Evaluation of the Antiproliferative Properties of CpRu Complexes Containing N-Methylated Triazaphosphaadamantane Derivatives

Piano-stool-{CpRu} complexes containing 1,3,5-triaza-7-phosphaadamantane (PTA), N-methyl-1,3,5-triaza-7-phosphaadamantane (mPTA), and 3,7-dimethyl-1,3,7-triaza-5-phosphabyciclo[3.3.1]nonane (dmoPTA) were evaluated as drugs against breast cancer. The evaluated compounds include two new examples of this family, the complexes [RuCp(DMSO-κS)(HdmoPTA)(PPh3)](CF3SO3)2 (8) and [RuCp(PPh3)2-µ-dmoPTA-1κP-2κ2N,N′-PdCl2](CF3SO3) (11), which have been synthesized and characterized by NMR, IR, and single-crystal X-ray diffraction. The cytotoxic activity of compounds was evaluated against MDA-MB-231 breast cancer cells, and the three most active complexes were further tested against the hormone-dependent MCF-7 breast cancer cell line. Their cell death mechanism and ruthenium uptake were also evaluated, as well as their binding ability to human serum albumin.


Introduction
Te development of new drugs for the treatment of cancer is certainly a topic of preeminent importance.Since the discovery of the anticancer activity of cis-[PtCl 2 (NH 3 ) 2 ] (cisplatin) by Rosenberg et al. in the 1960s [1,2], large research eforts have been focused on the development of new platinum-based drugs, some of which are currently used in numerous chemotherapeutic treatments [3][4][5].However, their side efects, lack of selectivity, and resistance-related issues pushed the scientifc community to explore complexes with similar geometry and chemical properties but containing other metals such as Pd(II) [6][7][8].Tis strategy has led to the recent regulatory approval in Europe of the Pd-porphyrin complex padeliporfn (WST11) for photodynamic therapy treatments [9].
Research eforts targeted to obtain alternatives to Pt(II) compounds have led to the development of new complexes containing metals that adopt diferent coordination geometries concerning the strictly square planar of Pt(II) and Pd(II).Among the transition metals that meet this requirement, ruthenium is one of the most promising [10][11][12].Several ruthenium-based compounds have shown outstanding encouraging results such as Ru(III) complexes KP1019 (replaced by NKP1339/IT-139) and NAMI-A, which entered phases I and II of clinical trials, respectively [12].Other examples are the Ru(II) complex TLD1433, which was evaluated in phase II [13,14], and the compounds of the family of RAPTAs, which were tested in preclinical trials [15][16][17].Tis latter group of complexes exhibits a Ru-(η 6 -arene) scafold and 1,3,5-triaza-7-phosphaadamantane (PTA, 1) together with two chlorides (Figure 1) [18].Te combination of ligands and their arrangement around the metal provides a stable platform with adequate hydrosolubility, stabilizing the Ru(II) oxidation state [19].
Te reasons that determine the signifcant cytotoxic activity of heterometallic complexes containing dmoPTA are not still envisaged.As a part of our eforts to shed light on this topic, some signifcantly active previously published complexes containing PTA and PTA derivatives (Figure 1) were compared with the two new members of this family: monometallic [RuCp(DMSO-κS)(HdmoPTA)(PPh 3 )](CF 3 SO 3 ) 2 (8) and bimetallic [RuCp(PPh 3 ) 2 -µ-dmoPTA-1κP-2κ 2 N,N′-PdCl 2 ] (CF 3 SO 3 ) (11).Complexes 1-11 were evaluated against the MDA-MB-231 breast cancer cell line, and the most active compounds (9)(10)(11) were further tested against the MCF-7 breast cancer cell line.In addition, their cell death mechanism and cell distribution (Ru quantifcation) were also determined.To gain insight into the possible role of human serum albumin (HSA) in their bloodstream distribution, their interaction with HSA was also quantifed using steady-state and time-resolved fuorescence.

Stability Studies.
Te assessment of the biological properties of the complexes requires their dissolution in a cell culture medium which is mainly constituted by water.When the complex is not hydrosoluble enough, the used protocol contemplates preliminary dissolution in DMSO before the addition to the culture medium [36].Tus, it is important to determine the stability and behaviour of the complexes in both DMSO and water/ DMSO mixtures to check whether diferent species have been formed when dissolved.Dissolutions in DMSO-d 6 and DMSO-d 6 /D 2 O of the new complexes 8 and 11 were monitored by 31 P{ 1 H} NMR over time at room temperature and 37 °C.Te stability studies of the rest of the complexes (1-7, 9, 10) were published previously.All experiments were performed by a similar procedure: the complex (0.01 g) was introduced into a 5 mm NMR tube and dissolved in 0.5 mL of degassed solvent (DMSO-d 6 and a 1 : 1 mixture of DMSO-d 6 /D 2 O).Te solution was left at room temperature and monitored by 31 P{ 1 H} NMR frst every 15 minutes, and later in longer periods.Stability was also monitored at incubation cell-based assay temperature (37 °C).

Cell Lines and Culture Conditions.
Te MDA-MB-231 and MCF-7 human breast cancer cells were purchased from ATCC.Te MDA-MB-231 and MCF-7 cells were grown in Dulbecco's modifed Eagles' medium (DMEM high glucose) (Capricorn Scientifc) at 37 °C in 5% CO 2 supplemented with 10% fetal bovine serum (Capricorn Scientifc).HDF cells were purchased from Merck and were grown in the fbroblast growth medium.All cells were adherent in monolayers and, upon confuence, were washed with phosphate bufer saline (PBS) and harvested by digestion with trypsin-EDTA 0.05% (v/v).Te cells were suspended and transferred into new, sterile, culture fasks for maintenance or seeded in sterile test microplates for diferent assays.All cells were manipulated under aseptic conditions in a fow chamber.

Compound Cytotoxicity Evaluated Using the MTT
Assay.Te cells were adherent in monolayers and, upon confuence, were harvested by digestion with trypsin.Te cytotoxicity of the complexes against the tumour cells was assessed using the colorimetric assay MTT (3-(4,5-2-yl)-2,5ditetrazolium bromide), which evaluates the metabolic activity of viable cells.Tis assay measures the conversion of yellow tetrazolium into purple formazan by an active mitochondrial dehydrogenase in living cells.For this purpose, the cells (10-20 × 10 3 in 200 μL of the medium) were seeded into 96-well plates and incubated in a 5% CO 2 incubator at 37 °C.Te cells were settled for 24 h followed by the addition of a dilution series of the complexes in a medium (200 μL).Te complexes were frst solubilized in 100% DMSO, given a 10 mM stock solution, and then in a medium within the concentration range of 0.1-100 µM.DMSO did not exceed 1% even for the higher concentration used and was without cytotoxic efects.After 24 h of incubation, the treatment solutions were removed by aspiration, and MTT solution (200 μL, 0.5 mg•mL −1 in PBS) was added to each well.After 3 h at 37 °C/5% CO 2 , the solution was removed, and the purple formazan crystals formed inside the cells were dissolved in DMSO (200 μL) by thorough shaking.Te cellular viability was evaluated by measuring the absorbance at 570 nm by using a microplate spectrophotometer.Te IC 50 values were obtained by dose-response curves using the GraphPad Prism software (vs.5).

Cell Death Measurement Using Flow Cytometry: Te
Annexin V/PI Assay.After 24 h treatment with compounds 9, 10, and 11, both suspended and attached cells were collected and washed with PBS.Te cells were resuspended in 200 μL of 1x binding bufer and were incubated with 5 μL of FITC annexin V (BD Biosciences, San Jose, CA, USA) and 10 μL of PI (50 mg•mL −1 ) for 20 min in the dark.Te samples were analysed by using fuorescence-activated cell sorting (FACS) using Beckman Coulter EPICS XL-MCL.All data were analysed using the FlowJo software (version 10, Tree Star Inc.).

Complex Uptake and Distribution by ICP-MS.
For the cellular uptake experiments, MDA-MB-231 cells (approx.5•10 6 cells/5 mL medium) were exposed to the complexes at their respective IC 50 concentrations and in two conditions (6 h and 24 h of incubation time) and then washed with ice-cold PBS and centrifuged to obtain a cellular pellet [37].Te cytosol, membrane/particulate, cytoskeletal, and nuclear fractions were extracted using a FractionPREP ™ cell fractionation system (BioVision, USA) and performed according to the manufacturer's protocol.Te Ru ( 101 Ru) and Pd ( 106 Pd) contents in diferent fractions were measured by using Termo X-Series Quadrupole ICP-MS (Termo Scientifc) after the digestion of the samples.Briefy, the samples were digested with ultrapure HNO 3 (65%), H 2 O 2 , and H 3 PO 4 in a closed pressurized microwave digestion unit (Mars5, CEM) with medium-pressure HP500 vessels and then diluted in ultrapure water to obtain 2.0% (v/v) nitric acid.Te instrument was tuned using a multielement ICP-MS 71C standard solution (Inorganic Venture).Indium (115In) at 10μML −1 was used as the internal standard.

Interaction with Human Serum Albumin.
Te stock solutions of HSA were freshly prepared for each experiment by gently dissolving protein in PBS pH 7.4 (tablets from Fisher) for 1 hour to allow protein to completely hydrate.Te concentration of the protein in the stock solutions was determined by spectrophotometry using the molar absorption coefcient at 280 nm ε 280 (HSA) � 36,850 M −1 •cm −1 [38].
In each essay, all spectroscopic measurements were carried out on individually prepared samples to ensure the same preincubation time at (37.0 ± 0.1) °C, the same % of DMSO in the fnal samples, and the same exposure to excitation light and to avoid the need for dilution corrections.Moreover, for a direct titration protocol, a highly concentrated stock solution of the compounds in the bufer would be required, which would raise solubility issues.Dimethylsulfoxide (DMSO, from Fisher) was used to prepare concentrated stock solutions of each complex, followed by appropriate dilution to obtain the desired concentration.Te DMSO content was kept at 2% (v/v) in PBS pH 7.4 in all samples.Dilutions were carried out immediately before sample preparation.
Fluorescence measurements were performed at 25.0 ± 0.1 °C.Te fnal protein concentration in the individually prepared samples (3.72 to 5 μM) was kept constant in each assay, and the complex concentration was varied to obtain desired HSA: Ru-complex molar ratios ranging from 1 : 0.25 to 1 : 10.A control sample containing the same amount of protein and fnal DMSO content and with no complex was also prepared for each experiment.Appropriate blank samples with no protein but with the same complex concentrations were prepared as well for background correction.
Te steady-state fuorescence intensity was measured with excitation at 295 nm, and emission spectra were collected with bandwidths of 4 nm for both excitation and emission.For Stern-Volmer plots, the fuorescence intensity with emission at 340 nm was selected because it is near the maximum emission wavelength (so the sensitivity is close to maximum), but it is further to red in relation to the water Raman scattering peak [39].Tese values were corrected for the absorption and emission inner flter efects [40] using the absorbance recorded for each sample at the excitation and emission wavelengths used for the steady-state quenching analysis.For time-resolved fuorescence measurements, the single-photon-counting technique was used with nanoLED N-280 (Horiba Jobin Yvon) as the pulsed excitation source (280 nm) and with emission collected at 350 nm (13 nm emission bandwidth), eliminating the contribution of the emission by tyrosine residues.
Te experimental fuorescence intensity decays of HSA in bufer solution can be described by a sum of exponentials: where p i and τ i are the pre-exponential factors and lifetime of component i, respectively.Te amplitude or normalized pre-exponential of each lifetime component α i is p i /Σ i p i with Σ i α i � 1. Fluorescence decays were analysed by an iterative 4 Bioinorganic Chemistry and Applications deconvolution method using the TRFA Data Processor software (version 1.4; Minsk, Belarus), and the instrument response function was obtained using scattering by a colloidal suspension of silica (Ludox ® , Sigma-Aldrich) diluted in water.Criteria for judging the quality of the ft were reduced χ 2 close to 1 and a random distribution of weighted residuals and residual autocorrelation.

Cytotoxicity Studies.
Te cytotoxicity of free-PTA ligands (1-3) and ruthenium complexes (4-11) was evaluated using the colorimetric MTTassay against human breast cancer cells MDA-MB-231 and, for the most active compounds, also the MCF-7 cancer cells (Table 1).Tese cells were selected considering their diferences in terms of aggressiveness: the MDA-MB-231 cell line is highly aggressive, invasive, and hormone-independent (estrogen insensitive), while the MCF-7 cell line is noninvasive and hormone-dependent (estrogen sensitive) [46].Te cells were treated with concentrations of compounds in the range of 0.1-100 mM.None of the ligands 1-3 were found to be cytotoxic against the evaluated cancer cells (Table 1).In marked contrast, all ruthenium complexes, but 4, display a notably much better activity (lower IC 50 value) than cisplatin which was used as the positive control.Te results obtained suggest a general trend that was seen for other related ruthenium-cyclopentadienyl compounds: cationic complexes are more cytotoxic than neutral ones (compounds 4-5 vs. 6-10) [47,48].In addition, and as previously observed, a PPh 3 ligand in the complex structure, but particularly in combination with methylated-PTA, improves cytotoxicity (Scheme 2) [23,27,49].Moderate cytotoxicity is observed for complex 5, whose structural diference with respect to 4 (noncytotoxic) is the presence of a PPh 3 ligand instead of one of the two PTA ligands.A 7-fold increase in cytotoxicity with respect to complex 5 is shown, when the PTA ligand is replaced by an 6 Bioinorganic Chemistry and Applications   Bioinorganic Chemistry and Applications mPTA ligand, as in cationic complex 6.A 4-fold increase in antiproliferative activity can be observed with respect to complex 6, when HdmoPTA is replaced by an mPTA ligand in the structure of the complex as it happens in 7. Observing the structural changes in which the antiproliferative activity has increased, complexes 9 and 10 were the most active.Teir structures present a combination of PPh 3 and dimethylated PTA, in place of Cl − and PTA.However, a marked decrease in antiproliferative activity is observed when there is DMSO in place of Cl − , as in complex 8, although the antiproliferative activity is still signifcant.Nevertheless, and interestingly, the coordination of one {PdCl 2 } moiety to the CH 3 N dmoPTA nitrogen atoms, which gives rise to Ru-Pd heterometallic complex 11, provides an activity quite similar to those of 9 and 10 [24,27].
Complexes 9-11, with the best antiproliferative activity in hormone-independent MDA-MB-231 cells, were further evaluated against the MCF-7 cells, showing even better cytotoxicity activity against this hormone-dependent cancer cell line: IC 50 values were ∼2-fold lower and very markedly surpassed cisplatin by more than one order of magnitude.In addition, complexes 9-11 were tested against normal human dermal fbroblasts (HDF) to evaluate the selectivity for cancer cells.Compared to fbroblasts, complexes 9, 10, and 11 are 1.5-fold and ∼2-3-fold less cytotoxic than for MDA-MB-231 cells and MCF-7 cells, respectively.Tese results are important although not clinically relevant.While further studies are needed, the overall results show that complexes 9-11 seem prospective candidates as metallodrugs for breast cancer showing remarkable activity (in the submicromolar range) against the aggressive, highly metastatic, and cisplatin-resistant MDA-MB-231 line, a model of triplenegative breast cancer, as well as for hormone-dependent MCF-7 cancer cells, largely surpassing the activity of cisplatin by ∼50 fold.

Cell Death Measurement Using Flow Cytometry: Te
Annexin V/PI Assay.To determine the cell death mechanism caused by 9-11, the annexin V/propidium iodide (AV/ PI) cytometry-based assay was carried out in the MDA-MB-231 cells after 24 h incubation with complexes at their IC 50 concentrations.Results showed that all compounds mostly induce apoptosis with less than 10% necrosis and that the cells appear at late apoptosis (Figure 3).Induced cell death by apoptosis, a controlled and programmed mechanism of disposal of damaged cells and their content, is a desirable feature for any drug and reinforces the interest of compounds 9-11 as prospective therapeutics.

Complex Cell Uptake and Subcellular Distribution.
Te intracellular distribution of complexes 9-11 in MDA-MB-231 cells was investigated after 6 h and 24 h incubations at their respective IC 50 values.Cytosol, membranes, nucleus, and cytoskeletal fractions were extracted using a commercial kit, and the ruthenium content in each fraction was quantifed by ICP-MS.Results are summarized in Figure 4.
Te results clearly show that all three complexes are mainly accumulated at cell membranes (>86% for 6 h incubation, >90% for 24 h incubation, Figure 4), while ca.10% or less in the nucleus and marginally in cytosol and cytoskeletal.Tese results agree with previously published related works on ruthenium-cyclopentadienyl-phosphanebased compounds [44,50].No relevant diferences in the ruthenium uptake between compounds are observed, possibly due to the very high structural resemblance at the Ru(II) coordination sphere.Despite complex 11 being constituted by Ru and Pd, the observed amount of this last metal in the membranes was only residual, and no evidence of its presence in other organelles of cells was found.

Binding Interaction with Human Serum Albumin by Steady-State and Time-Resolved Fluorescence Spectroscopy.
A desirable feature, included in the FDA requirements, for any metallodrug aimed at therapeutic application, is its ability to be transported and distributed within the system where required, at the target sites.Tis aspect was accessed for complexes 9-11: the possibility of their transport in blood was modelled by the interaction with human serum albumin, the most abundant protein in blood plasma and the major transport vehicle for endo/exogenous compounds in the human system.
Fluorescence spectroscopy was used to assess the interaction between compounds and HSA [50,51].HSA exhibits intrinsic fuorescence due to the presence of its phenylalanine, tyrosine, and tryptophan (Trp) residues, of which single Trp at position 214 is the dominant fuorophore [51][52][53][54][55]. Trp214 is located in protein subdomain IIA, near Sudlow's drug binding site I, and it is very sensitive to Bioinorganic Chemistry and Applications changes in its microenvironment, which can occur following drug binding in its vicinity or due to structural alterations of protein.Tus, it can also probe interactions at drug-binding site II [56][57][58].In this work, we used the intrinsic fuorescence of HSA and selectively monitored the fuorescence emission by Trp214 for constant protein concentration, in the presence of increasing concentrations of the complex [38,50,55,59].
Emission spectra of HSA-Trp214 were acquired in the presence of increasing concentrations of the compounds after an incubation period of 24 h.As can be observed in Figure 5, in the absence of the complex, the maximum emission intensity for Trp214 occurs at 334 nm (red line in Figure 5), in agreement with previous reports [51,56,58], and denoting that the indole side chain of Trp214 is not fully exposed to the aqueous solvent, where the maximum emission of Trp would occur at ca. 350 nm [53,61,62].Rucompounds 9 and 10 do not induce any spectral shift, unlike complex 11, which causes a small blueshift of the emission by Trp214 (Figure S22).Nonetheless, all complexes caused a marked and concentration-dependent decrease in the Trp214 fuorescence intensity.Te emission intensity of Trp214 after 24 h, at a 20 µM concentration of 9, 10, and 11 (corresponding to a 1 : 4 HSA:compound ratio), reached approximately 60%, 75%, and 40%, respectively, of the value measured in the absence of compound (Figure 5, inset).Tis clear quenching of Trp214 fuorescence indicates that there is a strong interaction between the protein and the three complexes.

Bioinorganic Chemistry and Applications
Te increase in absorbance for the longer wavelengths in the absorption spectra for concentrations higher than 10 µM of 11 is consistent with the formation of an aggregate (Figure S21) [62], which only occurs in the presence of HSA.Tus, high concentrations of 11 may induce HSA aggregation to some extent, which would also explain the previously noted small blueshift of the emission spectra induced by this complex (Figure S22).Te global environment of the Trp214 residue would become less polar in the aggregates as it is even more protected from the aqueous environment, leading to the blueshift in emission.In addition, the formation of protein aggregates would also lead to increased light scattering, which is evidenced by the rise in the tail of the absorption spectra of the sample (i.e., an increase in absorbance in a spectral region where none of the species present absorbs light).Nevertheless, it is important to point out that this phenomenon should be of little relevance to the biological efect of the complex because the plasma concentration of HSA is much higher than that used in this study and because the IC 50 values of 11 are clearly below 10 µM.
Figure 6 shows that the presence of 9 and 10 does not afect the mean fuorescence lifetime of HSA-Trp214 regardless of the concentration used.Tis fact indicates that these complexes quench the steady-state fuorescence of HSA without afecting its fuorescence lifetime.Tis result points to the formation of a nonfuorescent ground state complex through the binding of 9 and 10 to the protein near the Trp214 residue.Bioinorganic Chemistry and Applications In contrast, the amplitude-weighted mean fuorescence lifetime of Trp214 decreased in the presence of 11, which suggests that dynamic quenching might be occurring.Typical Stern-Volmer constants are in the order of 10 mM-100 mM for dynamic quenching resulting from random collisions between the compound and the indole sidechain of Trp214; however, for the concentration range here used, there could be no appreciable collisional quenching.Terefore, the observed behaviour should mean that fuorescence lifetime is due to a stronger and more specifc interaction related to a binding process.As both amplitude-weighted mean fuorescence lifetimes decrease to a larger extent in the steady-state fuorescence intensity, the presence of two independent binding sites should be considered: one of them in closer proximity to Trp214 than the other.However, more complex situations cannot be completely ruled out, such as two binding modes in the same binding site or two binding sites, both close to Trp214.
(2) Determination of the Compound-HSA-Binding Constants.Te frst approach to estimate the complex-HSAbinding constants based on fuorescence quenching is usually the inspection of the Stern-Volmer plots.No variation in the amplitude-weighted mean fuorescence lifetime was detected for compounds 9 and 10.However, this was not the case for the steady-state fuorescence intensity-based Stern-Volmer plots which are shown in Figure 7.
Considering the absence of the efect on the fuorescence lifetimes for 9 and 10, the slope of the Stern-Volmer plots in Figures 7(a) and 7(b) can be safely interpreted as a static quenching constant.In turn, in these two specifc situations, the static quenching constant for Trp214 by each compound can be taken as the binding constant (K B ) for the formation of a ground state 1 : 1 nonfuorescent complex between HSA and the compound (equation ( 4)), according to the equilibrium: where IF 0 and IF are, respectively, the fuorescence intensity of Trp214 of HSA in the absence and presence of the compound.Te determined binding constant for 9 of (4.59 ± 0.21) × 10 4 •M −1 and of (1.43 ± 0.05) × 10 4 •M −1 for 10 correspond to log K B values of (4.66 ± 0.04) for 9 and (4.16 ± 0.03) for 10, respectively.From a linear ft to the amplitude-weighted mean fuorescence lifetime of the Stern-Volmer plot for HSA-Trp214 quenching by 11 (Figure 6), it is possible to estimate conditional binding constant K′ B of (4.58 ± 0.33) × 10 4 •M −1 .Te two quenching processes, afecting the steady-state fuorescence intensity alone or together with the fuorescence lifetime, strongly suggest two binding modes as previously noted.Terefore, the following equation was ftted to the variation of the steady-state fuorescence intensity of Trp214 with an increasing concentration of 11 (Figure 7(c)): where K′ B was fxed to (4.58 ± 0.33) × 10 4 M −1 .A K B of (3.70 ± 0.33) × 10 4 M −1 was retrieved, corresponding to a log K B of (4.57± 0.07).Moreover, as previously stated, K′ B can be considered an additional binding constant for the equilibrium between 11 and HSA, being log K′ B of (4.66 ± 0.07).Interestingly, both K B and K′ B are in the same order of magnitude as all other binding constants determined.Note that concentrations of 11 above 10 µM were not considered for the estimation of either K B or K′ B (Figures 6 and 7(c)), due to the possible aggregation of HSA, which was reported in the previous section.

Bioinorganic Chemistry and Applications
Table 2 presents the summary of all the binding constants determined for the interaction of HSA with 9, 10, and 11.
Te K B values obtained for the interaction of HSA with each compound are very similar.Also, the strong interaction of ruthenium [51] and copper complexes [58,63] with HSA determined in previous works is comparable with the values of binding constants obtained herein for all three compounds.Interestingly, heterodimetallic complex 11 is the one with the strongest interaction with HSA, due to the two modes of interaction that are relatively efcient.Monometallic parent complexes 9 and fnally 10 show a similar interaction with HSA, which is smaller than that observed for 11.Log K B obtained for complexes 9-11 are of the same order of magnitude as the one found for the Ru(III) complex KP1019, known to be transported in blood by serum albumin and to bind the protein reversibly [57].Our results thus suggest that all three compounds, 9, 10 and 11, can be efciently transported by albumin in blood plasma.
Not only the cytotoxic activities of all the complexes (4-11) but also those of ligands PTA (1), mPTA (2), and dmPTA (3) were evaluated against the MDA-MB-231 and MCF-7 breast cancer cells using the MTT assay.Te most   active complexes against both breast cancer cells were 9, 10, and 11, showing IC 50 values in the micromolar range, suggesting that the highest cytotoxicity is achieved when two PPh 3 ligands and one dmoPTA ligand were combined around the metal in the {CpRu} moiety.Tese complexes were also tested against normal human dermal fbroblasts (HDFs) to evaluate the selectivity for cancer cells, being 1.5-fold and ∼2-3-fold less cytotoxic than for MDA-MB-231 cells and MCF-7 cells, respectively.Studies by fow cytometry showed that these three complexes induce mostly apoptosis in combination with a small percentage of necrosis (<10%) on MDA-MB-231 cells, which is very positive for their possible use as a drug.Cell distribution studies of the three complexes showed that Ru is mostly retained in the cell membrane (ca.90%) and a reduced quantity in the cell nucleus (<10%) of MDA-MB-231 cells.
It is important to stress that Pd metal was not found in a signifcant quantity in cells when 11 was evaluated.
Experiments to determine the binding interaction with human serum albumin were carried out, concluding that the presence or absence of an H-bridge between CH 3 N dmoPTA determines the interaction mechanism with the carrier blood protein.Also, an interesting conclusion is that Ru-Pd heterobimetallic complex 11 interacts with HSA to a higher extent and by a diferent mechanism with respect to monometallic complexes.Te obtained results show how the combination of ligands around Ru determines the antiproliferative activity of this family of complexes, but the fact that Ru is located mainly in the cell membrane suggests that these ligands should afect how the cell membrane works, being the factor that induces their cytotoxic activity.Te fact that Pd is not located in the cell also suggests that complex 11 decomposes, releasing the Pd atom before interacting with the cells, but the fact that this complex interacts with HSA by a diferent mechanism than 9 and 10 also suggests that this extra metal can determine how the complex arrives to the cell, being this also an important role related to the antiproliferative activity.Additional experiments are in progress to determine these suspicions.Overall, complexes 9 and 10 emerge from this work as interesting and highly promising to pursue to further studies as prospective ruthenium metallodrugs.

3. 2 . 1 .
Stability Studies.Te stability of RuCp complexes in DMSO and in DMSO/H 2 O mixtures has been studied quite extensively in recent years.In general, this family of complexes is reasonably stable in the abovementioned solvent mixtures, and in most cases, decomposition occurs at longer times in comparison with the antiproliferative assays.Nevertheless, typical decomposition pathways usually involve the release of ligands Cl − or PPh 3 , which are substituted by solvent molecules (H 2 O or DMSO) [24, 26, 28].Complex 8 is soluble in DMSO-d 6 and DMSO-d 6 /D 2 O (1 : 1) and stable in both media at room temperature and at 37 °C as no spectral changes were observed during 48 h (Figures S13-S16 in S.I.).Also, complex 11 is very stable in DMSO-d 6 at room temperature and 37 °C showing two sharp Bioinorganic Chemistry and Applications signals relative to the coordinated PPh 3 groups (39.93 ppm, d) and the {dmoPTA-PdCl 2 } unit (−12.07 ppm, t) (Figures S17-S18) during 48 h.Nevertheless, a small set of signals corresponding to complex 8 becomes visible after 4 h (<7%), remaining constant up to 48 h.Terefore, only a marginal fraction of 11 undergoes solvolysis in DMSO.In DMSO-d 6 /D 2 O (1 : 1), the 31 P{ 1 H} NMR spectrum of this complex displays broad bands centred at +38.86 ppm assigned to the coordinated PPh 3 ligands, and diferent signals, some of them broad, for the {dmoPTA-PdCl 2 } moiety (Figures

Figure 3 :
Figure 3: Ruthenium-based compounds potentiate apoptotic cell death in the MDA-MB-231 breast cancer cell line.Apoptotic cell death was analysed by the annexin V fuorescein isothiocyanate (AV-FITC) and propidium iodide (PI) assay in MDA-MB-231 cells, after incubation with IC 50 concentrations for 24 h graphical representation of the annexin V/PI dot plots of fow cytometry data analysed using Flowing software for control and compounds 9-11.

Figure 4 :
Figure 4: Cellular Ru distribution of 9-11 in MDA-MB-23 cells at two diferent time points: the amount of Ru determined by ICP-MS for each subcellular fraction (normalized, 10 6 cells) at 6 h (a) and 24 h (b); Ru accumulation (relative %) profle at 6 h (c) and 24 h (d).

Figure 6 :
Figure 6: Compound 11, but not 9 or 10, afects the amplitude-weighted mean fuorescence lifetime of HSA-Trp214 for a Stern-Volmer plot built from the fuorescence lifetimes of HSA in the presence of increasing concentrations of each compound: ratio of the amplitudeweighted mean fuorescence lifetime (see equation (2) in 2.4.6 Interaction with Human Serum Albumin) in the absence (τ 0 ) and presence (τ) of the complex (conditions: PBS pH 7.42% (v/v) DMSO; C HSA � 3.7 µM for 9 and 5.0 µM for 10 and 11 µM; λ em � 350 nm; the samples were incubated for 24 h at 37 °C; the fuorescence intensity decays were recorded at 25.0 ± 0.1 °C).

Table 2 :
Parameters determined for the binding of 9, 10, and 11 with HSA.HSA � 3.7 µM for 9 and 5.0 µM for 10 and 11 µM, kept constant; the samples were incubated for 24 h at 37 °C.