Ruthenium(II) Diphosphine Complexes with Mercapto Ligands That Inhibit Topoisomerase IB and Suppress Tumor Growth In Vivo.

pubs.acs.org/IC Article Ruthenium(II) Diphosphine Complexes with Mercapto Ligands That Inhibit Topoisomerase IB and Suppress Tumor Growth In Vivo Monize M. da Silva, Gabriel H. Ribeiro,* Mariana S. de Camargo, Antônio G. Ferreira, Leandro Ribeiro, Marília I. F. Barbosa, Victor M. Deflon, Silvia Castelli, Alessandro Desideri, Rodrigo S. Correâ , Arthur B. Ribeiro, Heloiza D. Nicolella, Saulo D. Ozelin, Denise C. Tavares, and Alzir A. Batista* Downloaded via UNIV OF GLASGOW on November 7, 2022 at 16:53:25 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. Cite This: Inorg. Chem. 2021, 60, 14174−14189 ACCESS Metrics & More Read Online Article Recommendations sı Supporting Information * ABSTRACT: Ruthenium(II) complexes (Ru1−Ru5), with the general formula [Ru(N-S)(dppe)2]PF6, bearing two 1,2-bis(diphenylphosphino)ethane (dppe) ligands and a series of mercapto ligands (N-S), have been developed. The combination of these ligands in the complexes endowed hydrophobic species with high cytotoxic activity against five cancer cell lines. For the A549 (lung) and MDA-MB-231 (breast) cancer cell lines, the IC50 values of the complexes were 288- to 14-fold lower when compared to cisplatin. Furthermore, the complexes were selective for the A549 and MDAMB-231 cancer cell lines compared to the MRC-5 nontumor cell line. The multitarget character of the complexes was investigated by using calf thymus DNA (CT DNA), human serum albumin, and human topoisomerase IB (hTopIB). The complexes potently inhibited hTopIB. In particular, complex [Ru(dmp)(dppe)2]PF6 (Ru3), bearing the 4,6-diamino-2-mercaptopyrimidine (dmp) ligand, effectively inhibited hTopIB by acting on both the cleavage and religation steps of the catalytic cycle of this enzyme. Molecular docking showed that the Ru1−Ru5 complexes have binding affinity by active sites on the hTopI and hTopI-DNA, mainly via π-alkyl and alkyl hydrophobic interactions, as well as through hydrogen bonds. Complex Ru3 displayed significant antitumor activity against murine melanoma in mouse xenograph models, but this complex did not damage DNA, as revealed by Ames and micronucleus tests. ■ INTRODUCTION Since the anticancer properties of the first ruthenium complexes were tested, several classes of these metal complexes that exert cytotoxic activity have been investigated both in vitro and in vivo.1−9 The well-established synthetic chemistry of ruthenium provides numerous approaches for the development of new complexes containing this metal, and their structural diversities reflect on their anticancer properties against different types of cancer cells.10 Cell death induced by ruthenium complexes can occur via different mechanisms of action given that these complexes can inhibit the action of various intracellular and extracellular pharmacological targets that play relevant roles in cellular processes.11−17 Within this theme, we had previously designed, synthesized, and studied the chemistry, reactivity, and cytotoxic properties of ruthenium(II) phosphine complexes bearing mercapto ligands (N-S).18−21 Herein, we will focus on the cytotoxicity of these complexes and their interaction with biological molecules, mainly DNA 22,23 and proteins, 24,25 to get information about their mechanism of action and to try to shed some light on possible structure−activity relationships in these systems. Remarkably, complexes with the general formulas [Ru(N-S)(bipy)(dppb)]PF6 and [Ru(N-S)(bipy)© 2021 American Chemical Society (PPh3)2]PF6 [where bipy = 2,2′-bipyridine, dppb = 1,4bis(diphenylphosphino)butane, and PPh3 = triphenylphosphine] exert promising cytotoxic activity against different cancer cell lines in vitro.19,21 The most active complexes of these series have IC50 values ranging from 10 to 0.05 μM, especially against the human lung (A549) and breast (MDAMB-231) cancer cell lines. In contrast, neutral complexes with the general formula [Ru(N-S)2(dppb)] display low cytotoxicity.23,26 The ruthenium(II) phosphine complexes exert cytotoxic effects by different mechanisms of action, which are distinct from the mechanisms of action of platinum-based drugs. For example, complex [Ru(mtz)(bipy)(dppb)]PF6 (mtz = 1,3-thiazolidine-2-thione) has a broad spectrum of anticancer properties.21,22 Detailed mechanistic studies have suggested that this complex induces apoptosis mediated by the MAPK ERK1/2 inhibitor pathway in HepG2 cells. This points to an Received: May 27, 2021 Published: September 3, 2021 14174 https://doi.org/10.1021/acs.inorgchem.1c01539 Inorg. Chem. 2021, 60, 14174−14189 Inorganic Chemistry pubs.acs.org/IC association with a prosurvival function, causing ERK1/2 activation induced by DNA damage and leading to apoptotic cell death. Notably, this complex shows highly promising antitumor activity. The complex inhibits HepG2 cell growth in C.B-17 SCID mice more effectively than doxorubicin, a commercial anticancer drug.22 Ruthenium(II) phosphine complexes present an important feature described in different works: lipophilicity,10,27,28 which is essential for their cytotoxic activity.10 Lipophilicity is believed to be crucial for efficient cellular uptake of these complexes, as well as for their interaction with biomolecules. The biological targets and the mechanism of actions of ruthenium(II) phosphine complexes have not been fully elucidated, but there is evidence that the complexes act as multitargets, such as topoisomerase, proteassoma 26S, and DNA. Ruthenium(II) phosphine complexes containing mercapto ligands have been reported as potent inhibitors of human DNA topoisomerase IB, but extensive studies on this subject are still necessary.24,29 Human topoisomerase IB (hTopIB) is a key nuclear enzyme involved in the replication, transcription, recombination, and chromosome condensation of the DNA; it is highly expressed in many tumors.30 Well-known hTopIB-targeting drugs, such as camptothecin and its derivatives, are among the most used chemotherapeutic drugs in cancer treatment.31−34 hTopIB catalyzes DNA relaxation by transiently cleaving one strand of the DNA double helix and religating the DNA strand.35 Mechanistically, formation of the cleaved complex is a critical event during the cell cycle because the cell is seriously compromised by poisoning of the DNA-hTopIB complex. The inhibitors that act in this process are known as “topoisomerase poisons”. The inhibitors that prevent religation of one DNA strand are called catalytic inhibitors. The stalled hTopIB can directly affect progression of the cell processes, producing double DNA strand damage and consequently inducing cell death.36,37 Bearing these facts in mind, we described the development of new ruthenium(II) complexes bearing two 1,2-bis(diphenylphosphino)ethane (dppe) ligands and different mercapto ligands. Here, we evaluate the cytotoxic activity of these new complexes, namely, [Ru(mtz)(dppe)2]PF6 (Ru1), [Ru(mmi)(dppe)2]PF6(Ru2), [Ru(dmp)(dppe)2]PF6 (Ru3), [Ru(mpca)(dppe)2]PF6 (Ru4), and [Ru(2mq)(dppe)2]PF6 (Ru5) [where mtz = 1,3-thiazolidine-2-thione, mmi = mercapto-1-methylimidazole, dmp = 4,6-diamino-2-mercaptopyrimidine, mpca = 6-mercaptopyridine-3-carboxylic acid, and 2mq = 2-mercapto-4(3H)-quinazoline] against a panel of cancer cell lines and a nontumor cell line. We also assess the multitarget [e.g., toward DNA and human serum albumin (HSA)] character of these complexes. Furthermore, we investigate the ability of these complexes to inhibit hTopIB during relaxation of supercoiled DNA. Moreover, we investigate whether complex Ru3 disrupts the cleavage and religation steps of the hTopIB catalytic cycle. Finally, we evaluate the mutagenicity of complex Ru3 and the effects of this complex on melanoma tumor growth in vivo in mouse xenograph models. Article Figure 1. General chemical structures of complexes Ru1−Ru5. under an argon atmosphere and reflux for 12 h. The complexes Ru1−Ru5 in good yields (82−95%), as yellow solids, were isolated and characterized by the usual techniques. The structures of complexes Ru1−Ru5 and their purities were confirmed in solution and the solid state. The single-crystal X-ray structures of complexes Ru1 (CCDC 2079011), Ru2 (CCDC 2079012), Ru4 (CCDC 2079013), and Ru5 (CCDC 2079014) (Figure 2) were determined. Tables S3 and S4 list the selected bond distances and bond angles. All of the complexes were in the sixcoordination mode, consistent with a distorted octahedral geometry around the RuII center, which was coordinated with two dppe ligands and one mercapto ligand. The mercapto ligands were coordinated to the metal center in a bidentate manner, through S and N atoms, with the N1 atom trans to the P3 atom and, the S1 atom trans to the P2 atom. The P1 and P4 atoms occupied positions in the equatorial plane and were situated trans to each other. Compared to the Ru−P bond distances (Table S1), the bond lengths involving the axial P atoms (P1 and P4) were slightly longer than those involving the equatorial P atoms (P2 and P3), which could be related to a competitive effect due to the trans influence between the P atoms, causing the Ru−P bond to elongate along the axial position. The geometric parameters (bond lengths, bond angles, and torsional angles) obtained for complexes Ru1−Ru5 were consistent with the expected values for related ruthenium(II) complexes bearing N,S-donor ligands.20,38 In the region 3200−3100 cm−1 of the IR spectra of complexes Ru1−Ru5, the absence of a broad band confirms the monoanionic coordination of the mercapto ligands to ruthenium. The spectra of the free mercapto ligands are characterized by the presence of a broad band referring to the ν(N−H) mode. Characteristic vibrations assigned to the ν(C− S) and δ(C−S) modes occur in the range 1225−1275 cm−1 (Figures S1−S5).6,11,14,19 The resolved patterns of resonances in the 1H and 13C {1H} NMR spectra are consistent with low symmetry of the ruthenium(II) complexes (Figures S6−S31). For all of the ruthenium(II) complexes, the 31P{1H} NMR spectra presented four distinct signals of double double doublets (ddd), consistent with ABMX pattern spin systems. Thus, the four ■ RESULTS AND DISCUSSION Characterization of the Complexes. Complexes Ru1− Ru5 (Figure 1) were prepared by reacting the precursor cis[RuCl2(dppe)2] with appropriate equivalents of the corresponding mercapto ligand in methanol/dichloromethane 14175 https://doi.org/10.1021/acs.inorgchem.1c01539 Inorg. Chem. 2021, 60, 14174−14189 Inorganic Chemistry pubs.acs.org/IC Article Figure 2. ORTEP view of the ruthenium(II) complexes showing the atom labels and 50% probability ellipsoids. All H atoms and anions have been omitted for clarity. Table 1. IC50 Valuesa (μmol L−1) of the In Vitro Cytotoxic Activity of Complexes Ru1−Ru5, Precursor cis-[RuCl2(dppe)2], and Cisplatin (Reference Drug) against the MCF-7, MDA-MB-231, A549, DU-145, and HepG2 Cancer Cell Lines and the MRC-5 Nontumor Human Lung Cell Line IC50 (μM) complex A549 MDA-MB-231 MCF-7 DU-145 HepG2 MRC-5 Ru1 Ru2 Ru3 Ru4 Ru5 precursor cisplatin 0.07 ± 0.03 0.12 ± 0.07 0.89 ± 0.09 1.02 ± 0.09 0.05 ± 0.01 0.40 ± 0.06 14.40 ± 1.45 0.03 ± 0.01 0.08 ± 0.02 0.20 ± 0.02 1.97 ± 0.03 0.30 ± 0.03 0.77 ± 0.04 2.44 ± 0.39 3.68 ± 0.89 5.24 ± 0.08 8.14 ± 0.45 9.01 ± 1.53 6.27 ± 0.25 24.26 ± 4.08 13.98 ± 2.02 0.35 ± 0.12 1.46 ± 0.05 3.90 ± 0.99 5.92 ± 0.75 6.02 ± 0.10 0.61 ± 0.20 2.33 ± 0.40 1.94 ± 0.06 5.80 ± 0.70 5.95 ± 0.09 25.45 ± 0.68 7.37 ± 0.72 44.99 ± 0.11 16.31 ± 0.74 0.79 ± 0.10 0.81 ± 0.12 2.62 ± 0.42 19.59 ± 0.51 5.72 ± 0.64 2.83 ± 0.06 29.09 ± 0.79 Data are expressed as mean ± SD (n = 3) with 95% confidence intervals. bFor the free ligands, IC50 > 50 μM; assay involved incubation for 48 h. a plexes exhibit lower-energy bands at about 350 nm, with weak intensity, which can be assigned as metal-to-ligand chargetransfer transitions, Ru(dπ) to ligand (π*). The cyclic voltammetry experiments of complexes Ru1−Ru5, carried out in CH2Cl2 solutions (Figure S33 and Table S5), presented a quasi-reversible process, corresponding to one-electron RuII/ P atoms were chemically and magnetically nonequivalent in the ruthenium(II) complexes. The UV−vis absorption spectra in solutions of CH2Cl2 for the complexes (Figure S32) are characterized by high-energy bands around 250 nm, which can be assigned to ligandlocalized, intraligand π−π* transitions. Moreover, the com14176 https://doi.org/10.1021/acs.inorgchem.1c01539 Inorg. Chem. 2021, 60, 14174−14189 Inorganic Chemistry pubs.acs.org/IC Article Table 2. SI Values of Complexes Ru1−Ru5 for Cancer Cell Lines SIb complex A549 MDA-MB-231 MCF-7 DU-145 HepG2 Ru1 Ru2 Ru3 Ru4 Ru5 precursor cisplatin 11 ± 5 7±4 2.9 ± 0.6 19 ± 2 114 ± 26 7±1 2.0 ± 0.2 26 ± 9 10 ± 3 13 ± 2 9.9 ± 0.3 19 ± 3 3.7 ± 0.2 11 ± 1 0.21 ± 0.06 0.15 ± 0.02 0.32 ± 0.05 2.2 ± 0.4 0.9 ± 0.1 0.12 ± 0.02 2.1 ± 0.3 2.3 ± 0.8 0.55 ± 0.08 0.7 ± 0.2 3.3 ± 0.4 0.9 ± 0.1 5±1 12 ± 2 0.41 ± 0.05 0.14 ± 0.03 0.44 ± 0.07 0.77 ± 0.03 0.78 ± 0.12 0.06 ± 0.01 1.78 ± 0.09 a The SI of each complex was calculated using the following formula: SI = IC50(MRC-5)/IC50(cancer cell line). bData are expressed with propagated uncertainties of the SI. RuIII, with oxidation-wave potentials, Epa (anodic peak potential), in the range 1116−1360 mV. The electrochemical behavior of these complexes, in cyclic voltammetric experiments, was similar to that found for other ruthenium compounds reported in the literature, such as the [Ru(pymS)(dppe)2]PF6 (Epa = 1.47 mV) and [Ru(pymS)(dppm)2]PF6 [pymS = 2-mercaptopyrimidine; dppm = 1,1(diphenylphosphino)methane] complexes (Epa = 1.42 mV).13 Stability in Aqueous Medium. Complexes Ru1−Ru5 are soluble and stable in solvents such as dimethyl sulfoxide (DMSO), N,N-dimethylformamide, and chlorinated solvents. However, the complexes are not soluble in a pure aqueous medium, but they are soluble and stable in a mixture of 1:99 DMSO/aqueous medium at micromolar concentrations, which were used for biological studies. For biological experiments, complexes Ru1−Ru5 were initially dissolved in the DMSO solvent and subsequently diluted in the respective medium. Therefore, the stability of the new ruthenium(II) complexes dissolved in DMSO were evaluated by 31P{1H} NMR spectroscopy in time course of 0, 48, and 72 h (Figures S34−S38). It is worth mentioning that we evaluated the cytotoxic activity of the ruthenium(II) complexes against cells for an incubation period of 48 h. From the 31P{1H} NMR spectra, the signals of the ruthenium(II) complexes remained unchanged over 72 h, which attested to their integrity in the DMSO solvent. Cytotoxicity Assays and Partition Coefficient (log P). We evaluated the cytotoxic activities (Table 1) of complexes Ru1−Ru5 against five cancer cell lines, namely, A549 (human lung epithelial), MDA-MB-231 (mesenchymal-like human triple-negative breast adenocarcinoma), MCF-7 (epitheliallike estrogen-dependent human breast adenocarcinoma), DU145 (human prostate carcinoma), and HepG2 (human hepatocellular carcinoma). To determine the selectivity index (SI; Table 2), we also assessed the cytotoxic activities of complexes Ru1−Ru5 against MRC-5 nontumor human lung cell line (Table 1). First, the precursor cis-[RuCl2(dppe)2] was active against all tested tumor lines, especially for the DU-145, A549, and MDA-MB-231 cells. The precursor bearing two dppe ligands was 36-, 3-, and 4-fold more active than cisplatin, respectively, with respect to these tumor cells. In general, replacement of the chlorido ligands in the precursor cis-[RuCl2(dppe)2] by mercapto ligands was highly advantageous: it afforded new cationic complexes containing two diphosphine ligands, leading to high cytotoxicity, and mainly most of the complexes were more cytotoxic compared to the precursor, cisplatin, and all free mercapto ligands. All of the tested ruthenium(II) complexes were more active against the A549 (lung) and MDA-MB-231 (breast) cancer cell lines than toward the other studied cancer cells. For the A549 cancer cell line, complexes Ru1−Ru5 exhibited 288−14-fold lower IC50 values than that cisplatin. Furthermore, the IC50 values for the MDA-MB-231 cell line were 81−1 times lower compared to that of the commercial drug. However, it is worth mentioning that, toward A549 cells, complexes Ru3 and Ru4 were approximately 2.2and 2.6-fold, respectively, less active than the precursor cis[RuCl2(dppe)2]. Similarly, for the MDA-MB-231 cells, complex Ru3 was less active compared to its respective precursor. Surprisingly, for the DU-147 cells, the precursor cis[RuCl2(dppe)2] was more effective than most of the new complexes. However, for the vast majority of the cases, the addition of the mercapto ligands to the precursors forms products that are more cytotoxic against the tumor cells. Complexes Ru1−Ru5 were more cytotoxic against the A549 and MDA-MB-231 cancer cell lines compared to the MRC-5 nontumor cell line. All of the ruthenium(II) complexes, including the precursor cis-[RuCl2(dppe)2], exhibited SIs (Table 2) in the range of 3−35 regarding the pair MRC-5/ MDA-MB-231. Also, SIs of the new complexes were greater than that of cisplatin for the pair MRC-5/A549. As far as we know, the SI value of Ru5 of 114 for the pair MRC-5/A549 is the highest described for ruthenium(II) phosphine complexes. These new results, combined with the data previously reported by our group, have provided more solid insight into ruthenium(II) phosphine complexes: (I) These complexes exhibited remarkable cytotoxic action against the A549 and MDA-MB-231 cancer cell lines, which are better than most of the complexes reported in the literature.39−44 This behavior probably resulted from the good lipophilicity of this class of complexes. (II) The cytotoxic activity of the ruthenium(II) complexes was dependent on the auxiliary mercapto ligands. In general, complexes Ru1 and Ru2 were more cytotoxic than that complexes Ru3−Ru5. Structurally, complexes Ru1 and Ru2 are characterized by mercapto ligands without functional groups or “electrophilic groups” attached to the rings. Complexes Ru3−Ru5 have mercapto ligands with “donor groups” in the mercapto ligand, such as amine and carbonyl moieties. This trend for mercapto ligands in ruthenium(II) complexes has been reported.10 However, no trend concerning the selectivity of the complexes has been observed. (III) The presence and quantity of phosphine ligands were key for the efficacy of the ruthenium(II) complexes.28,45,46 The complexes described herein were more active than the respective analogous complexes bearing the same mercapto ligands, [Ru(N-S)(bipy)(dppb)]PF 6 2 1 and [Ru(NS)2(dppb)].26 In the case of the A549 cancer cells, the 14177 https://doi.org/10.1021/acs.inorgchem.1c01539 Inorg. Chem. 2021, 60, 14174−14189 Inorganic Chemistry pubs.acs.org/IC Article Figure 3. (A) Electrophoresis mobility shift assays of plasmid pBlue-Script KSII(+) in the absence of DNA and CTL and treated with 0.5 (a), 1.0 (b), or 2.0 (c) equiv of Ru1−Ru5. CTL refers to untreated plasmid in Tris-HCl buffer with 10% DMSO. (B) Effect of increasing concentration of the ruthenium(II) complexes on the relative viscosity of CT DNA. [DNA] = 150 μM. (C) Square-wave voltammograms of 1.0 mM Ru1 in the absence (a) and presence of DNA at different concentrations: (a) 0, (b) 15, (c) 29, (d) 43, and (e) 57 μM. aspects of the drug−protein interactions to an initial understanding of drug pharmacokinetics.12,47−49 The HSA fluorescence intensity gradually decreased with increasing concentration of the ruthenium(II) complexes, indicating that the microenvironment of the HSA Trp-214 residue was affected (Figure S39). To evaluate the HSA fluorescence quenching mechanism, the Stern−Volmer constant was determined at different temperatures (Table S6). The Stern−Volmer constant values decreased with increasing temperature, indicating that the mechanism of HSA fluorescence quenching was static. Indeed, the bimolecular quenching constant (kq) values were higher than 1 × 1010 M−1 s−1, exceeding the maximum value of the kq constant for a mechanism to be considered pure dynamic quenching.50 We found binding constant values (Table S4) lying between 104 and 106 M−1 for complexes Ru1−Ru5 binding to HSA, which indicated a moderate-to-strong affinity between the species. Furthermore, the number of specific binding sites was approximately 1. Analysis of the thermodynamic parameters with positive ΔH and ΔS values51 showed that hydrophobic interactions were the main intermolecular forces involved in insertion of the ruthenium(II) complexes into the HSA framework. Negative ΔG values revealed that the HSA− ruthenium(II) complex binding affinities were spontaneous. Thus, the ruthenium(II) complexes would be transported into human plasma by HSA. Interactions with DNA. To investigate whether DNA is a biological target for the ruthenium(II) complexes, we conducted binding affinity studies by electrophoretic mobility plasmid pBlue-Script KSII(+) in gel agarose (Figure 3A), viscosity (Figure 3B), and square-wave voltammetry (Figures 3C and S40). Compared to untreated plasmid DNA, the patter of electrophoretic mobility of the circular (NC), linear (LC), and supercoiled (SC) conformations of plasmid DNA treated with different concentrations of the ruthenium(II) complexes was not affected (Figure 3A). Moreover, there were no alterations in the DNA viscosity in the presence of ruthenium- complexes containing bipyridine gave IC50 values ranging from 0.20 ± 0.03 to 11.74 ± 0.62 μM, whereas the new complexes Ru1−Ru5 afforded IC50 values below 1.02 ± 0.09 μM. In contrast, the IC50 values for complex [Ru(N-S)2(dppb)] were above 8.80 ± 1.44 μM. This behavior has also been observed for other cancer cell lines. Even if the diphosphine ligands are different in the series of complexes, the presence of the two diphosphine ligands was crucial for the cytotoxic activity of complexes Ru1−Ru5. We believe that these results stemmed from the lipophilic character of the ruthenium(II) complexes, conferred mainly by the diphosphine ligands. Thus, changing the lipophilicity or charge of the ruthenium(II) complexes, or both, might be an effective way of tuning their cytotoxicity. Having this in mind, we evaluated the partition coefficient of the ruthenium(II) complexes in water and n-octanol. The log P values determined for the ruthenium(II) complexes were 0.26 ± 0.04 (Ru1), 0.31 ± 0.01 (Ru2), 0.24 ± 0.11 (Ru3), 0.44 ± 0.02 (Ru4), and 0.36 ± 0.10 (Ru5). These positive values of log P reflected the relative affinity of these complexes for the lipid-like organic phase. Lipophilic compounds are expected to cross biological membranes more easily, which would justify the high cytotoxic activity of the ruthenium(II) complexes. However, on the basis of the aforementioned data, the lipophilicity was not directly correlated with the IC50 values of the ruthenium(II) complexes, probably because the lipophilicities of these complexes were practically the same within experimental error. Consequently, other parameters like the presence of peripheral groups in the mercapto ligand may also underline the activity of the ruthenium(II) complexes. HSA Binding Study by Fluorescence Quenching. The novel anticancer metal therapeutic candidates will probably be applied by an intravenous route, like most commercially available anticancer drugs. In this perspective, pharmacological characterization of ruthenium(II) complexes with the most abundant protein in human plasma, HSA, is of great importance. HSA plays a vital role in drug biodistribution, transport, release, and toxicity. Therefore, we evaluated some 14178 https://doi.org/10.1021/acs.inorgchem.1c01539 Inorg. Chem. 2021, 60, 14174−14189 Inorganic Chemistry pubs.acs.org/IC (II) complexes, at different concentrations, which contrasted with the effects of cisplatin (used as a positive control). Cisplatin significantly decreased the DNA viscosity because of irreversible covalent binding to DNA (Figure 3B). As for the voltammetric assay, the peak potential values corresponding to the RuIII/RuII couple were shift slightly toward lower potentials, after addition of CT DNA to the solutions of ruthenium(II) complexes (Figures 3C and S40). Increasing the electron density on the RuII center suggested reversible electrostatic interactions between the negatively charged DNA and the positively charged ruthenium(II) complexes. Therefore, the covalent and intercalation binding modes of the ruthenium(II) complexes to DNA were unlikely. Thus, these experiments indicated that complexes Ru1−Ru5 did not lead to significant conformational altercation in the tertiary and secondary DNA structures. The results were consistent with the structural features of the ruthenium(II) complexes and showed that the DNA binding affinity for the complexes did not depend on the auxiliary mercapto ligands. Also, ruthenium(II) complexes might not exert their cytotoxic action directly through DNA damage pathways. The “trigger” for cell death could be associated with other biological targets, such as enzymes overexpressed in tumor cells. Topoisomerase IB Activity. We evaluated hTopIB inhibition at different concentrations of complexes Ru1−Ru5 by supercoiled plasmid DNA relaxation assay (Figure 4). All of the ruthenium(II) complexes inhibited the ability of hTopIB to relax supercoiled plasmid DNA. The inhibition efficacy was concentration-dependent. As shown by the disappearance of several circular DNA forms, Ru2 (at 100 μM) and Ru3 (at 6 μM) totally inhibited the hTopIB activity. These results indicated that the hTopIB inhibition by ruthenium(II) complexes is dependent on the auxiliary mercapto ligands. Upon preincubation of the ruthenium(II) complexes with hTopIB, DNA relaxation was efficiently inhibited (Figure S41). After preincubation for 1 min, complexes Ru1, Ru2, Ru4, and Ru5, at low concentrations, completely inhibited hTopIB. These results suggested that the ruthenium(II) complexes can act directly on hTopIB, as well as on hTopIB in the presence of DNA (hTopIB-DNA). Complex Ru3 was the most potent topoisomerase IB inhibitor: it completely inhibited the hTopIB I activity at the lowest concentration among all of the ruthenium(II) complexes. To investigate whether complex Ru3 disrupts the hTopIB catalytic cycle, we carried out studies on cleavage and religation reactions. We assayed the kinetics of substrate cleavage in a timecourse reaction by using a suicide cleavage substrate, in the presence and absence of complex Ru3. The asymmetric CL14/ CP25 substrate has its 5′-end radiolabeled at the short strands. The enzyme preferentially cleaves the substrate at the site indicated by the arrow (Figure 5). The cleaved DNA fragments were resolved in a time-course experiment in agarose gel, and the amount of fragment was normalized and plotted as a function of time (Figure 5B). In the case of the negative control (10% DMSO), the kinetics of substrate cleavage by hTopIB was fast and efficient: about 80% of the substrate was cleaved within 45 s. After 20 min, the substrate had been completely cleaved. In contrast, complex Ru3 totally inhibited the hTopIB catalytic activity in the substrate cleavage. We studied the religation kinetics by testing the ability of hTopIB toward religate the complementary oligonucleotide R11 to the product of the cleaved substrate (Figure 6) in the absence and presence of complex Ru3. Initially, the cleaved Article Figure 4. Relaxation of supercoiled plasmid pBlue-Script KSII by topoisomerase IB at different concentrations of complexes Ru1−Ru5. *Concentration of ruthenium(II) complex of 300 μM. CTL refers to the negative control (10% DMSO). Concentrations (μM) of the ruthenium(II) complexes: (a) 0.75; (b) 1.50; (c) 3; (d) 6; (e) 12.5; (f) 25; (g) 50; (h) 100; (i) 200; (j) 300. substrate was obtained by incubating the substrate with hTopIB. The percentage of religated substrate was normalized and plotted as a function of time. As expected, hTopIB had a high religation rate, with a plateau being reached within approximately 20 min, with a religation rate of 100% (Figure 6B). The data showed that the presence of complex Ru3 slowed the religation kinetics compared to the negative control. The presence of complex Ru3 strongly decreased the rate of substrate religation by hTopIB, with the curve reaching a plateau with approximately 50% religation rate. 14179 https://doi.org/10.1021/acs.inorgchem.1c01539 Inorg. Chem. 2021, 60, 14174−14189 Inorganic Chemistry pubs.acs.org/IC Article religation step. Therefore, hTopIB inhibition may consequently lead to cell death from hTopIB-mediated DNA damage. Molecular Docking Studies of Ru1−Ru5 Complexes with hTopIB and hTopIB-DNA. The molecular docking technique was performed to evaluate the types of interactions between complexes Ru1−Ru5 and the binding sites of hTopIB and hTopIB-DNA biomolecules.51 The binding site was defined on the cocrystallized (PDB 1T8I),52 which on hTopIB consists of the important residues Arg364 and Asp532 and additionally on hTopIB-DNA of the DA113 and DT10 nucleobases. The main data from the molecular docking simulations are shown in Figures S42−S47. At the molecular level, all complexes exhibited a high affinity for the binding site of free hTopIB through hydrophobic interactions and hydrogen bonds (Table S7). The binding energies were from −29.3 to −32.1 kcal mol−1. First, because the phenyl rings of the dppe ligand are treated as hydrophobic centers, they play an important role in stabilizing the interactions of the complexes in the hydrophobic pocket of topoisomerase IB. Several hydrophobic interactions of the πalkyl and alkyl types are observed between the phenyl rings of the ruthenium complexes with acid amino residues (Figures 7 Figure 5. Kinetics of substrate cleavage by hTopIB in the presence of complex Ru3. (A) Time course of the CL14-U/CP25 substrate (at the top) cleavage reaction by hTopIB in the presence of complex Ru3 and 10% DMSO (negative control). CTL represents the DNA strand cleaved by the enzyme at the preferred cleavage site, as indicated by an arrow at the top of the figure. (B) Percentage of cleaved substrate, normalized to the maximum value of the CTL, plotted against time for reaction in the presence of complex Ru3 and DMSO (10%). The data are the mean ± SD of three independent experiments. Figure 6. Kinetics of substrate religation by hTopIB in the presence of complex Ru3. (A) Time course of the substrate religation reaction by hTopIB in the presence of complex Ru3 and 10% DMSO (negative control). The substrate was CL14-U/CP25, and the complementary oligonucleotide R11 was used for rewiring (at the top). CTL represents the DNA strand cleaved by hToPIB at the preferred cleavage site, as indicated by an arrow at the top of the figure. (B) Percentage of rewired product, normalized to the maximum value of the CTL, plotted against time for the reaction with complex Ru3 and 10% DMSO. The data are the mean ± SD of three independent experiments. Figure 7. (A) Molecular docking of complex Ru3 with hTopI-DNA and (B) expansion of the hTopIB-DNA active site with intermolecular interactions. and S42−S46 and Table S6). The mercapto ligands also play a relevant role due to hydrogen bonds of the complexes with the biomolecule. In the complexes, the S atom from the mercapto ligands acts as a hydrogen acceptor and the Lys532 residue acts as a H-donor atom. Molecular docking simulations were also performed using the hTopIB-DNA complex as a receptor in order to evaluate Therefore, complex Ru3 acts as a hTopIB catalytic and poison inhibitor; that is, it is a mixed inhibitor. Complex Ru3 can act by preventing hTopIB from binding to DNA, thereby inhibiting the cleavage reaction. Also, it can stabilize the intermediate state of the hTopIB-DNA complex in the 14180 https://doi.org/10.1021/acs.inorgchem.1c01539 Inorg. Chem. 2021, 60, 14174−14189 Inorganic Chemistry pubs.acs.org/IC Article the interactions with ruthenium complexes (Figures S42−S47 and Table S8). The best binding poses also demonstrated that complexes Ru1−Ru5 have binding affinities by binding sites on hTopIB-DNA mainly via π-alkyl and alkyl hydrophobic interactions. These interactions occur between the phenyl rings from the dppe ligands and the amino acid residues and nucleotides of hTopIB-DNA. For complexes Ru2−Ru4, the mercapto ligands also present hydrogen bonds with hTopIBDNA. Specifically, compound Ru3 is tightly fixed around the binding site of free hTopIB (Figure S45) through four alkyl hydrophobic interactions and a hydrogen bond (Table 3). The Table 3. Docking Interactions between the Complex Ru3 Cleavage/Religation Active Sites from hTop and hTopIDNA complex Ru3 Ru3 residue interaction type hTopI and Complex Ru3 Arg488 2× alkyl hydrophobic Lys532 hydrogen bond Lys532 alkyl hydrophobic Lys493 alkyl hydrophobic hTopI-DNA and Complex Ru3 thymine π-alkyl hydrophobic guanine π-alkyl/alkyl hydrophobic Leu721 2× alkyl hydrophobic Asp352 hydrogen bond Ala351 alkyl hydrophobic Pro431 alkyl hydrophobic complex Ru moiety P-phenyl S-dmp P-phenyl P-phenyl Figure 8. Assessment of the cell survival by clonogenic assay. Representative colony formation images: (A) MDA-MB-231 cancer and MRC-5 nontumor cells after treatment with different concentrations of complex Ru3; (B) A549 cancer and MRC-5 nontumor cells after treatment with different concentrations of complex Ru3. The negative control group was treated with the vehicle (0.05% DMSO). P-phenyl P-phenyl P-phenyl S-dmp P-phenyl P-phenyl the MDA-MB-231 cell colonies, whereas the survival of the MRC-5 nontumor cells was 73% (Figure 8A). However, none of the cells survived at 1.00 μM Ru3. Likewise, the number and size of the A549 cancer cell colonies also decreased upon exposure to complex Ru3 (Figure 8B). At a concentration of 0.9 μM, complex Ru3 allowed no A549 cancer cells to survive, while the survival of the MRC-5 nontumor cells was 48% (Figure 8B). For both the A459 cancer and MRC-5 nontumor cells, no cell colonies were observed in the presence of 2.0 μM complex Ru3. On the basis of this assay, complex Ru3 exhibited both cytotoxic and cytostatic activity against the tested cell lines. Importantly, the cytotoxic and antiproliferative activity of complex Ru3 on the MRC-5 nontumor cells was less pronounced compared to its activity against the A549 and MDA-MB-231 cancer cell lines, in accordance with the selectivity results for this complex. Mutagenic Activity. Ames Test. When therapeutic agents bind to DNA, they usually present mutagenicity. Several mutagenic complexes used in cancer treatment (e.g., cisplatin and carboplatin) have their mechanism of action associated with their interaction with DNA.54 This adverse characteristic is related to the carcinogenicity of such agents. The mutagenicity associated with complex Ru3 was investigated by the Ames test, in vitro, in the presence and absence of exogenous activation (Table 4). Compared to the negative control group, complex Ru3 did not increase the average number of revertants in the Salmonella typhimurium strains TA98, TA100, TA102, and TA97 at any of the tested concentrations. In contrast, the mean values increased significantly for all of the bacterial strains in the positive control groups. We concluded that complex Ru3 did not promote gene mutations in the tests with or without metabolic activation for any of the tested bacterial strains. For all of the tested concentrations, the mutagenicity index (MI) was less S atom on the dmp ligand was mapped as a hydrogen acceptor for hydrogen bonding from the NH2 group on the Arg488 amino acid residue. These interactions are able to stabilize the combination of complexes with hTopIB, inhibiting the binding of the enzyme to DNA. On the other hand, in the hTopIBDNA species (Figure 7), complex Ru3 binds to the active site via six hydrophobic interactions and one hydrogen bond. Among them, there are four π-alkyl/alkyl interactions between the phenyl rings of Ru3 and the amino acid residues from the hTopIB enzyme. Also, there are π-alkyl and alkyl hydrophobic interactions of the diphosphine ligands from Ru3 with the thymine and guanine moieties of the nucleobases, respectively (Table 3). The molecular docking results demonstrated that complex Ru3 is able to stabilize the binding sites of both free hTopIB and hTopIB-DNA, which justifies the ability of complex Ru3 to inhibit the enzymatic activity in the cleavage and religation steps, respectively. Biological Studies with Complex Ru3. Given that complex Ru3 is a potent hTopIB inhibitor and, shows excellent cytotoxic activity and selectivity for the cancer lung and breast cell lines, we investigated its ability to inhibit the size and number of cell colonies by clonogenic assay (Figure 8A,B).53 The long-term survival (Figure S48) of the A549 and MDA-MB-231 cancer cells, and the MRC-5 nontumor cell line was assessed at different concentrations of complex Ru3. For the three cell lines, the number and size of the cell colonies decreased upon treatment with complex Ru3 in a concentration-dependent manner. In the case of the MDA-MB-231 cancer cells treated with complex Ru3 (Figure 8A), cell survival reduced significantly at the Ru3 concentrations (0.02 and 0.08 μM) below its corresponding IC50. At a concentration of 0.20 μM, complex Ru3 completely inhibited formation of 14181 https://doi.org/10.1021/acs.inorgchem.1c01539 Inorg. Chem. 2021, 60, 14174−14189 Inorganic Chemistry pubs.acs.org/IC Table 4. Revertants/Plate, Standard Deviation, and MIa (in Brackets) in the S. typhimurium Strains TA1535 after Treatment with Complex Ru3, with (+S9) and without (−S9) Metabolic Activation compounds. We evaluated the chromosome damage induced by complex Ru3 by analyzing the frequencies of micronuclei (MNs), nucleoplasmatic bridges (NPBs), and nuclear buds (NBUDs) in binucleated HepG2 cells. The nuclear division index (NDI) was determined by the proportion of mono-, bi-, tri-, or multinucleated cells. The HepG2 cells treated with different concentrations of complex Ru3 did not differ in terms of the frequencies of MNs, NPBs, NBUDs, and NDI (Table 5) when compared to the negative control group. These results demonstrated that complex Ru3 did not induce chromosome damage in HepG2 cells under the employed experimental conditions. Doxorubicin, used as a positive control, significantly increased the frequencies of these parameters in the cells, which showed its genotoxicity. In Vivo Antitumor Assay B16-F10 Cells. The in vivo antitumor activity of complex Ru3 was evaluated in tumorbearing C57BL/6 mice engrafted with B16-F10 melanoma cells (Figure 9). The mice were randomly divided into three groups: treated with complex Ru3, with cisplatin (positive control), and without treatment (negative control). The doses were administered by intraperitoneal injections for five consecutive days. After treatment for 5 days, the mean tumor mass of the mice of the negative control group was 4.2 ± 0.7 g (Figure 9A). For the mice treated with complex Ru3 and cisplatin, the mean tumor masses were 0.9 ± 0.2 and 1.5 ± 0.2 g, respectively. The tumor mass growth inhibition rates were 79% and 64% for complex Ru3 and cisplatin, respectively, compared to the mice of the negative control group. The results demonstrated that complex Ru3 inhibited tumor development more efficiently than cisplatin. In addition, microscopic analysis revealed a significantly reduced frequency of mitoses in tumor tissue of the mice treated with complex Ru3 and cisplatin compared to the control group, indicating low proliferative activity of the melanoma cells (Figure 9B). Furthermore, some toxicological aspects were evaluated in all mouse groups (Figure 9C,D). The group of mice treated with cisplatin exhibited a significant loss of body weight3.8 ± 0.3 gcompared to healthy mice, while the group of mice treated with complex Ru3 exhibited no significant alterations in body weight compared to the vehicle and negative control groups. Also, the mean weight of tissues in all of the groups was analyzed (Figure 9D). Weight analysis of the liver, kidneys, spleen, heart, and lungs of the mice treated with complex Ru3 did not show any significant variations compared to the vehicle control group. For liver weight, the mice treated with cisplatin showed a weight loss of 47% compared to the vehicle control group. These results confirmed complex Ru3 as a novel anticancer drug candidate for melanoma cancer. This complex reduced B16-F10 cell growth in mice more efficiently than no. of revertants/plate (MI)a TA98 treatment (μg plate−1) negative controlb positive controlc Ru3 (0.78) Ru3 (1.56) Ru3 (3.12) Ru3 (6.25) Ru3 (9.37) TA100 +S9 −S9 +S9 −S9 20 ± 3 23 ± 2 111 ± 10 129 ± 15 878 ± 45 (43.9) 19 ± 2 (1.0) 18 ± 13 (0.9) 18 ± 3 (0.9) 20 ± 2 (1.0) 19 ± 1 (1.0) 1367 ± 50 (59.4) 20 ± 5 (0.9) 1109 ± 55 (9.99) 110 ± 7 (1.0) 99 ± 9 (0.9) 1374 ± 76 (10.6) 124 ± 10 (1.0) 128 ± 11 (1.0) 127 ± 9 (1.0) 127 ± 13 (1.0) 124 ± 10 (1.0) 25 ± 4 (1.1) 28 ± 2 (1.2) 100 ± 7 (0.9) 26 ± 6 (1.1) 104 ± 6 (0.9) 25 ± 3 (1.1) 102 ± 11 (0.9) no. of revertants/plate (MI)a TA102 TA97a treatment (μg plate−1) +S9 +S9 +S9 +S9 negative controlb positive controlc Ru3 (0.78) 320 ± 15 359 ± 15 100 ± 10 128 ± 15 1238 ± 41 (3.9) 317 ± 10 (1.0) 321 ± 12 (1.0) 328 ± 10 (1.0) 325 ± 15 (1.0) 320 ± 10 (1.0) 1347 ± 50 (26.9) 358 ± 12 (1.0) 356 ± 13 (1.0) 360 ± 12 (1.0) 359 ± 17 (1.0) 347 ± 20 (1.0) 1031 ± 67 (10.3) 98 ± 19 (1.0) 97 ± 17 (1.0) 100 ± 11 (1.0) 98 ± 14 (1.0) 96 ± 15 (1.0) 1471 ± 72 (11.5) 130 ± 10 (1.0) 132 ± 12 (1.0) 127 ± 15 (1.0) 131 ± 11 (1.0) 130 ± 10 (1.0) Ru3 (1.56) Ru3 (3.12) Ru3 (6.25) Ru3 (9.37) Article a MI was also calculated from the average number of revertants per plate with the test complex divided by the average number of revertants per plate with negative control. Values are the mean ± SD. b Negative control: DMSO (100 μL plate−1). cPositive control: sodium azide (1.25 μg plate−1) in the absence of S9 and c2anthramine in the presence of S9. than 2.0, which encourages further studies on their possible uses in anticancer therapy. Cytokinesis-Block Micronucleus Cytome (CBMN-cyt) Assay. Drug-metabolizing cells, such as cells derived from human liver, are used for the detection of genotoxic Table 5. Assessment of Mutagenic Effects of Complex Ru3 on HHO-K1 Cells by Means of CBMN-cyt Assay total in 1000 binucleated cells treatment MNs NPBs NBUDs IDN negative control positive control Ru3 (1.5 μM) Ru3 (3.0 μM) Ru3 (6.0 μM) 3.33 ± 1.56 59.33 ± 9.55d 2.00 ± 1.33 1.56 ± 0.44 1.79 ± 0.89 3.00 ± 0.67 21.33 ± 5.58d 5.2 ± 1.00 3.3 ± 0.67 1.97 ± 0.44 4.67 ± 2.89 11.33 ± 5.12d 6.63 ± 1.53 1.67 ± 0.89 1.00 ± 0.67 1.85 ± 0.05 1.78 ± 0.83 2.00 ± 0.18 1.78 ± 0.05 1.88 ± 0.11 a Values are the mean ± SD. Abbreviations: N, binucleated cell; MN, micronuclei; NPBs, nucleoplasmic bridges; NBUDs, nuclear buds; NDI, nuclear division index. The data are based on three independent experiments. bVehicle negative control: 1.0% DMSO. cPositive controls: 0.05 μM doxorubicin; 5 μM aflatoxin B1. dSignificantly different from the vehicle control (p < 0.05). 14182 https://doi.org/10.1021/acs.inorgchem.1c01539 Inorg. Chem. 2021, 60, 14174−14189 Inorganic Chemistry pubs.acs.org/IC Article Figure 9. In vivo antitumor activity of complex Ru3 and respective controls in C57BL/6 mice with melanoma cell xenografts, submitted to treatment for 5 days: (A) tumor weight (g); (B) number of mitoses of the tumor; (C) mean body weight variation; (D) organ weights. Tumor control: with tumor and treated with the vehicle DMSO (5%); cisplatin (5 mg kg−1 body weight). *Significantly different from the tumor control group (p < 0.05). cisplatin. As for the analyzed toxicological aspects, complex Ru3 was advantageous over cisplatin: in contrast to cisplatin, complex Ru3 did not change the body and tissue weights. characterized by mercapto ligands without functional groups or “electrophilic groups” attached to the rings, whereas complexes Ru3−Ru5 have mercapto ligands with “donor groups” in the mercapto ligand, such as amine and carbonyl moieties. The complexes bind to DNA through reversible electrostatic interactions, but none of them cause significant damage to the tertiary and secondary DNA structures. Thus, the results suggest that the “trigger” for cell death does not involve the “direct action” of complexes Ru1−Ru5 on DNA. By screening of the complexes with the hTopIB, it was demonstrated that they inhibit the hTopIB catalytic activity in a dose-dependent manner. Specifically, complex Ru3 at 6 μM completely inhibits hTopoIB. This complex acts not only by preventing the binding of hTopIB to DNA during the cleavage reaction, but also by stabilizing the intermediate state of the hTopIB-DNA complex. In accordance, molecular docking results demonstrated that complexes Ru1−Ru5 exhibit affinity by active sites for both free hTopIB and hTopIB-DNA, which can lead to inhibition of the enzymatic activity. The phenyl rings of the dppe phosphine ligands are responsible for π-alkyl/alkyl hydrophobic interactions and the mercapto ligands for hydrogen bonds. Additionally, the cytotoxic activity of this complex may be related to its interactions with hTopIB. Complex Ru3 significantly inhibits melanoma tumor growth in mice, without signs of systemic toxicity. Histopathological analysis revealed that this complex significantly reduces the number of mitoses when compared to the untreated group. In this sense, complex Ru3 acts by inhibiting cell division in melanoma tumor tissue implanted in mice. Furthermore, ■ CONCLUSIONS We have developed new ruthenium(II) complexes of the general formula [Ru(N-S)(dppe)2]PF6 obtained from of the precursor cis-[RuCl2(dppe)2] with five mercapto ligands. The precursor complex by itself already has excellent cytotoxic activity, especially against DU-145, A549, and MDA-MB-231 cancer cell lines, being more active than that cisplatin. Most of the complexes synthesized endowed hydrophobic species with high cytotoxic activity against five cancer cell lines. The IC50 values of the complexes are lower than the IC50 values of cisplatin against the A549 and MDA-MB-231 cancer cell lines. For the A549 cancer cell line, the IC50 value of the complexes is between 288- and 14-fold lower than the IC50 value of cisplatin. For the MDA-MB-231 cancer cell line, the IC50 value of the complexes is between 81 and 1 times lower compared to the commercial drug. Furthermore, the complexes are more selective for the A549 and MDA-MB-231 cancer cell lines than for nontumoral lung MRC-5 cell line. Complex Ru5 stands out for being 114 times more active against the A549 tumor cell line than against the MRC-5 nontumor cell line, whereas cisplatin has a MRC-5/A549 selectivity index of 2.08. In general, ruthenium(II) complexes containing two diphosphine ligands and mercapto ligand are highly hydrophobic complexes, which certainly facilitates their cellular uptake. Complexes Ru1 and Ru2 are more cytotoxic than complexes Ru3−Ru5. Structurally, complexes Ru1 and Ru2 are 14183 https://doi.org/10.1021/acs.inorgchem.1c01539 Inorg. Chem. 2021, 60, 14174−14189 Inorganic Chemistry pubs.acs.org/IC was filtered off, washed with water and ethyl ether, and dried under vacuum. [Ru(mtz)(dppe) 2 ]PF 6 (Ru1). Yield: 89%. Anal. Calcd for C57H58F6NP5RuS2 [exptl (calcd)]: C, 57.05 (56.89); H, 4.66 (4.51); N, 1.09 (1.21); S, 5.65 (5.52). Molar conductance (μS cm−1, CH2Cl2): 50.2. IR (cm−1): ν(C−H) 3065/3053, ν(CH2) 2949/ 2854, ν(CN) 1584, ν(CC + CN) 1514, ν(C−S) 1268, ν(P− Cring) 1093, ν(PF6−) 842, 739, δ(PF6) 559, ν(P−C) 522, ν(Ru−S) 426, ν(Ru−N) 406. UV−vis [CH2Cl2; λ/nm (ε/M−1 cm−1)]: 262 (69298; IL), 374 (2816; TCML). 31P{1H} NMR (161.98 MHz, CH2Cl2/D2O): δ 57.2 (1P, ddd, 2JPP = 304.5, 19.4, and 13.0 Hz), 47.8 (1P, ddd, 2JPP = 22.7, 19.4, and 9.7 Hz), 45.6 (1P, ddd, 2JPP = 304.5, 25.9, and 9.7 Hz), 39.5 (1P, ddd, 2JPP = 25.9, 22.7, and 13.0 Hz), −144.40 (1P, hept, 2JPF = 710.7 Hz, PF6−). [Ru(mmi)(dppe) 2]PF 6 (Ru2). Yield: 95%. Anal. Calcd for C59H59F6N4P5RuS [exptl (calcd)]: C, 58.41 (58.13); H, 4.89 (4.70); N, 2.54 (2.42); S, 2.77 (2.53). Molar conductance (μS cm−1, CH2Cl2): 51.4. IR (cm−1): ν(C−H) 3140/3055, ν(CH2) 2948/ 2848, ν(CN) 1587, ν(CC + CN) 1524, ν(C−S) 1283, ν(P− Cring) 1092, ν(PF6−) 844, δ(PF6−) 556, ν(P−C) 524, ν(Ru−S) 425, ν(Ru−N) 402. UV−vis [CH2Cl2; λ/nm (ε/M−1 cm−1)]: 262 (53372; IL), 354 (1244; TCML). 31P{1H} NMR (162 MHz, CH2Cl2/D2O): δ 59.2 (1P, ddd, 2JPP = 299.6, 19.4, and 16.2 Hz), 55.9 (1P, ddd, 2JPP = 24.3, 19.4, and 14.6 Hz), 53.4 (1P, ddd, 2JPP = 299.7, 24.3, and 14.6 Hz), 52.8 (1P, ddd, 2JPP = 24.3, 19.4, and 13.0 Hz), −144.40 (1P, hept, 2JPF = 710.7 Hz, PF6−). [Ru(dmp)(dppe) 2 ]PF 6 (Ru3). Yield: 94%. Anal. Calcd for C58H59F6N4P5RuS [exptl (calcd)]: C, 56.50 (56.81); H, 4.49 (4.51); N, 4.62 (4.73); S, 2.53 (2.71). Molar conductance (μS cm−1, CH2Cl2): 52.54. IR (cm−1): ν(NH2)3494/3394, ν(C−H) 3183/3051, ν(CH2) 2921/2852, ν(CN) 1585, ν(CC + CN) 1538, ν(C−S) 1248, ν(P−Cring) 1160, ν(PF6−) 841, δ(PF6−) 559, ν(P−C) 518, ν(Ru−S) 406, ν(Ru−N) 375. UV−vis [CH2Cl2; λ/nm (ε/M−1 cm−1)]: 268 (94476, IL), 324 (12130; TCML). 31P{1H} NMR (162 MHz, CH2Cl2/D2O): δ 58.9 (1P, ddd, 2JPP = 285.0, 24.3, and 13.0 Hz), 56.5 (1P, ddd, 2JPP = 24.3, 24.3, and 16.2 Hz), 52.6 (1P, ddd, 2JPP = 285.0, 19.4, and 16.2 Hz), 52.0 (1P, ddd, 2JPP = 24.3, 19.4, and 13.0 Hz), −144.40 (1P, hept, 2JPF = 710.7 Hz, PF6−). [Ru(mpca)(dppe)2]PF6 (Ru4). Yield: 92%. Anal. Calcd for C60H58F6NO2P5RuS [exptl (calcd)]: C, 58.38 (58.20); H, 4.41 (4.38); N, 1.26 (1.17); S, 2.82 (2.68). Molar conductance (μS cm−1, CH2Cl2): 52.8. IR (cm−1): ν(C−H) 3153/3051, ν(CH2) 2921/2850, νas(COOH) 1710, ν(CN) 1587, ν(CC + CN): 1534, νs(COOH) 1358, ν(C−S) 1254, ν(P−Cring) 1158, ν(PF6−) 843, δ(PF6−) 557, ν(P−C) 522, ν(Ru−S) 411, ν(Ru−N) 377. UV−vis [CH2Cl2; λ/nm (ε/M−1 cm−1)]: 264 (86566; IL), 382 (11660; TCML). 31P{1H} NMR (162 MHz, CH2Cl2/D2O): δ 62.2 (1P, ddd, 2 JPP = 278.6, 19.4, and 14.6 Hz), 51.0 (1P, ddd, 2JPP = 19.4, 19.4, and 8.1 Hz), 50.1 (1P, ddd, 2JPP = 278.6, 22.7, and 8.1 Hz), 47.7 (1P, ddd, 2 JPP = 22.7, 19.4, and 14.6 Hz), −144.40 (1P, hept, 2JPF = 710.7 Hz, PF6−). [Ru(2mq)(dppe) 2 ]PF 6 (5). Yield: 82%. Anal. Calcd for C62H59F6N2OP5RuS [exptl (calcd)]: C, 59.08 (59.07); H, 4.49 (4.38); N, 2.33 (2.30); S, 2.83 (2.63). Molar conductance (μS cm−1, CH2Cl2): 55.2. IR (cm−1): ν(C−H) 3144/3051, ν(CH2) 2925/2852, ν(CO) 1653, ν(CN) 1586, ν(CC + CN) 1530, ν(C−S) 1243, ν(P−Cring) 1097, ν(PF6) 841, δ(PF6) 555, ν(P−C) 525, ν(Ru− S) 425, 409, ν(Ru−N) 377. UV−vis [CH2Cl2; λ/nm (ε/M−1 cm−1)]: 272 (51927; IL), 318 (12485; TCML), 344 (11832; TCML). 31 P{1H} NMR (162 MHz, CH2Cl2/D2O): δ 57.2−56.5 (2P, multiplet), 53.7−53.0 (1P, multiplet), 52.8−52.2 (1P, multiplet), −144.40 (1P, hept, 2JPF = 710.7 Hz, PF6−). Water/n-Octanol Distribution Coefficient (log P). Water− octanol partition coefficients were determined using the shake-flask method.58 A total of 1 mg of each complex was solubilized in 100 μL of DMSO and posteriorly diluted in a mixture of equal volumes of water (750 μL) and n-octanol (750 μL). The solutions were continuous shaken for 24 h at 1000 rpm and 37 °C. Then the samples were centrifuged for 5 min at 300 rpm, and the organic and aqueous phases were separated. The concentration of drug in each phase was complex Ru3 does not promote gene mutations, as evaluated by the Ames test, and does not induce chromosome damage in drug-metabolizing cells. Thus, we hope that the results described herein constitute compelling evidence that the ruthenium(II) diphosphine complexes have therapeutic potential as candidate drugs for cancer treatment, encouraging the advance of in vivo biological assays with this class of complexes. ■ Article EXPERIMENTAL SECTION Instrumentation. The IR spectra were obtained using KBr pellets in a Bomem-Michelson 102 Fourier transform infrared spectrometer in the 4000−200 cm−1 region. Cyclic voltammetry experiments were performed in a BAS model 100B electrochemical analyzer and carried out at room temperature. The typical conditions were 0.10 mol L−1 Bu4NClO4 (TBAP) as a supporting electrolyte in CH2Cl2 and an electrochemical cell with a three-electrode system, where a glassy carbon was used as the working electrode, Ag/AgCl as the reference electrode, and a platinum plate as the auxiliary electrode. Elemental analyses were performed in the Microanalytical Laboratory at the Universidade Federal de São Carlos, São Carlos, Brazil, with an EA 1108 CHNS microanalyzer (Fisons Instruments). Conductivity values were obtained, at room temperature, using 1.0 × 10−3 M solutions of the complexes in CH2Cl2 in a Meter Lab CDM2300 instrument. 31 1 P{ H}, 1H, COSY (1H−1H), 13C{1H}, and HSQC (1H−13C) NMR were recorded on a Bruker DRX 400 MHz using chemical shifts, which are reported in relation to H3PO4, 85% CH2Cl2/D2O, or DMSO-d6. The UV−vis spectra of the complexes were recorded in CH2Cl2 on a Hewlett-Packard 8452A diode array. X-ray Crystallography. All single crystals for complexes Ru1, Ru2, Ru4, and Ru5 were obtained from solvent evaporation [a 1:1 (v/v) dichloromethane and methanol mixture]. X-ray diffraction was carried out at room temperature using a Bruker AXS-Mach3 diffractometer and an APEX II CCD area detector with Mo Kα radiation (λ = 0.71073 Å). All crystal structures were resolved by direct methods, and the models were refined by full-matrix least squares on F2 using SHELX.55 Anisotropic displacement parameters were applied for Ru, P, S, C, N, O, and F atoms. H atoms were located at their positions and refined with the appropriate riding model, considering the C−H distance fixed as 0.93 and 0.97 Å for C− H aromatic and methylene bonds, respectively, with Uiso(H) = 1.2Ueq(Caromatic/Cmethylene). Summaries of the crystal data collection procedures and refinement results for complexes are given in Tables S1 and S2. The representation of the structure was drawn with the Mercury 4.0 program.56 Materials for Synthesis. Reactions and chemicals were handled under an argon atmosphere. Solvents were purified by standard methods. All chemicals used were of reagent grade or comparable purity. RuCl3·3H2O, 1,2-bis- (diphenylphosphino)ethane (dppe) ligand; 1,3-thiazolidine-2-thione (mtz); mercapto-1-methylimidazole (mmi); 4,6-diamino-2-mercapto-pyrimidine (dmp); 6-mercaptopyridine-3-carboxylic acid (mpca) and 2-mercapto-4(3H)-quinazoline (2mq) ligands were used as received from Sigma-Aldrich. The cis[RuCl2(dppe)2] compound, used as a precursor for the synthesis of complexes Ru1−Ru5, was prepared according to published procedures.57 Synthesis of Complexes Ru1−Ru5. The [Ru(N-S)(dppe)2]PF6 complexes were obtained from the cis-[RuCl2(dppe)2] precursor. In a Schlenk flask with a mixture of 15 mL of methanol and 15 mL of CH2Cl2 previously degassed, 0.12 mmol of the respective mercapto ligand was added (mtz = 0.014 g; mmi = 0.014 g; dmp = 0.023 g; mpca = 0.019 g, and 2mq = 0.021 g) along with 32 μL (24 mmol) of triethylamine. Posteriorly, 0.10 mmol (0.097 g) of the precursor cis[RuCl2(dppe)2] and 0.13 mmol (0.024 g) of KPF6 were added to the flask. The system was kept under stirring and reflux for approximately 12 h. The volume of the solution was reduced to approximately 2 mL, and water was added to precipitate a yellow powder. The precipitate 14184 https://doi.org/10.1021/acs.inorgchem.1c01539 Inorg. Chem. 2021, 60, 14174−14189 Inorganic Chemistry pubs.acs.org/IC measured spectrophotometrically in order to determine the log P values = [complex(in n-octanol)]/[complex(in water)]. The experiments were carried out in triplicate. In Vitro Assay. Cell Culture. The ruthenium complexes were assayed against human breast cancer cells MCF-7 (ATCC No. HTB22) and MDA-MB-231 (ATCC No. HTB-26), the human lung tumor line A549 (ATCC No. CCL-185), the human prostate DU-145 (ATCC No. HTB-81), the human hepatocellular carcinoma HepG2, and the normal cell line MRC-5. The cells were routinely maintained with Dulbecco’s modified Eagle’s medium (DMEM; for HepG2, A549, and MRC-5) or RPMI 1640 (for MCF-7 and DU145), supplemented with 10% fetal bovine serum (FBS), at 37 °C in a humidified 5% CO2 atmosphere. Cytotoxicity Assay. The cytotoxic activity of the complexes on cell lines was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.59 Cells (1.5 × 104 well−1) were seeded in 200 μL of a complete medium in 96-well plates (Corning Costar). Each complex was dissolved in sterile DMSO (from 40 to 0.01 mM). A total of 1 μL of each complex sample was added to 200 μL of the medium. Cells were exposed to the complex for a 48 h period. Posteriorly for 48 h, 50 μL of MTT (1 mg mL−1) was added to each well. Cells were incubated again for 4 h, the medium was removed, and formazan crystals were solubilized in isopropyl alcohol. The conversion of MTT to formazan by metabolically viable cells was spectrophotometrically monitored by an automated microplate reader at 540 nm. The percent cell viability was calculated by dividing the average absorbance of cells treated with a ruthenium complex (Ru1− Ru5) by that of the control; The percent cell viability versus drug concentration (logarithmic scale) was plotted to determine IC50 (drug concentration at which 50% of the cells are viable relative to the control), with its estimated error derived from an average of three trials. Clonogenic Survival Assay. For clonogenic survival assay, the breast cancer strain MDA-MB-231, lung cancer A549, and normal lung cancer strain MRC-5 were used. A total of 300 cells were cultured per well in a six-well plate. After 24 h, with the cells already adhered, they were treated with different concentrations of the selected complex. The concentrations (negative control; 1/2 × IC50, IC50, and 2 × IC50) used were determined from the IC50 value of complex Ru3 in the cancer cell lines. The plates were stored in an oven (37 °C/5% CO2) for 48 h. Then the culture medium (4 mL) was changed and incubated for another 10 days in an oven. After this period, the culture medium was discarded, and the plates were washed with phosphate saline buffer (PBS). The colonies formed were fixed with a methanol/acetic acid solution (3:1) for 5 min and stained with violet crystal 0.5% water for 25 min. After this period, the plates were washed with water and dried at room temperature. Then the number of colonies formed was counted. The test was performed in triplicate. Complex/DNA Binding Experiments. Interaction Study of Ruthenium Complexes and Calf-Thymus DNA (CT-DNA) by Viscosity. Viscosity measurements were carried out using an Ostwald viscometer immersed in a water bath maintained at 25 °C. The DNA concentration in buffer Tris-HCl was kept constant in all samples (150 μM), while the complex concentration was increased from 0 to 60 μM. In the assay, the percent DMSO was also kept constant. The flow time was measured at least five times with a digital stopwatch, and the mean value was calculated. Data are presented as (η/η0)1/3 versus the [complex]/[DNA] ratio, where η and η0 are the specific viscosities of DNA in the presence and absence of the complex, respectively. The values of η and η0 were calculated using the expression (t − tDNA)/tDNA, where t is the observed flow time and tDNA is the flow time of the DNA in buffer (DMSO).60 Interaction Study of Ruthenium Complexes and CT-DNA by Square-Wave Voltammetry. The complex/CT-DNA interaction studies were performed by square-wave voltammetry. The complex/ CT-DNA interaction studies were carried out in a Tris-HCl buffer (pH 7.4) and 30% DMSO. Titration was performed by adding 30 μL aliquots of CT-DNA (0−120 μL; stock solution 1.00 mM) to an electrochemical cell, containing 2 mL of a 1.00 mM complex solution. The final concentrations of CT-DNA were 0, 15, 29, 43, and 57 μM. Article In this condition, any denaturation of the DNA was observed, and no change in the UV−vis spectra of the solution was detected for at least 1 h. Interaction Study of Ruthenium Complexes and CT-DNA by Agarose Gel Electrophoresis. Agarose gel electrophoresis studies were performed by incubating 1 μL (0.25 μg μL−1) of pBlue-Script KSII(+) plasmid DNA with 1 μL of stock solution (in DMSO) of different concentrations of the complexes and 28 μL of a Tris-HCl buffer, resulting in 30 μL of final volume. The concentrations of the complexes in the final solutions were after incubation at 37 °C for 20 h, and 20 μL of each sample was run in a 1% agarose gel at 30 V for 18 h using a Tris-borate−ethylenediaminetetraacetic acid (EDTA) buffer and stained with ethidium bromide (5 μL of ethidium bromide per 50 mL of agarose gel mixture). Samples of free DNA and DNA + DMSO (3.3%) were used as controls. The DNA bands were visualized in a UV-light transilluminator (ChemiDocMP, Bio-Rad). HSA Binding Experiments. Fluorescence spectroscopy is an effective method for exploring the interactions between small molecules and macromolecules. The fluorescence of HSA comes from its tryptophan, tyrosine, and phenylalanine residues, where the latter two contribute to its fluorescence to only a minor extent.61 The protein interaction was examined in 96-well plates used for fluorescence assays. HSA (5.0 μM) was prepared by dissolving the protein in Tris-HCl at pH 7.4, and the complexes were dissolved in sterile DMSO. For the fluorescence measurements, the HSA concentration (950 μL) in the buffer Tris-HCl was kept constant in all samples, while the complex concentration (50 μL) was increased by 2.5, 5.0, 7.5, 10.0, 12.5, 15.0, 17.5, and 20.0 μM, and quenching of the emission intensity of the HSA tryptophan residues at 305 nm (excitation wavelength of 270 nm) was monitored at different temperatures (298 and 310 K). The experiments were carried out in triplicate and analyzed using the classical Stern−Volmer equation as follows:62 F0/F = 1 + Kqτ0[Q] = 1 + KSV[Q] (1) where F0 and F are the fluorescence intensities in the absence and presence of quencher, respectively, [Q] is the quencher concentration, and KSV is the Stern−Volmer quenching constant, which can be written as Kq = KSV/τ0, where Kq is the bimolecular quenching rate constant and τ0 is the average lifetime of the fluorophore in the absence of quencher (6.2 × 10−9 s). Therefore, eq 1 was applied to determine KSV by linear regression of a plot of F0/F versus [Q]. The binding constant (Kb) and number of complexes bound to HSA (n) were determined by plotting the double-logarithmic graph of the fluorescence data using eq 2 as follows:63 log[(F0 − F )/F ] = log Kb + n log [Q] (2) The thermodynamic parameters were calculated from eqs 3 and 4: ln(K 2/K1) = [(1/T1) − (1/T2)]ΔH /R (3) ΔG = − RT ln K = ΔH − T ΔS (4) where K1 and K2 are the binding constants at temperatures T1 and T2, respectively, and R is the gas constant. Topoisomerase IB Assays. Purification of hTopIB. hTopIB was expressed by the galactose inducible promoter in a multicopy plasmid, YCpGAL1-e-wild and YCpGAL1-e-Y723F, used for the transformation of EKY3 cells, as described previously.64 The epitopetagged constructs contain the N-terminal sequence FLAG: DYKDDDY (indicated with “e”), recognized by the M2 monoclonal antibody. Purification was carried out using an ANTI-FLAG M2 affinity gel column (Sigma-Aldrich). The FLAG-fusion topoisomerase IB was eluted by competition with five column volumes of a solution containing a 100 μg mL−1 FLAG peptide in 50 mM Tris-HCl and 150 mM KCl (pH 7.4). Glycerol was added to each fraction up to a final concentration of 40%. All of the fractions were stored at −20 °C. The integrity of the protein was verified by immunoblot assay. The purified protein was resolved on sodium dodecyl/sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane, and immunoblotted with a specific monoclonal antibody 14185 https://doi.org/10.1021/acs.inorgchem.1c01539 Inorg. Chem. 2021, 60, 14174−14189 Inorganic Chemistry pubs.acs.org/IC Article preliminary toxicity test. In all subsequent assays, the upper limit of the dose range tested was either the highest nontoxic dose or the lowest toxic dose determined in this preliminary assay. All experiments were analyzed in triplicate. The results were analyzed using the statistical software package Salanal 1.0 (Monitoring Systems Laboratory, U.S. Environmental Protection Agency, Las Vegas, NV, from the Research Triangle Institute, Research Triangle Park, NC), adopting the Bernstein et al. model.69 The data (revertants/plate) were assessed by analysis of variance (ANOVA), followed by linear regression. MI was also calculated for each concentration tested, which was the average number of revertants per plate with the test complex divided by the average number of revertants per plate with the negative control. A test solution was considered mutagenic when a dose−response relationship was detected and a 2-fold increase in the number of mutants (MI > 2) was observed for at least one concentration.68 The standard mutagens used as positive controls in experiments without a S9 mix were 4-nitro-o-phenylenediamine (NOPD; 10 mg plate−1) for TA98 and TA97a, sodium azide (1.25 μg plate−1) for TA100, and mitomycin (0.5 μg plate−1) for TA102. In experiments with S9 activation, 2-anthramine (1.25 μg plate−1) was used for TA98, TA97a, and TA100, and 2-aminofluorene (10 μg plate−1) for TA102. DMSO (100 μL plate−1) served as the (solvent) negative control. CBMN-cyt Assay. The mutagenicity was evaluated as described by Fenech.70 Three different concentrations (IC50 and two lower concentrations) were used for CBMN-cyt analysis. A total of 5 × 105 HepG2 cultures, as previously described, were incubated in 25 cm2 culture flasks for 24 h and then treated with three different concentrations of the ruthenium complexes or 0.05 μM doxorubicin. After 20 h of treatment (44 h after initiation of the culture), the cells were washed with PBS, the culture media were changed, and cytochalasin B (final concentration of 3.0 μg mL−1) was added. The cells were then incubated for an additional 28 h, harvested, treated with a cold hypotonic solution (0.01% sodium citrate), and fixed with formaldehyde and methanol/acetic acid (3:1). The slides were stained immediately before analysis using 40 μg mL−1 acridine orange, and the binucleated cells with 1−4 MN were scored at 1000× magnification. Additionally, the frequencies of NPBs and NBUDs were evaluated using the criteria of Fenech et al.41 The NDI was also calculated to evaluate the altered mitotic activity and/or cytostatic effects according to eq 5:71 (Sigma A9469). An immunoreactive band, corresponding to topoisomerase I, was detected with a 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate (Sigma B3804). Topoisomerase IB Activity In Vitro: DNA Relaxation Assay. The TopIB activity was assayed by preparing the samples with 1 μL (0.25 μg μL−1) of pBlue-Script KSII(+) plasmid DNA, 1 μL of stock solution (in DMSO) of different concentrations of the complexes and 28 μL of reaction buffer (20 mM Tris-HCl, 0.1 mM EDTA, 10 mM MgCl2, 50 μg mL−1 acetylated BSA, and 150 mM KCl, pH 7.4), resulting in 30 μL of final volume. The concentrations of the complexes in the final solutions were 0.75, 1.5, 3.0, 6.0, 12.5, 25.0, 50.0, 100.0, 200.0, and 300.0 μmol L−1. The reaction was incubated for 30 min at 37 °C and interrupted by the addition of 7.5 μL of SDS 4X STOP (25% Ficol 400, 2.5% SDS, 25 mM EDTA, 0.03% bromophenol blue, and 0.03% xylenocyanol). Samples were electrophoresed in 1% agarose gel in 50 mM Tris, 45 mM boric acid, and 1 mM EDTA. The gel was stained with ethidium bromide (5 μg mL−1), destained with water, and photographed under UV illumination. Wherever indicated, the enzyme and inhibitor were preincubated at 37 °C for 5 min before the DNA substrate was added. Assays were performed at least three times, but only one representative gel is shown. Cleavage Kinetics. The oligonucleotide CL14 (5′-GAAAAAAGACTTAG-3′), radio-labeled with [γ-32P] adenosine triphosphate (ATP) at its 5′ end, and the CP25 complementary strand (5′TAAAAATTTTTCTAAGTCTTTTTTC-3′), phosphorylated at its 5′ end, with unlabeled ATP, were annealed at a 2-fold molar excess of CP25 over CL14, creating the so-called “suicide substrate”, which contains only a partial duplex. The suicide cleavage reactions were carried out by incubating 20 nM suicide substrate with the enzyme in a reaction buffer at 37 °C and in the presence of 25 μM complex. DMSO (10%) was added to the no-drug control. Before the enzyme was added, a 5 μL sample of the reaction mixture was removed and used as the control. At different time points, 5 μL aliquots were removed and the reactions stopped with 0.5% SDS. After the ethanol precipitation, samples were resuspended in 6 μL of 1 μg mL−1 trypsin and incubated at 37 °C for 1 h. Samples were analyzed using denaturing urea/poly acrylamide gel electrophoresis. Religation Kinetics. A suicide CL14/CP25 substrate (20 nM), prepared as above, was incubated with the topoisomerase IB enzyme for 30 min at 37 °C in a reaction buffer. A 5 μL aliquot of the reaction mixture was removed and used as the zero time point. Religation reactions were initiated by adding a 200-fold molar excess of R11 oligonucleotide (5′-AGAAAAATTTT-3′) over the duplex CL14/ CP25 in the presence or absence of 25 μM complex Ru3. Moreover, 5 μL aliquots were removed, and the reactions were stopped with 0.5% SDS at different times. Afterward, ethanol precipitation samples were resuspended in 5 μL of 1 μg mL−1 trypsin and incubated at 37 °C for 1 h. Samples were analyzed by denaturing urea/polyacrylamide gel electrophoresis. The experiment was replicated three times, and a representative gel is shown. Molecular Docking Procedure. The docking simulations were implemented using the tools GOLD v 5.8.0 program.65 The structure of the human DNA topoisomerase I in complex with different ruthenium complexes was downloaded from the Protein Data Bank (PDB 1T8I),52 removing the ligant molecule before starting the docking calculation and making a note coordinates of binding site. In the preparation of protein for use in optimization calculations, the protein-preparation wizard in Maestro66 was used in this study. The validation study was carried out using the Chemscore and Goldscore functions for pose selection. The prepared protein and results were visualized and analyzed using Biovia Discovery Studio Visualizer.67 Mutagenicity Assays. Ames Test. The mutagenic activity was evaluated by the salmonella/microsome assay, using the S. typhimurium tester strains TA98, TA100, TA97a, and TA102, kindly provided by Dr. B. N. Ames (Berkeley, CA), with (+S9) and without (−S9) metabolization, by the preincubation method.68 To determine the mutagenic activity, five different concentrations of complex Ru3 (0.78−75 μg plate−1), diluted in DMSO, were assayed. The concentrations of the complex were selected on the basis of a NDI = (M1 + 2M2 + 3M3 + 4M4)/N (5) where M1, M2, M3, and M4 are the number of cells with one, two, three, and four nuclei and N is the number of cells assayed. A total of 500 cells per treatment were analyzed or NDI calculated and 1000 binucleated cells for the MN, NPB, and NBUD frequencies. A total of three independent experiments were performed. In Vivo Assay. Antitumor Assay Using Syngeneic Murine Melanoma Tumor Model. In vivo studies were carried out using treatment protocols approved by the Ethics Committee on Animal Use of the University of Franca (No. 2639070412). Male C57BL/6 mice, weighing 25−30 g, were obtained from the animal house of the University of São Paulo, Ribeirão Preto Campus, São Paulo, Brazil. The B16-F10 cell line (murine melanoma) was maintained in a RPMI culture medium (Sigma-Aldrich), supplemented with 10% FBS (Nutricell), 1.2 g mL−1 sodium bicarbonate (Sigma-Aldrich), 0.1 g mL−1 streptomycin (Sigma-Aldrich), and 0.06 g mL−1 penicillin (Sigma-Aldrich) in a 5% CO2/air atmosphere at 37 °C. The B16-F10 cells were counted, and their viability was assessed in a Muse Cell Analyzer (Merck). The procedures for obtaining the tumors, as well as the experimental design, were conducted according to Carnizello et al.72 The groups of animals with tumor induction were divided as follows: tumor control (5% DMSO; Sigma-Aldrich), compound Ru3 (5 mg kg−1 body weight), and cisplatin (5 mg/ kg−1 body weight). A vehicle control group (without tumor and treated with 5% DMSO) was also included. The tumor growth inhibition rate (TGIR) was calculated by eq 7, as described Qin et al.:73 14186 https://doi.org/10.1021/acs.inorgchem.1c01539 Inorg. Chem. 2021, 60, 14174−14189 Inorganic Chemistry pubs.acs.org/IC Silvia Castelli − Dipartimento di Biologia, Università Tor Vergata di Roma, 00133 Rome, Italy Alessandro Desideri − Dipartimento di Biologia, Università Tor Vergata di Roma, 00133 Rome, Italy Rodrigo S. Corrêa − Departamento de Química, Universidade Federal de Ouro Preto, CEP 35400-000 Ouro Preto, Minas Gerais, Brazil Arthur B. Ribeiro − Universidade de Franca, CEP 14404-600 Franca, São Paulo, Brazil; orcid.org/0000-0002-40569571 Heloiza D. Nicolella − Universidade de Franca, CEP 14404600 Franca, São Paulo, Brazil Saulo D. Ozelin − Universidade de Franca, CEP 14404-600 Franca, São Paulo, Brazil; orcid.org/0000-0003-17694347 Denise C. Tavares − Universidade de Franca, CEP 14404600 Franca, São Paulo, Brazil; orcid.org/0000-00034646-5914 Complete contact information is available at: https://pubs.acs.org/10.1021/acs.inorgchem.1c01539 TGIR = tumor weight of tumor control − tumor weight of treatment tumor weight of tumor control × 100 (7) Mice were euthanized on day 6, 24 h after the last treatment. The liver, kidneys, spleen, heart, and lungs were collected and weighed to evaluate the toxicological potential of the treatments. For evaluation of the antitumor activity, the tumors were collected, weighed, and stored for subsequent histopathological analysis. For this, the excised tumors were fixed in 10% neutral formalin and embedded in paraffin blocks. Five segments (5 μm thick) were cut from random parts of each tumor tissue, and the sections were dewaxed and stained with hematoxylin and eosin. Five random fields were analyzed under a microscope regarding the number of mitoses in 400× magnification, for a total of 25 areas per group. ■ ASSOCIATED CONTENT sı Supporting Information * The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.inorgchem.1c01539. Figures and tables providing IR, UV−vis, and NMR spectra, cyclic voltammetry, and X-ray crystallographic data of the complexes (PDF) Notes The authors declare no competing financial interest. ■ ACKNOWLEDGMENTS The authors are thankful for financial support from the following Brazilian Research Agencies: FAPESP (Processo 2018/19342-2), FAPEMIG, CNPq (Processo 422367/20184), and CAPES (this study was partially funded by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brasil (CAPES); Finance Code 001). R.S.C. is thankful for financial support by FAPEMIG (APQ-01674-18) and CNPq (Grants 403588/2016-2 and 308370/2017-1). Accession Codes CCDC 2079011−2079014 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033. ■ Article ■ AUTHOR INFORMATION Corresponding Authors Gabriel H. Ribeiro − Departamento de Química, Universidade Federal de São Carlos, CEP 13565-905 São Carlos, São Paulo, Brazil; orcid.org/0000-0003-07381638; Email: gabrielhenri10@hotmail.com Alzir A. Batista − Departamento de Química, Universidade Federal de São Carlos, CEP 13565-905 São Carlos, São Paulo, Brazil; Phone: +55 1633518285; Email: daab@ ufscar.br; Fax: +55 1633518350 REFERENCES (1) Allardyce, C. S.; Dyson, P. J. Metal-Based Drugs That Break the Rules. Dalt. Trans. 2016, 45 (8), 3201−3209. (2) Mari, C.; Pierroz, V.; Ferrari, S.; Gasser, G. Combination of Ru(II) Complexes and Light: New Frontiers in Cancer Therapy. Chem. Sci. 2015, 6, 2660−2686. 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Ferreira − Departamento de Química, Universidade Federal de São Carlos, CEP 13565-905 São Carlos, São Paulo, Brazil Leandro Ribeiro − Departamento de Química, Universidade Federal de São Carlos, CEP 13565-905 São Carlos, São Paulo, Brazil Marília I. F. Barbosa − Departamento de Química, Universidade Federal de São Carlos, CEP 13565-905 São Carlos, São Paulo, Brazil Victor M. Deflon − Instituto de Química de São Carlos, Universidade de São Paulo, CEP 13565-905 São Carlos, São Paulo, Brazil; orcid.org/0000-0002-5368-6486 14187 https://doi.org/10.1021/acs.inorgchem.1c01539 Inorg. Chem. 2021, 60, 14174−14189 Inorganic Chemistry pubs.acs.org/IC Chelating Proline and Threonine Ligands Cause Selective Cytotoxicity by the Induction of Genomic Instability, Cell Cycle Arrest and Apoptosis in Breast and Prostate Tumor Cells. Toxicol. In Vitro 2020, 62, 104679. (10) Ribeiro, G. H.; Guedes, A. P. M.; de Oliveira, T. D.; de Correia, C. R. S. T. B.; Colina-Vegas, L.; Lima, M. A.; Nóbrega, J. 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