Piano-stool ruthenium(II) complexes with delayed cytotoxic activity: Origin of the lag time

Piano-Stool Ruthenium(II) Complexes with Delayed Cytotoxic Activity: Origin of the Lag Time Laia Rafols, Dana Josa, David Aguila, LeoníA. Barrios, Olivier Roubeau, Jordi Cirera, Vanessa Soto-Cerrato, Ricardo Pérez-Tomás, Manuel Martínez, Arnald Grabulosa,*and Patrick Gamez* Cite This: Inorg. Chem. 2021, 60, 7974−7990 Read Online ACCESS Metrics & More Article Recommendations * sıSupporting Information ABSTRACT: We have recently reported a series of piano-stool ruthenium(II) complexes of the general formula [RuCl2(η6-arene)(P- (1-pyrenyl)R2R3)] showing excellent cytotoxic activities (particularly when R2=R 3= methyl). In the present study, new members of this family of compounds have been prepared with the objective to investigate the effect of the steric hindrance of a bulky phosphane ligand, namely diisopropyl(1-pyrenyl)phosphane (L), on exchange reactions involving the coordinated halides (X = Cl, I). Two η6-arene rings were used, i.e. η6-methyl benzoate (mba) and η6-p-cymene (pcym), and four complexes were synthesized, namely [RuCl2(mba)(L)] (1Cl2 iPr), [RuI2(mba)(L)] (1I2 iPr), [RuCl2(p-cym)(L)] (2Cl2 iPr), and [RuI2(pcym)(L)] (2I2 iPr). Unexpectedly, all of the complexes exhibited poor cytotoxic activities after 24 h of incubation with cells, in contrast to the related compounds previously reported. However, it was observed that aged DMSO solutions of 2I2 iPr (from 2 to 7 days) exhibited better activities in comparison to freshly prepared solutions and that the activity improved over “aging”time. Thorough studies were therefore performed to uncover the origin of this lag time in the cytotoxicity efficiency. The data achieved clearly demonstrated that compounds 2I2 iPr and 2Cl2 iPr were undergoing a series of transformation reactions in DMSO (with higher rates for the iodido complex 2I2 iPr), ultimately generating cyclometalated species through a mechanism involving DMSO as a coordinated proton abstractor. The cyclometalated complexes detected in solution were subsequently prepared; hence, pure [RuCl(p-cym)(κ2C-diisopropyl(1pyrenyl)phosphane)] (3Cl iPr), [RuI(p-cym)(κ2C-diisopropyl(1-pyrenyl)phosphane)] (3I iPr), and [Ru(p-cym)(κS-dmso)(κ2Cdiisopropyl(1-pyrenyl)phosphane)]PF6(3dmso iPr ) were synthesized and fully characterized. Remarkably, 3Cl iPr,3I iPr, and 3dmso iPr are all very efficient cytotoxic agents, exhibiting slightly better activities in comparison to the chlorido noncyclometalated complexes [RuCl2(η6-arene)(P(1-pyrenyl)R2R3)] described in an earlier report. For comparison purposes, the iodido compounds [RuI2(mba)(dimethyl(1-pyrenyl)phosphane)] (1I2 Me) and [RuI2(p-cym)(dimethyl(1-pyrenyl)phosphane)] (2I2 Me), bearing the less hindered dimethyl(1-pyrenyl)phosphane ligand, have also been prepared. The cytotoxic and chemical behaviors of 1I2 Me and 1I2 Me were comparable to those of their chlorido counterparts reported previously. ■INTRODUCTION Cancer has a major impact on society worldwide, as it represents one of the leading causes of death. 1,2 Cisplatin is one of the most used drugs to treat various types of cancer. 3 The remarkable chemotherapeutic properties of cisplatin have instigated tremendous research efforts in the area of platinum drugs. 4−6 Nevertheless, cisplatin suffers from some severe side effects, 7 and a decrease in its effectiveness may be observed with platinum-resistant tumors. 8 Therefore, the development of more efficient and less toxic therapeutic agents is essential in this area of investigation. In that context, alternative transition metals have been used to generate new compounds. 9−14 For instance, some ruthenium complexes have been reported that exhibit remarkable anticancer properties, 15 making them potential drug candidates. 16−22 Actually, two ruthenium compounds are currently undergoing clinical trials, namely BOLD-100 (Na[trans-RuCl4(Ind)2], Ind = indazole) 23−25 and TLD1433 ([Ru(bpy)(IP-TT)]2+(IP-TT = 2-(2′,2″:5′′,2‴- terthiophene)imidazo[4,5-f][1,10]phenanthroline). 26,27 To date, there are no efficient molecules capable of targeting most types of disseminated tumor cells. NAMI-A shows Received: February 19, 2021 Published: May 12, 2021 Articlepubs.acs.org/IC © 2021 American Chemical Society 7974 https://doi.org/10.1021/acs.inorgchem.1c00507 Inorg. Chem. 2021, 60, 7974−7990 Downloaded via CSIC on April 1, 2022 at 11:00:09 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. interesting antimetastatic properties, 28 as do some Ru(II)- arene complexes from the RAPTA family; 29,30 for instance, RAPTA-T exerts antimetastatic activities. 31,32 Hence, Ru-based compounds are increasingly being seen as potential nextgeneration anticancer metallodrugs. 33,34 Recently, we have reported a series of half-sandwich ruthenium(II) complexes of the general formula [RuX2(η6arene)(P(1-pyrenyl)R2R3)] (with η6-arene = p-cymene, methyl benzoate, R2= methyl, phenyl, and R3= methyl, phenyl) displaying valuable cytotoxic behaviors. 35 In that previous study, the effect of the nature of the η6-arene on the cytotoxic activity was examined (viz. p-cymene vs. methyl benzoate), as well as that of different R groups on the monophosphane P(1pyrenyl)R2R3ligand; a significant effect of these two parameters on cell toxicity was observed. 35 Therefore, we decided to investigate the role played by the halide, i.e. X, on the biological activity of [RuX2(η6-arene)(P(1-pyrenyl)R2R3)] compounds and thus study and compare the cytotoxic properties of the four complexes, depicted in Scheme 1: viz., 1Cl2 iPr,1I2 iPr,2Cl2 iPr, and 2I2 iPr. These Ru compounds all contain the ligand diisopropyl(1pyrenyl)phosphane (L), purposely chosen for its steric hindrance that would favor the displacement of the halide ligands, for instance by water molecules. 36,37 Two different arene rings were used, namely methyl benzoate and p-cymene, which have produced piano-stool ruthenium complexes with drastically distinct cytotoxic properties in a previous study. 35 Surprisingly, cell viability assays with various cancer lines revealed that 1Cl2 iPr,1I2 iPr,2Cl2 iPr, and 2I2 iPr were poorly active (mostly inactive) compounds after an incubation time of 24 h. However, complex 2I2 iPr,namely[RuI 2(η6-p-cymene)- (diisopropyl(1-pyrenyl)phosphane)] (Scheme 1), exhibited a drastic improvement in its activity with time; for instance, 2I2 iPr was 4 times more active against lung adenocarcinoma cells after 7 days (in comparison to its activity after 1 day). This highly interesting behavior was thoroughly investigated to elucidate the origin of this time-dependent enhancement of the cytotoxic properties; the mechanistic studies carried out showed that 2I2 iPr (as well as its corresponding chlorido complex 2Cl2 iPr) was gradually converted into a highly cytotoxic, cyclometalated species, through a three-step process (whose rate was halide-dependent: i.e., the conversion process was faster with iodido complex 2I2 iPr than with chlorido complex 2Cl2 iPr). ■RESULTS AND DISCUSSION Preparation of the Ru Compounds 1Cl2 iPr,1 I2 iPr,2 Cl2 iPr, and 2I2 iPr.The ligand, namely diisopropyl(1-pyrenyl)phosphane (L), was prepared by the reaction of lithiated 1-bromopyrene with chlorodiisopropylphosphane in THF at −78 °C(Scheme S1). Ligand Lis unstable in air (an oxide of the phosphane is produced); therefore, Lwas protected by the formation of its borane adduct. L·BH3can be deprotected, just before use (to prepare the Ru compounds), by reaction with the tetrafluoroboric acid diethyl ether adduct in dichloromethane (Scheme S1). The chlorido complexes [RuCl2(η6-methyl benzoate)(L)] (1Cl2 iPr) and [RuCl2(η6-p-cymene)(L)] (2Cl2 iPr) were obtained in good yields by the reaction of ligand Lwith the corresponding ruthenium dimeric precursors: namely, [RuCl(μ-Cl)(η6-methyl benzoate)]2for 1Cl2 iPr and [RuCl(μ-Cl)(η6-p-cymene)]2for 2Cl2 iPr (Figure 1). The iodido compounds [RuI2(η6-methyl benzoate)(L)] (1I2 iPr) and [RuI2(η6-p-cymene)(L)] (2I2 iPr) can be generated in good yields from 1Cl2 iPr and 2Cl2 iPr in the presence of an excess of sodium iodide in refluxing technical acetone (Figure 1). 38 It should be pointed out that the conversion of 2Cl2 iPr to 2I2 iPr was achieved in 1 h, whereas 16 h was required for the chloride to iodide exchange that generated 1I2 iPr from 1Cl2 iPr (see the Experimental Section). Thus, it appears that the bulkier and more electron donating p-cymene ring significantly favors the chloride to iodide substitution. This substitution can Scheme 1. Representation of the Structure of the PianoStool Ruthenium Complexes Designed and Synthesized in the Present Study to Evaluate the Effect of Chloride and Iodide on the Cytotoxic Activity Figure 1. Synthetic procedures used to prepare the half-sandwich Ru(II) chlorido 1Cl2 iPr and 2Cl2 iPr and iodido complexes 1I2 iPr and 2I2 iPr. Inorganic Chemistry pubs.acs.org/IC Article https://doi.org/10.1021/acs.inorgchem.1c00507 Inorg. Chem. 2021, 60, 7974−7990 7975 easily be monitored by 31P NMR spectroscopy. For instance, the 31P{1H} NMR spectrum of 1Cl2 iPr shows a singlet at +38.9 ppm, whereas the corresponding singlet is observed at +34.5 ppm for 1I2 iPr (Δδ−4.4 ppm). Similarly, the 31P chemical shift is observed at +36.3 ppm for chlorido 2Cl2 iPr and at higher field for the iodido complex 2I2 iPr: viz., +31.3 ppm (Δδ−5.0 ppm). The lower electronegativity of iodine, in comparison to that of chlorine, may explain why the P atoms of the iodido complexes are more shielded. All of the ruthenium compounds were characterized by common techniques, including X-ray diffraction, which confirmed their identity (see the Experimental Section for details). Crystal Structures of Ru Compounds 1Cl2 iPr,1 I2 iPr,2 Cl2 iPr, and 2I2 iPr.Single crystals of the four compounds, suitable for X-ray diffraction studies, were obtained (see Experimental Section). Compounds 1Cl2 iPr and 1I2 iPr crystallize in the monoclinic space group P21/c, compound 2Cl2 iPr crystallizes in the monoclinic space group C2/c, and 2I2 iPr crystallizes in the triclinic space group P1(see Tables S1 and S2). The solid-state structures of the four complexes are shown in Figure 2; selected (coordination) bonds and angles are given in Table S3 (see also Figure S1). The four ruthenium compounds exhibit the expected, typical “three-legged piano-stool”geometry for such systems. The centroid to metal distance varies from 1.69 to 1.72 Å, and the Ru−P length is in the range 2.39−2.41 Å (Table S3). The Ru− Cl bond distances are 2.41 and 2.42 Å for 1Cl2 iPr and 2.40 and 2.43 Å for 2Cl2 iPr. As could be predicted, the Ru−I bonds are longer (of about 0.3 Å), with values at around 2.72 Å for 1I2 iPr, and 2.72 and 2.73 Å for 2I2 iPr (Table S3). The coordination angles are similar for all four complexes (Table S3), and they are in the range expected for such molecules. 35,39,40 Effects of Compounds 1Cl2 iPr,1 I2 iPr,2 Cl2 iPr, and 2I2 iPr on Cell Viability. The ability of the compounds to inhibit cell growth was evaluated next. Hence, their cytotoxic properties were first assessed in A549 cells (lung adenocarcinoma) at a fixed complex concentration of 10 μM. The corresponding cell viabilities (in percent) are given in Table 1. Surprisingly, the cytotoxic activities of the Ru compounds are mostly poor; for instance, 1Cl2 iPr and 1I2 iPr are inactive (cell viability of around 100%; Table 1), and 2Cl2 iPr only gives rise to some cell growth inhibition (73% cell viability; Table 1). Only compound 2I2 iPr is efficiently capable of eradicating A549 cells (37% cell viability; Table 1). An unusual phenomenon was observed during the replication experiments with 2I2 iPr. Indeed, clearly different cell viability values after 24 h of incubation were obtained for the same stock solution of 2I2 iPr in DMSO, used after 1, 5 and 7 days after preparation. A significant improvement in the cytotoxic properties of 2I2 iPr was indeed noticed from day 0 of the preparation of the stock solution to, for instance, days 5 and 7; actually, the value of 37 ±17% (Table 1) corresponds to the average value from three replicates carried out with a stock solution of 2I2 iPr used after 0, 5, and 7 days. The large deviations observed among the replicates are due to the significantly different activities of the aging solution. It was also noted that the aging DMSO solution of 2I2 iPr became slightly darker after a few days (see the graphical abstract). It thus appears that 2I2 iPr is gradually converted into a new and more active, “unknown”species. Subsequently, half-maximum inhibitory concentrations (IC50) were determined for compounds 2Cl2 iPr and 2I2 iPr, considering this feature (viz. the observed time-dependent evolution of the biological properties of 2I2 iPr). IC50 values were not determined for 1Cl2 iPr and 1I2 iPr, which did not show any cytotoxic behavior (see Table 1). The IC50 data obtained are given in Table 2. Compound 2Cl2 iPr gives an IC50 value of 24 μM after 24 h incubation with A549 (lung adenocarcinoma) human cells (Table 2), using a stock solution of Ru compound (in DMSO) prepared the same day (viz. the day in which the compound was incubated with the cells, day 0). As already noticed with the cell viability studies, the use of a 5-day-old or 7-day-old stock solution of 2Cl2 iPr did not lead to different IC50 values (Table 2), suggesting that the integrity of the Ru compound is most likely maintained (in DMSO solution). A completely different behavior was observed for 2I2 iPr. Indeed, a freshly prepared solution of 2I2 iPr in DMSO (day 0) gave an IC50 value of 48 μM; hence, 2I2 iPr was 2 times less Figure 2. Representation of the crystal structures of complexes 1Cl2 iPr, 1I2 iPr,2Cl2 iPr, and 2I2 iPr. The atoms bonded to the metal center are labeled, and Crepresents the centroid of the η6-arene ring. Hydrogen atoms are omitted for clarity. Table 1. Cell Viability Values (%) of Complexes 1Cl2 iPr,1 I2 iPr, 2Cl2 iPr and 2I2 iPr (Fixed Concentration of 10 μM) for A549 (Lung Adenocarcinoma) Human Cells, after Incubation for 24 h 1Cl2 iPr 1I2 iPr 2Cl2 iPr 2I2 iPr cell viability (%) a , b 108 ±2 104 ±12 73 ±18 37 ±17 a After 24 h incubation at 37.5 °C. b The results are expressed as mean values ±SD out of three independent experiments. Inorganic Chemistry pubs.acs.org/IC Article https://doi.org/10.1021/acs.inorgchem.1c00507 Inorg. Chem. 2021, 60, 7974−7990 7976 cytotoxic than 2Cl2 iPr. However, if the stock solution of 2I2 iPr is used 5 days after its preparation, then an IC50 value of 26 μMis obtained; the cytotoxic behavior of 2I2 iPr is comparable with that of 2Cl2 iPr(day 5, Table 1). Even more interestingly, after 7 days in DMSO, 2I2 iPr becomes very active, as illustrated by the IC50 value of 9.5 μM (day 7, Table 2). Clearly, the initial compound 2I2 iPr is slowly converted into a significantly more active species (the new “unknown”species is indeed 5 times more cytotoxic than the original Ru complex against A549 cells). The cytotoxicity of 2I2 iPr against MCF7 (breast carcinoma) human cells was evaluated as well, which gave an interesting IC50 value of 12 μM with a 7-day-old solution of the complex (Table 2). The remarkable behavior of 2I2 iPr in DMSO, namely its progressive activation (i.e., its increased cytotoxicity), was subsequently investigated with the objective of elucidating the nature of the formed, more cytotoxic species. NMR Studies. Since the stock solutions of the complexes used for the cytotoxicity assays were prepared in DMSO, the potential modification/alteration of 2I2 iPr in this solvent was monitored by 31P{1H} NMR spectroscopy. The time-dependent corresponding spectra obtained after a period of 48 h are shown in Figure 3. Already after 5 min, two new peaks, in addition to that corresponding to 2I2 iPr(+30.7 ppm), are found at −8.3 and +80.0 ppm. The chemical shift at −8.3 ppm is due to the free ligand diisopropyl(1-pyrenyl)phosphane Lthat is released from the complex (in CDCl3δis found at −8.8 ppm; see the Experimental Section). The second new peak at +80.0 ppm arises from the development of a new species, denoted N1 (Figure 3; X = I). On the basis of the chemical shift, a cyclometalation reaction involving the pyrenyl ring may be considered to explain the observed low-field value; indeed, a number of related cyclometalated (phosphane)ruthenium(II) complexes have been reported with chemical shifts in the range +70−90 ppm. 41−44 After 30 min, the intensity of the peak corresponding to N1 increases, whereas those of 2I2 iPr and L slightly decrease. After 24 h, two new peaks are detected at +55.7 and +86.6 ppm (Figure 3). The chemical shift at +55.7 ppm is attributable to the oxide of the ligand, i.e. diisopropyl- (1-pyrenyl)phosphane oxide (LO). Actually, LOwas purposely synthesized by oxidation of Lwith dihydrogen peroxide (see Figures S2 and S3 and Table S4 in the Supporting Information), and its 31P{1H} NMR spectrum gave a single signal at δ+56.9 ppm in CDCl3(Figure S4). The second new peak at δ+86.6 ppm can be ascribed to another cyclometalated species, denoted N2 (Figure 3). After 48 h in DMSO, the signals corresponding to Land 2I2 iPr disappeared completely, while traces of LOcould still be seen. The peak due to species N1 slightly decreased, whereas that of N2 clearly increased, hence suggesting that N2 may be formed from N1. The same time-course study was carried out for the chlorido complex 2Cl2 iPr, for comparison. The corresponding NMR spectra in DMSO-d6are shown in Figure S5. The same behavior is observed for this compound. An intense broad peak at −8.3 ppm, corresponding to free phosphine L, is detected instantly; such peak broadness can be explained by rapid exchange processes between different species in solution. The chemical Table 2. Half-Maximum Inhibitory Concentrations (IC50, μM) of Compounds 2Cl2 iPr and 2I2 iPr for A549 (Lung Adenocarcinoma) Human Cells, after Incubation of 24 h, Using Freshly Prepared Stock Solutions of the Complexes (Day 0), and 5and 7-Day Aged Solutions of the Complexes a cell line compound day 0 day 5 day 7 A549 2Cl2 iPr 24 ±126±10 27 ±12 A549 2I2 iPr 48 ±826±8 9.5 ±1.5 MCF7 2I2 iPr 12 ±2 a The IC50 value (μM) of a 7-day-old DMSO solution of 2I2 iPr for MCF7 (breast carcinoma) human cells, after incubation for 24 h, is also included. The results are expressed as mean values ±SD out of three independent experiments. Figure 3. 31P{1H} NMR spectra of complex 2I2 iPr in DMSO-d6recorded during a period of 48 h, illustrating the progressive formation of new species. Inorganic Chemistry pubs.acs.org/IC Article https://doi.org/10.1021/acs.inorgchem.1c00507 Inorg. Chem. 2021, 60, 7974−7990 7977 shift of 2Cl2 iPr is found at +36.3 ppm and the oxidized ligand, viz. the phosphane oxide LO, is detected at +55.7 ppm. As for 2I2 iPr, two signals are observed above +80 ppm, in the “cyclometalated region”. By analogy with 2I2 iPr (see above), the peak observed at +80.8 ppm is attributed to N1 (with X = Cl) and that at +86.6 ppm to N2. It can be stressed here that the N2 species is formed in significantly lower amounts for 2Cl2 iPr in comparison to 2I2 iPr (see Figure 3 and Figure S5) for the same aging time. Also, in contrast to 2I2 iPr, the development of a third new species, labeled N3, is observed at δ+45.7 ppm (Figure S5). On the basis of its chemical shift, N3 may be associated with the cationic [RuCl(η6-p-cymene)(diisopropyl(1-pyrenyl)- phosphane)(dmso)]+species resulting from the substitution of one of the chlorido ligands of 2Cl2 iPr by a DMSO molecule. Actually, when the time-dependent NMR experiments are carried out for 2Cl2 iPr in pure CDCl3, the peaks corresponding to N1 (X = Cl), N2 and N3 (X = Cl) are not observed (Figure S6), suggesting that DMSO is actively involved in the formation of these species. Similarly, the time-dependent study for 2I2 iPr in pure CDCl3only shows the presence of the starting ruthenium(II) complex together with oxidized ligand LO(Figure S7), in higher amounts than for 2Cl2 iPr (Figure S6). It thus appears that stable DMSO-containing species (Scheme 2, right) are generated from the cyclometalated complexes [RuX(η6-p-cymene)(κ2C-diisopropyl(1-pyrenyl)phosphane)] (X = Cl, I; Scheme 2, left) in the presence of DMSO and that the process is slower with the chlorido complex (see Study of the Solvation of the Cyclometalated Complexes). For comparison purposes, 31P{1H} NMR time-resolved studies in DMSO-d6were performed for η6-methyl benzoate containing complexes 1Cl2 iPr and 1I2 iPr. The corresponding spectra are shown in Figures S8 and S9. After 24 h, 1Cl2 iPr, free ligand L and LOare observed together with two new compounds that are most likely cyclometalated species (Figure S8). After 48 h, the same species are present in solution. For 1I2 iPr, the formation of a cyclometalated species is immediately observed (Figure S9). After 24 h, 1I2 iPr has completely disappeared; as for the η6-p-cymene-containing compounds, it appears that the cyclometalation reaction is favored for the iodido complex. Subsequently, the effect of water on DMSO stock solutions of 2Cl2 iPr and 2I2 iPr was investigated by 31P{1H} NMR, as biological studies are performed in aqueous media. Therefore, 24 h aged, concentrated solutions of complexes 2Cl2 iPr and 2I2 iPr in DMSO-d6 were mixed with D2O, using a DMSO-d6to D2O ratio of 25:75. Under these conditions, precipitation was observed; the solids were filtered off, and the NMR spectra of the filtrates were recorded. For both samples (i.e. prepared from 2Cl2 iPr and 2I2 iPr), the same single peak was detected in the cyclometalated region, corresponding to that obtained for the N2 intermediate (see above). Using 48 h aged, concentrated solutions of complexes 2Cl2 iPr and 2I2 iPr, comparable data were obtained; an increase in dissolved sample over time was observed (Figure S10), hence suggesting both the polar nature of the new complex [Ru(η6-p-cymene)(κ2C-diisopropyl(1-pyrenyl)- phosphane)(dmso)]+in solution and its relatively slow formation process. The formation of an aquo complex of the type [Ru(η6-p-cymene)(κ2C-diisopropyl(1-pyrenyl)- phosphane)(H2O)]+could not be detected under the conditions applied for these NMR experiments. Cyclometalated Compounds. The cyclometalated complexes [RuX(η6-p-cymene)(κ2C-diisopropyl(1-pyrenyl)- phosphane)] (X = Cl, I) and [Ru(η6-p-cymene)(κ2Cdiisopropyl(1-pyrenyl)phosphane)(dmso)]+(depicted in Scheme 2) are thus clearly important end species formed in solution from 2Cl2 iPr and 2I2 iPr. Therefore, these compounds were purposely prepared in their pure form. The reaction of dichloro(p-cymene)ruthenium(II) dimer with diisopropyl(1-pyrenyl)phosphane (L) in methanol in the presence of sodium acetate 42,45 produces the cycloruthenated complex [RuCl(η6-p-cymene)(κ2C-diisopropyl(1pyrenyl)- phosphane)] (3Cl iPr) with a yield of 52% (Figure 4). The iodido complex [RuI(η6-p-cymene)(κ2C-diisopropyl(1-pyrenyl)- phosphane)] (3I iPr) is obtained from 3Cl iPr with a yield of 82%, by halide exchange in the presence of an excess of sodium iodide in refluxing technical acetone 38 (Figure 4). Finally, the cationic cyclometalated complex [Ru(η6-p-cymene)(κS-dmso)- (κ2C-diisopropyl(1-pyrenyl)phosphane)]PF6(3dmso iPr ) can be prepared in nearly quantitative yield, viz. 95%, by reaction of an excess of DMSO with complex 3Cl iPr in the presence of thallium hexafluorophosphate in dichloromethane solution at room temperature (Figure 4). It can be pointed out here that the 31P{1H} chemical shifts of 3Cl iPr,3I iPr, and 3dmso iPr in CDCl3, respectively +80.8, +79.3, and +86.6 ppm (Experimental Section), are in the range of those mentioned in NMR Studies (see Figures 3 and 4), therefore corroborating the assumptions thus made regarding the potential nature of the “unknown”species labeled N1 (X = Cl, I) and N2. Single crystals of the three cyclometalated compounds, suitable for X-ray diffraction analyses, were obtained (see the Experimental Section); all three complexes crystallize in the monoclinic space group P21/c(Tables S5 and S7). The crystal structures of the cyclometalated compounds are shown in Figure 5; selected (coordination) bonds and angles are given in Table S6 for 3Cl iPr and 3I iPr and Table S8 for 3dmso iPr . The pseudooctahedral geometry of the Ru center in the three compounds is significantly distorted due to the cyclometalation; for instance, the C−Ru−P angles for all complexes are close to 80°(Tables S5 and S7), while the corresponding X−Ru−P angles in 2Cl2 iPr and 2I2 iPr are closer to the ideal value, namely 90° (Table S3). The coordination bond lengths are in the range of those found for similar complexes. 34,42 Study of the Solvation of the Cyclometalated Complexes. Kinetic studies of the solvation of 3Cl iPr,3I iPr, and 3dmso iPr by different solvents were carried out by UV−vis Scheme 2. Possible Cyclometalated N1 and N2 Species Generated from 2Cl2 iPr (X = Cl) and 2I2 iPr (X = I) Inorganic Chemistry pubs.acs.org/IC Article https://doi.org/10.1021/acs.inorgchem.1c00507 Inorg. Chem. 2021, 60, 7974−7990 7978 spectroscopy at distinct temperatures and pressures. Saturated solutions of 3Cl iPr and 3I iPr were prepared in the solvents to be investigated: namely, DMSO, DMSO saturated with NaCl, nbutanol (chosen for comparative purposes), and water. Generally, suspensions were obtained, which were sonicated for 5−6 min and filtered over glass wool (to eliminate any remaining insolubilized compound); the filtrates were then transferred into UV−vis cells that were subsequently placed in a thermostated UV−vis spectrophotometer. After temperature equilibration, time-resolved spectra were collected at different time scales and intervals to warrant total conversion (4−6 halflives) to the expected solvato complexes. The first-order rate constants obtained for all solvation experiments carried out are given in Tables S9 and S10. These constants were determined by fitting the time-resolved spectral data with Specfit 46 or ReactLab, 47 considering an A →B process. It should be noted that, upon dilution of the initial solution (1:1 and 1:5 dilutions), no variations in rate constants were observed, therefore indicating the nonactuation of multinuclear species during the process. Moreover, when DMSO saturated with NaCl was used (Figure 6a), no noticeable differences in kobs values were found, hence suggesting the nonequilibrium nature of the solvation process under the conditions applied. Examples of the temperature and pressure dependence of the kobs values for representative systems are shown in Figure 6b,c, respectively. The values determined for the thermal and pressure activation parameters for a series of solvation experiments with 3Cl iPr,3I iPr, and 3dmso iPr are given in Table 3; the extrapolated kobs and 6 ×t1/2 values at 37 °C (310 K) are also given in Table 3. It should be noted that the data provided for 3Cl iPr in water were obtained from the time-resolved appearance of definite UV−vis spectra, resulting from the progressive aquation of the chlorido ligand producing polar ionic species, most likely [Ru(η6-p-cymene)(κ2Cdiisopropyl(1-pyrenyl)phosphane)(H2O)]+, exhibiting higher (but still limited) water solubility in comparison to 3Cl iPr (see below). Wide ranges of enthalpies (52−113 kJ mol−1) and entropies (−162 to +25 J K−1mol−1) of activation are observed for these solvation processes (Table 3; compounds 3Cl iPr and 3I iPr). These data suggest the operation of a rather uniform substitution mechanism showing a complete compensation between the activation entropies and enthalpies (Figure 7, black squares), Figure 4. Synthetic procedures to prepare cyclometalated complexes 3Cl iPr,3I iPr, and 3dmso iPr . Figure 5. Representation of the crystal structures of cyclometalated 3Cl iPr,3I iPr, and 3dmso iPr . The atoms bonded to the Ru center are labeled, and Cstands for the centroid of the p-cymene ring. Hydrogen atoms are omitted for clarity. Inorganic Chemistry pubs.acs.org/IC Article https://doi.org/10.1021/acs.inorgchem.1c00507 Inorg. Chem. 2021, 60, 7974−7990 7979 with a clear isokinetic temperature, i.e. 79 °C, outside of the range experimentally used (3Cl iPr,Table S9;3I iPr,Table S6). 48−50 The process is therefore explained by a rather pure interchange mechanism with a certain degree of dissociation and ordering, as indicated by the general trend of the set of ΔV⧧(>0) and ΔS⧧(<0) (see Table 3). On consideration that a neat charge separation is taking place during the process and that DMSO is less polar than n-butanol, 51 the larger negative values of ΔS⧧ for the latter can be explained by solvent ordering of charge formation. Similarly, the more positive values of ΔV⧧for nbutanol can be justified by a higher number of solvent molecules involved in the process. Furthermore, the more positive ΔV⧧values for iodido complex 3I iPr in comparison with chlorido complex 3Cl iPr (Table 3) suggest that higher volume changes on charge separation occur with the less polar compound, as one would expect. 52 The results obtained are rather surprising, given the accepted associativeness of substitution mechanisms observed with ruthenium(II) complexes, 53,54 which is normally accepted as a positive factor for biological applications. The important covalent character of the M−L bonds in organometallic 3Cl iPr and 3I iPr most likely decreases the Lewis acidity of the metal center, by comparison with classical Werner-type complexes; consequently, the associative demand in the substitution process is reduced. Figure 6. (a) Representative set of time-resolved spectral changes observed for a solution of 3Cl iPr in NaCl-saturated DMSO at 70 °C for 10 h. (b) Selected Eyring plots of the temperature dependence of kobs for 3Cl iPr in n-butanol (black ■) and 3I iPr in DMSO (red ●). (c) Selected plots of the pressure dependence of ln kobs for 3Cl iPr in DMSO (black ■) and 3Cl iPr in n-butanol (red ●). Table 3. Summary of the Kinetic, Thermal, and Pressure Activation Parameters for the Solvation of 3Cl iPr,3 I iPr, and 3dmso iPr with Various Solvents a compound solvolysis by 105×310kobs/s−1(6 ×t1/2/days) ΔH⧧/kJ mol−1ΔS⧧/J K−1mol−1ΔV⧧/cm3mol−1 3Cl iPr DMSO d 0.35 (330) 85 ±5−78 ±14 6.2 ±0.6 n-butanol 2.4 (48) 63 ±1−133 ±112±2 H2O b 5.2 (22) 52 ±2−162 ±5nd c 3I iPr e DMSO d 1.6 (72) 113 ±325±813±0.5 n-butanol 12 (9.6) 62 ±2−123 ±520±1 3dmso iPr H2O f 2.0 (58) 71 ±4−110 ±13 −14 ±2 a [Complex] = 10−50 μM. b 3Cl iPr is poorly soluble in water; the values are derived from the rate of appearance of definite UV−vis spectra. c Not determined. d DMSO containing 0.005% of water was used ([H2O] = 2.77 ×10−3M). e 3I iPr is completely insoluble in water. f Experiments performed using a saturated aqueous solution; [3dmso iPr ]≈10 μM (such a low concentration had to be used due to the very low solubility of this compound in water). Inorganic Chemistry pubs.acs.org/IC Article https://doi.org/10.1021/acs.inorgchem.1c00507 Inorg. Chem. 2021, 60, 7974−7990 7980 Similar features were observed for substitution reactions with organometallic platinum(IV) 55−57 and platinum(II) compounds. 58,59 The behavior of 3dmso iPr in water was investigated next. As 3dmso iPr is very poorly soluble in water, 10 μm solutions of this compound were prepared by sonication and the higher temperatures given in Table S9, i.e. 40−80 °C, were used (since the compound was hardly water soluble at room temperature). Despite these solubility issues, the large values of the extinction coefficients of the absorptions in the range of 350−400 nm allowed us to record satisfactory time-resolved spectra, even at variable pressures. The values of the kinetic and activation parameters obtained (with larger associated errors, compared with those of 3Cl iPr and 3I iPr) are given in Table 3. The data point shown in Figure 7 (red square) suggests that a similar substitution mechanism for the DMSO/water exchange is taking place in 3dmso iPr . However, the values of both the volume and entropy of activation are negative, hence suggesting that the ordering in this reaction is associated with a volume decrease; for 3dmso iPr , no charge variation is occurring during the DMSO/water exchange, in contrast to the halide/ DMSO exchanges in 3Cl iPr and 3I iPr. As a result, the ordering and contraction go in the same direction in the case of 3dmso iPr . Furthermore, from the data obtained, a very interesting shift to the associative activation side of the interchange process seems to apply for these cationic ruthenium(II) species. In summary, once in solution, and especially in the presence of DMSO, compounds 2CI2 iPr and 2I2 iPr undergo a series of transformations, which are illustrated in Figure 8. Partial degradation of the compounds is observed, as the free phosphane ligand Lis detected together with its oxidized form, namely phosphane oxide LO(Figure 9, degradation pathway). In the absence of DMSO, cyclometalated species are not generated; therefore, this solvent clearly plays an important role in the transformation pathway. It is believed that the first step consists of the substitution of an halido ligand by a DMSO molecule (k1), converting 2CI2 iPr or 2I2 iPr into intermediate A. This intermediate is readily converted into 3Cl iPr or 3I iPr(k2;Figure 8), through a cyclometalation reaction with DMSO acting as a Lewis base, in the so-called concerted metalation−deprotonation (CMD) mechanism. 60,61 It is indeed proposed that coordinated DMSO makes an intramolecular hydrogen bond with the pyrenyl group, hence fostering the cyclometalation (Figure S11). 3Cl iPr and 3I iPr then undergo substitution of the second halido ligand by a DMSO molecule, producing the Figure 7. Isokinetic (79 °C) compensation plot (black squares) for the thermal activation parameters obtained for the solvolysis reactions of 3Cl iPr and 3I iPr (Table 3). The red square corresponds to the aquation of 3dmso iPr . Figure 8. Representation of the different species generated in solution upon dissolution of 2CI2 iPr or 2I2 iPr in the presence of DMSO. Inorganic Chemistry pubs.acs.org/IC Article https://doi.org/10.1021/acs.inorgchem.1c00507 Inorg. Chem. 2021, 60, 7974−7990 7981 cationic cyclometalated compound 3dmso iPr (k3), which is finally aquated (i.e. DMSO is replaced with water, k4;Figure 8). It should be noted that the k1and k2rate constants could not be determined. First, the degradation path masks the determination of the value of k1. Second, as k2is clearly significantly higher than k1(as indicated by the 31P{1H} NMR experiments), once 2Cl2 iPr and 2I2 iPr are converted into their respective intermediates A, the subsequent cyclometalation reaction to yield 3Cl iPr and 3I iPr is immediate (k2≫k1), not allowing a determination of the value of k2under the experimental conditions applied in the present study. It can be pointed out here that k2Iis most likely superior to k2Cl; indeed, NMR studies with 2Cl2 iPr have shown (see above) that a species labeled N3 was forming in DMSO (δ+45.7 ppm; Figure S5), which may be ascribed to the chlorido intermediate A(X = Cl; Figure 8). In the case of 2I2 iPr, such a species was not observed by NMR (Figure 3), suggesting that when it is formed it is very rapidly converted into compound 3I iPr (hence k2I>k2Cl). The rate constants for the conversion of 3Cl iPr and 3I iPr into 3dmso iPr , respectively k3Cl and k3I, could be obtained (see Table 3), which showed that the iodido to DMSO substitution was almost 4.6 times faster than the chlorido to DMSO substitution (k3I>k3Cl;Table 3). It should be stressed here that one may expect an analogous trend for the first halido to DMSO substitution, namely k1I>k1Cl (2Cl2 iPr or 2I2 iPr → intermediate A;Figure 8); though additional in-depth studies are required to confirm this. Finally, the rate constant for the aquation of 3dmso iPr , namely k4(3dmso iPr →aquo complex B;Figure 8), was also determined (Table 3), showing that it was a relatively slow process. The changes in free energy (ΔG°) have been calculated for intermediates A(for X = Cl and X = I), 3Cl iPr and 3I iPr, and 3dmso iPr (Figure 8), with respect to the corresponding starting compounds 2Cl2 iPr and 2I2 iPr; these are shown in Figure 9. The energy profile is clearly more favorable when X = I (orange profile in Figure 9), corroborating the experimental data. For instance, the cyclometalation step (intermediate A→3X iPr) costs only 0.75 kcal mol−1for X = I, whereas it is 2.17 kcal mol−1for X = Cl. However, it can be noted that in both cases the cyclometalation is energetically inexpensive (although more feasible for X = I). The step 3X iPr →3dmso iPr is costless for X = I (energy difference of only 0.21 kcal mol−1), while a difference in energy of 6.9 kcal mol−1is found between 3Cl iPr and 3dmso iPr (Figure 9). It is noteworthy to stress that the geometry optimization of intermediate A(Figure 8) shows a clear orientation of the oxygen atom of the DMSO ligand toward the hydrogen atom involved in the cyclometalation of the pyrenyl group (the O··· H distances being 2.18 and 2.38 Å for X = Cl, I, respectively), as proposed in Figure S11, thus confirming the crucial involvement of the solvent in the cyclometalation pathway. Cytotoxicity Behaviors of Cyclometalated Compounds 3Cl iPr,3 I iPr,and3 dmso iPr .IC50 values were then determined for compounds 3Cl iPr,3I iPr, and 3dmso iPr at increasing “aging times”, using A549 (lung adenocarcinoma) human cells for comparison with the time-dependent IC50 values obtained for 2Cl2 iPr and 2I2 iPr for the same cell line (see Table 2). The results achieved are given in Table 4. It can be pointed out that a concentration range of 0.4−50 μM was used for these assays, because precipitation of the cyclometalated ruthenium compounds was observed at concentrations above 100 μM. Remarkably, in each case, comparable IC50 values were obtained for the samples with different “aging times”. These data indicate that the cyclometalated complexes remain unchanged in solution (in contrast to 2Cl2 iPr and 2I2 iPr). The cytotoxic activities of compounds 3Cl iPr,3I iPr, and 3dmso iPr are clearly better than those of the “parent compounds”2Cl2 iPr and 2I2 iPr.Itis interesting to note that the cytotoxic activities of 3dmso iPr are comparable to those of 3Cl iPr and 3I iPr, suggesting that replacement of the halide by DMSO has no effect on the biological properties. Surprisingly, compound 3I iPr is less cytotoxic than 3Cl iPr; from the IC50 data obtained with 2Cl2 iPr and 2I2 iPr (see Table 2), one would have expected compound 3I iPr to be more active than 3Cl iPr. This can be justified by the significantly higher conversion rates for 2I2 iPr to form 3I iPr and ultimately 3dmso iPr (see above, Figure 8;k1,k2, and k3). Hence, the formation of the active cyclometalated species is significantly faster with 2I2 iPr; thus, the little amounts of 3I iPr (and of 3dmso iPr ) progressively generated in solution are enough to give the increasing cytotoxicity observed for 2I2 iPr at days 5 and 7 (Table 2). Conversion of 2CI2 iPr to a cyclometalated species is much slower; therefore, evolution of the IC50 value is not observed within a period of 7 days (see Table 2). It can be pointed out that the observed IC50 values, ranging from 1.72 to 5.82 μM, are comparable to data reported in the literature for various types of piano-stool ruthenium(II) complexes. 62−65 Figure 9. Energy profiles (in kcal mol−1) for intermediate A→3X iPr → 3dmso iPr processes: orange, X = I; green, X = Cl. Table 4. Half-Maximum Inhibitory Concentrations a (IC50, μM) of Compounds 3Cl iPr,3 I iPr, and 3dmso iPr for A549 (Lung Adenocarcinoma) Human Cells, after Incubation for 24 h (Day 0) and after 1, 2, and 7 days of Aging (before IC50 Determination) compound day 0 b day 1 day 2 day 7 3Cl iPr 2.26 ±0.34 2.77 ±0.38 4.92 ±0.59 2.67 ±1.61 3I iPr 5.82 ±1.89 5.58 ±0.47 5.61 ±2.16 4.32 ±2.54 3dmso iPr 1.72 ±0.67 2.47 ±0.57 2.56 ±0.44 2.34 ±0.61 a The results are expressed as mean values ±SD out of three independent experiments. b Day 0 corresponds to the first determination of IC50, after 24 h of incubation with freshly prepared stock solutions of the compounds. Inorganic Chemistry pubs.acs.org/IC Article https://doi.org/10.1021/acs.inorgchem.1c00507 Inorg. Chem. 2021, 60, 7974−7990 7982 (32) Lee, R. F. S.; Escrig, S.; Maclachlan, C.; Knott, G. W.; Meibom, A.; Sava, G.; Dyson, P. J. The Differential Distribution of RAPTA-T in Non-Invasive and Invasive Breast Cancer Cells Correlates with Its Anti-Invasive and Anti-Metastatic Effects. Int. J. Mol. Sci. 2017,18 (9), 1869. (33) Thota, S.; Rodrigues, D. A.; Crans, D. C.; Barreiro, E. J. Ru(II) Compounds: Next-Generation Anticancer Metallotherapeutics? J. Med. Chem. 2018,61 (14), 5805−5821. (34) Rafols, L.; Torrente, S.; Aguila, D.; Soto-Cerrato, V.; PerezTomas, R.; Gamez, P.; Grabulosa, A. Expanding the Range of Pyrenylphosphines and Their Derived Ru(II)-Arene Complexes. Organometallics 2020,39 (16), 2959−2971. (35) Brissos, R. F.; Clavero, P.; Gallen, A.; Grabulosa, A.; Barrios, L. A.; Caballero, A. B.; Korrodi-Gregorio, L.; Perez-Tomas, R.; Muller, G.; Soto-Cerrato, V.; Gamez, P. Highly Cytotoxic Ruthenium(II)- Arene Complexes from Bulky 1-Pyrenylphosphane Ligands. Inorg. Chem. 2018,57 (23), 14786−14797. (36) Wang, F. Y.; Habtemariam, A.; van der Geer, E. P. L.; Fernandez, R.; Melchart, M.; Deeth, R. J.; Aird, R.; Guichard, S.; Fabbiani, F. P. A.; Lozano-Casal, P.; Oswald, I. D. H.; Jodrell, D. I.; Parsons, S.; Sadler, P. J. Controlling ligand substitution reactions of organometallic complexes: Tuning cancer cell cytotoxicity. Proc. Natl. Acad. Sci. U. S. A. 2005,102 (51), 18269−18274. (37) Chen, H. M.; Parkinson, J. A.; Morris, R. E.; Sadler, P. J. Highly selective binding of organometallic ruthenium ethylenediamine complexes to nucleic acids: Novel recognition mechanisms. J. Am. Chem. Soc. 2003,125 (1), 173−186. (38) Hodson, E.; Simpson, S. J. Synthesis and characterisation of (eta(6)-cymene)Ru(L)X-2 compounds: single crystal X-ray structure of (eta(6)-cymene)Ru(P{OPh}(3))Cl-2 at 203 K. Polyhedron 2004, 23 (17), 2695−2707. (39) Pettinari, R.; Marchetti, F.; Condello, F.; Pettinari, C.; Lupidi, G.; Scopelliti, R.; Mukhopadhyay, S.; Riedel, T.; Dyson, P. J. Ruthenium(II)-Arene RAPTA Type Complexes Containing Curcunnin and Bisdemethoxycurcumin Display Potent and Selective Anticancer Activity. Organometallics 2014,33 (14), 3709−3715. (40) Babak, M. V.; Meier, S. M.; Huber, K. V. M.; Reynisson, J.; Legin, A. A.; Jakupec, M. A.; Roller, A.; Stukalov, A.; Gridling, M.; Bennett, K. L.; Colinge, J.; Berger, W.; Dyson, P. J.; Superti-Furga, G.; Keppler, B. K.; Hartinger, C. G. Target profiling of an antimetastatic RAPTA agent by chemical proteomics: relevance to the mode of action. Chem. Sci. 2015,6(4), 2449−2456. (41) Sun, R. C.; Chu, X. D.; Zhang, S. W.; Li, T. Y.; Wang, Z.; Zhu, B. L. Synthesis, Structure, Reactivity, and Catalytic Activity of Cyclometalated (Phosphine)- and (Phosphinite)ruthenium Complexes. Eur. J. Inorg. Chem. 2017,2017 (25), 3174−3183. (42) Grabulosa, A.; Granell, J.; Font-Bardia, M. Cyclometallated ruthenium complexes with P-stereogenic monophosphines containing a polycyclic aromatic substituent. J. Organomet. Chem. 2019,896,51− 58. (43) Kuwabara, T.; Kato, T.; Takano, K.; Kodama, S.; Manabe, Y.; Tsuchida, N.; Minami, Y.; Hiyama, T.; Ishii, Y. P-C reductive elimination in Ru(ii) complexes to convert triarylphosphine ligands into fiveor six-membered phosphacycles. Chem. Commun. 2018,54 (42), 5357−5360. (44) Zhang, S. W.; Chu, X. D.; Li, T. Y.; Wang, Z.; Zhu, B. L. Synthesis, Structures, and Reactivity of Single and Double Cyclometalated Complexes Formed by Reactions of [Cp*MCl2](2) (M = Ir and Rh) with Dinaphthyl Phosphines. ACS Omega 2018,3(4), 4522−4533. (45) Sun, R.; Chu, X.; Zhang, S.; Li, T.; Wang, Z.; Zhu, B. Synthesis, Structure, Reactivity, and Catalytic Activity of Cyclometalated (Phosphine)- and (Phosphinite)ruthenium Complexes. Eur. J. Inorg. Chem. 2017,2017 (25), 3174−3183. (46) Binstead, R. A.; Jung, B.; Zuberbuhler, A. D. Specfit/32; Spectrum Software Associates: 2000. (47) King, P.; Maeder, M. Reactlab Kinetics, 1.1; J PlusConsulting Pty Ltd: Karawarra, Western Australia, 2009. (48) Tobe, M. L.; Burgess, J. Inorganic Reaction Mechanisms; Longman: New York, 1999. (49) Wilkins, R. G. Kinetics and Mechanisms of Reactions of Transition Metal Complexes; VCH: 1991. (50) Crespo, M.; Martinez, M.; Sales, J. Effect of Fluorine Substituents in Intramolecular Activation of C−F and C−H Bonds by Platinum(II). Organometallics 1993,12 (11), 4297−4304. (51) Reichardt, C. Solvents and Solvent Effects in Organic Chemistry, 3rd ed.; Wiley-VCH: 2003. (52) Burgess, J. Ions in Solution; Woodhead Publishing: 1999. (53) Merbach, A. E. In High Pressure Chemistry and Biochemistry; van Eldik, R., Jonas, J., Eds.; Reidel: 1987; p 311. (54) Vazquez, M.; Martinez, M. Kinetico-mechanistic Studies on the Substitution Reactivity on the {Ru-II(bpy)(2)} Core with Nucleosides and Nucleotides at Physiological pH. Inorg. Chem. 2016,55 (13), 6731−6738. (55) Bernhardt, P. V.; Gallego, C.; Martinez, M. Mechanisms of substitution reactions on cyclometallated platinum(IV) complexes: “Quasi-labile”systems. Organometallics 2000,19 (23), 4862−4869. (56) Bernhardt, P. V.; Gallego, C.; Martinez, M.; Parella, T. Substitution reactions on cyclometalated Pt(IV) complexes. Associative tuning by fluoro Ligands and fluorinated substituents. Inorg. Chem. 2002,41 (7), 1747−1754. (57) Gallego, C.; Gonzalez, G.; Martinez, M.; Merbach, A. E. Activation volumes of substitution reactions on neutral and cationic organometallic platinum(IV) complexes: Definite proof of selective associative activation. Organometallics 2004,23 (10), 2434−2438. (58) Plutino, M. R.; Scolaro, L. M.; Albinati, A.; Romeo, R. Atropisomerization, C-H activation, and dissociative substitution at some biphenyl platinum(II) complexes. J. Am. Chem. Soc. 2004,126 (20), 6470−6484. (59) Frey, U.; Helm, L.; Merbach, A. E.; Romeo, R. Dissociative Substitution in Four-Coordinated Planar Platinum(II) Complexes As Evidenced by Variable-Pressure High-Resolution Proton NMR Magnetization Transfer Experiments. J. Am. Chem. Soc. 1989,111 (21), 8161−8165. (60) Yuan, B. F.; Wan, J. W.; Guo, X. G.; Gong, Y. M.; Zhang, F. L.; Li, Q.; Wang, G. Z.; Chen, J. Y.; He, R. X. Theoretical investigation on the Cu(i)-catalyzed N-carboxamidation of indoles with isocyanates to form indole-1-carboxamides: effects of solvents. New J. Chem. 2020,44 (23), 9878−9887. (61) Bjelopetrovic, A.; Barisic, D.; Duvnjak, Z.; Dzajic, I.; Juribasic Kulcsar, M.; Halasz, I.; Martinez, M.; Budimir, A.; Babic, D.; Curic, M. A Detailed Kinetico-Mechanistic Investigation on the Palladium C-H Bond Activation in Azobenzenes and Their Monopalladated Derivatives. Inorg. Chem. 2020,59 (23), 17123−17133. (62) Harringer, S.; Wernitznig, D.; Gajic, N.; Diridl, A.; Wenisch, D.; Hejl, M.; Jakupec, M. A.; Theiner, S.; Koellensperger, G.; Kandioller, W.; Keppler, B. K. Introducing N-, P-, and S-donor leaving groups: an investigation of the chemical and biological properties of ruthenium, rhodium and iridium thiopyridone piano stool complexes. Dalton Trans. 2020,49 (44), 15693−15711. (63) Ballester, F. J.; Ortega, E.; Porto, V.; Kostrhunova, H.; DavilaFerreira, N.; Bautista, D.; Brabec, V.; Dominguez, F.; Santana, M. D.; Ruiz, J. New half-sandwich ruthenium(ii) complexes as proteosynthesis inhibitors in cancer cells. Chem. Commun. 2019,55 (8), 1140− 1143. (64) Gatti, A.; Habtemariam, A.; Romero-Canelon, I.; Song, J. I.; Heer, B.; Clarkson, G. J.; Rogolino, D.; Sadler, P. J.; Carcelli, M. HalfSandwich Arene Ruthenium(II) and Osmium(II) Thiosemicarbazone Complexes: Solution Behavior and Antiproliferative Activity. Organometallics 2018,37 (6), 891−899. (65) Schmid, W. F.; John, R. O.; Arion, V. B.; Jakupec, M. A.; Keppler, B. K. Highly antiproliferative ruthenium(II) and osmium(II) arene complexes with paullone-derived ligands. Organometallics 2007, 26 (26), 6643−6652. (66) Clavel, C. M.; Paunescu, E.; Nowak-Sliwinska, P.; Griffioen, A. W.; Scopelliti, R.; Dyson, P. J. Modulating the Anticancer Activity of Inorganic Chemistry pubs.acs.org/IC Article https://doi.org/10.1021/acs.inorgchem.1c00507 Inorg. Chem. 2021, 60, 7974−7990 7989 Ruthenium(II)-Arene Complexes. J. Med. Chem. 2015,58 (8), 3356− 3365. (67) Marchetti, F.; Pettinari, R.; Di Nicola, C.; Pettinari, C.; Palmucci, J.; Scopelliti, R.; Riedel, T.; Therrien, B.; Galindo, A.; Dyson, P. J. Synthesis, characterization and cytotoxicity of areneruthenium(II) complexes with acylpyrazolones functionalized with aromatic groups in the acyl moiety. Dalton Trans. 2018,47 (3), 868− 878. (68) Tabrizi, L.; Olasunkanmi, L. O.; Fadare, O. A. Experimental and theoretical investigations of cyclometalated ruthenium(ii) complex containing CCC-pincer and anti-inflammatory drugs as ligands: synthesis, characterization, inhibition of cyclooxygenase and in vitro cytotoxicity activities in various cancer cell lines. Dalton Trans. 2019,48 (2), 728−740. (69) Chen, F.; Romero-Canelon, I.; Soldevila-Barreda, J. J.; Song, J. I.; Coverdale, J. P. C.; Clarkson, G. J.; Kasparkova, J.; Habtemariam, A.; Wills, M.; Brabec, V.; Sadler, P. J. Transfer Hydrogenation and Antiproliferative Activity of Tethered Half-Sandwich Organoruthenium Catalysts. Organometallics 2018,37 (10), 1555−1566. (70) Pettinari, R.; Pettinari, C.; Marchetti, F.; Skelton, B. W.; White, A. H.; Bonfili, L.; Cuccioloni, M.; Mozzicafreddo, M.; Cecarini, V.; Angeletti, M.; Nabissi, M.; Eleuteri, A. M. Arene-Ruthenium(II) Acylpyrazolonato Complexes: Apoptosis-Promoting Effects on Human Cancer Cells. J. Med. Chem. 2014,57 (11), 4532−4542. (71) de la Cueva-Alique, I.; Sierra, S.; Munoz-Moreno, L.; PerezRedondo, A.; Bajo, A. M.; Marzo, I.; Gude, L.; Cuenca, T.; Royo, E. Biological evaluation of water soluble arene Ru(II) enantiomers with amino-oxime ligands. J. Inorg. Biochem. 2018,183,32−42. (72) Armarego, W. L. F.; Chai, C. L. L. Purification of Laboratory Chemicals., 7th ed.; Butterworth Heinemann: 2013. (73) Gabbai, F. P.; Chirik, P. J.; Fogg, D. E.; Meyer, K.; Mindiola, D. J.; Schafer, L. L.; You, S.-L. An Editorial About Elemental Analysis. Organometallics 2016,35 (19), 3255−3256. (74) SAINT and SADABS; Bruker AXS Inc.: Madison, WI, USA. (75) Sheldrick, G. M. SHELXT - Integrated space-group and crystalstructure determination. Acta Crystallogr., Sect. A: Found. Adv. 2015, 71,3−8. (76) Sheldrick, G. M. Crystal structure refinement with SHELXL. Acta Crystallogr., Sect. C: Struct. Chem. 2015,71,3−8. (77) Dolomanov, O. V.; Bourhis, L. J.; Gildea, R. J.; Howard, J. A. K.; Puschmann, H. OLEX2: a complete structure solution, refinement and analysis program. J. Appl. Crystallogr. 2009,42, 339−341. (78) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M. J.; Heyd, J.; Brothers, E. N.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A. P.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, N. J.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, Rev. D01; Gaussian Inc.: 2009. (79) Perdew, J. P.; Burke, K.; Ernzerhof, M. Generalized gradient approximation made simple. Phys. Rev. Lett. 1996,77 (18), 3865− 3868. (80) Perdew, J. P.; Burke, K.; Ernzerhof, M. Generalized gradient approximation made simple. Phys. Rev. Lett. 1997,78 (7), 1396− 1396. (81) Schafer, A.; Huber, C.; Ahlrichs, R. Fully optimized contracted Gaussian basis sets of triple zeta valence quality for atoms Li to Kr. J. Chem. Phys. 1994,100 (8), 5829−5835. (82) Haussermann, U.; Dolg, M.; Stoll, H.; Preuss, H.; Schwerdtfeger, P.; Pitzer, R. M. Accuracy of energy-adjusted quasirelativistic ab initio pseudopotentials. Mol. Phys. 1993,78 (5), 1211−1224. (83) Kuchle, W.; Dolg, M.; Stoll, H.; Preuss, H. Energy-adjusted pseudopotentials for the actinides. Parameter sets and test calculations for thorium and thorium monoxide. J. Chem. Phys. 1994,100 (10), 7535−7542. (84) Leininger, T.; Nicklass, A.; Stoll, H.; Dolg, M.; Schwerdtfeger, P. The accuracy of the pseudopotential approximation.2. A comparison of various core sizes for indium pseudopotentials in calculations for spectroscopic constants of InH, InF, and InCl. J. Chem. Phys. 1996,105 (3), 1052−1059. (85) Marenich, A. V.; Cramer, C. J.; Truhlar, D. G. Universal Solvation Model Based on Solute Electron Density and on a Continuum Model of the Solvent Defined by the Bulk Dielectric Constant and Atomic Surface Tensions. J. Phys. Chem. B 2009,113 (18), 6378−6396. Inorganic Chemistry pubs.acs.org/IC Article https://doi.org/10.1021/acs.inorgchem.1c00507 Inorg. Chem. 2021, 60, 7974−7990 7990