Synthesis and In Vitro ( Anticancer ) Evaluation of η 6-Arene Ruthenium Complexes Bearing Stannyl Ligands

Treatment of the known half-sandwich complexes of the type [(η-C6H6)RuCl2(P(OR)3)] (R = Me or Ph) with SnCl2 yielded three new half-sandwich ruthenium complexes (C1–C3): [(η-C6H6)RuCl(SnCl3)(P(OMe)3)] (C1), [(η-C6H6)RuCl(SnCl3)(P(OPh)3)] (C2) and the bis-stannyl complex [(η-C6H6)Ru(SnCl3)2(P(OMe)3)] (C3) by facile insertion of SnCl2 into the Ru–Cl bonds. Treatment of the known complexes [(η-C6H6)RuCl(SnCl3)(PPh3)] and [(η-C6H6)RuCl2(PPh3)] with 4-dimethylaminopyridine (DAMP) and ammonium tetrafluoroborate afforded the complex salts: [(η-C6H6)Ru(SnCl3)(PPh3)(DAMP)]BF4 (C4) and [(η-C6H6)RuCl(PPh3)(DAMP)]BF4 (C5) respectively. Complexes C1–C5 have been fully characterized by spectroscopic means (IR, UV–vis, multinuclear NMR, ESI–MS) and their thermal behaviour elucidated by thermal gravimetric analysis (TGA). Structural characterization by single crystal X-ray crystallography of the novel complex C2 and [(η-C6H6)RuCl2(P(OPh)3)], the latter having escaped elucidation by this method, is also reported. Finally, the cytotoxicity of the complexes was determined on the A2780 (human ovarian cancer), A2780cisR (human ovarian cis-platin-resistant cancer), and the HEK293 (human embryonic kidney) cell lines and discussed, and an attempt is made to elucidate the effect of the stannyl ligand on cytotoxicity.

The reaction of half-sandwich ruthenium(II) arene complexes [(η 6 -C6H6)RuCl2(PR3)] (R = aryl or O-Aryl, O-alkyl) with SnCl2 is also known to yield a Ru(II) complex exhibiting a strong covalent Ru-Sn bond via facile insertion of the SnCl2 moiety into the Ru-Cl bond [35,36].While the reaction of SnX2 (X = halide) with other metals, such as palladium and platinum, has been extensively studied [37,38], the analogous reaction with ruthenium derivatives has received far less attention.The addition of trichlorostannyl ligands to the coordination sphere of the ruthenium centre is known to enhance the anticancer properties of the complexes from earlier investigations [39], possibly due to the enhanced σ-donor properties of the ligand, which might facilitate and promote the binding of the agent to potential biomolecular targets.Although there is known to be an increase in cytotoxicity, only a few examples of this class, i.e., those bearing stannyl groups, have been tested.
In this work we report the synthesis and characterisation of a series of complexes of formula [(η 6 -C6H6)RuX(SnCl3)(P(OR)3)] (X = Cl, SnCl3 and R = Me, Ph), and some cationic derivatives [(η 6 -C6H6)RuX(PPh3)(DAMP)]BF4 (X = SnCl3, Cl), with a view of attempting to delineate the effect of a trichlorostannyl group on cytotoxicity against several cancer cell-lines.Hence, the cytotoxicity of these new complexes against A2780 and A2780cisR (cis-platin resistant) human ovarian carcinoma cells and non-cancerous HEK293 embryonic kidney cells are reported, along with the known  [31,32].A stable phosphine complex, reported in the 1970s [(η 6 -C 6 H 6 )RuCl 2 (PPh 3 )] [31,32], which is obtained in high yields as a product via a reaction of the afore-mentioned ruthenium dimer with triphenylphosphine.Similarly the phosphite derivatives [(η 6 -C 6 H 6 )RuCl 2 (P(OMe) 3 )] and [(η 6 -C 6 H 6 )RuCl 2 (P(OPh) 3 )] are afforded by reaction of [(η 6 -C 6 H 6 )Ru(µ-Cl)Cl] 2 with trimethyl phosphite and triphenyl phosphite in an analogous fashion [32][33][34].Surprisingly, despite the fact that these easily accessible phosphite complexes have been known since the early 1970s, they have not undergone rigorous in vitro cytotoxic testing with respect to cancer cell lines.This encouraged us to prepare and evaluate their cytotoxic activity.Moreover, to the best of our knowledge, the complex [(η 6 -C 6 H 6 )RuCl 2 (P(OPh) 3 )] has also not been structurally characterised by single crystal X-ray diffraction analysis, which prompted us to carry out such an investigation, and this is also reported herein.
The reaction of half-sandwich ruthenium(II) arene complexes [(η 6 -C 6 H 6 )RuCl 2 (PR 3 )] (R = aryl or O-Aryl, O-alkyl) with SnCl 2 is also known to yield a Ru(II) complex exhibiting a strong covalent Ru-Sn bond via facile insertion of the SnCl 2 moiety into the Ru-Cl bond [35,36].While the reaction of SnX 2 (X = halide) with other metals, such as palladium and platinum, has been extensively studied [37,38], the analogous reaction with ruthenium derivatives has received far less attention.The addition of trichlorostannyl ligands to the coordination sphere of the ruthenium centre is known to enhance the anticancer properties of the complexes from earlier investigations [39], possibly due to the enhanced σ-donor properties of the ligand, which might facilitate and promote the binding of the agent to potential biomolecular targets.Although there is known to be an increase in cytotoxicity, only a few examples of this class, i.e., those bearing stannyl groups, have been tested.
Scheme 2. Synthesis of the cationic complexes C4 and C5.

Spectroscopic Characterisation
Complexes C1-C3 all exhibit an upfield shifted resonance signal, for the arene protons associated with the η 6 -coordinated ring, in the 1 H NMR spectra: δ = 6.31 ppm (C1 and C3), 5.82 (C2).In complexes C1 and C3, a doublet is observed in the 1 H NMR spectrum corresponding to the P(OMe) 3 groups due to coupling to the phosphorus atom: The 31 P{ 1 H} NMR spectrum of complex C1 is shown in Figure 1.Both 119 Sn and 117 Sn satellites are visible, along with rotational side-bands on the main signal, the latter of which is typical in solution 31 P NMR spectra.Inspection of the experimental solution UV-vis spectra of the complexes C1-C3 reveal that, for the bis-trichlorostannyl complex C3, a much higher wavelength of absorption (λ = 459 nm) is observed compared to C1: λ = 348 and C2: λ = 351 nm, indicating pertubation in the electronic situation upon bis SnCl2-insertion.This is most likely due to the enhanced σ-donor capacity of SnCl3 − vs. Cl − .For the ionic complex the UV-vis spectra reveal absorptions at λ = 364 nm (C4) and λ = 335 nm (C5), comparable to that of C1 and C2.All complexes were also subjected to a TGA analysis to obtain information on their thermal behaviour and stability.In all cases the complexes are thermally robust with the first onset of mass loss occurring well in excess of 100 °C: (C1: 122 °C, C2: 186 °C, C3: 223 °C, C4: 184 °C, and C5: 190 °C), which is in accord with the melting point (decomposition temperature) determinations.An exact assignment of the mode of decomposition, i.e., according to which fragments are lost at which temperature was undertaken, and in all cases one decomposition step can be tentatively traced to the loss of the η 6 coordinated ring.Figure 2 shows the TGA trace of complex C1.The approximately 12% mass loss can be roughly correlated to the loss of the arene ring.Inspection of the experimental solution UV-vis spectra of the complexes C1-C3 reveal that, for the bis-trichlorostannyl complex C3, a much higher wavelength of absorption (λ = 459 nm) is observed compared to C1: λ = 348 and C2: λ = 351 nm, indicating pertubation in the electronic situation upon bis SnCl 2 -insertion.This is most likely due to the enhanced σ-donor capacity of SnCl 3 − vs. Cl − .For the ionic complex the UV-vis spectra reveal absorptions at λ = 364 (C4) and λ = 335 nm (C5), comparable to that of C1 and C2.All complexes were also subjected to a TGA analysis to obtain information on their thermal behaviour and stability.In all cases the complexes are thermally robust with the first onset of mass loss occurring well in excess of 100 An exact assignment of the mode of decomposition, i.e., according to which fragments are lost at which temperature was undertaken, and in all cases one decomposition step can be tentatively traced to the loss of the η 6 coordinated ring.Figure 2 shows the TGA trace of complex C1.The approximately 12% mass loss can be roughly correlated to the loss of the arene ring.

X-ray Crystallography
Single crystals of complex C2 and [(η 6 -C6H6)RuCl2(P(OPh)3)] were obtained and single crystal X-ray diffraction studies were undertaken and their structures are shown in Figures 3 and 4, respectively with selected metric parameters provide in the figure captions (other bond angles and lengths are available in the supporting information).It is somewhat surprising that the complex [(η 6 -C6H6)RuCl2(P(OPh)3)] has eluded structural characterisation by X-ray diffraction, despite being reported in the 1970s.

X-ray Crystallography
Single crystals of complex C2 and [(η 6 -C 6 H 6 )RuCl 2 (P(OPh) 3 )] were obtained and single crystal X-ray diffraction studies were undertaken and their structures are shown in Figures 3 and 4, respectively with selected metric parameters provide in the figure captions (other bond angles and lengths are available in the supporting information).It is somewhat surprising that the complex [(η 6 -C 6 H 6 )RuCl 2 (P(OPh) 3 )] has eluded structural characterisation by X-ray diffraction, despite being reported in the 1970s.

X-ray Crystallography
Single crystals of complex C2 and [(η 6 -C6H6)RuCl2(P(OPh)3)] were obtained and single crystal X-ray diffraction studies were undertaken and their structures are shown in Figures 3 and 4, respectively with selected metric parameters provide in the figure captions (other bond angles and lengths are available in the supporting information).It is somewhat surprising that the complex [(η 6 -C6H6)RuCl2(P(OPh)3)] has eluded structural characterisation by X-ray diffraction, despite being reported in the 1970s.ORTEP view of (C2) with atom-labelling scheme and thermal ellipsoids drawn at the 50% probability level.

Cytotoxicity Studies
The antiproliferative activity of the neutral complexes C1-C3, cationic complexes C4 and C5 and the three known compounds [(η 6 -C6H6)RuCl2(PPh3)], [(η 6 -C6H6)RuCl2(P(OPh)3)], and [(η 6 -C6H6)RuCl(SnCl3)(PPh3)] were investigated in vitro against human ovarian cancer cells A2780 and the A2780cisR variant with aquired cis-platin resistance, as well as against non-cancerous human embryonic kidney (HEK293) cells (Table 1).The cytotoxicity of the latter three complexes has not been reported previously and are shown together with cis-platin for comparison (Table 2).IC50 values of the compounds were determined after exposure of the cells to the compounds for 72 h using the MTT assay.
Complexes C1 and C3 with trimethylphosphite ligands did not induce cytotoxicity even at concentrations as high as 500 μM and 200 μM, respectively, whereas all complexes with triphenylphosphite or triphenylphosphine ligands exhibit considerable cytotoxicity in A2780, A2780cisR and HEK293 cells.This is somewhat surprising as the presence of the SnCl3 moiety would have been expected to enhance the cytotoxic effect of the complex (see above).In case of complex C3, this may be due to its rather low solubility due to the presence of two trichlorostannyl groups attached to the Ru centre.Notably, the cationic complexes C4 and C5 display IC50 values in the low micromolar concentration range and, compared to cis-platin, showed even high efficacy in A2780cisR cells.Whereas complex C5 bearing a chloride ligand showed similar activity in all three cell lines, complex C4 with the chlorine replaced by the SnCl3 moiety, showed slight cancer cell selectivity.This phenomenon was not observed for [(η 6 -C6H6)RuCl2(PPh3)] and [(η 6 -C6H6)RuCl(SnCl3)(PPh3)], where the tin congener induced generally a two-fold higher Both complexes exhibit the typical piano-stool geometry with the metal centre being coordinated by the arene in η 6 fashion.

Cytotoxicity Studies
The antiproliferative activity of the neutral complexes C1-C3, cationic complexes C4 and C5 and the three known compounds [(η 6 -C 6 H 6 )RuCl 2 (PPh 3 )], [(η 6 -C 6 H 6 )RuCl 2 (P(OPh) 3 )], and [(η 6 -C 6 H 6 )RuCl(SnCl 3 )(PPh 3 )] were investigated in vitro against human ovarian cancer cells A2780 and the A2780cisR variant with aquired cis-platin resistance, as well as against non-cancerous human embryonic kidney (HEK293) cells (Table 1).The cytotoxicity of the latter three complexes has not been reported previously and are shown together with cis-platin for comparison (Table 2).IC 50 values of the compounds were determined after exposure of the cells to the compounds for 72 h using the MTT assay.
Complexes C1 and C3 with trimethylphosphite ligands did not induce cytotoxicity even at concentrations as high as 500 µM and 200 µM, respectively, whereas all complexes with triphenylphosphite or triphenylphosphine ligands exhibit considerable cytotoxicity in A2780, A2780cisR and HEK293 cells.This is somewhat surprising as the presence of the SnCl 3 moiety would have been expected to enhance the cytotoxic effect of the complex (see above).In case of complex C3, this may be due to its rather low solubility due to the presence of two trichlorostannyl groups attached to the Ru centre.Notably, the cationic complexes C4 and C5 display IC 50 values in the low micromolar concentration range and, compared to cis-platin, showed even high efficacy in A2780cisR cells.Whereas complex C5 bearing a chloride ligand showed similar activity in all three cell lines, complex C4 with the chlorine replaced by the SnCl 3 moiety, showed slight cancer cell selectivity.This phenomenon was not observed for [(η 6 -C 6 H 6 )RuCl 2 (PPh 3 )] and [(η 6 -C 6 H 6 )RuCl(SnCl 3 )(PPh 3 )], where the tin congener induced generally a two-fold higher cytotoxicity, but did not contribute to cancer cell selectivity.In contrast, the complexes with triphenylphosphite ligands [(η 6 -C 6 H 6 )RuCl 2 (P(OPh) 3 )] and its tin congener C2 show the opposite behaviour with C2 being >20-fold less potent than [(η 6 -C 6 H 6 )RuCl 2 (P(OPh) 3 )].

Crystallographic Structure Determination
Crystals of X-ray diffraction quality were obtained by slow evaporation of a dichloromethane-diethyl ether 1:1 mixture of (C2) and [(η 6 -C 6 H 6 )RuCl 2 (P(OPh) 3 )] at room temperature using a vial with a narrow opening.For X-ray structure analyses the crystals are mounted onto the tip of glass fibers, and data collection was performed with a BRUKER-AXS SMART APEX CCD diffractometer using graphite-monochromated Mo Kα radiation (0.71073 Å) (Table 2).The data were reduced to F o 2 and corrected for absorption effects with SAINT [43] and SADABS [44,45], respectively.The structures were solved by direct methods and refined by full-matrix least-squares method (SHELXL97) [46].If not noted otherwise all non-hydrogen atoms were refined with anisotropic displacement parameters.All hydrogen atoms were located in calculated positions to correspond to standard bond lengths and angles.All diagrams are drawn with 30% probability thermal ellipsoids and all hydrogen atoms were omitted for clarity.Figures of solid state molecular structures were generated using Ortep-3 as implemented in WINGX [47] and rendered using POV-ray 3.6 [48].

Cell Cultures and Cytotoxicity Measurements
Human A2780 and A2780cisR ovarian carcinoma cells were obtained from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK) and non-cancerous HEK293 cells were obtained from ATCC (Sigma, St. Gallen, Switzerland).A2780 were routinely grown in RPMI (Roswell Park Memorial Institute) medium: 1640 GlutaMAX (Lifetechnologies, Zug, Switzerland), while HEK293 were maintained in DMEM medium (Dulbecco's modified media), both containing 10% heat-inactivated fetal bovine serum (FBS, Pan Biotech, Aidenbach, Germany) and 1% antibiotics (penicillin/streptomycin), at a humidified atmosphere with 5% CO 2 at 37 • C. Cytotoxicity was determined using the MTT assay (MTT = 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide).Cells were seeded in 96-well plates as monolayers with 100 µL of cell solution per well and were pre-incubated for 24 h in the cell culture medium.Compounds were prepared as DMSO stock solutions that were dissolved in the culture medium and two-fold serially diluted to the appropriate concentration to give a final DMSO concentration of maximum 0.5%.100 µL of the compound solution were added to each well and the plates were incubated for 72 h.Subsequently, MTT (5 mg/mL solution, 20 µL per well) was added to the cells and the plates were incubated for another 4 h.The culture medium was aspirated, and the purple formazan crystals formed by the mitochondrial dehydrogenase activity of vital cells were dissolved in DMSO (100 µL).The optical density, directly proportional to the number of surviving cells, was quantified at 590 nm using a multiwell plate reader (Molecular Devices).The fraction of surviving cells was calculated from the absorbance of untreated control cells.The IC 50 values for the inhibition of cell growth were determined by fitting the plot of the logarithmic percentage of surviving cells against the logarithm of the drug concentration using a linear regression function.Evaluation is based on means (±SD) from at least two independent experiments, each comprising four tests per concentration level.

Conclusions
A series of novel, neutral, and cationic η 6 -arene ruthenium(II) complexes, some bearing one or two trichlorostannyl groups, have been synthesized, characterized, and tested in vitro for antiproliferative activity against human ovarian cancer cells and a non-tumorigenic cell line.Complexes C1 and C3 exhibit rather poor cyctotoxic activity, whilst complex C2 exhibits moderate activity.The lack of potency of complexes C1 and C3 may be linked to solubility in aqueous media, despite the presence of stannyl ligands expected to enhance the cytotoxicity.The ionic complexes C4 and C5 are cytotoxic, with an activity similar to cis-platin, with C4 even showing a degree of cancer cell selectivity.We are currently attempting to further delineate the effect of the SnCl 3 − moiety on related complexes, taking solubility into consideration, and will report these endeavours in due course.

Chart 1 .
Examples of anti-cancer ruthenium-based agents.
3 J(H,P): C1: 12.0 Hz, C3: 12.3 Hz.Complexes C1-C3 exhibit singlet resonance signals in their31 P{1 H} NMR spectra: (C1: δ = 131.2,C2: δ = 122.1,C3: δ = 136.5 ppm).Notaby, the presence of both119 Sn and 117 Sn satellites, flanking the main resonance signals in all three complexes (C1-C3) are visible in these spectra due to 2 J(Sn,P) coupling.The presence of the Sn satellites in the31 P{ 1 H} NMR spectra suggest, that in DMSO(dimethyl sulfoxide), the complexes are stable and dynamic SnCl 3 − exchange is unlikely to occur.The formation, in DMSO solutions, of [(η 6 -C 6 H 6 )Ru(SnCl 3 )(DMSO)(PR 3 )] + Cl − can be ruled out for the mono-insertion products C1 and C2 over the time periods of the NMR measurements in DMSO-d 6 (12 h).The cationic complexes C4 and C5 exibit dramatically shielded chemical shift positions in their respective 31 P{ 1 H} NMR spectra (C4: δ = 26.7,C5: δ = 36.0ppm) compared with the neutral complexes C1-C3, owing to their cationic nature.Unfortunately 119 Sn NMR spectroscopy could not be carried out on the tin compounds due to the lack of a suitable probe in our laboratories.The 31 P{ 1 H} NMR spectrum of complex C1 is shown in Figure 1.Both 119 Sn and 117 Sn satellites are visible, along with rotational side-bands on the main signal, the latter of which is typical in solution 31 P NMR spectra.Inorganics 2017, 5, 44 4 of 13 In complexes C1 and C3, a doublet is observed in the 1 H NMR spectrum corresponding to the P(OMe)3 groups due to coupling to the phosphorus atom: 3 J(H,P): C1: 12.0 Hz, C3: 12.3 Hz.Complexes C1-C3 exhibit singlet resonance signals in their 31 P{ 1 H} NMR spectra: (C1: δ = 131.2,C2: δ = 122.1,C3: δ = 136.5 ppm).Notaby, the presence of both 119 Sn and 117 Sn satellites, flanking the main resonance signals in all three complexes (C1-C3) are visible in these spectra due to 2 J(Sn,P) coupling.The presence of the Sn satellites in the 31 P{ 1 H} NMR spectra suggest, that in DMSO(dimethyl sulfoxide), the complexes are stable and dynamic SnCl3 − exchange is unlikely to occur.The formation, in DMSO solutions, of [(η 6 -C6H6)Ru(SnCl3)(DMSO)(PR3)] + Cl − can be ruled out for the mono-insertion products C1 and C2 over the time periods of the NMR measurements in DMSO-d6 (12 h).The cationic complexes C4 and C5 exibit dramatically shielded chemical shift positions in their respective 31 P{ 1 H} NMR spectra (C4: δ = 26.7,C5: δ = 36.0ppm) compared with the neutral complexes C1-C3, owing to their cationic nature.Unfortunately 119 Sn NMR spectroscopy could not be carried out on the tin compounds due to the lack of a suitable probe in our laboratories.

Figure 1 .
Figure 1.The 31 P NMR spectrum of complex C1 in which the main resonance signal is flanked with 117 Sn (inner) and 119 Sn (outer) satellites.

Figure 1 .
Figure 1.The 31 P NMR spectrum of complex C1 in which the main resonance signal is flanked with 117 Sn (inner) and 119 Sn (outer) satellites.

Figure 2 .
Figure 2. TGA trace of complex C1 with the onset of decomposition occuring at 121.51 °C.

Figure 2 .
Figure 2. TGA trace of complex C1 with the onset of decomposition occuring at 121.51 • C.

Figure 2 .
Figure 2. TGA trace of complex C1 with the onset of decomposition occuring at 121.51 °C.