-p-cym)Os(L)Cl]-
Cl (1 and 2) and [(η6-p-cym)Ru(L)Cl]Cl (3 and 4), where L
= N-(2-hydroxy)-3-methoxybenzylidenethiosemicarbazide
(L1) or N-(2,3-dihydroxybenzylidene)-3-phenylthiosemicarba-
zide (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.
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 antiprolifer-
ative 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
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 Thiosemicarba-
zones (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 studies21
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 scaffold23 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
Received: December 7, 2017
© XXXX American Chemical Society
A
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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 104 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
micarbazide (L2) were synthesized according to previously
reported procedures.54,55 The reactions between [(η6-p-cym)-
MCl2]2 (M = Os and Ru) and the corresponding
thiosemicarbazone ligands were carried out in a mixture of
dry CH3OH and CH2Cl2 at ambient temperature and led to the
isolation of pseudo-octahedral complexes 1−4 of general
formula [(η6-p-cym)M(L)Cl]Cl in good yields. The identity
1
of the complexes was verified by 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 η6-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 [(η6-p-cym)Os(L1)Cl]Cl (1),
[(η6-p-cym)Os(L2)Cl]Cl·(CH3)2CO (2·(CH3)2CO), [(η6-p-
cym)Ru(L1)Cl]Cl (3), and [(η6-p-cym)Ru(L2)Cl]Cl·
(CH3)2CO (4·(CH3)2CO) 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 P212121, while complexes 2 and 4 crystallize in
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 [(η6-p-cym)Os(L)Cl]Cl (1 and 2) and
two analogous Ru(II) complexes [(η6-p-cym)Ru(L)Cl]Cl (3
and 4), where L = N-(2-hydroxy)-3-methoxybenzylidenethio-
semicarbazide (L1) or N-(2,3-dihydroxybenzylidene)-3-phenyl-
thiosemicarbazide (L2), respectively (Figure 1). This type of
triclinic system with centrosymmetric space group P1. 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 η6-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.
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
Figure 1. Schematic representation of ligands L1 and L2 and
corresponding osmium(II) and ruthenium(II) complexes 1−4.
ligands, which could in principle be tridentate, can confer
solution stability on their metal complexes; moreover they have
shown interesting cytotoxic properties54 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.
RESULTS AND DISCUSSION
■
Synthesis and Characterization of the Complexes.
Ligands N-(2-hydroxy)-3-methoxybenzylidenethiosemicarba-
zide (L1) and N-(2,3-dihydroxybenzylidene)-3-phenylthiose-
B
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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)
Os1−Cl1
Os1−S1
Os1−N4
S1−C2
H14−CE1
Os1−Cl1
Os1−S8
Os1−N10
S8−C8
H21−CE1
Ru1−Cl1
Ru1−S1
Ru1−N4
S1−C2
H14−CE1
Ru1−Cl1
Ru1−S8
Ru1−N10
S8−C8
2.4113(12)
2.3551(13)
2.118(4)
1.695(5)
2.563
S1−Os1−Cl1
N4−Os1−Cl1
N4−Os1−S1
86.52(4)
81.63(10)
81.63(11)
1
2.4030(5)
2.3527(5)
2.1227(17)
1.693(2)
2.500
S8−Os1−Cl1
N4−Os1−Cl1
N10−Os1−S8
87.81(2)
83.44(5)
81.79(5)
2·(CH3)2CO
2.4046(11)
2.3501(10)
2.125(3)
1.695(4)
2.548
S1−Ru1−Cl1
N4−Ru1−Cl1
N4−Ru1−S1
86.90(4)
83.06(9)
81.95(10)
3
2.3993(3)
2.3508(3)
2.1256(9)
1.6923(12)
2.486
S8−Ru1−Cl1
N10−Ru1−Cl1
N10−Ru1−S8
88.338(11)
84.77(3)
81.94(3)
4·(CH3)2CO
H21−CE1
thiosemicarbazone ligands, are observed (distances from 2.50 to
2.86 Å, Figure 2).
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
Solution Studies. 1H NMR studies were used to investigate
the stability of the four complexes in various solvents. 1H NMR
spectra of 1−4 were first recorded in MeOD-d4, 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
1
complexes contain chiral metal centers and in the 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
C
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and the aromatic ring of the TSC ligand in the E form, as
observed previously in analogous systems.21,63
1
Figure 3. Aromatic region of the time-dependent H NMR spectrum
of 1 in MeOD-d4 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.
1
The time dependence of the H NMR spectra of 1−4 (5
mM) in MeOD-d4 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
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.
1
reached over a period of 21 days. Variable-temperature 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).
coordinated ligand either with E or Z conformation have been
reported, confirming the possibility of having both isomers in
organometallic complexes.65
Analysis of the data provides evidence that the interconver-
sion 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,
1
Figure S2). However, the 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
1
paramagnetic species which broaden signals in the 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 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
1
mechanism is supported by the 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 1H NMR spectra of the
same crystals recorded in MeOD-d4 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
1
the catechol moiety is not involved in redox processes. 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-
d6 at 298 K for all the complexes (Figure 5). Comparison with
the 1H NMR obtained in MeOD at t = 0 indicates that one set
D
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Scheme 1. Proposed Mechanism for the E/Z Interconversion Process of the Coordinated Ligand L1 for Metal Complexes 1 and
3 in Methanol
1
Figure 5. Aromatic region of the H NMR spectrum of 2 recorded in acetone-d6 at 298 K and followed over 7 days. Red circles indicate proton
resonances related to the species with a coordinated solvent molecule.
Figure 6. Comparison of the aromatic region of the 1H NMR of complex 1 (upper spectrum) and that of the corresponding free ligand L1 (lower
spectrum) in DMSO-d6 at t = 0 and 298 K.
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 1H NMR spectrum of L2
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).
1
recorded in acetone-d6. It is notable that the H NMR spectra
Due to the limited aqueous solubility of the metal complexes,
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
antiproliferative cell assays were performed using stock
E
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Table 2. IC50 Values (μM) for L1 and L2 and Related Metal Complexes 1−4 towards Human Ovarian (A2780), Cisplatin-
a
Resistant Ovarian (A2780Cis), Lung (A549), Colon (HCT116), and Prostate (PC3) Cancer Cell Lines
cell lines IC50 (μM)
resistance factors
A2780Cis/A2780
compound
A2780
A2780Cis
A549
HCT116
PC3
L1
0.85 0.03
0.27 0.02
1.60 0.02
0.75 0.08
4.2 0.3
0.12 0.02
1.23 0.08
6.6 0.9
42
23
2
1
30.6 0.5
6.1 0.1
4.6 0.2
0.14
4.55
4.12
9.60
1.33
3.47
11.25
L2
33
24
5
2
1
2.4 0.2
17
21
1.60 0.08
19
1
2
7.2 0.1
1
2.7 0.2
10.5 0.3
1.64 0.08
5.2 0.1
3
5.6 0.8
1
4
0.36 0.03
1.2 0.2
1.25 0.06
13.5 0.3
1.38 0.04
9.8 0.4
CDDP
3.1 0.2
a
Clinical drug cisplatin (CDDP) is used as positive control.
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
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. IC50 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.
Both thiosemicarbazones L1 and L2 are highly potent toward
ovarian cell lines A2780 and A2780Cis. L1 in particular exhibits
IC50 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 IC50 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 −NH2. 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 IC50 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
1
investigated also in DMSO-d6. In the H NMR spectra of 1
and 3 in DMSO-d6 recorded at 298 K, three different sets of
signals were observed. A comparison with the 1H 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(η6-p-
cym)(DMSO)2Cl]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(η6-p-cym)(L)Cl2]
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.
Complexes 2 and 4 in DMSO-d6 gave a complex pattern of
1H 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(η6-p-cym)(DMSO)2Cl]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 1H NMR spectrum. A
possible explanation for this set of signals is the presence of a
monosolvated species of the type [Os(η6-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 D2O−(10%)DMSO, monitored for 24 h to
1
mimic the biological test conditions. The 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
F
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5% CO2 humidified atmosphere and passaged at ca. 70−80%
confluence.
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.
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. IC50 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 Thiosemi-
carbazone 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.
CONCLUSIONS
■
Two new osmium(II) and two ruthenium(II) half-sandwich
complexes [(η6-p-cym)M(L)Cl]Cl containing a thiosemicarba-
1
zone ligand (L) were synthesized and characterized by 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
1
appear in the 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.
N-(2-Hydroxy)-3-methoxybenzylidenethiosemicarbazide (L1).
1
White powder, yield: 87%. H NMR (DMSO-d6): δ 11.39 (s, 1H,
NH), 9.17 (s, 1H, OH), 8.40 (s, 1H, CHN), 8.10−7.88 (2s, 1H
+1H, NH2), 7.52 (d, 1H, J = 7.5 Hz, CHAr), 6.95 (d, 1H, J = 7.5 Hz,
CHAr), 6.75 (t, 1H, J = 7.5 Hz, CHAr), 3.81 (s, 3H, OCH3). ESI-MS
(C9H11N3SO2, MeOH): m/z= 225 [M + H]+.
N-(2,3-Dihydroxybenzylidene)-3-phenylthiosemicarbazide (L2).
1
White powder, yield: 81%. H NMR (DMSO-d6): 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, CHAr), 7.49 (d, 2H, J = 8 Hz,
CHAr), 7.34 (t, 2H, J = 7.5 Hz, CHAr), 7.17 (t, 1H, J = 7.5 Hz, CHAr),
6.81 (d, 1H, J = 8 Hz, CHAr), 6.64 (t, J = 8 Hz, CHAr). ESI-MS
(C14H13N3SO2, 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%.
[(η6-p-cym)MCl2]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.
EXPERIMENTAL SECTION
■
Materials. All commercial reagents were used as received. 2-
Hydroxy-3-methoxybenzaldehyde, 2,3-dihydroxybenzaldehyde, thiose-
micarbazide, and 4-phenylthiosemicarbazide were purchased from
Sigma-Aldrich; OsCl3·nH2O and RuCl3·nH2O 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. [(η6-p-cym)OsCl2]2 and [(η6-p-
cym)RuCl2]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
[Os(η6-p-cym)Cl(L1)]Cl (1). Orange powder, yield: 98%. Anal. Calcd
for C19H25Cl2N3O2OsS: C, 36.77; H, 4.06; N, 6.77. Found: C, 36.51;
H, 4.56; N, 6.70. 1H NMR (MeOD-d4): δ 8.76 (s, 1H, CHN), 7.86
(d, 1H, J = 8 Hz, CHAr), 7.25 (d, 1H, J = 8 Hz, CHAr), 7.01 (t, 1H, J =
8 Hz, CHAr), 5.87 (d, 1H, J = 5.5 Hz, CHp‑cym), 5.44 (d, 1H, J = 5.5
Hz, CHp‑cym), 5.31 (d, 1H, J = 5.5 Hz, CHp‑cym), 4.90 (d, 1H, J = 5.5
Hz, CHp‑cym), 3.99 (s, 3H, OCH3), 2.54 (m, 1H, J = 7 Hz, CHi‑prop),
2.16 (s, 3H, CH3), 1.20−1.11 (2d, 3H+3H, J = 7 Hz, CH3i‑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(η6-p-cym)Cl(L2)]Cl (2). Orange powder, yield: 72%. Anal. Calcd
for C24H27Cl2N3O2OsS·H2O: C, 41.14; H, 4.17; N, 6.00. Found: C,
G
DOI: 10.1021/acs.organomet.7b00875
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Article
1
40.81; H, 4.16; N, 6.23. H NMR (MeOD-d4): δ 8.87 (s, 1H, CH
N), 7.75 (d, 1H, J = 7 Hz, CHAr), 7.48 (t, 2H, J = 7 Hz, CHAr), 7.43
(d, 2H, J = 7 Hz, CHAr), 7.35 (t, 1H, J = 7 Hz, CHAr), 7.08 (dd, 1H, J
= 8 Hz, CHAr), 6.88 (t, 1H, J = 8 Hz, CHAr), 5.86 (d, 1H, J = 5.5 Hz,
CHp‑cym), 5.49 (d, 1H, J = 5.5 Hz, CHp‑cym), 5.31 (d, 1H, J = 5.5 Hz,
CHp‑cym), 4.93 (d, 1H, J = 5.5 Hz, CHp‑cym), 2.55 (m, 1H, J = 7 Hz,
CHi‑prop), 1.21−1.13 (2d, 3H+3H, J = 7 Hz, CH3i‑prop). ESI-MS
(positive ions, CH3OH): m/z= 648 [M − Cl]+. Crystals suitable for X-
ray analysis were obtained by slow evaporation of a saturated acetone
solution.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.organo-
met.7b00875.
X-ray crystallographic data, 1H-1H 2D NMR NOESY and
1H NMR spectra, UV−vis spectra, dose−response curves
(PDF)
[Ru(η6-p-cym)Cl(L1)]Cl (3). Red powder, yield: 78%. Anal. Calcd for
C19H25Cl2N3O2RuS·CH3OH: C, 42.63; H, 5.19; N, 7.46. Found: C,
Accession Codes
CCDC 1584383−1584386 contain the supplementary crystal-
lographic data for this paper. These data can be obtained free of
charge via www.ccdc.cam.ac.uk/data_request/cif, or by email-
ing data_request@ccdc.cam.ac.uk, or by contacting The
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
1
41.92; H, 5.21; N, 7.34. H NMR (MeOD-d4): δ 8.79 (s, 1H, CH
N), 8.06 (d, 1H, J = 8 Hz, CHAr), 7.28 (d, 1H, J = 8 Hz, CHAr), 7.07
(t, 1H, J = 8 Hz, CHAr), 5.71 (d, 1H, J = 6 Hz, CHp‑cym), 5.17 (d, 1H, J
= 6 Hz, CHp‑cym), 5.04 (d, 1H, J = 6 Hz, CHp‑cym), 4.00 (s, 3H, OCH3),
2.64 (m, 1H, J = 7 Hz, CHi‑prop), 2.10 (s, 3H, CH3), 1.20−1.14 (2d,
3H+3H, J = 7 Hz, CH3i‑prop). ESI-MS (positive ions, CH3OH): 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.
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: P.J.Sadler@warwick.ac.uk. Phone: (+44) 024 7652
3818.
*E-mail: mauro.carcelli@unipr.it. Phone (+33) 0521 905427.
ORCID
Dominga Rogolino: 0000-0003-2295-5783
Peter J. Sadler: 0000-0001-9160-1941
Mauro Carcelli: 0000-0001-5888-4556
[Ru(η6-p-cym)Cl(L2)]Cl (4). Red powder, yield: 87%. Anal. Calcd for
C24H27Cl2N3O2RuS·CH3COCH3: C, 49.77; H, 5.10; N, 6.45. Found:
C, 49.54; H, 5.23; N, 7.01. 1H NMR (MeOD-d4): δ 8.90 (s, 1H, CH
N), 7.95 (dd, 1H, J = 8 Hz, J′ = 1 Hz, CHAr), 7.48 (t, 2H, J = 7.5 Hz,
CHAr), 7.41 (d, 2H, J = 7.5 Hz, CHAr), 7.37 (d, 1H, J = 7 Hz, CHAr),
7.09 (td, 1H, J = 8 Hz, J′ =1 Hz, CHAr), 6.94 (t, 1H, J = 7.5 Hz, CHAr),
5.86 (d, 1H, J = 5.5 Hz, CHp‑cym), 5.49 (d, 1H, J = 5.5 Hz, CHp‑cym),
5.31 (d, 1H, J = 5.5 Hz, CHp‑cym), 4.93 (d, 1H, J = 5.5 Hz, CHp‑cym),
2.55 (m, 1H, J = 7 Hz, CHi‑prop), 1.21−1.13 (2d, 3H+3H, J = 7 Hz,
CH3i‑prop). ESI-MS (positive ions, CH3OH): 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 ShelXT71 structure solution
program using Direct Methods and refined with the ShelXL
refinement package using least-squares minimization.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank The Wellcome Trust (107691/Z/15/Z), EPSRC
(EP/F034210/1) and the University of Warwick Development
Trust for their support for this work. “Centro Interdiparti-
mentale Misure Giuseppe Casnati” of the University of Parma
is thanked for facilities.
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DOI: 10.1021/acs.organomet.7b00875
Organometallics XXXX, XXX, XXX−XXX