"Half-Sandwich" Ruthenium Complexes with Alizarin as Anticancer Agents: <i>In Vitro</i> and <i>In Vivo</i> Studies.

{"full_text": " pubs.acs.org/IC Article\n\n\n\n \u201cHalf-Sandwich\u201d Ruthenium Complexes with Alizarin as Anticancer\n Agents: In Vitro and In Vivo Studies\n Joa\u0303o Honorato de Araujo-Neto,* Adriana P. M. Guedes, Celisnolia M. Leite, Carlos Andr\u00e9 F. Moraes,\n Andressa L. Santos, Rafaella da S. Brito, Thiago L. Rocha, Francyelli Mello-Andrade, Javier Ellena,\n and Alzir A. Batista*\n Cite This: Inorg. Chem. 2023, 62, 6955\u22126969 Read Online\nSee https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.\n\n\n\n\n ACCESS Metrics & More Article Recommendations *\n s\u0131 Supporting Information\n\n\n ABSTRACT: Upon exploration of the chemistry of the combination of ruthenium/arene\n Downloaded via MOSCOW STATE UNIV on May 12, 2026 at 11:19:34 (UTC).\n\n\n\n\n with anthraquinone alizarin (L), three new complexes with the general formulas\n [Ru(L)Cl(\u03b76-p-cymene)] (C1), [Ru(L)(\u03b76-p-cymene)(PPh3)]PF6 (C2), and [Ru(L)(\u03b76-\n p-cymene)(PEt3)]PF6 (C3) were synthesized and characterized using spectroscopic\n techniques (mass, IR, and 1D and 2D NMR), molar conductivity, elemental analysis,\n and X-ray diffraction. Complex C1 exhibited fluorescence, such as free alizarin, while in C2\n and C3, the emission was probably quenched by monophosphines and the crystallographic\n data showed that hydrophobic interactions are predominant in intermolecular contacts.\n The cytotoxicity of the complexes was evaluated in the MDA-MB-231 (triple-negative\n breast cancer), MCF-7 (breast cancer), and A549 (lung) tumor cell lines and MCF-10A\n (breast) and MRC-5 (lung) nontumor cell lines. Complexes C1 and C2 were more\n selective to the breast tumor cell lines, and C2 was the most cytotoxic (IC50 = 6.5 \u03bcM for\n MDA-MB-231). In addition, compound C1 performs a covalent interaction with DNA,\n while C2 and C3 present only weak interactions; however, internalization studies by flow cytometry and confocal microscopy\n showed that complex C1 does not accumulate in viable MDA-MB-231 cells and is detected in the cytoplasm only after cell\n permeabilization. Investigations of the mechanism of action of the complexes indicate that C2 promotes cell cycle arrest in the Sub-\n G1 phase in MDA-MB-231, inhibits its colony formation, and has a possible antimetastatic action, impeding cell migration in the\n wound-healing experiment (13% of wound healing in 24 h). The in vivo toxicological experiments with zebrafish indicate that C1\n and C3 exhibit the most zebrafish embryo developmental toxicity (inhibition of spontaneous movements and heartbeats), while C2,\n the most promising anticancer drug in the in vitro preclinical tests, revealed the lowest toxicity in in vivo preclinical screening.\n\n\n \u25a0 INTRODUCTION\n The search for new and necessary alternatives for the treatment\n well as in vivo, leading to, for instance, a 50% reduction of the\n number of lung metastases in mice.4\u22126 Furthermore, RAPTA-\n of different types of diseases is one of the researchers\u2019 main C can perform the blockage of T-cell-associated proteins,\n tasks from different areas of knowledge. Among these diseases, enabling experimentation of this compound as an immuno-\n therapeutic drug.7,8\n cancer is one of the most prominent because it caused about\n The potency demonstrated by the RAPTA complexes has\n 10 million deaths in 2020.1 In this context, the investigation of\n led to the exploration of several other structural combinations,\n new, more efficient, and cheaper alternatives for cancer\n aiming to use (in this system) different ligands that would\n treatment, such as new drugs for chemotherapy, or inclusive\n trigger other modes of interaction with different biomolecular\n new forms of treatment has provided hope for an attempt to\n targets. Among these structures, we can highlight complexes\n mitigate the effects of the pandemic or other causes and reduce\n containing bidentate ligands that coordinate to the metallic\n the number of deaths caused by cancer. Thus, one prominent\n center through O,O-coordinating ligands.9 These combina-\n and promising drug candidate class is the \u201chalf-sandwich\u201d\n tions resulted in the obtainment of metal chelates with four-,\n ruthenium/arene compounds, mainly due to the emphasis\n five-, or six-membered chelate rings with different classes of\n received by the compound [Ru(\u03b76-p-cymene)Cl2PTA] (PTA =\n 1,3,5-triaza-7-phosphaadamantane), called RAPTA-C.2 Differ-\n ent from other metal complexes that tend to bind to DNA, Received: January 16, 2023\n RAPTA-C tends to be more interactive toward proteins, Published: April 26, 2023\n focusing on different targets in the cell and consequently\n different modes of action.3 This complex appears to be a\n promising candidate to reach clinical evaluation, exhibiting\n anticancer efficacy, as demonstrated by experiments in vitro as\n\n \u00a9 2023 American Chemical Society https://doi.org/10.1021/acs.inorgchem.3c00183\n 6955 Inorg. Chem. 2023, 62, 6955\u22126969\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 1. Examples of ruthenium/arene complexes with O,O-coordinating ligands such as benzoic acid and analogues,10 naphthoquinones,11,20\nflavonoids,13 and curcumins.19\n\nScheme 1. Synthesis of the Ruthenium(II)/Arene/Alizarin Complexes\n\n\n\n\norganic molecules, as exemplified in Figure 1A. Our research complexes, resulting in five-membered chelate rings. These\ngroup has already explored the chemistry of \u201chalf-sandwich\u201d complexes containing lapachol (naphthoquinone) display in\nruthenium/arene compounds containing benzoic acid and vitro anticancer activity, whose mechanism involves the\nanalogues as ligands (four-membered chelate ring), resulting in generation of reactive oxygen species and consequent oxidative\ncomplexes that are selective for breast cancer cells (MDA-MB- stress. The ruthenium/arene/flavonoid complexes trigger cell\n231) and capable of accumulating in tumor cells.10 Natural death mechanisms by topoisomerase II\u03b1 inhibition and\nproducts, such as naphthoquinones (Figure 1B) and flavonoids covalent DNA binding.11\u221215 Another powerful combination\n(Figure 1C), have also been coordinated to ruthenium/arene with natural products is complexes containing curcumins\n 6956 https://doi.org/10.1021/acs.inorgchem.3c00183\n Inorg. Chem. 2023, 62, 6955\u22126969\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nTable 1. Carbonyl IR Stretching and NMR Chemical Shift Values for Complexes C1\u2212C3 and Free Alizarin and Fluorescence\nQuantum Yield of Complexes C1\u2212C3 (TPyP as a Standard)\n 13\n IR C NMR\n \u03bd(C1\ufffdO1) \u03bd(C8\ufffdO8) \u03b4(C1\ufffdO1) \u03b4(C3\u2212O3) \u03b4(C4\u2212O4) \u03b4(C8\ufffdO8) fluorescence quantum yield\n (cm\u22121) (cm\u22121) (ppm) (ppm) (ppm) (ppm) (%)\n C1 1610 1660 182.2 155.9 158.4 181.2 12.0\n C2 1610 1656 182.2 157.2 157.2 180.5 0.5\n C3 1612 1651 184.5 158.5 156.6 180.5 0.9\n alizarin 1633 1664 181.5 151.0 149.3 189.5 16.2\n\n(Figure 1D), resulting in the formation of six-membered To obtain [Ru(L)Cl(\u03b76-p-cymene)] (C1; CCDC 2161723),\nchelate rings and high inhibition of the proliferation of breast the neutral complex P1 was dissolved in methanol, in the\nand ovarian tumor cells and also covalently binding to DNA presence of the unprotonated alizarin (1:2 ratio, P1/L, L =\n(Ru/N7-guanine) and suppressing the proteasome activ- alizarin), which results in a dark-blue precipitate C1 powder.\nity.16\u221219 Using this same procedure, but using P2 and P3 as precursors\n An underexplored class of ligands that can form six- in an attempt to synthesize the monocationic [Ru(L)(\u03b76-p-\nmembered chelated rings, as those described above, is cymene)(PPh3)]PF6 (C2) and [Ru(L)(\u03b76-p-cymene)(PEt3)]-\nanthraquinones, such as alizarin, which can be found in the PF6 (C3; CCDC 2161724), the reactions reach equilibrium,\nroots of Rubia tinctorum and is used as a fluorescent organic not consuming all precursors, even with the presence of the\nred dye.21 Studies on the antitumor action of alizarin reported counterion (PF6\u2212), and reflux. Therefore, to obtain pure C2\nthat this molecule exhibits an inhibitory effect on cells derived and C3 complexes, the chlorido ligands were extracted by\nfrom human colon carcinoma22 and osteosarcoma.23 An adding a silver salt (AgBF4), resulting in the instant formation\nimportant characteristic exhibited by alizarin is the absence of AgCl precipitate and consequent coordination of alizarin to\nof stimulation of cell proliferation, mutagenicity, or malignant ruthenium, resulting in pure C2 or C3 complexes, in high yield\ntransformation,24,25 showing no tumor-promoting activity, (>95%). Previous studies carried out by our research group\nmaking this molecule an interesting ligand for the synthesis adopted this same method for the coordination of bidentate\nof new complexes. O\u2212O binders to RuII.10,40\n Zebrafish (Danio rerio) is an appropriate animal preclinical Elemental analysis (C, N, H, and S %) of complexes C1\u2212C3\nmodel system for in vivo toxicological screening drugs for agreed with their suggested structures, as in Scheme 1, and the\npotential use in the treatment of human diseases, such as molar conductivity, in dichloromethane, was close to 60.0 S m2\ncancer.26 In addition, zebrafish has been an experimental mol\u22121 for C2 and C3, which is compatible with monocationic\nmodel widely adopted in scientific research in several areas, species. Compound C1 did not exhibit conductivity,\nincluding pharmacology and toxicology.27,28 Some key features confirming the neutrality of this complex. In the mass\nthat make it an excellent model for use in toxicology include spectrometry experiments, the signals of the main molecular\nexternal fertilization, rapid development, high reproduction ion [M]+ were observed for C2 and C3, while for C1, the main\nrate, the possibility of assessing early development due to signal was observed for the complex without the chloride\nchorion translucency, genome already sequenced, and high ligand [M \u2212 Cl]+, as presented in Figure S1A. The IR spectra\nhomology with humans.29\u221231 The zebrafish model system has of free alizarin displayed a broad band around 3400 cm\u22121,\nbeen recommended and adopted for toxicological preclinical assigned to \u03bd(O\u2212H), which decreases considerably its energy\nscreening to reduce the time and cost of the drug discovery after its coordination to the metal center, forming the C1\u2212O1\nprocess32\u221234 as an alternative model based on 10Rs ethical binding, leaving only C4\u2212O4 in the protonated form (Figure\nprinciples.35 S1B). The two \u03bd(C\ufffdO) bands of free alizarin appeared at\n Accordingly, the current study aimed to evaluate the 1664 cm\u22121 [\u03bd(C8\ufffdO8)] and 1633 cm\u22121 [\u03bd(C1\ufffdO1)], as\nantitumoral (in vitro) and toxicological safety (in vivo) presented in Table 1.41 For the three complexes, the band of\ncharacteristics of new \u201chalf-sandwich\u201d ruthenium/alizarin \u03bd(C8\ufffdO8) undergoes little change (around 1660 cm\u22121), while\ncomplexes, using breast and lung cancer cells, investigating the band of \u03bd(C1\ufffdO1) shifts approximately 20 cm\u22121,\nthe DNA interaction and in vitro antimetastatic effect of these appearing around 1610 cm\u22121. The decrease in the frequency\ncomplexes. In a complementary way, we will check the of \u03bd(C1\ufffdO1) indicates an increase in its single-bond character\ntoxicological safety using the zebrafish model associated with a because of its coordination to the metal center.\nmultiple biomarker response, such as the mortality rate, C2 and C3 formation was followed by the 31P{1H} NMR\nhatching rate, morphological change, neurotoxicity, and technique using aliquots of the reaction media. As usual, the\ncardiotoxicity, aiming to explore the potential of these unique P atom of the coordinated monophosphine ligand\ncomplexes as anticancer metallodrugs. exhibits just one singlet signal in the NMR spectrum (CH2Cl2/\n D2O) for both precursors and the final complexes (Figure S2).\n\n\u25a0 RESULTS AND DISCUSSION\n Synthesis and Characterization. Complexes C1\u2212C3\n Complex P2 exhibits this singlet signal at \u03b4 24.2 ppm, while for\n P3, it is at \u03b4 22.5 ppm, showing a P2 higher chemical shift as a\n consequence of the \u03c0 acidity of PPh3.\nwere synthesized following the procedure shown in Scheme 1. All of the complexes were characterized by 1D/2D 1H and\n 13\nStarting from the binuclear complex [{RuCl(\u03b76-p-cym- C NMR spectroscopy, with the 1D 1H NMR spectra showing\nene)} 2 (\u03bc-Cl) 2 ] (P1), 36 the mononuclear precursors the same profile, with three peak regions: the first region, at\n[RuCl2(\u03b76-p-cymene)(PPh3)] (P2) and [RuCl2(\u03b76-p-cymene)- high field, corresponds to the methyl/propyl H atoms (\u03b4 1.0\u2212\n(PEt3)] (P3) were obtained.37\u221239 3.0 ppm) of coordinated p-cymene and the ethyl groups from\n 6957 https://doi.org/10.1021/acs.inorgchem.3c00183\n Inorg. Chem. 2023, 62, 6955\u22126969\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nPEt3 for C3. The second region is related to the aromatic H 85.8(1), 84.1(2), and 84.8(2)\u00b0, respectively, and the O1\u2212Ru1\u2212\natoms from the coordinated p-cymene (\u03b4 5.0\u22126.0 ppm). The O3, O1\u2212Ru1\u2212P1, and O3\u2212Ru1\u2212P1 bond angles of C3 were\nthird region, at low field, shows aromatic H atoms from the 85.3(2), 88.3(2), and 85.0(2)\u00b0, respectively. The principal\nPPh3 ligand for C2 and from the alizarin ligand for all final bond lengths of the complexes are shown in Table 2. The main\nproducts (\u03b4 6.5\u22129.0 ppm). All registered 1D/2D spectra are change in the alizarin bond lengths is observed for C3\u2212O3,\npresented in Figures S3\u2212S17. which after coordination acquires greater double-bond\n Aiming to explore the fluorescence from the coordinated character, as was also observed in the 13C NMR experiments.\nalizarin molecule, in the final products, the emissive character- To better understand the crystal structure and rationalize\nistics of complexes C1\u2212C3 and free alizarin were evaluated in possible intermolecular interactions, we calculated the full\ndimethyl sulfoxide (DMSO) solvent. Free alizarin displays an interaction maps around the complex molecules using the\nintense fluorescence emission (\u03bbex) at approximately 650 nm, Mercury program.45,46 Parts B and E of Figure 3 show the\nresulting in absorption at 420 nm (\u03c0 \u2192 \u03c0*), as previously interaction maps for C1 and C3, respectively. The blue regions\ndescribed in the literature.42 In the spectra of the complexes, highlight the hydrophilic region (uncharged NH nitrogen\nonly C1 showed fluorescence when excited at \u03bbex 414 nm probe), the red regions indicate the hydrophobic positions\n(maximum intensity found for \u03c0 \u2192 \u03c0* present in the spectra of (carbonyl oxygen probe), and the green regions indicate likely\nthe complexes), resulting in a broad band emission, such as positions of aromatic interactions (aromatic CH carbon\nthat observed for alizarin, in the region of 550\u2212800 nm (Figure probe). The profiles obtained for the maps in both molecules\n2). This effect is a consequence of the phosphine ligands in the were similar, showing the same regions with a probability of\n intermolecular interactions. The regions in blue stand out,\n mainly around the uncoordinated O atoms, where there is a\n great chance of acting as a hydrogen acceptor, while the red\n regions dominate in the vicinity of the H atoms. The main\n difference between these maps is the red region around the\n OH group of C1, which is blocked in C3 due to steric\n hindrance caused by phosphine. The most abundant\n intermolecular contacts are of the H\u00b7\u00b7\u00b7H type, corresponding\n to practically half of the existing contacts (Figure 3F).\n In the fingerprint 2D plots of C1 (Figure 3C) and C3\n (Figure S18), the H\u00b7\u00b7\u00b7H contacts for C3 are approximately 0.8\n \u00c5 (di/de), while in C1, they are approximately 1.1 \u00c5 (di/de),\n showing that the hydrophobic interactions involving C3\n molecules are shorter and more intense, contributing to the\n formation of aggregates in solution and consequently\n cooperating with the suppression of fluorescence observed\n for C3. These abundant hydrophobic interactions are also\n characteristic of substances with predominant lipophilic\n character and are important in interactions with biomolecules.\n The stability of the complexes in solutions, such as those\nFigure 2. Normalized emission and absorption spectra of C1 in used in the biological tests, was evaluated using the 1H NMR\nDMSO (\u03bbex = 420 nm, 100 \u03bcM).\n technique in DMSO-d6. Thus, the spectrum of complex C1\n exhibits three specific regions, which can be seen by following\ncoordination sphere of the complexes, which promote the signal referring to the propyl group from p-cymene (Figure\nsuppression of the fluorescent emission processes when S19 of SI), which presents three independent groups of signals.\nassociated in the same complex (fluorescent dye and This may be the result of labilization of the chlorido ligand,\nphosphine).43,44 Quantum yield calculations confirmed the which in the presence of other coordinating molecules (DMSO\nsame trend as the observed in the recorded spectra, using or water) can be replaced by them. This process is widely\n5,10,15,20-tetra(4-pyridyl)porphyrin (TPyP) as a standard discussed in the literature because this fact allows coordination\n(DMSO, 100 \u03bcM), where complex C1 showed a quantum of the metal to biomolecules, such as DNA or proteins.47,48\nyield 20 times higher than those of the other complexes and Complexes C2 and C3 were also evaluated in terms of their\nsimilar to that of free alizarin, as seen in Table 1. stability in the cell culture medium (RPMI) and DMSO (2:1)\n The crystal structures of complexes C1 (Figure 3A) and C3 and pure DMSO, adopting the 31P{1H} NMR technique. Both\n(Figure 3D) were determined by single-crystal X-ray complexes were stable in pure DMSO (48 h of experiment),\ndiffraction. Both complexes crystallized in the monoclinic and in the DMSO/RPMI mixture, at the beginning of the\nP21/c space group, presenting one molecule per asymmetric experiment, the formation of two additional signals at \u03b4 24 and\nunit and the PF6\u2212 counterion in the C3 structure. The C3 28 ppm for C2 and at \u03b4 22 and 25 ppm for C3 wwa observed,\nasymmetric unit showed positional disorders in the p-cymene which remained as the minority species up to 48 h\nligand (refined in two positions with 50% occupancy), (approximately 20%). The original complexes remained as\ntriethylphosphine (refined in two positions with occupancies the majority species, as shown in Figure S20. The signals at \u03b4\nof 45% and 55%), and the PF6\u2212 counterion (refined in two 24 ppm for C2 and \u03b4 22 ppm for C3 can be related to the\npositions with 50% occupancy). The structure confirms the six- precursors P2 and P3, respectively, which have the same\nmembered ring resulting from the coordination of alizarin to chemical shifts. The appearance of signals referring to\nruthenium besides \u03b76-coordinated p-cymene. The O1\u2212Ru1\u2212 precursors P2 and P3 can be favored by two factors: The\nO3, O1\u2212Ru1\u2212Cl1, and O3\u2212Ru1\u2212Cl1 bond angles of C1 were first is due to labilization of a O,O-coordinated ligand, as was\n 6958 https://doi.org/10.1021/acs.inorgchem.3c00183\n Inorg. Chem. 2023, 62, 6955\u22126969\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 3. (A) Crystal structure of C1 (30% of thermal ellipsoids). (B) Interaction map for complex C1. (C) Full fingerprint 2D plot for complex\nC1. (D) Crystal structure of C3 (30% of thermal ellipsoids). (E) Interaction map for complex C3. (F) Percentage of each contact type for\ncomplexes C1 and C3.\n\nTable 2. Selected Bond Lengths of C1, C3, and the Alizarin Based on UV\u2212vis absorption spectroscopy, the \u03c0 \u2192 \u03c0*\nCrystal Structure transition bands exhibited by the complexes were monitored to\n observe a possible decrease in intensity (hypochromism) or\n C1 C3 alizarina\n displacement (bathochromism) of these bands with successive\n Ru1\u2212O1 2.075(3) 2.068(6)\n additions of calf thymus DNA (ct-DNA) solutions. The affinity\n Ru1\u2212O3 2.057(2) 2.063(5)\n between the complex and DNA can be measured by calculating\n Ru1\u2212P1 2.350(2)\n the binding constant (kb, from neighbor-exclusion equation),\n Ru1\u2212Cl1 2.439(1)\n which is dependent on the maximum absorption of the\n Ru1-cpb 1.652 1.694\n transition bands present in the spectrum. As can be seen in\n C1\u2212O1 1.252(5) 1.25(1) 1.245(7)\n C3\u2212O3 1.295(5) 1.29(1) 1.347(5)\n Figure 4, when DNA aliquots are added to a solution\n C4\u2212O4 1.339(7) 1.35(1) 1.343(5)\n containing the complexes, a decrease in the absorption\n C8\u2212O8 1.239(7) 1.21(1) 1.224(7)\n intensity (hypochromic effect) of the bands is observed. This\na effect is due to dilution of the solution and to variations in the\n CCDC 800614. bcp = centroid of p-cymene.\n complex electronic transition energies, causing changes in the\n absorption intensity due to interactions between the complex\nalready reported for ruthenium/arene complexes.49 The and DNA. To evaluate the contribution of the dilution effect\nsecond is due to the fact that the culture medium used in for each complex, an experiment was carried out separately by\nthis experiment has a high free chloride content, which also adding aliquots of buffer, determining the hypochromism that\nfavors the formation of precursors in solution. However, in the resulted from complex dilution. The hypochromism values\npresent study and under the conditions tested for C2 and C3, (discounting the dilution effect) resulting from this experiment\nthis speciation occurred only on a small scale, with C2 and C3 show that practically only complex C1 interacts with DNA,\nremaining as the majority species in solution. presenting 25% hypochromism, while C2 and C3 present\n DNA Interaction Experiments. Knowing the strength and irrelevant hypochromism values. Complex C1 displays a kb\ntype of binding of metal complexes with DNA is relevant constant value equal to 2.5 \u00d7 104 \u00b1 0.6 \u00d7 104, with a\ninformation for investigating possible mechanisms of action magnitude of 104 corresponding from a medium/strong\nagainst tumor cells. The cytotoxic activity of cisplatin is partly interaction with DNA.52\u221255\nattributed to its ability to bind to DNA, interfering in the Because complexes such as C1, with a labile chlorido ligand,\nmechanisms of repair of DNA and triggering cell death establish their anticancer effects by coordinating to the N7-\npathways.50,51 guanine units in DNA, the moderate/strong interaction\n 6959 https://doi.org/10.1021/acs.inorgchem.3c00183\n Inorg. Chem. 2023, 62, 6955\u22126969\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n MB-231 (breast cancer), MCF-7 (breast cancer), and A549\n (lung cancer) and nontumor cell lines MCF-10A (breast) and\n MRC-5 (lung). The tests were carried out using cell treatment\n protocols and using DMSO as a solvent but without exceeding\n the limit of 0.5% of the final volume, in which the period of\n exposure of cells to the complexes is equal to 48 h. The IC50\n values (concentration of the complex capable of inhibiting 50%\n cell proliferation) found for the complexes, alizarin, and\n precursors against the different cell lines cited above are shown\n in Table 3.\n Precursors P1 and P3 and alizarin (L) did not present\n cytotoxicity toward the cells at the highest concentration tested\n (100 \u03bcM), while precursor P2 showed considerable cytotox-\n icity, even with values close to those shown by cisplatin in\n nontumor cells. Complex C2 exhibited greater inhibition of\n cytotoxicity against the cells, presenting in all cases IC50 values\n at least 3 times lower than those of C1 and C3 and slightly\n lower than that of cisplatin in MDA-MB-231 and MCF-10A\n cells. When complexes and precursors are compared, it can be\nFigure 4. Absorption spectra in the UV\u2212vis region obtained for\ncomplex C1 with the addition of successive aliquots of ct-DNA.\n observed that in all cases the compounds containing\n triphenylphosphine as the ligand are the most active (C2\n and P2), showing considerably lower values than the others, in\nobserved for C1 may be a result of the existence of covalent all cell lines tested. This fact may be correlated with the higher\ncomplex/DNA interaction. To confirm this observation, we lipophilicity exhibited by triphenylphosphine complexes, giving\nperformed assays with plasmid DNA (PBR322), using the gel complex C2 a high value of partition coefficient n-octanol/\nelectrophoresis technique, adopting solutions with [complex]/ water (log P = 0.61 \u00b1 0.02), while in the complex with\n[DNA] molar ratios of 0.25, 0.5, 1.0 and 1.5, in which all of the triethylphosphine (C3), this value is equal to 0.30 \u00b1 0.04;\nsamples were preincubated, under stirring for 24 h, at 37 \u00b0C. therefore, PPh3 confers additional lipophilicity to the complex\nFigure S21 presents the results obtained for these experiments. than the corresponding PEt3-containing complex, which, in\nComparing the bands displayed in the experiments containing turn, was more lipophilic than complex C1, which lacks\nthe complexes with those of the negative control (DNA + monophosphine and exhibits a lower log P value (0.17 \u00b1\nDMSO), we observed that the C2 and C3 compounds did not 0.08).59,60\ninduce changes in the position and intensity of the supercoiled It is known that lipophilic compounds have greater affinity\nDNA bands, showing that there are no interactions between with the membrane proteins of eukaryotic cells, where through\nthe complexes and DNA, as seen in the spectrophotometric interactions with these proteins (including the hydrophobic\ntitration experiments. Complex C1 exhibited a different result, interactions cited in the crystallographic discussion), these\nshowing complete disappearance of the bands at higher molecules become more likely to undergo active transport\nconcentrations, which is characteristic of a strong interaction from the extracellular to intracellular environment and are able\nbetween the complex and DNA, as we observed for the to trigger mechanisms of cell death in the interior of cells.\npositive control (cisplatin). Thus, on the basis of the results of Thus, the lower IC50 value displayed by the C2 compound may\nthe spectroscopic titrations and electrophoresis, we suggest be correlated with this characteristic. This compound is more\nthat the existence of interactions of the covalent type for lipophilic, which can lead to a greater ability to accumulate\ncomplex C1 may be predominant in relation to other types of inside the cells and inhibit the proliferative ability, and this fact,\ninteraction, which is able to compose its mechanism of action to be confirmed, requires the performance of other cell\nagainst cancer cells, similar to other ruthenium/arene/chlorine permeation experiments. Table 3 also shows the selectivity\ncomplexes described in the literature.56,57 values found for the complexes, where only in the breast cells\n Biological In Vitro Experiments. The cytotoxic profile of was there selectivity for tumor lineages, ranging from 0.9 to\nthe complexes was evaluated in human tumor cell lines MDA- 1.7. However, the selectivity exhibited by the complexes is\n\nTable 3. IC50 Values (\u03bcM) and Selectivity Indexesa Obtained for C1\u2212C3, Alizarin (L), Precursors P1\u2212P3, and Cisplatin (CP)\nagainst Tumor and Nontumor Cell Lines in the Period of 48 h\n MDA-MB-231 MCF-7 MCF-10A A549 MRC-5 IS1 IS2 IS3\n C1 42.2 \u00b1 3.6 32.8 \u00b1 1.2 57.0 \u00b1 0.6 >100 53.9 \u00b1 0.5 1.3 1.7\n C2 6.5 \u00b1 0.1 9.0 \u00b1 0.1 10.0 \u00b1 0.3 17.8 \u00b1 0.8 7.7 \u00b1 0.1 1.5 1.1 0.4\n C3 45.4 \u00b1 1.4 >100 41.6 \u00b1 0.1 52.6 \u00b1 1.2 25.0 \u00b1 0.1 0.9 0.5\n L >100 >100 >100 >100 >100\n P1 >100 >100 >100 >100 >100\n P2 46.8 \u00b1 0.6 >100 18.2 \u00b1 0.6 41.2 \u00b1 0.3 23.9 \u00b1 0.1 0.4 0.6\n P3 >100 >100 >100 >100 >100\n CP58 10.2 \u00b1 0.2 8.6 \u00b1 1.8 14.0 \u00b1 2.0 14.4 \u00b1 1.4 29.1 \u00b1 0.8 1.4 1.6 2.0\n\na\n IS1 = IC50(MCF-10A)/IC50(MDA-MB-231); IS2 = IC50(MCF-10A)/IC50(MCF-7); IS3 = IC50(MRC-5)/IC50(A549).\n\n 6960 https://doi.org/10.1021/acs.inorgchem.3c00183\n Inorg. Chem. 2023, 62, 6955\u22126969\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 5. (A) Micrographs of clonogenic assay showing MDA-MB-231 cells treated with complex C1 and graphs of quantifications of the colony\narea. (B) Effects of complex C2 on the cell morphology of MDA-MB-231, after exposure of 0, 24, and 48 h. (C) Effect of complex C1 on MDA-\nMB-231 cell migration by using an inverted microscope (4\u00d7) and graph of the wound-healing closure percentages for treatment with C1 and C2.\nThese experiments were representative of three independent assays. Significance at (****) p < 0.0001 using ANOVA. Solvent control experiments\nwere performed with DMSO (1%).\n\nlower than that found for cisplatin because they had a strong cells to lose their clonogenicity with an increase of the\ncytotoxic effect on nontumor cell lines. concentration of the complexes, where in a concentration of 2\n Aiming to explore the influence of the substances on the \u00d7 IC50, complete inhibition of the ability of cells to form\nmorphology of MDA-MB-231 cells, we selected complexes C1 colonies (Figure 5A) was observed. This result is interesting\nand C2 to be evaluated because they were the ones that because, even maintaining the morphology of the cells (as\npresented the highest selectivity indexes among the complexes observed in the morphological assay), at the concentration of 2\ndescribed in this work. The cells were treated with \u00d7 IC50, the cells appear to suffer irreversible damage, directly\nconcentrations of complexes proportional to the IC50 values affecting their ability to form colonies, suggesting a possible\n(1/2 \u00d7 IC50, IC50, 2 \u00d7 IC50, and 4 \u00d7 IC50), and the images were antimetastatic action of the C1 and C2 compounds. The\nrecorded at times of 0, 24, and 48 h. In addition, a similar concentration-dependent effect can be seen in Figure 5A,\nexperiment was performed with solvent control (1% DMSO). which shows the resulting colony area as a function of the total\nAnalyzing the resulting images (Figure 5B), we observed that area of the well, where the decrease in the colony area with\nC2 did not cause significant changes in concentrations equal to increasing concentration is considerable and significant.\n1\n /2 \u00d7 IC50, IC50, and 2 \u00d7 IC50, demonstrating that under these Another important information that contributes to the\nconditions the cells maintain their morphological integrity and antimetastatic activity of the complexes is their ability to inhibit\nare indistinguishable from the control experiment. The most the migration of cells, which in this work was done following\nsignificant change is observed only at the concentration equal the collective migration of cells in an empty strip made in a\nto 4 \u00d7 IC50, where already in 24 h of experiment the cells show monolayer of the cells. To do this, we used MDA-MB-231 cells\nchanges in their morphology and a loss of adhesion to the treated with complexes at concentrations equal to 1/4IC50,\n 1\nbottom of the plate. Besides these observations, complex C1 in /2IC50, and IC50, recording the images at 0, 24, and 48 h of\n48 h at a concentration of 2 \u00d7 IC50 displays a loss of cell experiment. When the results displayed in Figure 5C are\nconfluence (Figure S22). analyzed, it can be concluded that the negative control\n To evaluate the cytostatic effect of the complexes on the experiment (1% DMSO), in 48 h, practically closed the empty\nMDA-MB-231 cell line, we performed clonogenic survival space, while at concentrations equal to 1/2IC50 and IC50, a\nassays, where it is possible to determine the ability of a cell to similar pattern was not observed. This observation becomes\nproliferate, forming a colony of cells (important step in the clearer upon analysis of the percentage of the closure presented\nmetastatic process), after being exposed to different concen- in the graph of Figure 5C, where in the control, the empty area\ntrations of the complexes (1/4 \u00d7 IC50, 1/2 \u00d7 IC50, IC50, and 2 \u00d7 decreases by 50% (24 h), while in the C1 complex (IC50\nIC50) for 48 h. After this period, the culture medium concentration), this decrease is 24%, and with C2, it is even\ncontaining the complexes was discarded and a new medium lower, closing at just 13%. In 48 h, the control displays the\nwas added, and the surviving cells were incubated again, for 10 wound healing practically completed; however, in all treat-\ndays, for growth of the resulting colonies.61,62 Both complexes ments, migration inhibition was observed, where the C2\n(C1 and C2) display antiproliferative activity, inducing the compound exhibited the highest inhibition. It is important to\n 6961 https://doi.org/10.1021/acs.inorgchem.3c00183\n Inorg. Chem. 2023, 62, 6955\u22126969\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 6. (A) Cell count distribution for phases G2/M, S, G1/G0, and Sub-G1 of the negative control and complex C2. (B) Percentage of cells in\neach phase of the cell cycle at different concentrations of complex C2: (*) p < 0.05; (**) p < 0.01; (***) p < 0.001; (****) p < 0.0001. Solvent\ncontrol experiments were performed with DMSO (1%).\n\npoint out that the migration inhibition effect was also observed the nucleus and cytoplasm of the cells. Thus, upon exploration\nat concentrations lower than the IC50 concentration, which of the fluorescence of complex C1, the treatment of MDA-MB-\nindicates that the observed effect is mainly due to the 231 cells using the concentration of IC50 (40 \u03bcM) was\ninhibition of cell migration and not to the cytotoxic effect of performed to visualize the complex uptake by the cells.\nthe tested compound. These results place the compounds As shown in Figure 7A, the flow cytometry obtained for cells\namong an important class of substances that have antimeta- MDA-MB-231 treated with complex C1 showed that this\nstatic activity, such as NAMI-A and the ruthenium(II)/arene complex does not accumulate in viable cells, either at 24 h or at\nanalogue RAPTA-C, which exhibit in vivo and in vitro 48 h, without altering the fluorescence profile displayed by the\nantimetastatic activity, in addition to having already been cells. Free alizarin is also not internalized by the cell, as shown\nevaluated in advanced clinical trials.63\u221265 in Figure 7B. When a fluorescent compound is internalized by\n Knowing that the complexes have antiproliferative and the cells, the distribution of fluorescence displayed by the cells\nprobable antimetastatic activity against MDA-MB-231 cells, is altered as the fluorophore accumulates, unlike that observed\nanalysis of the effect of complex C2 (1/4IC50, 1/2IC50, IC50, and for alizarin and complex C1. Using confocal microscopy, we\n2 \u00d7 IC50) on the cell cycle distribution was performed. Figure sought to confirm what was observed in the previous\n6A shows the distribution of cells in the phases of the cell cycle experiment. To delimit the nucleus of the cells, we used the\nfor the control experiments and in the concentration of 2 \u00d7 DNA marker DAPI, which when irradiated at 360 nm,\nIC50 of complex C2. When these graphs are compared, it can exhibited fluorescence at 460 nm, showing blue staining. To\nbe observed that complex C2 triggers changes in the cell cycle capture the fluorescence from complex C1, the cells were\nof MDB-MB-231 cells, leading to an increase in the number of irradiated at 420 nm (maximum absorption of the band\ncells in the Sub-G1 phase and a decrease in the G2/M and G1/ responsible for fluorescence). As shown in Figure 7E, at first,\nG0 phases. In Figure 6B, these trends become clearer when a internalization of the complex by the cells was not observed,\nsignificant increase in the number of cells in the Sub-G1 phase showing only the fluorescence inherent to the cell nucleus\nwith an increase in the concentration of the complexes is marker (DAPI). Internalization of the complex was only\nvisualized. The decrease in the number of cells in G2/M is an observed after permeabilization of the cells (Figure 7F),\nindication that the damage caused to cellular structures is exhibiting fluorescence in the red spectral region (650 nm), as\ndifficult to repair, leading to a consequent increase in cells in is seen in the quantum yield calculation experiments. This\nSub-G1, which is a natural cell response to the damage caused, information shows that this complex is internalized in the cells\nleading to the activation of cell death mechanisms by only after the damage done to the cell membranes, not being\napoptosis.66\u221270 able to be internalized by viable cells. These data provide\n Although there are mechanisms of cell death that can be important information, showing us that the complex is unable\ntriggered through interactions with membrane proteins or with to be internalized by the cell probably due to the low\ncomponents of the extracellular matrix, there are several other lipophilicity exhibited by this complex compared with the\nknown mechanisms that occur involving molecules present in others with phosphines. Consequently, because the C1\n 6962 https://doi.org/10.1021/acs.inorgchem.3c00183\n Inorg. Chem. 2023, 62, 6955\u22126969\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 7. Flow cytometric analysis of MDA-MB-231 cells incubated with (A) a blank solution and C1 for 24 and 48 h and (B) a blank solution and\nalizarin for 24 and 48 h. (C) Quantification of the mean fluorescence intensity (geometric mean) of the histograms. Data represent mean \u00b1 SD of\nthe assays in triplicate. Significance at the levels (ns) was determined using one-way ANOVA and Dunnet\u2019s test. Fluorescent imaging of (D) MDA-\nMB-231 cells incubated with a blank solution (negative control), (E) nonpermeabilized MDA-MB-231 cells incubated with C1, and (F) MDA-MB-\n231 cells permeabilized with 01% Triton-X 100 in PBS and incubated with C1. The nuclei were stained with Hoechst 33342 (blue channel). All\nimages were acquired for both channels (blue and red).\n\ncomplex is not internalized, the interactions with molecules in the end of 96 h in the group exposed to 7.5 \u03bcM C2. Zebrafish\nthe intracellular environment do not occur, thus triggering embryos exposed to the highest concentrations of C2 (15 and\ninternal cell death mechanisms, maintaining the normal 30 \u03bcM) presented mortality rates of 60 \u00b1 0% and 93 \u00b1 11% at\nfunctioning of the cell and justifying the high IC50 value found. 96 h (p < 0.001), respectively (Figure S23B). Similarly,\n Biological In Vivo Experiments. In this study, an early exposure of animals to complex C3 at low concentration did\ntoxicological screening of new ruthenium-based anticancer not lead to embryo mortality; however, at high concentrations\ndrugs using the zebrafish model was performed as a strategy to of C3 (30 and 60 \u03bcM), the mortality rates were 66 \u00b1 15% and\navoid late-stage failures of the development of new drugs and 93 \u00b1 11%, respectively (96 h) (Figure S23C). Exposure of the\nreduce cost and time. Differential mortality rates were animals to free alizarin at both concentrations (15 and 30 \u03bcM)\nobserved in the zebrafish embryo\u2212larval stages after exposure caused 33 \u00b1 15% and 83 \u00b1 11%, respectively, of embryonic\nto the C1\u2212C3 compounds and free alizarin, which were mortality (p < 0.001) at 96 h (Figure S23D).\ncompared to negative and solvent groups (Figures S23A\u2212D). The hatching rate was also evaluated in zebrafish after\nAs expected, zebrafish embryos exposed to negative control exposure to complexes C1\u2212C3 and free alizarin (Figures\nand solvent control (0.5% DMSO) groups did not present S23E\u2212H). Exposure of the zebrafish embryos to the complexes\nmortality, which demonstrates a valid test. On the other hand, inhibited hatching, while the embryos from the control groups\nexposure of animals to ruthenium complexes showed that hatched normally. At all concentrations tested, exposure of the\nanimal mortality is concentration-dependent. Thus, exposing animals to complexes C1 and C3 and free alizarin caused\nthe zebrafish embryos to complex C1 caused the highest hatching inhibition in zebrafish. On the other hand, exposure\nmortality among the complexes tested, within the first 24 h. At of the animals to complex C2 at the lowest concentration\nall concentrations tested for complex C1, mortality rates caused hatching delays. Thus, while 100% zebrafish hatched\nbetween 76 \u00b1 11 and 100 \u00b1 0.0% (p < 0.001) were observed until 96 h, for the group exposed to 7.5 \u03bcM C2, 76 \u00b1 15%\n(Figure S23A). On the other hand, 7 \u00b1 5% of embryos died at hatched embryos at 96 h (p < 0.05) was observed. However,\n 6963 https://doi.org/10.1021/acs.inorgchem.3c00183\n Inorg. Chem. 2023, 62, 6955\u22126969\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 8. Morphological changes in the zebrafish embryos after 48 h of exposure to C1\u2212C3 and L: (A) negative control group (without treatment\nor solvent); (B) solvent control group; (C) C1 at 15 \u03bcM; (D) C2 at 7.5 \u03bcM; (E) C2 at 15 \u03bcM; (F) C2 at 30 \u03bcM; (G) C3 at 15 \u03bcM; (H) C3 at 30\n\u03bcM; (I) C3 at 60 \u03bcM; (J) alizarin at 15 \u03bcM and alizarin (L) at 30 \u03bcM. Arrow = head changes; arrowhead = tail changes; asterisk (*) = yolk sac\nchanges; BA = blood accumulation; PE = pericardial edema.\n\nthe other concentrations of complex C2 tested also caused complexes in zebrafish chorion (Figure 8C,E\u2212L). Besides\nhatching inhibition in zebrafish. edema in the pericardium and yolk sac and delayed growth,\n Besides mortality, both C1 and C3 caused a higher hatching exposure to alizarin caused head, somite, and tail malformation\nfailure of zebrafish embryos. Interestingly, the hatching failure in zebrafish alive at 15 \u03bcM.\nwas a common end point reported in other toxicological Yolk sac edema has been considered to be a sensitive\nscreening of ruthenium complexes.71\u221277 In this model, toxicity toxicological response for embryo toxicity. It is noteworthy\nevaluation considers hatching success to be a sensitive end that the yolk sac is metabolically active and highly lipophilic.\npoint. Hatching of all embryos until 96 h is expected; however, However, exposure to chemical substances can decrease yolk\nwhen hatching failure occurs, it means that the compounds utilization/mobilization, resulting in impaired embryonic\nunder testing can interfere with the normal biological nutrition that affect its entire development.79 Moreover,\ndevelopment of zebrafish. Moreover, embryos that did not osmoregulation of the zebrafish embryos can also be altered,\nhatch evolve to death.78 resulting in excessive uptake of the substance to which it was\n Morphological changes were recorded in zebrafish upon exposed. As a consequence, edema is formed, and the\nexposure to the ruthenium complexes (Figure 8). Exposure to substance may accumulate in the embryo.80 Gills, the digestive\nboth compounds, C2 and C3, induced several malformation system, and kidney are responsible for osmoregulation of\nphenotypes in zebrafish embryos, such as head malformation, zebrafish.81 Until these structures become functionally active,\nsomite malformation, tail malformation, delayed growth, osmatic balance is maintained for two water permeability\npericardial edema, yolk sac edema, and interaction with barriers on the surface of the embryo and one surrounding the\nchorion (Table S2). The exposure at 15 \u03bcM C1 caused yolk sac.80 Therefore, the tested compounds may impair the\nedema in the pericardium and yolk sac of 17 \u00b1 6% zebrafish embryonic nutrition and/or maintenance of the permeability\nalive (p < 0.05) and 20% of them interacted with chorion (p < barrier, causing edema in the yolk sac and pericardium and\n0.05). For the group exposed to C2 at the lowest bioaccumulation.\nconcentration, there were no significant alterations in the Anticancer agents present clinical concerns with their\ndevelopment of the zebrafish embryos. However, delayed systemic toxicity. Among the side effects, neurotoxicity,\ngrowth, pericardial edema, yolk sac edema, and interaction vascular toxicity, nephrotoxicity, and ototoxicity are the most\nwith chorion were observed in zebrafish exposed to C2 at high common.82 Therefore, overcoming the unpleasant side effects\nconcentrations (15 and 30 \u03bcM; p < 0.001). On the other hand, is a major challenge concerning new anticancer agents.\nall C3 concentrations induced morphological changes in live Besides the classic biomarker (mortality, hatching rate, and\nzebrafish, such as chorion alteration (15\u221260 \u03bcM; p < 0.001), morphological change), multiple biomarker responses can be\npericardial edema (15 and 30 \u03bcM; p < 0.001), yolk sac edema explored using a zebrafish model system in early preclinical\n(30 \u03bcM; p < 0.001), and growth retardation (60 \u03bcM; p < toxicological screening, including neurotoxicity and cardiotox-\n0.001). Interaction with chorion includes changing its color, icity. The neurotoxic effect of ruthenium/alizarin complexes\nwhich probably suggests the accumulation of these metal was determined in zebrafish embryos at 24 h by analyzing the\n 6964 https://doi.org/10.1021/acs.inorgchem.3c00183\n Inorg. Chem. 2023, 62, 6955\u22126969\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 9. (A) SCF (no. min\u22121) and (B) heartbeat rate (beats min\u22121) of zebrafish embryos alive from negative control and solvent groups and after\nexposure to C1\u2212C3 and alizarin for 24 h (SCF) and 48 h (heartbeat rate). Dots touching the x axis at 0 indicate embryos alive but lacking\nmovement during the analysis. (*) p < 0.05 versus negative control, (**) p < 0.01 versus negative control, and (***) p < 0.001 versus negative\ncontrol.\n\nfrequency of spontaneous movements (SCF). As shown in min\u22121, was observed in zebrafish exposed to C2 (7.5 and 15\nFigure 9A, only exposure of the zebrafish embryos to C2 at low \u03bcM, respectively) compared to the control group (p < 0.05).\nconcentration did not cause neurotoxicity. The SCF is an The heartbeat frequency can be obtained at 48 hpf because\nimportant parameter of neurotoxicity because spontaneous tail major heart structures have been formed in zebrafish.\ncoiling is the first motor activity as a consequence of However, because its heart is still immature, cardiac function\ninnervation of the muscle by the developing nervous\n can influence the development of the whole animal.83\nnetwork,82 which could reflect a morphological effect or even\n Furthermore, a decrease of the heartbeat in the early\nmortality.82 Therefore, hatching failure and morphological\nchange, including delayed growth, somite, and tail malforma- embryonic development of zebrafish can lead to animal\ntion, in embryos and mortality can be related to the neurotoxic death.78 In this study, the cardiotoxicity classified as\neffect of the compounds. bradycardia may be associated with the mortality rate and\n The cardiotoxicity, one of the most common side effects of morphological change because pericardial effusion can cause\nanticancer agents,82 was analyzed in the zebrafish embryos at blood congestion, decreasing blood flow and consequently\n48 h after exposure to the compounds (Figure 9B). As decreasing the heartbeats. In fact, pericardial edema consisted\nexpected, a normal embryonic heart rate was recorded for the of the main morphological alterations observed. It is important\ncontrol (without complexes and solvent) and solvent groups,\n to note that embryos exposed to 7.5 \u03bcM C2 did not present\nat 162 \u00b1 24 and 167 \u00b1 21 beats min\u22121, respectively. In\n significant morphological change.\ncontrast, all exposure of the zebrafish embryos to the\n Taken together, C1 and C3 showed greater toxicity to the\nruthenium complexes causes alterations in the heartbeat rate\nof zebrafish compared to embryos from control groups. development of the zebrafish embryo. On the other hand, C2,\nZebrafish embryos exposed to C1 and C3 and free alizarin the most promising anticancer drug in in vitro preclinical tests,\nshowed the lowest heart rates, from 52 to 93 beats min\u22121. A revealed the lowest toxicity in in vivo preclinical screening\ndecrease in the heartbeats, at 106 \u00b1 30 and 105 \u00b1 21 beats using zebrafish as a model system.\n 6965 https://doi.org/10.1021/acs.inorgchem.3c00183\n Inorg. Chem. 2023, 62, 6955\u22126969\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\u25a0 CONCLUSION\nIn this work, a new series of three ruthenium/arene/alizarin\n Rafaella da S. Brito \u2212 Instituto de Patologia Tropical e Sau\u0301de\n Pu\u0301blica, Universidade Federal de Goi\u00e1s, Goia\u0302nia, Goi\u00e1s\ncomplexes containing chlorido or monophosphine ligands 74605-050, Brazil\nwere synthesized and characterized. Complex C1 exhibits Thiago L. Rocha \u2212 Instituto de Patologia Tropical e Sau\u0301de\nstrong covalent DNA interaction due to the presence of the Pu\u0301blica, Universidade Federal de Goi\u00e1s, Goia\u0302nia, Goi\u00e1s\nlabile chlorido ligand in its structure. Complex C2 was the 74605-050, Brazil\nmost selective and cytotoxic toward breast cancer cells (MDA- Francyelli Mello-Andrade \u2212 Instituto de Patologia Tropical e\nMB-231), exhibiting IC50 values comparable to those of Sau\u0301de Pu\u0301blica, Universidade Federal de Goi\u00e1s, Goia\u0302nia,\ncisplatin. Fluorescence studies showed that complex C1 Goi\u00e1s 74605-050, Brazil; Instituto Federal de Educac\u0327a\u0303o\nexhibits fluorescence like free alizarin does and it cannot be Cie\u0302ncia e Tecnologia (IFG), Goia\u0302nia, Goi\u00e1s 74055-110,\ninternalized in MDA-MB-231 cancer cells probably due to its Brazil; orcid.org/0000-0001-7389-6125\nlow lipophilicity. Complexes C1 and C2 were selected for Javier Ellena \u2212 Instituto de F\u00edsica de Sa\u0303o Carlos, Universidade\ndetailed studies on MDA-MB-231 cells, and it was found that de Sa\u0303o Paulo (USP), Sa\u0303o Carlos, Sa\u0303o Paulo 13566-590,\nthese complexes inhibit colony formation and induce cell cycle Brazil; orcid.org/0000-0002-0676-3098\narrest in the Sub-G1 phase in a concentration-dependent Complete contact information is available at:\nmanner. Complex C2 showed quite encouraging outcomes in https://pubs.acs.org/10.1021/acs.inorgchem.3c00183\nthe in vitro setting, as well as in vivo toxicological screening at\nlow concentration using the zebrafish model. Therefore, Notes\ncomplex C2 is the most suitable candidate for drug The authors declare no competing financial interest.\ndevelopment to treat triple-negative breast cancer.\n\n\u25a0 ASSOCIATED CONTENT \u25a0 ACKNOWLEDGMENTS\n We thank the Group of Nanomedicine and Nanotoxicology,\n*\ns\u0131 Supporting Information\n Physics Institute of Sa\u0303o Carlos (IFSC-USP), for carrying out\nThe Supporting Information is available free of charge at the flow cytometry and fluorescence experiments. The authors\nhttps://pubs.acs.org/doi/10.1021/acs.inorgchem.3c00183. are grateful for financial support provided by CNPq (306329/\n Measurements, tables, and figures providing the NMR 2020-4), CAPES (finance code 001), and FAPESP (17/15850-\n data of the complexes, X-ray crystallographic data, and 0, 21/02522-0, and 21/04876-4).\n DNA interaction studies of all complexes (PDF)\nAccession Codes\n \u25a0 REFERENCES\n (1) World Health Organization. Estimated number of deaths in\nCCDC 2161723 and 2161724 contain the supplementary 2020, all cancers, both sexes, all ages. https://gco.iarc.fr/today/home\ncrystallographic data for this paper. These data can be obtained (accessed Dec 19, 2022).\nfree of charge via www.ccdc.cam.ac.uk/data_request/cif, or by (2) Lee, S. Y.; Kim, C. Y.; Nam, T.-G. Ruthenium Complexes as\nemailing data_request@ccdc.cam.ac.uk, or by contacting The Anticancer Agents: A Brief History and Perspectives. Drug Des. Devel.\nCambridge Crystallographic Data Centre, 12 Union Road, Ther. 2020, 14, 5375\u22125392.\nCambridge CB2 1EZ, UK; fax: +44 1223 336033. (3) Babak, M. V.; Meier, S. M.; Huber, K. V. M.; Reynisson, J.;\n Legin, A. A.; Jakupec, M. A.; Roller, A.; Stukalov, A.; Gridling, M.;\n\n\u25a0 AUTHOR INFORMATION\nCorresponding Authors\n Bennett, K. L.; Colinge, J.; Berger, W.; Dyson, P. J.; Superti-Furga, G.;\n Keppler, B. K.; Hartinger, C. G. 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Chem. 2023, 62, 6955\u22126969\n\f", "pages_extracted": 15, "text_length": 103361}

"Half-Sandwich" Ruthenium Complexes with Alizarin as Anticancer Agents: In Vitro and In Vivo Studies.