A novel benzothiazole-1,2,3-triazole-based arene osmium(ii) complex as an effective rhabdomyosarcoma cancer stem cell agent

{"full_text": " INORGANIC CHEMISTRY\n FRONTIERS\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n View Article Online\n RESEARCH ARTICLE View Journal | View Issue\n\n\n\n\n A novel benzothiazole-1,2,3-triazole-based\n arene osmium(II) complex as an e\ufb00ective\nOpen Access Article. Published on 10 January 2025. Downloaded on 5/12/2026 12:50:56 PM.\n\n\n\n\n Cite this: Inorg. Chem. Front., 2025,\n 12, 1693 rhabdomyosarcoma cancer stem cell agent\u2020\n So\ufb01a Sharkawy, \u2021a,b Alba Hern\u00e1ndez-Garc\u00eda,\u2021c Hana Kostrhunova, a\n\n Delia Bautista,d Lenka Markova, a Mar\u00eda Dolores Santana, c\n Jana Kasparkova, a,e Viktor Brabec *a,e and Jos\u00e9 Ruiz *c\n\n We designed a series of pseudo-octahedral arene Os(II) complexes (Os1\u2013Os5) with the general formula\n [(\u03b76-p-cym)Os(BTAT)Cl]+, where BTAT represents chelating N^N\u2019 ligands based on the 1-aryl-4-ben-\n zothiazolyl-1,2,3-triazole sca\ufb00old. The structures of Os3 and Os5 were con\ufb01rmed by X-ray di\ufb00raction,\n and Os5 exhibits a bathochromic shift in its absorption band compared to the other complexes, likely due\n to the electron-donating properties of the substituent NMe2. Os5 also hydrolyzed without losing its BTAT\n ligand and exhibited the highest cellular accumulation in Rhabdomyosarcoma (RD) cancer cells. The\n investigated Os(II) complexes demonstrated moderate antiproliferative activity across six cancer cell lines,\n with Os5 being the most potent, showing activity comparable to or better than conventional cisplatin.\n Cellular accumulation was a key factor in\ufb02uencing their antiproliferative e\ufb00ect, though binding to human\n serum albumin did not play a signi\ufb01cant role. Further studies with Os5 in RD cells, the most responsive cell\n line, revealed that its mechanism of action includes mitochondrial dysfunction, apoptosis via a caspase-\n dependent pathway, and cell cycle arrest at the G1 phase. Os5 also increased the production/generation of\n reactive oxygen species (ROS) in RD cells, implicating ROS production as a contributor to its activity.\n Received 29th October 2024, Importantly, Os5 was e\ufb00ective against cancer stem cells (CSCs) in 3D spheroid models, marking the \ufb01rst\n Accepted 9th January 2025\n report of an osmium-based compound targeting CSC-enriched RD cells. This highlights the potential of\n DOI: 10.1039/d4qi02737j Os5 as a CSC-targeted therapy, addressing the need for treatments that prevent relapse and metastasis. The\n rsc.li/frontiers-inorganic study underscores the promising role of metal-based complexes in cancer stem cell chemotherapy.\n\n\n Introduction Over the last few decades, the prognosis for children with\n localized Rhabdomyosarcoma (RMS) has significantly improved,\n Cancer is the second leading cause of mortality worldwide. with a 5-year overall survival rate of >70%.3 At the same time,\n According to data provided by the International Agency for despite aggressive combination therapy, no further significant\n Research on Cancer (IARC), it is estimated that by 2022, improvements have been made for the treatment of children\n approximately 20 million new cancer cases and 9.7 million with high-risk disease or recurrent disease (5-year survival <30%\n cancer-related deaths occurred.1,2 and 17%, respectively).4 Conventional therapies usually aim to\n attack rapidly dividing non-stem (di\ufb00erentiated) cancer cells, ulti-\n mately forming the bulk of a tumor. However, by adopting slow\n a\n Czech Academy of Sciences, Institute of Biophysics, CZ-61200 Brno, Czech Republic. proliferation states, cancer stem cells (CSCs) can evade antiproli-\n E-mail: vbrabec44@gmail.com ferative treatments due to their stem cell-like properties.5 The sur-\n b\n Faculty of Science, Department of Biochemistry, Masaryk University, CZ-62500 vival of a small number of CSCs within the tumor population\n Brno, Czech Republic can regenerate the original tumor and/or produce invasive cancer\n c\n Departamento de Qu\u00edmica Inorg\u00e1nica, Universidad de Murcia, and Murcia\n cells that can colonize distant organs.6 Therefore, CSCs are\n BioHealth Research; Institute (IMIB-Arrixaca), E-30100 Murcia, Spain.\n E-mail: jruiz@um.es thought to be responsible for cancer relapse and metastasis.7\u201310\n d\n ACTI, Universidad de Murcia, Murcia E-30100, Spain To overcome this clinical problem, novel therapeutics and modal-\n e\n Department of Biophysics, Faculty of Science, Palacky University, Slechtitelu 27, ities for treatments that can eradicate both fast-growing cancer\n 783 71 Olomouc, Czech Republic cells and quiescent CSCs are urgently needed.\n \u2020 Electronic supplementary information (ESI) available. CCDC 2393536 and\n The discovery of anticancer properties of some ruthenium\n 2393537 for Os5 and Os3. For ESI and crystallographic data in CIF or other elec-\n tronic format see DOI: https://doi.org/10.1039/d4qi02737j compounds led to the development and research of osmium\n \u2021 These authors contributed equally to this work. analogs. The application of osmium compounds as therapeutic\n\n\n This journal is \u00a9 the Partner Organisations 2025 Inorg. Chem. Front., 2025, 12, 1693\u20131715 | 1693\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\n\n agents was initially met with hesitation because osmium com- Sadler et al. have recently reported a few interesting half-\n plexes have been generally considered either toxic11 or substi- sandwich sixteen electron Os(II) transfer hydrogenation cata-\n tution-inert,12 which may explain the limited exploration of lysts (OsB Scheme 1) to achieve in-cell pyruvate and quinone\n their therapeutic potential. Nevertheless, subsequent research reduction as a novel anticancer mechanism.23,24 Os(II) com-\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n revealed that these compounds present significant anticancer plexes bearing a pH-sensitive reversible tether between the \u03b76\n potential. The first osmium compounds considered as potential ligand and the monodentate coordination site have been\n organometallic anticancer drugs were closely related congeners shown to improve aqueous solution stability. They can catalyze\n of RuII-arene-pta compounds (pta = phosphine 1,3,5-triaza-7- the in-cell reduction of pyruvate to lactate.25 Recently, it has\n phosphatricyclo[3.3.1.1]decane).13 Other categories of organo- been shown that a family of arene osmium(II) complexes with\n metallic Os complexes that show promising in vitro activity oxoglaucine (OsC in Scheme 1) can induce glucose metab-\nOpen Access Article. Published on 10 January 2025. Downloaded on 5/12/2026 12:50:56 PM.\n\n\n\n\n against cancer cells and adjustable reactivity in aqueous olism reprogramming and exhibit good anti-tumor activity\n environments include arene complexes containing a series of in vivo with low side e\ufb00ects under hypoglycemia.26\n di\ufb00erent bidentate nitrogen chelators,14 picolinate derivatives,15 Some of us have synthesized other noteworthy osmium\n or paullone-derived ligands,16 and also osmium analogs of half-sandwich complexes.27\u201329 Brabec et al. have developed an\n ruthenium-based antimetastatic agent NAMI-A type compound Os(II) compound incorporating bathophenanthroline, OsD\n [(NAMI-A) = (ImH)[trans-RuCl4(dmso-S)(Im)], Im = imidazole].17 (Scheme 1), which exhibits antimetastatic properties against\n Lippard et al. reported the first osmium compound [osmium(VI) triple-negative breast cancer.27 Furthermore, recent studies have\n nitrido complex] to exhibit selective toxicity against breast CSC- demonstrated its superior e\ufb03cacy against human breast cancer\n enriched HMLERtax cell populations.18 A selection of reported stem cells compared to salinomycin and RuD (Scheme 1).28\n Os(II) arene anticancer complexes are shown in Scheme 1. The Importantly, RuD hydrolyzed very fast, whereas OsD did not.\n one synthesized by Sadler et al., known as FY26 (OsA in Additionally, Ruiz et al. synthesized the Os(II) compounds [(\u03b76-p-\n Scheme 1A), is particularly noteworthy.19 FY26 demonstrated cym)Os(C^N)(4-NMe2-py)]+ (C^N = ppy OsE and ppy-CHO OsF,\n substantially greater potential than cisplatin, and its ruthenium Scheme 1), which have shown activity against ovarian cancer\n analog, RuA, across various cell lines, and recently, in vivo in vivo in Caenorhabditis elegans in the tumoral strain JK1466.29\n studies were conducted in mice to ascertain its pharmacoki- In the last series, the activity of OsF and the analogous RuF was\n netics and tolerability in the treatment of liver cancer.20\u201322 similar, with no hydrolysis observed for either of them.29,30\n\n\n\n\n Scheme 1 A selection of reported examples of Ru(II) and Os(II) arene anticancer complexes by Sadler\u2019s lab (A and B), Liang\u2019s lab (C) Tr\u00e1vn\u00ed\u010dek\u2019s lab\n (D) and Ruiz\u2019s lab (E), highlighting their biological properties.\n\n\n\n 1694 | Inorg. Chem. Front., 2025, 12, 1693\u20131715 This journal is \u00a9 the Partner Organisations 2025\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\n\n On the other hand, benzothiazole and 1,2,3-triazole hetero- S23\u2020). ESI-MS spectra from HPLC-MS displayed a peak due to\n cycles are chemical structures found in various pharmaceuti- the ion [M \u2212 PF6]+ with the expected isotopic distribution.\n cals and biologically active molecules. BLZ945 (sotuletinib), a The 1H NMR spectra were recorded in CD3CN, and in all\n derivative of 2-aminobenzothiazole, is currently undergoing cases, they show resonances due to the aromatic and aliphatic\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n clinical trials.31 Additionally, they are components of ligands protons of the p-cymene ligand. Thus, the three characteristic\n in metal complexes with biological activity, as exemplified by resonances of the isopropyl group (two doublets at 0.9 and\n certain Ru(II) compounds.32\u201334 Notably, both a series of ben- 1 ppm, and a septuplet around 2.5 ppm), the characteristic\n zothiazolyl-1,2,3-triazole (BTAT) derivatives with push\u2013pull resonance of the methyl group appearing as a singlet around\n architecture and bioactive properties35 and a family of photo- 2.3 ppm, and the expected ABCD spin system for the aromatic\n activatable BTAT-based octahedral ruthenium(II) complexes of resonances were observed in the p-cymene derivatives. Due to\nOpen Access Article. Published on 10 January 2025. Downloaded on 5/12/2026 12:50:56 PM.\n\n\n\n\n general formula [Ru( phen)2(BTAT)]2+ has been recently devel- the coordination of the N,N\u2032 chelating BTAT ligands L1\u2013L5,\n oped by some of us (Ru1-Ru5 in Scheme 2A).36 It is noteworthy some characteristic changes in the splitting pattern of the\n that, as reported, the cytotoxicity of these octahedral heterolep- p-cymene ring protons were observed. The C2v symmetry of the\n tic BTAT Ru complexes towards several di\ufb00erentiated human complexes was disrupted, and the peaks corresponding to the\n cancer cells under dark conditions was very low, even at high four aromatic protons of the p-cymene ring were observed as\n concentrations. These complexes exhibited significant photo- four distinct doublets between 6.2 and 6.5 ppm. Moreover, the\n toxicity indexes toward several cancer cells only after exposure frequencies of the H6+7, H8+9, and H5 proton resonances\n to blue light irradiation. It was further shown by HPLC-MS (doublets and singlet, respectively) depend on the substituent\n spectrometry and UV\u2013vis spectroscopy that these heteroleptic (\u2013R), resulting in lower frequencies for Os5 (containing the\n Ru complexes photoreleased the BTAT ligand upon irradiation \u2013NMe2 group) and higher frequencies for Os3 and Os4 (whose\n in water with light (as illustrated in Scheme 2A). substituents are \u2013CF3 and \u2013NO2, with electron-withdrawing\n Herein, we present the synthesis and characterization of nature) than the other osmium complexes, Os1 and Os2. All\n novel osmium(II) arene compounds of the type [Os(\u03b76-p- other proton resonances of the complexes Os1\u2013Os5 were\n cymene)(N^N\u2032)Cl]PF6, where the N^N\u2032 ligands are BTAT assigned based on their coupling profiles in the 1H\u20131H COSY\n ligands with di\ufb00erent substituents in the R4 position of the and 1H\u20131H-NOESY spectra obtained and by comparing these\n phenyl ring (Scheme 2B). Our goal was to develop potential profiles between di\ufb00erent complexes within the series (Fig. 1\n osmium-based anticancer drugs that can inhibit both the pro- and Fig. S8\u2013S16\u2020).\n liferation of di\ufb00erentiated tumor cells and reduce the tumori-\n genic potential of cancer stem cells (CSCs). Our research Crystal structure by X-Ray di\ufb00raction\n demonstrates the antiproliferative e\ufb00ects of these compounds Suitable single crystals of Os3 and Os5 for X-ray di\ufb00raction\n in both monolayer and 3D spheroid assays across a variety of analysis were obtained by slow di\ufb00usion of diethyl ether into a\n cancer cell lines under dark conditions. This work contributes saturated acetonitrile solution at 5 \u00b0C for 3 days. The mole-\n to understanding the structure\u2013activity relationship and key cular structures of both complexes are shown in Fig. 2.\n aspects of the mechanism of action of these novel osmium Crystallographic data are given in Tables S2 and S5.\u2020 The\n compounds, particularly in rhabdomyosarcoma (RD) cells, structures of complexes were unambiguously confirmed by\n which are associated with one of the most challenging and X-ray di\ufb00raction, supporting our predicted molecular struc-\n hard-to-treat pediatric tumors. ture. Both complexes present the pseudo-octahedral \u201cthree-\n legged piano-stool\u201d geometry, with the metal atom \u03c0-bonded\n to the \u03b76-p-cymene ligand, whereas the benzothiazole-1,2,3-tri-\n Results and discussion azole ligands assume a bidentate chelate coordination mode\n (\u03ba2-N,N\u2032), occupying two coordination positions. The Os\u2013N\n Synthesis and characterization of osmium complexes (Os1\u2013Os5) bond distances (2.064\u20132.112 \u00c5) and Os\u2013Cl (2.3949 \u00c5) are\n The BTAT ligands L1\u2013L5 were obtained following a synthetic within the range reported for osmium half sandwich\n three-steps procedure recently reported (see Schemes S1 and complexes.19,37\u201339 Apart from the cation-hexafluorophosphate\n S2 in ESI\u2020 for further details).35 Yellow solids Os1\u2013Os5 were anion coulombic interaction, the packing in Os3 is organized\n synthesized from the reaction between the dimeric precursor by intermolecular interactions C\u2013H\u22efF (Table S3 and\n [{Os(\u03b76-p-cymene)Cl(\u03bc-Cl)}2] and the corresponding BTAT che- Fig. S24\u2020) and C\u2013H\u22ef\u03c0 interactions (Table S4 and Fig. S25\u2020).\n lating ligand, following similar procedures to those reported However, the packing in Os5 is organized not only by inter-\n for other half-sandwich arene-complexes (Scheme 2B).19,25,37 molecular interactions C\u2013H\u22efX (X = F and Cl, Table S6 and\n Osmium half-sandwich complexes were isolated as pure hexa- Fig. S26\u2020) and C\u2013H\u22ef\u03c0 interactions (Table S8 and Fig. S28\u2020),\n fluorophosphate salts and fully characterized by multinuclear but also by \u03c0\u2013\u03c0 interactions (Table S7 and Fig. S27\u2020).\n NMR spectroscopy (Fig. S1\u2013S16\u2020). The purities of complexes\n were assessed by elemental analysis of C, H, N, and S, Photophysical characterization of the compounds\n ESI-HRMS ( positive mode, Fig. S17\u2013S21\u2020), and it was also con- The UV/Vis absorption spectra of complexes Os1\u2013Os5 (50 \u03bcM)\n firmed that the purities of complexes were higher than 95% were recorded in DMSO and acetonitrile (Fig. 3A, B and\n through RP-HPLC/MS in ACN/H2O (Table S1 and Fig. S22, Table S9\u2020) at room temperature. As can be observed in Fig. 3A\n\n\n This journal is \u00a9 the Partner Organisations 2025 Inorg. Chem. Front., 2025, 12, 1693\u20131715 | 1695\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\nOpen Access Article. Published on 10 January 2025. Downloaded on 5/12/2026 12:50:56 PM.\n\n\n\n\n Scheme 2 (A) Previously reported photoactivable octahedral Ru BTAT complexes (B) synthesis of new osmium arene BTAT complexes Os1\u2013Os5\n investigated in this work.\n\n\n\n and B, all osmium half-sandwich complexes show intense extinction coe\ufb03cients around 5000 M\u22121 cm\u22121. Noteworthy, the\n sharp bands between 260 and 330 nm, with extinction coe\ufb03- Os5 complex undergoes bathochromic displacement of that\n cients around 20 000 M\u22121 cm\u22121 in both solvents, that can be band compared to the rest of the osmium complexes, possibly\n mainly assigned to the 1\u03c0\u2013\u03c0* transition aromatic system of the due to the electron donor nature of its substituent (\u2013NMe2).\n ligands, and other less intense broad bands around 380 nm, The emission spectra of Os1\u2013Os5 (50 \u03bcM) compounds were\n due to metal\u2013ligand charge transfer transitions (1MLCT), with also recorded in DMSO (Fig. 3C). Except for Os4, all the com-\n\n\n 1696 | Inorg. Chem. Front., 2025, 12, 1693\u20131715 This journal is \u00a9 the Partner Organisations 2025\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\nOpen Access Article. Published on 10 January 2025. Downloaded on 5/12/2026 12:50:56 PM.\n\n\n\n\n 1\n Fig. 1 H-NMR stacking spectra (aromatic region) for compounds Os1\u2013Os5 in acetonitrile-d3 along with proton assignment.\n\n\n\n\n Fig. 2 (A) ORTEP plot of the cation of complex Os3. Selected bond lengths (\u00c5) and angles (\u00b0) for Os3: Os\u2013N2: 2.064(2), Os\u2013N1: 2.112(2), Os\u2013Cl:\n 2.3949(7), Os\u2013Cg( p-cymene): 1.6808(12), N2\u2013Os1\u2013N1: 75.15(8), N1\u2013Os\u2013Cl: 81.96(6), N2\u2013Os\u2013Cl: 83.98(6). CCDC reference number is: 2393537. (B)\n ORTEP plot of the cation of complex Os5. Selected bond lengths (\u00c5) and angles (\u00b0) for Os5: Os\u2013N2: 2.072 (2), Os\u2013N1: 2.114(2), Os\u2013Cl: 2.3958(6),\n Os\u2013Cg( p-cymene): 1.6774(11), N2\u2013Os1\u2013N1: 75.29(8), N1\u2013Os\u2013Cl: 82.61 (6), N2\u2013Os\u2013Cl: 83.05(6). CCDC reference number is: 2393536. Cg = the cen-\n troid of the p-cymene ring de\ufb01ned by C1\u2013C6 atoms.\n\n\n\n plexes were found to be emitters in DMSO, with compound the free BTAT ligands.35 To understand the intramolecular\n Os5 highlighted for presenting an intense and broad emission charge transfer (ICT) character of the excited state, the emis-\n band, whose maximum is shifted to longer wavelengths sion spectra of Os5 were recorded in di\ufb00erent solvents at room\n (529 nm). These emission results agree with those obtained for temperature (Fig. 3D). Large and positive solvatofluorochro-\n\n\n This journal is \u00a9 the Partner Organisations 2025 Inorg. Chem. Front., 2025, 12, 1693\u20131715 | 1697\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\nOpen Access Article. Published on 10 January 2025. Downloaded on 5/12/2026 12:50:56 PM.\n\n\n\n\n Fig. 3 UV/Vis absorption spectra of Os1\u2013Os5 (50 \u03bcM) in acetonitrile (A) and DMSO (B) at room temperature. (C) Emission spectra of complexes\n Os1\u2013Os3 (\u03bbex = 310 nm), Os4 (\u03bbex = 320 nm), and Os5 (\u03bbex = 360 nm) in aerated DMSO at room temperature. Normalized emission spectra of Os5 in\n di\ufb00erent solvents (D). Emission spectra of Os5 (\u03bbex = 360 nm, 50 \u03bcM) in DMSO/water mixtures with di\ufb00erent fw (E).\n\n\n\n\n mism indicates considerable ICT characteristics of the com- disappearance of H5 of the corresponding BTAT ligand, which\n pound.40 Os5 shows strong solvatochromic behavior; as was attributed to substituting a proton with deuterium\n solvent polarity increases (e.g., toluene, chloroform, aceto- (Fig. S30\u2013S34\u2020). It is worth mentioning that the ESI-MS spec-\n nitrile, and DMSO), the emission maxima are red-shifted (\u03bbem trum of Os5 in 1 : 2 D2O : DMSO-d6 mixture displayed a peak\n 413, 445, 515, and 529 nm, respectively). On the other hand, corresponding to the ion [M + 1\u2013PF6]+ (Fig. S35\u2020). To further\n the aggregation-caused quenching (ACQ) e\ufb00ect of Os5 was next confirm that the disappearance of the proton in the aforemen-\n evaluated in DMSO/water mixtures with varied water volu- tioned NMR spectra is due to the substitution by deuterium, a\n 1\n metric fractions ( fw). As shown in Fig. 3E, Os5 shows classic H-NMR experiment was performed for Os3, showing the rever-\n ACQ properties, as at 30% water, there has been complete sibility of the process, with details provided in the ESI\n emission extinction.41,42 (Fig. S36\u2020). Based on these results, we can conclude that all\n new half-sandwich Os(II) complexes remained stable and did\n Stability and hydrolysis not hydrolyze over that period under those conditions.\n We next investigated the stability and hydrolysis behavior of Furthermore, hydrolysis of the Os complexes was also studied\n the new BTAT-based osmium(II) since this is one of the poss- by HPLC-MS in mixtures containing a higher percentage of\n ible mechanisms for activating halide osmium arene com- water than previously used. For this purpose, solutions of Os1\u2013\n plexes in their interaction with biological targets.43 Therefore, Os5 were prepared in water (5% DMSO), and HPLC-MS spectra\n we first evaluated the stabilities of Os1\u2013Os5 in dimethyl- were recorded at the initial time and after 24 h of incubation\n sulfoxide (DMSO), a frequently used solvent, to ensure the at room temperature. The results reveal that compounds Os1\u2013\n solubility of compounds in biological media. Stabilities in Os4 are hardly hydrolyzed (Fig. S37\u2013S40\u2020), whereas Os5\n DMSO were checked by UV/Visible spectra, which remained (Fig. S41\u2020) hydrolyzes readily, achieving up to 76% hydrolysis\n unchanged after 48 h. Stability in aqueous media was then at 24 h under these conditions. This is observed by MS from\n evaluated by 1H-NMR spectroscopy by dissolving each Os the peak of interest extracted from HPLC. However, the for-\n complex (1 mM) in 1 : 2 D2O : DMSO-d6 (v/v) and recording mation of adducts with any other biological nucleophiles\n spectra at di\ufb00erent times. The 1H-NMR spectra of complexes cannot be discarded in the cell culture medium. To clarify this\n Os1\u2013Os5 remained almost unaltered after 24 h, except for the point a bit further, we have performed the HPLC-MS studies of\n\n\n 1698 | Inorg. Chem. Front., 2025, 12, 1693\u20131715 This journal is \u00a9 the Partner Organisations 2025\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\n\n the Os complexes (Fig. S42\u2013S46\u2020) dissolved in RPMI culture Table 1 KSV, KB, and n-values of Os1\u2013Os5 complexes with HSA\n medium (5% DMSO). As shown in Fig. S46,\u2020 complex Os5\n hydrolyzed to a lesser extent in these conditions, likely due to Complex KSV \u00d7 105 (M\u22121) KB (M\u22121) \u00d7 105 n\n the high concentration of sodium chloride (6 g L\u22121) present in Os1 0.96 28.58 1.33\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n the RPMI-1640 medium. Os1\u2013Os4 remained unaltered also in Os2 1.13 0.77 0.97\n Os3 1.24 1.39 1.01\n these conditions. Notably, Os5, which undergoes faster hydro- Os4 1.76 15.09 1.21\n lysis, turned out to be the best performer of the series for bio- Os5 2.08 23.85 1.09\n logical applications (vide infra), so it can be concluded that the\n hydrolysis process could play a crucial role in its cancer treat-\n ment properties. Os5 is the complex that incorporates the elec- quenching mechanism. To establish whether the extinction\nOpen Access Article. Published on 10 January 2025. Downloaded on 5/12/2026 12:50:56 PM.\n\n\n\n\n tron donor and steric bulk NMe2 group in the BTAT ligand. mechanism is due to a static or dynamic process, the variation\n in the absorption intensity of HSA in the presence of the com-\n Interaction of half-sandwich osmium complexes with human plexes was studied, as can be seen in Fig. 4A for Os3 and\n serum albumin Fig. S49\u2020 for the rest. The absorption intensity of HSA\n As proteins are present in biological membranes, our next decreased drastically in the presence of complexes Os1\u2013Os5,\n investigations focused on assessing the impact of the investi- suggesting a disturbance in the secondary structure of HSA.\n gated Os compounds on proteins, with Human Serum Thus, the addition of the complexes to the HSA produces\n Albumin (HSA) being the most abundant protein in blood changes in the excited state of the fluorophore and also\n serum.44 The fluorescence of HSA is mainly due to tryptophan induces changes in the absorption spectrum, so the possible\n residues and, to a lesser extent, tyrosine and phenylalanine quenching mechanism induced by the complexes would be a\n residues. Changes in the conformation of HSA, resulting in a static mechanism that has been previously described in the lit-\n noticeable change in fluorescence emission intensity, can erature.45 When the static mechanism takes place, it is\n occur upon binding with the interacting compounds. Given assumed that the complex interacts independently with a set\n the high lipophilicity of the investigated Os compounds, it is of equivalent binding sites in the HSA.46 The binding para-\n reasonable to anticipate that hydrophobic interactions would meters can be determined according to the Scatchard\n be predominant in protein-compound interactions. Previous equation, where KB is the binding constant and \u201cn\u201d is the\n studies have revealed that the surface of HSA contains both number of binding sites per HSA molecule. The linear fit of\n hydrophobic and hydrophilic domains, rendering it a suitable the plot of log(F0 \u2212 F/F) versus log[C] (Fig. S50\u2020) at room temp-\n model for investigating compound interactions. erature allows obtaining KB for the complexes and the values\n To determine the possible interaction of the complexes of n (0.9\u20131.3) indicating that there is only one binding site\n Os1\u2013Os5 with HSA, luminescence quenching experiments were accessible in HSA for complexes Os1\u2013Os5.47\u201349\n carried out by exciting at 295 nm and adding variable amounts As shown in Table 1, complexes Os1 and Os5, which\n (0\u201327 \u00b5M) of complexes Os1\u2013Os5 and HSA (2.7 \u00b5M) at room contain the donor substituents Me and NMe2, respectively, in\n temperature (Fig. 4B for Os3 and Fig. S47\u2020 for the rest) and fol- the R4 position of the phenyl ring of the BTAT ligands, exhibit\n lowing the decrease in luminescence intensity, which usually the highest binding constants to HSA.\n fits the Stern Volmer (SV) equation (Fig. S48\u2020). The Ksv values,\n obtained from the slope of the representation of F0/F against Site-selective binding of complexes on HSA\n [C], are collected in Table 1. The linear fit obtained for the SV To improve the future design of new drugs, there is growing\n quenching analysis for all complexes indicates a single interest in understanding the mechanism of action of metallo-\n\n\n\n\n Fig. 4 Interaction of Os3 with HSA. (A) UV/Visible absorption spectrum of HSA (1 \u03bcM) with the addition of di\ufb00erent equivalents of Os3 (0\u20132 eq.). (B)\n Emission spectrum of HSA (2.7 \u03bcM) in the presence of increasing amounts of Os3 (0\u201327 \u03bcM) (top to bottom gradual increments).\n\n\n\n This journal is \u00a9 the Partner Organisations 2025 Inorg. Chem. Front., 2025, 12, 1693\u20131715 | 1699\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\n\n drugs, including the nature of the binding of metallodrugs to Table 2 Stern\u2013Volmer constant values in the interaction with HSA and\n biomolecules. To identify the location of the binding site of the displacement of warfarin or ibuprofen\n new osmium compounds in the HSA region, competitive\n KSV \u00d7 105 (M\u22121)\n binding experiments were carried out. Albumins have two\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n main drug-binding sites characterized as sites I and II.50 These Interaction with WF IBU\n sites bind drugs selectively. Site I is located in the subdomain Complex HSA displacement displacement\n IIA of HSA and involves the lone tryptophan of the protein Os1 0.96 0.38 0.69\n (214Trp), whereas site II is positioned in subdomain IIIA and Os2 1.13 0.49 0.59\n Os3 1.24 0.48 0.68\n primarily involves the individual amino acid residues 410Arg\n Os4 1.76 0.82 0.94\n and 411Tyr.51 We used warfarin as a marker substance for the Os5 2.08 1.15 1.11\nOpen Access Article. Published on 10 January 2025. Downloaded on 5/12/2026 12:50:56 PM.\n\n\n\n\n specific binding of the investigated Os complexes on the HSA\n molecule site I52 and ibuprofen on-site II.53\n Warfarin fluorescence at \u03bb = 320 nm increases considerably of Os5 is the most advantageous and that this compound dis-\n upon binding to serum albumin due to its interaction with plays the highest inhibitory e\ufb00ect in the cancer cells tested.\n Trp214. This fluorescence decreases if a second compound With the IC50 values ranging from 4.3 to 8.3 \u00b5M, its activity is\n competes for the same site. Thus, the binding of the com- comparable or, in some cases, better than that of the estab-\n plexes is assessed by monitoring the changes in the fluo- lished anticancer drug cisplatin. Representative dose\u2013response\n rescence spectrum when increasing amounts of each of the curves are shown in Fig. S54.\u2020\n complexes are added (0\u201325 \u03bcM) (Fig. 5A for Os3 and Fig. S51\u2020 Besides the tumor cell lines, the human lung fibroblast\n for the rest). Similarly, a decrease in the fluorescence of the MRC5, a non-cancerous cell line, was included in the experi-\n [HSA-IBU] complex upon the addition of increasing amounts ment. Selectivity indexes (SIs) for the osmium complexes were\n of each osmium compound indicates the binding of the com- calculated and are shown in Table 4. The SIs determined for\n plexes to site II of HSA (Fig. 5B and Fig. S52\u2020 for the rest). As Os5 were noticeably higher than those calculated for Os1\u2013Os4,\n observed in Table 2 and Fig. S53,\u2020 compounds Os1\u2013Os4 prefer except Os1 in RD cells. This result indicates that the selectivity\n binding to site II of HSA, as indicated by the higher SV fluo- of Os5 for the chosen cancer cell lines over MRC5 is better\n rescence quenching constants in all instances. In contrast, than that of the remaining agents, including clinically used\n compound Os5 binds indiscriminately to both binding sites, cisplatin. Nevertheless, the SIs of all the compounds and all\n displaying no preference for either. cell lines are higher than 1, i.e., the complexes inhibit the pro-\n liferation of the chosen cancer cells to a greater extent than the\n E\ufb00ect on growth and viability of cancer cells non-cancerous lung fibroblasts. Hence, exceptional SI values\n A panel of six cancer cell lines was employed to examine the obtained especially for Os5 (SI = 5.4\u201310.4) reveal its remarkable\n antiproliferative potential of the new half-sandwich Os com- selectivity for cancer over noncancerous cells, thereby alleviat-\n pounds. The anticancer activity was determined with MTT ing possible toxic side e\ufb00ects \u2013 a foundational prerequisite in\n assay after a 72 h treatment and expressed as IC50 values developing anticancer therapies.\n shown in Table 3. The osmium complexes Os1\u2013Os4 manifested\n medium cytotoxicity with IC50 ranging from 15 to 66 \u00b5M. The Cellular accumulation\n best compound was Os1, carrying the methyl group with IC50 The series of processes leading to cell death commences with\n of 15 \u00b5M in rhabdomyosarcoma (RD) cells. It has shown that the cellular uptake of a compound and its intracellular\n the presence of the dimethylamine substituent in the molecule accumulation. The amounts of osmium localized inside RD\n\n\n\n\n Fig. 5 Emission spectra of HSA-WF (A) and HSA-IBU (B) in the presence of increasing amounts of complex Os3. \u03bbex = 320 nm, [HSA\u2013WF] = 1 : 1 \u03bcM;\n \u03bbex = 280 nm, [HSA\u2013IBU] = 1 : 1 \u03bcM. [Complex Os3]: 0\u201325 \u03bcM (top to bottom gradual increments).\n\n\n\n 1700 | Inorg. Chem. Front., 2025, 12, 1693\u20131715 This journal is \u00a9 the Partner Organisations 2025\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\n\n Table 3 IC50 a values (\u00b5M) determined with MTT assay\n\n RD HCT116 PSN1 MCF7 MDA-231 HeLa MRC5\n\n Os1 15 \u00b1 5 25 \u00b1 2 27 \u00b1 4 27 \u00b1 8 27 \u00b1 5 23 \u00b1 1 91 \u00b1 18\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n Os2 38 \u00b1 3 53 \u00b1 8 66 \u00b1 5 42 \u00b1 5 60 \u00b1 16 46 \u00b1 10 >100\n Os3 24 \u00b1 6 34 \u00b1 3 47 \u00b1 2 45 \u00b1 9 40 \u00b1 1 43 \u00b1 7 76 \u00b1 12\n Os4 43 \u00b1 8 45 \u00b1 9 43 \u00b1 9 45 \u00b1 4 44 \u00b1 7 36 \u00b1 7 69 \u00b1 4\n Os5 4\u00b11 6.6 \u00b1 0.6 7\u00b12 6\u00b11 6\u00b11 8\u00b12 45 \u00b1 3\n Cisplatin 3.1 \u00b1 0.2 8.8 \u00b1 0.6 3.3 \u00b1 0.4 21 \u00b1 2 19 \u00b1 3 15 \u00b1 3 9\u00b12\n a\n Data represents MEAN \u00b1 SD from at least three independent measurements. The cells were treated for 72 h.\nOpen Access Article. Published on 10 January 2025. Downloaded on 5/12/2026 12:50:56 PM.\n\n\n\n\n Table 4 Selectivity indexes (SI)a damental role in their transport and accumulation in tumor\n cells.\n RD HCT116 PSN1 MCF7 MDA-231 HeLa It should also be noted that according to HPLC (Fig. S22\u2020),\n Os1 6.07 3.64 3.37 3.37 3.37 3.96 Os5 is one of the more lipophilic complexes of the series; Os5\n Os2 >2.63 >1.89 >1.52 >2.38 >1.67 >2.17 is the complex that incorporates the electron donor and steric\n Os3 3.17 2.24 1.62 1.69 1.90 1.77\n Os4 1.60 1.53 1.60 1.53 1.57 1.92\n bulk NMe2 group in the BTAT ligand.\n Os5 10.47 6.82 6.34 7.03 7.38 5.42\n Cisplatin 2.90 1.02 2.73 0.43 0.47 0.60 Mechanism of action\n a\n The SIs were calculated as IC50 (MRC5)/IC50 (indicated cancer cell Since Os5 proved to be the most active compound of the new\n line). group of osmium(II) half-sandwich complexes, we chose this\n agent to study the mechanism of action in cancer cells.\n Rhabdomyosarcoma (RD) was selected as a model cell line for\n cells following a 5 h treatment with the new Os complexes two reasons. First, rhabdomyosarcomas are one of the most\n were determined. The results are shown in Table 5, and the frequent sarcomas in childhood and adolescence; they are\n data inspection confirms that the compounds are accumulated highly aggressive and belong to poorly treatable cancers with\n in RD cells with various e\ufb00ectivity. The most abundant intra- survival rates of metastatic and relapsed cases of less than\n cellular osmium was found in the cells exposed to Os5. 20%.54,55 Therefore, it is of high importance to find a satisfac-\n According to Os uptake, the agents may be organized in the tory cure for this kind of cancer. Second, from the selected\n order Os5 > Os1 > Os3 > Os2 > Os4. A comparison of the panel of cancer cells, the RD cell line was the most sensitive to\n results from Table 3 (RD column) and Table 5 discloses a the treatment with the new group of Os(II) complexes, with the\n strong correlation between cellular accumulation of the highest sensitivity to Os5.\n osmium complexes and their antiproliferative activity. It is,\n therefore, reasonable to suggest that cellular accumulation of Localization of Os5 in rhabdomyosarcoma cells\n the investigated osmium complexes plays a significant role in We took advantage of the fact that Os5 possesses fluorescence\n the mechanism of their antiproliferative activity. properties to monitor its cellular accumulation and localiz-\n Transport into cells and accumulation in cells of several ation (Fig. 6). The cells were treated with 12.9 \u00b5M (3 \u00d7 IC50)\n known drugs are mediated by their binding to HSA.44 The Os5 for 3 h, and then confocal imaging was performed using\n comparison of the binding constants of the investigated Os UV laser (Ex/Em = 355 nm/400\u2013485 nm). Brightfield images\n complexes when they bind to HSA (Table 1) and their accumu- were taken to provide information on cell morphology. As\n lation in RD cells (Table 5) shows no correlation. This compari- shown in Fig. 6, the fluorescence signal from Os5 was localized\n son may, therefore, be interpreted to mean that the interaction mainly in the cytoplasm, partly as a weak blue background\n of the investigated Os complexes with HSA does not play a fun- and partly as discrete puncta emanating a strong signal.\n Control non-treated cells produced no fluorescence signal\n under the same laser and detector settings. Thus, the observed\n Table 5 Accumulation of Os complexes in Rhabdomyosarcoma cells. fluorescence may be attributed to Os5, not cellular autofluores-\n The cells were treated with Os complexes at 50 \u00b5M concentrations for\n cence. In the confocal images, some cells gradually adopted\n 5h\n di\ufb00erent morphology (Fig. 6(C)). They lost stretched, adhered\n ng Os/106 cellsa shape and became rounded with apparent blebbing mem-\n branes. At the same time, the discrete blue puncta dis-\n Os1 618 \u00b1 20\n Os2 128 \u00b1 4 appeared, and only a weak blue background remained.\n Os3 462 \u00b1 26 To better reveal the mechanism of action of Os5, we aimed\n Os4 62 \u00b1 15 to detect the specific cellular component with which Os5\n Os5 1645 \u00b1 37\n associates. Understanding the intracellular distribution of a\n a\n The data represent MEAN \u00b1 SD, n = 3. drug is essential for elucidating its mechanism of action, as\n\n\n This journal is \u00a9 the Partner Organisations 2025 Inorg. Chem. Front., 2025, 12, 1693\u20131715 | 1701\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\nOpen Access Article. Published on 10 January 2025. Downloaded on 5/12/2026 12:50:56 PM.\n\n\n\n\n Fig. 6 Localization of Os5 in Rhabdomyosarcoma cells. The cells were exposed to Os5 (12.9 \u00b5M) for 3 h, and then confocal images were taken\n using a UV laser (\u03bbex/\u03bbem = 355/400\u2013485 nm). The signal from Os5 (blue) is merged with the bright\ufb01eld channel. (A) \u2013 control (non-treated cells); (B)\n and (C) \u2013 cells were treated with Os5.\n\n\n\n the drug must localize within the specific intracellular com- that the complex is not selectively accumulated in any particu-\n partment containing its target to exert its therapeutic e\ufb00ect. lar cell organelles. Still, it is distributed among di\ufb00erent orga-\n Therefore, a colocalization assay with Lyso-Tracker and nelles, including partial localization freely in the cytoplasm\n ER-Tracker dyes was performed. The results are shown in (Fig. 6). This suggests that Os5 could a\ufb00ect multiple cellular\n Fig. S55.\u2020 Pearson correlation coe\ufb03cients were calculated from targets simultaneously.\n 30\u201340 images using ImageJ software and are shown in In the attempt to determine the degree of colocalization of\n Fig. S56.\u2020 The correlation coe\ufb03cient for Os5 with Lyso-Tracker Os5 with mitochondria, we obtained poor results since, upon\n (0.65 \u00b1 0.09) was the highest found. Even lower correlation the addition of Os5 to Mito-Tracker-stained cells, the Mito-\n coe\ufb03cient values (0.57 \u00b1 0.07) were found for colocalization of Tracker signal gradually disappeared (not shown). We repeated\n Os5 with endoplasmic reticulum. Both findings indicate a the attempt with TMRE-stained cells. The strong TMRE signal\n moderate/weak correlation.56 The fact that the fluorescence enabled us to capture better the mitochondria in cells exposed\n signal associated with the Os5 complex does not strongly cor- to Os5 (Fig. 7). The correlation coe\ufb03cient for Os5 and TMRE\n relate with any of the trackers used (Fig. S55 and S56\u2020) shows calculated from the images showed only very weak (0.35 \u00b1\n\n\n\n\n Fig. 7 Colocalization of Os5 and TMRE-stained mitochondria in RD cells. The cells were prestained with TMRE and then exposed to Os5 (12.9 \u00b5M)\n for 1 h. Os5 (blue) \u2013 the signal of Os5; TMRE (green) \u2013 the signal of TMRE stained mitochondria; bright\ufb01eld \u2013 bright\ufb01eld channel; merge \u2013 the\n merged signal of the three channels; detail \u2013 the magni\ufb01ed image of merged channels, the cropped area is marked with a square in merge images.\n (A) \u2013 cells exposed to Os5 only; (B) \u2013 the cells prestained with TMRE, non-treated; (C) \u2013 the TMRE prestained cells treated with Os5.\n\n\n\n 1702 | Inorg. Chem. Front., 2025, 12, 1693\u20131715 This journal is \u00a9 the Partner Organisations 2025\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\n\n 0.05) colocalization of Os5 with mitochondria. However, a total Os5 was bound within 24 h). These results further\n closer look at Fig. 7, especially at the detailed images, discloses support the view that the biological action of Os5 may likely be\n that mitochondria undergo significant changes in the pres- related to its binding to proteins and phospholipids and\n ence of Os5. The characteristic tubular network visible in non- damage to these molecules.\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n treated cells (yellow arrow) rapidly disappears upon the\n addition of Os5, and punctate spheres appear (red arrow), Loss of mitochondrial membrane potential\n which finally disappear in time (not shown). The results from colocalization experiments suggest that mito-\n To determine which biomolecules Os5 is associated with, chondria in RD cells are a\ufb00ected by the presence of Os5.\n the cells treated with Os5 were fractionated using a commer- Therefore, we decided to analyze the changes in mitochondrial\n cial FractionPREP\u2122 Cell Fractionation Kit. The amount of Os membrane potential (MMP) inflicted by Os5. In one experi-\nOpen Access Article. Published on 10 January 2025. Downloaded on 5/12/2026 12:50:56 PM.\n\n\n\n\n in each fraction was determined by ICP-MS. Most Os (71 \u00b1 6%) ment (Fig. 8A), the cells were prestained with TMRE and then\n was associated with a membrane/particulate fraction contain- analyzed with flow cytometry immediately after adding Os5 or\n ing phospholipids and membrane proteins. Cytosolic fraction staurosporine (for comparison). The analysis was repeated\n contained 21 \u00b1 4% of Os, whereas 6.3 \u00b1 0.9% was found in the several times within an hour. The graph (Fig. 8A) shows that\n fraction comprising nuclear proteins and nuclear membrane. the presence of Os5 rapidly causes the loss of mitochondrial\n Only a negligible portion of Os was found in the cytoskeleton membrane potential, while the e\ufb00ect of staurosporine is much\n and nuclear DNA containing fraction (1.7 \u00b1 0.3%) (Table S10\u2020). lower (or slower). In the second experiment, the RD cells were\n The results are in agreement with the data from confocal incubated with a series of Os5 concentrations for periods of 1,\n microscopy and indicate that the antiproliferative e\ufb00ect of Os5 2, 5.5, and 24 h and then stained with TMRE. TMRE fluo-\n can be mainly caused by damage to cell membranes ( plasma rescence was analyzed with the flow cytometer. The results\n membrane or membranes of intracellular organelles). shown in Fig. 8B reveal that even at concentrations lower than\n However, interactions with cytosolic biomolecules ( presumably IC50, there is a significant loss of mitochondrial membrane\n proteins) may also play a significant role. potential. Moreover, the graph shows that the MMP decreases\n In pursuit of revealing target biomolecule(s), further experi- within the first two hours following the treatment and then\n ments were performed using cell-free media. Since the vast remains steady for up to 24 h, and that the MMP decrease is\n majority of Os is located in membranes and the cytoplasm of proportional to the Os5 concentration applied. These results,\n the cell, the binding ability of Os5 to biomolecules present in together with the information provided by confocal\n these cellular compartments, i.e., phospholipids (in mem- microscopy, indicate that following the RD cells\u2019 exposure to\n branes), RNA (in the cytoplasm), and proteins (in both mem- Os5, mitochondria in some cells lose their potential and\n brane and cytosol) was assessed as described in Experimental characteristic morphology.\n section. Model molecules HSA, t-RNA, and 18 : 0 DSPC (1,2-dis-\n tearoyl-sn-glycero-3-phosphocholine) were used as models for Comparison of cytotoxicity determination with MTT and\n proteins, RNA, and phospholipids, respectively. We found that neutral red (NR)\n Os5 binds most rapidly to proteins (57.6% of total Os5 present The knowledge that mitochondria are a\ufb00ected by the presence\n in the reaction was bound within 24 h) and phospholipids of Os5 prompted us to assess whether there is a di\ufb00erence in\n (54.5% of total Os5 was bound within 24 h), whereas binding cytotoxicity evaluation by MTT (a method based on the activity\n to RNA was significantly slower, though still notable (18% of of mitochondrial succinate dehydrogenase) and a mitochon-\n\n\n\n\n Fig. 8 The loss of mitochondrial membrane potential. (A) RD cells were prestained with TMRE (100 nM; 30 min) and then aliquoted into three parts.\n The \ufb01rst part remained untreated, staurosporine (2 \u00b5M) was added to the second part, and Os5 (20 \u00b5M) was added to the third part. Flow cytometry\n analysis of TMRE \ufb02uorescence was performed immediately, and further analyses were performed several times within an hour. The graph (A) displays\n relative TMRE \ufb02uorescence depending on the treatment time. (B) RD cells were treated with several concentrations of Os5 (indicated in the graph)\n for 1, 2.5, 5.5, and 24 h, stained with TMRE, and analyzed using \ufb02ow cytometry. The graph (B) displays relative TMRE \ufb02uorescence depending on the\n treatment time.\n\n\n\n This journal is \u00a9 the Partner Organisations 2025 Inorg. Chem. Front., 2025, 12, 1693\u20131715 | 1703\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\n\n Table 6 Cytotoxicity (IC50 valuesa; \u00b5M) of Os5 in RD cells determined RealTime-Glo\u2122 Annexin V apoptosis and necrosis assay. To\n after 24 h and 72 h with MTT or NR gain insight into the chronological aspect of cell death\n induced in RD cells by Os5, we employed the RealTime-Glo\u2122\n 24 h 72 h\n Annexin V Apoptosis and Necrosis Assay. This assay enables\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n MTT 13 \u00b1 2 4.3 \u00b1 0.6 the real-time monitoring of the progression of phosphatidyl-\n NR 13 \u00b1 3 4.0 \u00b1 0.2\n serine exposure on the outer cell membrane during apoptosis\n a and loss of membrane integrity during necrosis. In Fig. 9E, the\n Data represents MEAN \u00b1 SD from at least three independent\n measurements. real-time apoptosis (green) and necrosis (blue) signals for cells\n exposed to necrosis inducer (10% EtOH), apoptotic inducer\n (2 \u00b5M Staurosporine), and four concentrations of Os5 are\nOpen Access Article. Published on 10 January 2025. Downloaded on 5/12/2026 12:50:56 PM.\n\n\n\n\n dria-independent assay based on the uptake of neutral red dye. shown. Neither signal increase was observed in non-treated\n The results of both assays are shown in Table 6 and Fig. S57,\u2020 control cells. The increase in necrotic signal corresponding to\n and they reveal that there is no significant di\ufb00erence between the loss of cell membrane integrity documents ethanol-\n the IC50 values determined by either method after 24- and 72 h induced necrosis. Staurosporine, on the other hand, rapidly\n treatment with Os5. caused apoptosis. The profiles recorded for cells treated with\n Os5 indicate an apoptotic phenotype with the onset of phos-\n Detection of cell death phatidylserine exposure approximately 400 min following the\n Flow cytometry analysis of Annexin V/propidium iodide compound addition. Necrosis appears to be secondary to\n staining. To probe whether apoptosis is the mode of cell death apoptosis. The graph of Os5-treated cells further shows that\n triggered by cell exposure to Os5, we performed the flow cyto- the beginning of phosphatidylserine appearance on the\n metry analysis of RD cells treated with Os5 for 24 h and dual outside of the cell membrane is independent of the complex\n stained with Annexin V and propidium iodide. The densito- concentration. The extent of apoptosis, on the other hand, is\n grams of flow cytometry measurements are shown in Fig. 9A\u2013 concentration-dependent. Along with the previous experi-\n D. Following a 24 h treatment, the cell population gradually ments, the RealTime-Glo\u2122 Annexin V Apoptosis and Necrosis\n translocates from the left bottom quadrant, corresponding to Assay confirm that apoptosis is the dominant mode of cell\n living cells, to the bottom right quadrant, corresponding to death caused in RD cells by Os5.\n early apoptosis, and finally to the upper right quadrant, corres- Activation of caspase-3. Translocation of phosphatidylserine\n ponding to dead/late apoptotic/necrotic cells, in a dose-depen- to the outer cellular surface, as detected by Annexin V binding,\n dent manner. is a feature that is usually predictive of apoptosis. To further\n\n\n\n\n Fig. 9 Mode of cell death. (A\u2013D) Flow cytometry analysis of Annexin V/Propidium iodide dual staining of rhabdomyosarcoma cells treated with Os5\n complex. The cells were treated with several concentrations of Os5 for 24 h. X- and Y-axes indicate Annexin V negative/positive (A\u2212/A+) and\n Propidium iodide negative/positive (PI\u2212/PI+) populations. Left bottom quadrant (A\u2212/PI\u2212) \u2013 living cells; right bottom quadrant (A+/PI\u2212) \u2013 early apop-\n totic cells; left upper quadrant (A\u2212/PI+) \u2013 necrotic cells and right upper quadrant (A+/PI+) \u2013 late apoptotic/necrotic cells. (A) control non-treated\n cells; (B\u2013D) Os5 treated cells; (B) 5 \u00b5M; (C) 10 \u00b5M (D) 20 \u00b5M. (E) RealTime Annexin V Apoptosis and Necrosis Assay. RD cells were seeded in a\n 96-well plate, and the next day, EtOH (10%), Staurosporine (2 \u00b5M), or Os5 (at indicated concentrations) were added to the wells immediately fol-\n lowed by the addition of the kit components. Luminescence (apoptosis) and Fluorescence (necrosis) were measured in a kinetic mode for cca\n 1000 min at 37 \u00b0C.\n\n\n\n 1704 | Inorg. Chem. Front., 2025, 12, 1693\u20131715 This journal is \u00a9 the Partner Organisations 2025\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\n\n characterize the mode of cell death, we determined caspase-3 experiments based on phosphatidylserine exposure on the\n activation in RD cells treated with Os5. As shown in Fig. S58,\u2020 outer leaflet of the plasma membrane (Fig. 9) reveal that apop-\n the treatment of RD with Os5 resulted in the activation of tosis progression to this step is slower than in the case of\n caspase-3 in a concentration-dependent manner. The results staurosporine, which is again dose-dependent. These findings\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n support the finding that Os5 induces apoptotic cell death and suggest that Os5 triggers apoptosis in RD cells by mitochon-\n executes that to a significant extent through a caspase-depen- drial damage.\n dent pathway.\n From the results obtained, it is evident that apoptosis is the Generation of reactive oxygen species (ROS)\n leading cause of death induced in RD cells with Os5. Confocal We examined the generation of ROS in RD cells exposed to the\n images of TMRE-stained mitochondria (Fig. 7) demonstrate Os5 complex. The level of ROS was detected with CellROX\nOpen Access Article. Published on 10 January 2025. Downloaded on 5/12/2026 12:50:56 PM.\n\n\n\n\n notable changes in mitochondrial morphology. Kinetic studies Green reagent. The method is based on the probe emitting\n of the loss of mitochondrial membrane potential (MMP) fluorescence after ROS oxidation. The results are shown in\n (Fig. 8) showed that the mitochondrial membrane integrity is Fig. 10. The RD cells treated with Os5 exhibited elevated fluo-\n disrupted within minutes following the addition of Os5. This rescence compared to the untreated control. As shown in the\n phenomenon is strictly compound dose-dependent. The graph, the production of ROS induced with Os5 was concen-\n tration-dependent. Os5 at a concentration corresponding to its\n 3 \u00d7 IC50 value induced ROS generation approximately twice the\n level of untreated control, whereas menadione used as positive\n control triggered ROS to a significantly higher level (cca 6-fold\n of control level). Overall, this result indicates a relatively low\n ROS production induced by Os5 in RD cells that might, to a\n certain extent, contribute to its anticancer activity.\n\n The e\ufb00ect of Os5 on cell cycle\n Drug treatment often induces modulation of cell cycle pro-\n gression. Hence, we analyzed the cell cycle perturbation in\n RD cells exposed to Os5 (Fig. 11). The cells were treated with\n Os5 at concentrations corresponding to 1\u00d7 and 2 \u00d7 IC50\n values for 24 h. A close inspection of the distribution of the\n treated RD cells into individual cell cycle phases indicates\n that a significant cell count is arrested in the G1 phase com-\n Fig. 10 ROS production. Quanti\ufb01cation of ROS produced in RD cells\n pared to untreated control cells. While 40% of untreated cells\n exposed to Os5 and menadione ( positive control). The cells were\n treated with three concentrations of Os5 \u2013 4.3 \u00b5M (1 \u00d7 IC50), 8.6 \u00b5M (2 \u00d7 are in the G1 phase, 69 and 72% of cells treated with Os5 at\n IC50), and 12.9 \u00b5M (3 \u00d7 IC50) for 3 h or with 100 \u00b5M menadione for 1 h. the two concentrations are in the G1 phase, respectively. The\n The ROS production was evaluated with CellRox\u00ae-green reagent on BD increase in the G1 population occurs at the expense of both\n FACS Verse \ufb02ow cytometer. The bars in the graph show relative \ufb02uor- the S and G2/M phases but mostly the S phase, where there\n escence (vs. untreated control) and represent MEAN \u00b1 SD from three\n are only 18 and 13% cells (vs. 41% of untreated control),\n independent experiments. The results were subjected to statistical ana-\n lysis using an unpaired Student t-test, and statistically signi\ufb01cant di\ufb00er- respectively. The results reveal that Os5 arrests RD cells in the\n ences from untreated control are marked with asterisks with *p \u2264 0.05 G1 phase and significantly reduces cell portion (vs. control)\n or **p \u2264 0.01. in the S phase.\n\n\n\n\n Fig. 11 Cell cycle distribution. RD cells were treated with Os5 at concentrations corresponding to 1\u00d7 and 2 \u00d7 IC50 for 24 h. Propidium iodide was\n used as a nuclear staining. (A) nontreated control; (B) cells treated with 4.3 \u00b5M Os5; (C) cells treated with 8.6 \u00b5M Os5; (D) bar graph of cell popu-\n lations (%) in individual cell cycle phases. Red \u2013 G1; blue striped \u2013 S; green \u2013 G2/M. (A\u2013C) representative images, (D) \u2013 the graph shows the results\n of 3 measurements (MEAN \u00b1 SD). The results were subjected to statistical analysis using an unpaired Student t-test, and statistically signi\ufb01cant di\ufb00er-\n ences from untreated control are marked with asterisks with *p \u2264 0.05 or **p \u2264 0.01.\n\n\n\n This journal is \u00a9 the Partner Organisations 2025 Inorg. Chem. Front., 2025, 12, 1693\u20131715 | 1705\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\n\n In the mechanistic section (vide supra), we focused on clari-\n fying the mechanism of action of Os5. At the reviewer\u2019s\n request, we clarify why we did not use cisplatin for positive\n control in the mechanistic studies in the following paragraph.\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n The results described in the present study clearly show that\n the mechanism of action of the investigated Os complexes is\n markedly di\ufb00erent from that of cisplatin. The major mecha-\n nism of cisplatin activity is generally accepted to be related to\n damage to nuclear DNA, to which cisplatin binds covalently\n (coordinatively). The resulting adducts then inhibit replication\nOpen Access Article. Published on 10 January 2025. Downloaded on 5/12/2026 12:50:56 PM.\n\n\n\n\n and especially transcription.57 We did not observe this mecha-\n nism for Os complexes; on the contrary, it is clear that the\n action is related to damage to mitochondrial membranes, thus\n impacting mitochondrial function in cancer cells. This mecha-\n nism was not observed for cisplatin.\n\n Cytotoxic/antiproliferative e\ufb00ect in RD\u00b7CD133\u2212 and\n RD\u00b7CD133+ spheroids\n In cancer treatment, a population of cells called cancer stem\n cells (CSC) represents a small but dangerous cell fraction.58\n Whereas the bulk of tumor cells is e\ufb00ectively eradicated, CSCs\n escape the treatment and serve as a reservoir of resistant\n cancer cells that reinitiate secondary or tertiary tumors. The\n ine\ufb03ciency of current anticancer drugs to successfully and Fig. 12 Anticancer activity in RD\u00b7CD133+ and RD\u00b7CD133\u2212 cells\n expressed as IC50 values (\u00b5M). 96-Hour-old spheroids were incubated\n permanently eliminate tumors is, to a major extent, due to with the Os compounds for 72 h, and cell viability was then determined\n their failure to eliminate CSC. It is, therefore, worth the e\ufb00ort with Cell TiterGlo 3D assay. SAL \u2013 salinomycin; CF \u2013 cyclophosphamide.\n to detect compounds that can kill CSCs to the same or even The asterisks denote signi\ufb01cant di\ufb00erences as determined with the\n higher extent than the non-stem cells.59\u201363 In this work, we Student t-test ( p < 0.05).\n showed that the Os5 complex significantly damages mitochon-\n dria (Fig. 7 and 8). The ability of the complexes to e\ufb00ectively\n damage mitochondria suggests that they might also be obtained for salinomycin and cyclophosphamide. Although\n e\ufb00ective in CSCs since mitochondria are the determinant of salinomycin was e\ufb00ective at lower concentrations than the\n the function and fate of CSCs, playing core roles in the regu- tested Os complexes, this finding does not necessarily dimin-\n lation of CSC stemness maintenance and di\ufb00erentiation,64 ish the potential importance of Os5 for treating tumor cells,\n likely due to the higher mitochondrial load. Therefore, we including CSCs.65\u201367 Furthermore, it should be pointed out\n decided to examine the e\ufb03cacy of the new osmium com- that Os5 is about two orders of magnitude more e\ufb00ective than\n pounds in inhibiting the growth and viability of the sarcoma cyclophosphamide, a drug used to treat rhabdomyosarcoma in\n stem cell subpopulation. The isolation of sarcoma CSCs was clinic.68,69 Moreover, cancer stem cells are known to be inher-\n based on the CSC\u2019s expression of the CD133 marker. CD133 is ently resistant to cisplatin.70,71 The finding that osmium com-\n a transmembrane protein known as Prominin1 and has been plexes demonstrated comparable activity in both CSC-depleted\n previously used as a distinct marker for selecting CSCs in and CSC-enriched RD cells suggests their potential to target\n rhabdomyosarcoma.63 We sorted the RD cells into CD133+ both di\ufb00erentiated cancer cells and cancer stem cells concur-\n (expressing CD133) and CD133\u2212 (nonexpressing CD133) rently. This characteristic could represent a significant advan-\n groups. Both populations were cultured for 96 h until rhabdo- tage in chemotherapy, as it may reduce the reliance on che-\n spheres of the desired size (211 \u00b1 23 nm in diameter) were motherapeutics tailored to specific tumor cell subtypes when\n grown. The spheroids were then treated with the new osmium used in combination.\n complexes for another 72 h. Viability at the end of the incu-\n bation period was determined with Cell TiterGlo 3D assay. Confocal microscopy of RD\u00b7CD133+ spheroids\n Representative dose\u2013response curves are shown in Fig. S59.\u2020 To visualize the e\ufb00ect of Os5 on RD\u00b7CD133+ spheroids, we\n Cyclophosphamide, salinomycin, and cisplatin were stained untreated and treated representative spheroids with\n included in the assessment for comparative purposes. The Calcein AM (live-cells label), propidium iodide (stains cells\n results expressed as IC50 values in Fig. 12 and Table S10\u2020 indi- with disturbed membrane integrity) and Hoechst 33258 (stains\n cate that both RD populations (CD133+ and CD133\u2212) were sen- all cell nuclei). Representative images are shown in Fig. 13.\n sitive to the newly synthesized Os compounds to a similar The images reveal that spheroids exposed to Os5 di\ufb00er from\n extent. In some cases (Os1 and Os3), the agent was more untreated control in size, integrity, and propidium iodide\n potent in the RD CD133+ fraction. Similar results were intensity. The treated samples are smaller than the control,\n\n\n 1706 | Inorg. Chem. Front., 2025, 12, 1693\u20131715 This journal is \u00a9 the Partner Organisations 2025\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\nOpen Access Article. Published on 10 January 2025. Downloaded on 5/12/2026 12:50:56 PM.\n\n\n\n\n Fig. 13 Confocal microscopy of RD spheroids exposed to Os5. The spheroids were stained with Hoechst 33258 dye (Hoechst), Calcein AM, and pro-\n pidium iodide. Merge \u2013 all three channels merged. (A) \u2013 Untreated control, (B) \u2013 spheroid treated with Os5 (5 \u00b5M, 72 h), (C and D) \u2013 spheroids\n treated with Os5 (10 \u00b5M, 72 h). Individual pictures were obtained as the maximal projection from 10 z-stacks. Representative images.\n\n\n\n and spheroid disintegration is apparent in the case of the N^N\u2032 ligands based on the 1-aryl-4-benzothiazolyl-1,2,3-triazole\n 10 \u00b5M concentration applied. Meanwhile, in untreated spher- sca\ufb00old. The molecular structures of Os3 and Os5 were con-\n oid, a small portion of cells displays propidium iodide positiv- firmed via X-ray di\ufb00raction. Notably, Os5 exhibits a bathochro-\n ity, and markedly increased cell count with compromised mic shift in its absorption band compared to the other\n membrane integrity (PI) is present in treated spheroids. The osmium complexes, likely due to the electron-donating pro-\n quantification of the ratios of Calcein AM fluorescence inten- perties of its substituent (\u2013NMe2). Additionally, HPLC-MS\n sity to PI fluorescence intensity is shown in Fig. S60.\u2020 The studies show that Os5 undergoes hydrolysis without losing its\n graph shows that while the green and red channel intensity BTAT ligand and has the highest cellular accumulation in RD\n ratio for untreated control is 3.2, it is only cca 1.3 for treated cancer cells.\n spheroids. Significantly more dead cells are present in treated The new Os(II) half-sandwich complexes demonstrated\n spheroids than in control, and the treated spheroids are moderate antiproliferative activity across six cancer cell lines,\n smaller, and their integrity is impaired. with greater potency compared to non-cancerous lung fibro-\n blasts. Os5, which contains a dimethylamine substituent, was\n the most active compound, showing comparable or superior\n Conclusions activity to cisplatin, a widely used anticancer drug. Our find-\n ings also highlight that cellular accumulation is a crucial\n In conclusion, we have designed a series of pseudo-octahedral factor influencing the antiproliferative activity of these Os com-\n arene Os(II) complexes, Os1\u2013Os5, with the general formula [(\u03b76- plexes. Although it is known that several drugs rely on binding\n p-cym)Os(BTAT)Cl]+, where BTAT represents various chelating to HSA for cellular uptake and accumulation, our data suggest\n\n\n This journal is \u00a9 the Partner Organisations 2025 Inorg. Chem. Front., 2025, 12, 1693\u20131715 | 1707\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\n\n that HSA does not play a significant role in the transport of underscores the largely untapped potential of metal-based\n these Os complexes into tumor cells. complexes for CSC-targeted chemotherapy and o\ufb00ers insights\n Further investigations focused on the mechanism of action into the mechanisms and targets involved in osmium-induced\n (MoA) of the investigated Os complexes, with Os5 serving as toxicity.\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n the representative molecule. We selected RD cells for these\n studies, as they were the most responsive to Os complexes, and\n rhabdomyosarcoma is a highly aggressive cancer with a poor Experimental section\n prognosis for metastatic and relapsed cases, with survival rates\n below 20%.54,55 Reagents and chemicals\n Our initial goal was to identify the specific cellular com- Synthesis-grade solvents were employed in all cases.\nOpen Access Article. Published on 10 January 2025. Downloaded on 5/12/2026 12:50:56 PM.\n\n\n\n\n ponent with which Os5 interacts. Colocalization assays Deuterated solvents were purchased from Euriso-top.\n revealed that Os5 may target multiple cellular components OsCl3\u00b73H2O, \u03b1-terpinene, and ammonium hexafluoro-\n simultaneously, potentially providing an advantage in its bio- phosphate were obtained from Merck (Madrid, Spain). [Os(\u03b76-\n logical e\ufb00ects by making it more di\ufb03cult for cells to develop p-cymene)Cl2]2 was prepared as previously reported,75 and ben-\n resistance. However, these experiments also indicated that Os5 zothiazolyl-1,2,3-triazole derivatives (L1\u2013L5) were synthesized\n impacts mitochondrial function in RD cells, causing loss of following a synthetic procedure published by some of us\n membrane potential and changes in morphology. (Scheme S1\u2020).29 The purities \u226595% of the synthesized com-\n Annexin V/propidium iodide staining of RD cells treated plexes used for biological evaluation were determined by\n with Os5 confirmed that apoptosis, driven by a caspase-depen- RP-HPLC.\n dent pathway, is the primary mode of cell death induced by Preparation of new Os(II) complexes. [Os(\u03b76-p-cymene)Cl2]2\n Os5. Additionally, cell cycle analysis in RD cells revealed that (79 mg, 0.1 mmol) and the corresponding N,N ligand\n Os5 increases the G1 phase population while reducing the S (0.2 mmol), previously synthesized, were dissolved in MeOH\n and G2/M phase populations, indicating that Os5 may inhibit and stirred at reflux for 2 h. Then, ammonium hexafluoro-\n cancer cell proliferation by preventing progression through the phosphate was added to the solution and stirred at room\n DNA synthesis and mitosis phases. temperature for 30 minutes. After some time, the solvent was\n Previous studies on osmium-based anticancer complexes removed under reduced pressure, and the crude product was\n have shown their antiproliferative e\ufb00ects can be mediated recrystallized with dichloromethane/ethylic ether to obtain a\n through pathways involving reactive oxygen species (ROS) pro- pure yellow solid.\n duction. In our study, elevated ROS levels were detected in RD [Os(\u03b76-p-cymene)L1Cl]PF6 (Os1). Yellow solid. Isolated yield:\n cells treated with Os5, suggesting that ROS production may 53%. 1H NMR (401 MHz, Acetonitrile-d3) \u03b4 9.26 (s, 1H),\n contribute to its mechanism of action. 8.30\u20138.23 (m, 2H), 7.92\u20137.83 (m, 3H), 7.79 (ddd, J = 8.2, 7.2,\n The activity of these Os complexes was further demon- 1.1 Hz, 1H), 7.56\u20137.51 (m, 2H), 6.55\u20136.50 (m, 1H), 6.47 (dd, J =\n strated in both 2D monolayer cultures and 3D spheroids, the 5.8, 1.1 Hz, 1H), 6.35\u20136.28 (m, 1H), 6.25\u20136.18 (m, 1H), 2.49 (m,\n latter of which more closely resemble solid tumors and serve 4H), 2.29 (s, 3H), 1.01 (d, J = 6.9 Hz, 3H), 0.92 (d, J = 7.0 Hz,\n as a promising in vitro model. One challenge with convention- 3H). 13C NMR (101 MHz, CD3CN) \u03b4 160.3, 150.2, 144.6, 142.9,\n al anticancer metallopharmaceuticals is their ine\ufb03cacy 134.7, 134.5, 131.7, 130.3, 129.4, 126.3, 125.2, 124.1, 122.2,\n against cancer stem cells (CSCs), which often survive chemo- 98.6, 97.5, 79.8, 76.0, 74.0, 73.0, 32.0, 23.0, 22.1, 21.3, 19.2.\n therapy, leading to relapse and metastasis. For this reason, we ESI-MS ( positive ion. mode): m/z = 653.1226 [M \u2212 PF6]+, calcd\n prepared 3D spheroids from CSC-enriched RD cells and found m/z: 653.1182. Anal. calcd for C26H26ClF6N4OsPS: C, 39.17; H,\n that the Os complexes were not only e\ufb00ective against di\ufb00eren- 3.29; N, 7.03; S, 4.02. Found: C, 39.24; H, 3.11; N, 6.96; S, 4.09\n tiated RD cells but also against RD CSCs. This may be related (%).\n to the ability of the investigated Os complexes to increase [Os(\u03b76-p-cymene)L2Cl]PF6 (Os2). Yellow solid. Isolated yield:\n intracellular ROS levels. CSCs have been shown to be hypersen- 82%. 1H NMR (600 MHz, Acetonitrile-d3) \u03b4 9.25 (s, 1H),\n sitive to ROS, as they maintain lower ROS levels than bulk 8.28\u20138.24 (m, 2H), 8.04\u20137.97 (m, 2H), 7.89 (ddd, J = 8.5, 7.1, 1.2\n di\ufb00erentiated cancer cells,72 which helps them elude thera- Hz, 1H), 7.81\u20137.76 (m, 1H), 7.51\u20137.44 (m, 2H), 6.53 (d, J = 5.7\n peutic interventions. Targeting the redox state within CSCs Hz, 1H), 6.47 (d, J = 6.0 Hz, 1H), 6.31 (d, J = 5.7 Hz, 1H), 6.22 (d,\n with drugs capable of elevating the ROS level and thus disrupt- J = 5.8 Hz, 1H), 2.50 (sept, J = 6.9 Hz, 1H), 2.28 (s, 3H), 1.02 (d, J\n ing their survival advantage is, therefore, considered a promis- = 6.9 Hz, 3H), 0.92 (d, J = 6.9 Hz, 3H). 13C NMR (151 MHz,\n ing strategy for cancer treatment.73,74 CD3CN) \u03b4 165.5, 163.8, 160.2, 150.2, 144.8, 134.7, 133.2, 133.1,\n Importantly, Os5 exhibited anti-CSC properties, marking 130.3, 129.4, 126.4, 125.2, 125.0, 124.9, 124.1, 98.6, 97.7, 79.8,\n the first report of an osmium-based compound showing 76.1, 74.1, 73.2, 32.1, 23.0, 22.1, 19.2. ESI-MS (positive ion\n activity against CSC-enriched RD cells. Notably, the CSC-target- mode, CH3CN): m/z = 657.0977 [M \u2212 PF6]+, calcd m/z: 657.0931.\n ing potency of Os5 rivals some of the most CSC-selective com- Anal. calcd for C25H23ClF7N4OsPS: C, 37.48; H, 2.89; N, 6.99; S,\n pounds identified thus far. Given the critical need for CSC- 4.00. Found: C, 37.43; H, 2.70; N, 6.67; S, 3.93 (%).\n specific therapies to prevent cancer relapse and metastasis, the [Os(\u03b76-p-cymene)L3Cl]PF6 (Os3). Yellow solid. Isolated yield:\n preclinical potential of Os5 is highly promising. This study 62%. 1H NMR (300 MHz, Acetonitrile-d3) \u03b4 9.40 (s, 1H),\n\n\n 1708 | Inorg. Chem. Front., 2025, 12, 1693\u20131715 This journal is \u00a9 the Partner Organisations 2025\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\n\n 8.31\u20138.28 (m, 1H), 8.28\u20138.24 (m, 1H), 8.20 (d, J = 8.6 Hz, 2H), Photophysical characterization. UV/Vis spectroscopy was\n 8.11\u20138.04 (m, 2H), 7.90 (ddd, J = 8.5, 7.2, 1.3 Hz, 1H), 7.80 performed on a PerkinElmer Lambda 750 S spectrometer with\n (ddd, J = 8.2, 7.2, 1.1 Hz, 1H), 6.54 (d, J = 5.7 Hz, 1H), 6.49 (d, J operating software. Solutions of all complexes were prepared\n = 5.8 Hz, 1H), 6.33 (d, J = 5.5 Hz, 1H), 6.24 (d, J = 5.8 Hz, 1H), in acetonitrile, DMSO at 10 \u03bcM. The emission spectra were\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n 2.50 (sept, J = 6.8 Hz, 1H), 2.29 (s, 3H), 1.02 (d, J = 6.9 Hz, 3H), obtained with a Horiba Jobin Yvon Fluorolog 3-22 modular\n 0.93 (d, J = 6.9 Hz, 3H). 13C NMR (75 MHz, CD3CN) \u03b4 156.0, spectrofluorometer with a 450 W xenon lamp. Measurements\n 150.2, 145.0, 139.4, 134.7, 133.0, 132.6, 130.4, 129.5, 128.7, were performed in a right-angled configuration using 10 mm\n 128.6, 128.6, 128.5, 126.4, 125.2, 124.1, 122.9, 98.7, 97.8, 79.8, quartz fluorescence cells for solutions at 298 K.\n 76.2, 74.2, 73.2, 32.0, 23.0, 22.1, 19.1. ESI-MS ( positive ion X-Ray structure determinations. Single crystals of Os3 and\n mode, CH3CN): m/z = 707.0938 [M \u2212 PF6]+, calcd m/z: Os5 suitable for X-ray di\ufb00raction were obtained by slow\nOpen Access Article. Published on 10 January 2025. Downloaded on 5/12/2026 12:50:56 PM.\n\n\n\n\n 707.0899. Anal. calcd for C26H23ClF9N4OsPS: C, 36.69; H, 2.72; di\ufb00usion of diethyl ether in acetonitrile. Intensities were regis-\n N, 6.58; S, 3.77 (%). Found: C, 36.54; H, 2.58; N, 6.67; S, 3.68 tered at low temperatures on a Bruker D8QUEST di\ufb00ractometer\n (%). using monochromated Mo K\u03b1 radiation (\u03bb = 0.71073 \u00c5) for the\n [Os(\u03b76-p-cymene)L4Cl]PF6 (Os4). Yellow solid. Isolated yield: compounds. Absorption corrections were based on multi-\n 71%. 1H NMR (401 MHz, Acetonitrile-d3) \u03b4 9.43 (s, 1H), scans ( program SADABS).76 Structures were refined anisotropi-\n 8.63\u20138.49 (m, 2H), 8.31\u20138.25 (m, 2H), 8.25\u20138.21 (m, 2H), 7.91 cally using SHELXL-2018.77 Hydrogen atoms were included\n (ddd, J = 8.4, 7.2, 1.2 Hz, 1H), 7.81 (ddd, J = 8.3, 7.2, 1.1 Hz, using rigid methyl groups or a riding model.\n 1H), 6.55 (d, J = 5.7 Hz, 1H), 6.49 (d, J = 5.9 Hz, 1H), 6.33 (d, J = Special features. The CF3 ligand in Os3 is disordered over\n 5.7 Hz, 1H), 6.25 (d, J = 5.8 Hz, 1H), 2.51 (sept, J = 6.9 Hz, 1H), two positions, ca.: 44 : 56%.\n 2.29 (s, 3H), 1.03 (d, J = 6.8 Hz, 3H), 0.93 (d, J = 6.9 Hz, 3H).\n 13\n C NMR (101 MHz, CD3CN) \u03b4 159.9, 150.2, 149.8, 145.2, 140.7, Interaction with biomacromolecules\n 134.8, 130.4, 129.6, 126.8, 126.5, 125.3, 124.2, 123.2, 98.8, 98.0, Reaction with human serum albumin (HSA). The stock solu-\n 79.9, 76.3, 74.3, 73.4, 32.1, 23.0, 22.1, 19.2. ESI-MS ( positive tion of HSA was prepared by dissolving the HAS in 50 mM\n ion mode, CH3CN): m/z = 684.0900 [M \u2212 PF6]+, calcd m/z: Tris-HCl, 100 mM NaCl bu\ufb00er of pH 7.4. The concentration of\n 684.0876. Anal. calcd for C25H23ClF6N5O2OsPS: C, 36.26; H, HSA was determined by using \u03b5278 = 36 000 M\u22121 cm\u22121.78\n 2.80; N, 8.46; S, 3.87 (%). Found: C, 36.42; H, 2.68; N, 8.47; S, Quantitative analyses of the interaction between Os1\u2013Os5 com-\n 3.74 (%). plexes and HSA were performed by fluorimetric titration. A\n [Os(\u03b76-p-cymene)L5Cl]PF6 (Os5). Yellow solid. Isolated yield: 3.0 mL portion of aqueous protein solution (2.5 \u03bcM) was\n 79%. 1H NMR (401 MHz, Acetonitrile-d3) \u03b4 9.11 (s, 1H), titrated by successive additions of complex solution (0\u201325 \u03bcM).\n 8.29\u20138.21 (m, 2H), 7.89 (ddd, J = 8.6, 7.3, 1.2 Hz, 1H), The mixing solution was stirred for each addition and allowed\n 7.81\u20137.73 (m, 3H), 6.96\u20136.90 (m, 2H), 6.51 (d, J = 5.7 Hz, 1H), to stand for 5 min. Fluorescence intensities were then\n 6.47 (d, J = 5.8, 1H), 6.30 (d, J = 5.7, 1H), 6.19 (d, J = 5.7, 1H), measured at excitation wavelengths of \u03bb = 295 nm. The width\n 3.07 (s, 6H), 2.49 (sept, J = 6.8 Hz, 1H), 2.29 (s, 3H), 1.00 (d, J = of the excitation and emission slit was set at 5 nm, and the\n 6.9 Hz, 3H), 0.92 (d, J = 6.9 Hz, 3H). emission fluorescence spectra were recorded in the wavelength\n 13\n C NMR (151 MHz, CD3CN) \u03b4 160.5, 153.0, 150.2, 144.3, range \u03bb = 305\u2013570 nm.\n 134.5, 130.2, 129.3, 125.5, 125.2, 125.0, 124.0, 123.2, 113.1, Site marker competitive experiments. Using fluorescence\n 98.6, 97.3, 79.7, 75.9, 73.9, 72.9, 40.5, 32.1, 23.0, 22.2, 19.2. titration techniques, binding location studies between com-\n ESI-MS (positive ion mode, CH3CN): m/z = 682.1460 [M \u2212 PF6]+, plexes and HSA were quantified in the presence of two site\n calcd m/z: 682.1447. Anal. calcd for C25H23ClF6N5O2OsPS: C, markers (ibuprofen and warfarin). Warfarin as Marker for Site\n 39.25; H, 3.46; N, 8.48; S, 3.88. Found: C, 39.26; H, 3.46; N, 8.36; I. Warfarin was used as the site probe in the displacement\n S, 3.82 (%). studies. To begin, equimolar mixes of protein and warfarin\n were prepared, fully mixed, and allowed to equilibrate for one\n hour at room temperature. After transferring 3 mL aliquot of\n Methods and instrumentation the solution to the spectrofluorimetric cell, it was titrated by\n Nuclear magnetic resonance (NMR) spectroscopy. The 1H, successive additions of complexes Os1\u2013Os5. At each titration\n 13\n C{1H}, and bidimensional NMR spectra were recorded on a stage, the resulting solutions were thoroughly mixed and then\n Bruker AC 300E, Bruker AV 400, or Bruker AV 600 NMR spectro- let stand for 20 minutes. A 320 nm excitation wavelength was\n meter, and chemical shifts were determined by reference to chosen. The emission and excitation slit widths were adjusted\n the residual 1H and 13C{1H} solvent peaks. to 5 nm, and the wavelength range in which the emission fluo-\n Elemental analysis. The C, H, N, and S analyses were per- rescence spectra were recorded was 340\u2013550 nm. Ibuprofen as\n formed with a Carlo Erba model EA 1108 microanalyzer with Marker for Site II. The procedure was the same as in the dis-\n EAGER 200 software. placement experiments using the site probe warfarin. A\n Mass spectrometry (MS). ESI mass ( positive mode) analyses 280 nm excitation wavelength was chosen, the emission and\n were performed on an RP/MS TOF 6220. The isotopic distri- excitation slit widths were adjusted to 5 nm, and the wave-\n bution of the heaviest set of peaks matched very closely to that length range in which the emission fluorescence spectra were\n calculated for formulating the complex cation in every case. recorded was 290\u2013540 nm.\n\n\n This journal is \u00a9 the Partner Organisations 2025 Inorg. Chem. Front., 2025, 12, 1693\u20131715 | 1709\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\n\n Cell lines, culture conditions, and stock solution Colocalization studies were conducted under similar con-\n Human rhabdomyosarcoma cells (RD) were obtained from the ditions. The cells were prestained with LysoTracker\n ATCC (Manassas, VA, USA), human colorectal carcinoma cells (LysoTracker\u2122 Green DND-26, Invitrogen; 75 nM, 37 \u00b0C, 2 h),\n (HCT116) and human breast cancer cells (MCF7) were kindly ER-Tracker (ER-Tracker\u2122 Red, Invitrogen; 1 \u00b5M/HBSS, 37 \u00b0C,\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n provided by Prof. B. Keppler from University of Vienna 30 min), or Tetramethylrhodamine ethyl ester (TMRE,\n (Austria), human pancreatic adenocarcinoma cells (PSN1), Invitrogen; 0.1 \u00b5M, 37 \u00b0C, 30 min) and then Os5 was added to\n human highly invasive breast carcinoma cells (MDA-MB-231), indicated concentrations and the cells were subjected to con-\n human cervix carcinoma cells (HeLa) and human cells derived focal microscopy with Leica CM SP5 confocal microscope.\n from normal lung tissue (MRC5) were purchased from ECACC Sequential scanning was applied (Os5 \u2013 \u03bbex/\u03bbem = 355/\n (Salisbury, UK). RD and PSN1 cells were cultured in RPMI 400\u2013485 nm; LysoTracker \u2013 \u03bbex/\u03bbem = 504/511\u2013585 nm;\nOpen Access Article. Published on 10 January 2025. Downloaded on 5/12/2026 12:50:56 PM.\n\n\n\n\n 1640 medium (Biosera, Boussens, France) and the other cells ER-Tracker (\u03bbex/\u03bbem = 587/615\u2013660 nm); or TMRE \u2013 \u03bbex/\u03bbem =\n were grown in DMEM medium (high glucose, 4.5 g L\u22121, PAA, 549/574\u2013620 nm).\n Pasching, Austria); both media were supplemented with genta- Fractionation of rhabdomyosarcoma cells\n mycin (50 mg mL\u22121, Serva, Heidelberg, Germany) and 10%\n heat-inactivated fetal bovine serum (PAA, Pasching, Austria). RD cells were seeded in Petri dishes (2 \u00d7 06 cells per dish),\n Medium for MRC5 cells was further supplemented with 1% incubated overnight, and exposed to the tested compound for\n nonessential amino acids (Sigma-Aldrich, Prague, Czech 5 h. The cells were then harvested, washed, and pelleted. The\n Republic). The cells were cultured in a humidified atmosphere pellets were consequently processed with a FractionPREP\u2122\n (37 \u00b0C; 5% CO2) and subcultured twice a week. For in cellulo Cell Fractionation kit (BioVision, CA, USA) following the\n experiments, the stock solutions of Os complexes were freshly manufacturer\u2032s instructions. The individual fractions were\n prepared in DMSO and subsequently diluted to the respective lyzed with HCl, and osmium content was measured using the\n cell-culture medium. The final concentration of DMSO in bio- ICP-MS system.\n logical experiments did not exceed 0.5% (v/v). For the negative Cell-free determination of Os5 binding to HSA, tRNA, and\n controls, a matched DMSO concentration was used. phospholipid\n\n Cytotoxic/antiproliferative e\ufb03ciency The experiment was performed in PBS. Os5 (200 \u00b5M) was incu-\n bated with HSA (1 : 1), t-RNA (Os : nucleotide ratio = 0.1) and\n The cells were seeded at a proper density \u2013 2 \u00d7 10 cells per\n 3\n 18 : 0 DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) (1 mg\n well (RD, HCT116, PSN1), 3 \u00d7 103 cells per well (MCF7,\n mL\u22121) at 37 \u00b0C for 24 h. Os5 (200 \u00b5M) sample was used as a\n MDA-MB-231, HeLa), and 5 \u00d7 103 cells per well (MRC5) in\n control. Control, HSA, and t-RNA samples were then filtered\n 96-well plates one day before the treatment. The cells were\n through an Amicon\u00ae Ultra centrifugal filter device (3 K). DSPC\n treated with a series of concentrations of the tested com-\n from the DSPC sample was extracted with chloroform. Os5\n pounds in DMEM or RPMI medium. After 72 h of treatment,\n content in the filtrates and water phase was determined spec-\n an MTT assay was performed to determine the inhibition\n trophotometrically. Os5 bound to HSA, t-RNA, or DSPC was\n e\ufb00ect of the compounds (0.125 mg mL\u22121 MTT, 3\u20134 h; the\n calculated as A327 nm (control) \u2212 A327 nm (HSA, t-RNA, or\n medium was then removed and replaced with 100 \u00b5L DMSO).\n DSPC)/A327 nm (control) \u00d7 100.\n Absorbance was read at 570 nm (vs. 620 nm). IC50 values refer\n to compound concentrations corresponding to 50% absor- Loss of mitochondrial membrane potential\n bance (vs. control).\n Pre-treatment TMRE staining. RD cells were grown in a Petri\n dish, harvested with trypsinization, stained with TMRE (100\n Cellular accumulation\n nM, 37 \u00b0C, 30 min) in DMEM, and then aliquoted into three\n Rhabdomyosarcoma cells were seeded in Petri dishes at 2 \u00d7 parts. The first remained untreated, the second was treated\n 106 cells per dish density and incubated overnight. Cells were with staurosporine (apoptosis inducer, Merck, 2 \u00b5M), and the\n treated with 50 \u00b5M compounds for 5 h. Following the treat- third was treated with Os5 (20 \u00b5M). Flow cytometry analysis\n ment, the cells were harvested, washed, counted, and pelleted. was performed immediately and several times within an hour.\n Cell pellets were lyzed with the microwave acid (HCl) digestion Post-treatment TMRE staining. RD cells were grown in a\n system (CEM Mars\u00ae). The metal content was measured with 6-well plate and then treated with indicated concentrations of\n the ICP-MS system. Os5. After indicated periods (1\u201324 h), the cells were stained\n with TMRE and analyzed with flow cytometry.\n Localization of Os5 in rhabdomyosarcoma cells\n RD cells were seeded in confocal dishes (35 mm, MatTek Neutral red assay\n (Ashland, MA, USA)) at 1.5 \u00d7 105 cells per dish density and The cells were cultured and treated in the same way as for the\n incubated overnight. Os5 or vehicle (DMSO) was added to the MTT assay mentioned above. The final volume at the treat-\n indicated concentration, and the cells were visualized with a ment was 200 \u00b5L per well. Following 24 or 72 h of cell incu-\n Leica CM SP5 confocal microscope. Fluorescence of Os5 was bation with the tested compounds, 20 \u00b5L of Neutral red\n obtained using a UV laser (\u03bbex/\u03bbem = 355/400\u2013485 nm). reagent (Sigma, Prague, Czech Republic) was added, and the\n\n\n 1710 | Inorg. Chem. Front., 2025, 12, 1693\u20131715 This journal is \u00a9 the Partner Organisations 2025\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\n\n cells were incubated for another 3 h. The medium was then The next day, the cells were rinsed twice with PBS and\n removed, the cells were briefly washed with PBS, and 100 \u00b5L stained with propidium iodide (50 \u00b5g mL\u22121 with 100 \u00b5g\n solubilization solution (1% Acetic acid in 50% Ethanol) was mL\u22121 RNase A) in Vindel\u2019s solution (10 mM Tris-Cl ( pH =\n added. The resulting absorbance was read at 540 nm (vs. 8.0), 10 mM NaCl, 0.1% Triton X-100) for 30 min at 37 \u00b0C.\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n 690 nm) on an absorbance reader (Tekan Spark, Switzerland). Cell cycle profiles were gathered with FACS Verse flow cyt-\n ometer, and data were analyzed with FCS Express 6 (DeNovo\n Detection of cell death Software, Glendale, CA).\n Flow cytometry analysis of Annexin V/Propidium iodide\n staining. Mode of cell death was detected with Annexin-V and Preparation of CSC-enriched rhabdomyosarcoma Cells\n Propidium iodide staining assay. Rhabdomyosarcoma cells (RD\u00b7CD133+)\nOpen Access Article. Published on 10 January 2025. Downloaded on 5/12/2026 12:50:56 PM.\n\n\n\n\n were treated with Os5 at indicated concentrations for 24 h. CSC marker antibodies with CD133/1-APC (Miltenyi Biotec,\n After the treatment, the cells were trypsinized, washed, and Reutlingen, Germany) were used to stain RD CD133-positive\n resuspended in Annexin V binding bu\ufb00er (BD Pharmingen) cells (10 min, 4 \u00b0C). The cells were then washed and coupled\n containing Annexin V (Pacific Blue conjugate, 1 : 20 dilution) with anti-APC microbeads (15 min, 4 \u00b0C). Following another\n and Propidium iodide (10 \u00b5g mL\u22121). The cells were analyzed washing, the cells were magnetically sorted on an LS column\n on BD FACS Verse Flow Cytometer and evaluated with FCS using magnets on a MACS stand. CD133\u2212 and CD133+ frac-\n Express 6 (DeNovo Software, Glendale, CA). tions were consequently eluted.\n RealTime-Glo\u2122 Annexin V apoptosis and necrosis assay\n 3D rhabdospheres\n RD cells were grown in a 96-well plate (Corning, ME, USA) and\n Sorted RD\u00b7CD133\u2212 and RD\u00b7CD133+ cells were seeded into\n the next day, EtOH (10%), Staurosporine (2 \u00b5M) or Os5 (at indi-\n ultralow attachment U-shape 96-well plates (Corning, NY, USA)\n cated concentrations) were added to the wells immediately fol-\n at 800 cells per well density. The cells were grown for 4 days in\n lowed by the addition of the kit (RealTime-Glo\u2122 Annexin V\n a non-di\ufb00erentiation medium DMEM/F12 Ham (Merck,\n Apoptosis and Necrosis Assay, Promega, Madison, WI, USA)\n Sigma-Aldrich, Germany) supplemented with 2% B27\n components following the manufacturer\u2019s instructions.\n (Invitrogen, Thermofisher Scientific, Inc., MA, USA), fibroblast\n Luminescence (apoptosis) and Fluorescence (necrosis) were\n growth factor (FGF, 10 ng mL\u22121), epidermal growth factor\n measured in a kinetic mode for cca 1000 min at 37 \u00b0C with a\n (EGF, 20 ng mL\u22121) and 0.4% bovine serum albumin (BSA).\n Fluorescence reader (Tecan Spark, Switzerland).\n\n Activation of caspase-3 Cytotoxic/antiproliferative e\ufb00ect in RD\u00b7CD133\u2212 and\n RD\u00b7CD133+ spheroids\n Caspase-3 activation was detected with CellEvent\u00ae Caspase3/7\n Green Detection Reagent (Invitrogen). RD cells were treated The spheroids were treated with a series of concentrations of\n with vehicle or Os5 at indicated concentrations for 24 h. STAU the tested compounds for 72 h. After this period, CellTiter-\n \u2013 cells were treated with staurosporine (2 \u00b5M) for 3 h. Glo\u00ae Cell Viability Assay (Promega) was performed. The result-\n Following the staining with the green reagent (10 \u00b5M, 37 \u00b0C, ing luminescence was measured with a SPARK\u00ae microplate\n 30 min), the cells were analyzed with flow cytometry. 2 \u00d7 104 reader. IC50 values correspond to a 50% inhibition e\ufb00ect (vs.\n cells were analyzed per run; the experiment was performed control).\n twice.\n Confocal microscopy of RD\u00b7CD133+ spheroids\n Detection of ROS\n Representative RD\u00b7CD133+ spheroids were transferred to con-\n RD cells were seeded on 6-well plates at 2 \u00d7 105 cells per well focal dishes (MatTek) and stained with Calcein AM\n density and incubated for 16 h. The cells were then exposed to (Invitrogen, 2 \u00b5M) and propidium iodide (PI, Invitrogen, 8 \u00b5g\n the tested compounds for 24 h at given concentrations. 5 \u00b5M mL\u22121) at 37 \u00b0C for 2 h. Hoechst 33342 (Enzo, Farmingdale, NY,\n CellROX\u00aeGreen reagent was added to the cells, and the cells USA; 20 \u00b5g mL\u22121) was added to help define the shape of the\n were incubated for another 30 min at 37 \u00b0C. The cells were spheroids. Confocal images were recorded with a Leica SP8\n then harvested, washed in PBS, and analyzed with a flow cyt- SMD laser scanning confocal microscope (Leica Microsystems,\n ometer (BD FACS Verse). Data were analyzed with FCS Express Wetzlar, Germany). Individual pictures were obtained as the\n 6 (DeNovo software, Glendale, CA). 100 \u00b5M Menadione was maximal projection from 10 z-stacks. The fluorescence inten-\n used as a positive control. sity of the Calcein AM channel and PI channel was measured\n with ImageJ software.\n Cell cycle\n RD cells were seeded in 6-well plates (3 \u00d7 105 cells per well)\n and incubated for 24 h. The cells were treated with the Data availability\n tested compounds at given concentrations for 24 h. After\n harvesting and washing, the cells were pelleted (250 g, The data supporting this article have been included as part of\n 3 min), and the pellets were resuspended in 70% ethanol. the ESI.\u2020\n\n\n This journal is \u00a9 the Partner Organisations 2025 Inorg. Chem. Front., 2025, 12, 1693\u20131715 | 1711\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\n\n Crystallographic data for Os5 and Os3 has been deposited 10 M. R. Chang, D. A. Rusanov, J. Arakelyan, M. Alshehri,\n at the CCDC under accession numbers 2393536 and 2393537,\u2020 A. V. Asaturova, G. S. Kireeva, M. V. Babak and W. H. Ang,\n respectively. Targeting emerging cancer hallmarks by transition metal\n complexes: Cancer stem cells and tumor microbiome. Part\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n I, Coord. Chem. Rev., 2023, 477, 214923.\n Con\ufb02icts of interest 11 C. C. Konkankit, S. C. Marker, K. M. Knopf and\n J. J. Wilson, Anticancer activity of complexes of the third\n There are no conflicts to declare. row transition metals, rhenium, osmium, and iridium,\n Dalton Trans., 2018, 47, 9934\u20139974.\n 12 A. L. No\ufb00ke, A. Habtemariam, A. M. Pizarro and\n Acknowledgements\nOpen Access Article. Published on 10 January 2025. Downloaded on 5/12/2026 12:50:56 PM.\n\n\n\n\n P. J. Sadler, Designing organometallic compounds for\n The research of S. S., H. K., L. M., J. K. and V. B. was supported catalysis and therapy, Chem. Commun., 2012, 48, 5219\u2013\n by the Czech Science Foundation (grant 23-06316S). The 5246.\n research of A. H-G., M. D. S., J. R. was supported by the 13 A. Dorcier, W. H. Ang, S. Bola\u00f1o, L. Gonsalvi, L. Juillerat-\n Spanish Ministerio de Ciencia e Innovaci\u00f3n-Agencia Estatal de Jeannerat, G. Laurenczy, M. Peruzzini, A. D. Phillips,\n Investigaci\u00f3n (grant PID2021-122850NB-I00) and Fundaci\u00f3n F. Zanobini and P. J. Dyson, In vitro evaluation of rhodium\n S\u00e9neca-CARM ( project 21989/PI/22). A.H.-G. thanks Fundaci\u00f3n and osmium RAPTA analogues: The case for organo-\n S\u00e9neca-CARM for a grant ( project 21426/FPI/20). The graphical metallic anticancer drugs not based on ruthenium,\n abstract was created with BioRender.com. Organometallics, 2006, 25, 4090\u20134096.\n 14 A. F. A. Peacock, A. Habtemariam, S. A. Moggach,\n A. Prescimone, S. Parsons and P. J. Sadler, Chloro half-\n References sandwich osmium(II) complexes: Influence of chelated N,N-\n ligands on hydrolysis, guanine binding and cytotoxicity,\n 1 F. Bray, M. Laversanne, H. Sung, J. Ferlay, R. L. Siegel, Inorg. 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Front., 2025, 12, 1693\u20131715 | 1715\n\f", "pages_extracted": 23, "text_length": 253054}