Cyclometalated half-sandwich iridium(iii) and rhodium(iii) complexes as efficient agents against cancer stem-cell mammospheres

{"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 Cyclometalated half-sandwich iridium(III) and\n Cite this: Inorg. Chem. Front., 2025,\n rhodium(III) complexes as e\ufb03cient agents against\n 12, 2404 cancer stem-cell mammospheres\u2020\nOpen Access Article. Published on 28 2025. Downloaded on 2026/5/12 12:45:15.\n\n\n\n\n Dana Josa,a,b Piedad Herrera-Ram\u00edrez,a Xiao Feng,c Albert Guti\u00e9rrez, a,b\n\n David Aguil\u00e0,a,b Arnald Grabulosa, *a,b Manuel Mart\u00ednez, a\n Kogularamanan Suntharalingam *c and Patrick Gamez *a,d,e\n\n Four cyclometalated complexes, namely [IrCl(\u03b75-pentamethylcyclopendadienyl)(k2C-diphenyl(1-pyrenyl)\n phosphane)] (1) and [RhCl(\u03b75-pentamethylcyclopendadienyl)(k2C-diphenyl(1-pyrenyl)phosphane)] (2), and\n their DMSO-coordinated counterparts [Ir(\u03b75-pentamethylcyclopendadienyl)(kS-dmso)(k2C-diphenyl(1-\n pyrenyl)phosphane)]PF6 (1\u00b7DMSO) and [Rh(\u03b75-pentamethylcyclopendadienyl)(kS-dmso)(k2C-diphenyl(1-\n pyrenyl)phosphane)]PF6 (2\u00b7DMSO), were synthesized and fully characterized, including their single-crystal\n X-ray structures. DNA-interacting 1 and 2 exhibits IC50 values in the range 0.53\u20130.79 \u00b5M against bulk\n breast cancer cells and breast cancer stem cells (CSCs), i.e., HMLER and HMLER-shEcad cells. The com-\n plexes are up to seven times more active than salinomycin and up to nine times more active than cisplatin.\n Moreover, 1 and 2 are very e\ufb00ective (in the micromolar range) against mammospheres obtained from\n Received 31st October 2024, single cell suspensions of HMLER-shEcad cells, 1 being thrice more toxic than 2 and up to 4.5-fold more\n Accepted 20th January 2025\n potent than cisplatin and salinomycin. In depth mechanistic studies revealed that 1 induces necrosis,\n DOI: 10.1039/d4qi02763a which is potentially dependent on necrosome formation and independent of ROS concentration. The\n rsc.li/frontiers-inorganic e\ufb03cacy of 1 against breast CSCs can be enhanced by co-treatment with PARP-1 inhibitors.\n\n\n\n 10th anniversary statement\n I had the honour of writing the first \u2018review\u2019 for the journal, the theme of which was \u2018anion\u2013\u03c0 interactions\u2019. This \u2018review\u2019\n was very important to me because this type of supramolecular contact was not fully accepted by the scientific community\n at the time. Inorganic Chemistry Frontiers agreed to publish my discussion on this \u2018new non-bonding contact\u2019, which is\n now considered one of the possible \u03c0 interactions.\n\n\n\n Introduction\n The global cancer burden has been growing over the past few\n decades, and the World Health Organization (WHO) predicts\n over 35 million new cancer cases in 2050, which represents a\n a\n Departament de Qu\u00edmica Inorg\u00e0nica i Org\u00e0nica, Secci\u00f3 Qu\u00edmica Inorg\u00e0nica, 77% increase compared to incidences in 2022.1 Among the poss-\n Facultat de Qu\u00edmica, Universitat de Barcelona, Mart\u00ed i Franqu\u00e8s 1-11, E-08028 ible cancer treatments, chemotherapy is the second most com-\n Barcelona, Spain. E-mail: arnald.grabulosa@ub.edu, patrick.gamez@ub.edu monly used option.2 Cisplatin is a chemotherapeutic drug that is\n b\n Institute of Nanoscience and Nanotechnology (IN2UB), Universitat de Barcelona,\n widely used against solid tumours of various cancer types.3 Since\n 08028 Barcelona, Spain\n c\n School of Chemistry, University of Leicester, Leicester, UK.\n the discovery of the anticancer properties of cisplatin,4,5 metal-\n E-mail: k.suntharalingam@leicester.ac.uk containing drugs have received a great deal of attention from the\n d\n Institute of Biomedicine of the University of Barcelona (IBUB), Avda. Diagonal, 643, medicinal inorganic chemistry community.6 Thus, numerous\n Edifici Prevosti, planta-1, Barcelona, Spain cytotoxic coordination compounds have been designed,7\u201310 as\n e\n Catalan Institution for Research and Advanced Studies (ICREA), Passeig Llu\u00eds\n possible alternatives to clinically used metal-based drugs that\n Companys 23, 08010 Barcelona, Spain\n \u2020 Electronic supplementary information (ESI) available. CCDC 2390209\u20132390211.\n may exhibit undesirable side e\ufb00ects or/and are prone to resis-\n For ESI and crystallographic data in CIF or other electronic format see DOI: tance.11 In this context, a number of ruthenium complexes with\n https://doi.org/10.1039/d4qi02763a promising cytotoxic properties have been reported,12\u201314 including\n\n\n 2404 | Inorg. Chem. Front., 2025, 12, 2404\u20132416 This journal is \u00a9 the Partner Organisations 2025\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\n\n half-sandwich ruthenium(II)\u2013arene complexes,15 such as\n RAPTA-C (i.e., [RuCl2(\u03b76-p-cymene)(pta)] where pta = 1,3,5-triaza-\n 7-phosphaadamantane),16 [RuCl(\u03b76-fluorene)(ethylenediamine)]\n PF6 17 and [RuCl(\u03b76-5,6-dihydrophenanthrene)(ethylenediamine)]\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n PF6.18\n Over the last few years, we have developed a series of piano-\n stool Ru(II) and Os(II) complexes of formula [MX2(\u03b76-arene)(P(1-\n pyrenyl)R2R3)] (M = Ru or Os; \u03b76-arene = p-cymene or methyl ben-\n zoate; R2 and R3 = alkyl or aryl groups), which are based on bulky\n monophosphane ligands (viz., containing a 1-pyrenyl moiety).19\n Most of these organometallic compounds exhibited remarkable\n cytotoxic properties towards various cancer cell lines grown in\nOpen Access Article. Published on 28 2025. Downloaded on 2026/5/12 12:45:15.\n\n\n\n\n monolayers, with IC50 values in the submicromolar range.20\u201322\n Interestingly, the corresponding cyclometalated complexes of the\n type [MCl(\u03b76-arene)(k2C-P(1-pyrenyl)R2R3)]PF6 (M = Ru or Os) were\n more potent than the parent non-cyclometalated compounds.20,21\n In the present study, the sterically hindered ligand diphenyl\n (1-pyrenyl)phosphane (L), previously used to prepare Ru(II) and Scheme 1 Synthetic pathways for the preparation of complexes 1 and\n 2 and their DMSO derivatives 1\u00b7DMSO and 2\u00b7DMSO.\n Os(II) complexes, was employed to generate cyclometalated Ir\n (III) and Rh(III) complexes of formulae [MClCp*(k2C-L)] and\n [MCp*(kS-dmso)(k2C-L)]PF6 (with M = Ir or Rh; Cp* = penta- complexes containing an S-coordinated DMSO molecule, i.e.\n methylcyclopentadienyl), to add two additional members of 1\u00b7DMSO and 2\u00b7DMSO, were also prepared by reaction of\n the \u201cplatinum metals\u201d to our family of complexes, and evalu- respectively 1 and 2 with 10 equivalents of DMSO and 1.1\n ate the metal influence on the biological properties. It can be equivalents of thallium hexafluorophosphate in dichloro-\n stressed here that some cyclometalated half-sandwich iridium methane overnight at room temperature (Scheme 1, bottom).\n (III) and rhodium(III) complexes with interesting cytotoxic beha-\n viours have been reported in the literature.23\u201326\n Crystal structures\n The interaction of compounds [IrClCp*(k2C-L)] (1) and\n [RhClCp*(k2C-L)] (2) with DNA was investigated and solution Single crystals of 1 and 2, suitable for X-ray di\ufb00raction analysis,\n studies in water and DMSO were carried out. As 1 and 2 are were obtained by layering n-hexane onto concentrated solutions\n very slowly converted in their DMSO solvato complexes of the complexes in dichloromethane. Rhodium(III) complex 2\n [1\u00b7DMSO]+ and [2\u00b7DMSO]+, respectively, at room temperature was already described in the literature;27 hence, its crystal struc-\n in DMSO, biological studies were conducted with freshly pre- ture was not determined. Crystal data and structure refinement\n pared DMSO solutions of 1 and 2. The cytotoxic e\ufb00ects of 1 parameters for 1 are given in Table S1.\u2020 Selected bond distances\n and 2 towards bulk breast cancer cells and breast cancer stem and coordination angles for this iridium(III) compound are listed\n cells (i.e., CSCs, which clearly play an important role in breast in Table S2.\u2020 1 crystallizes in the monoclinic space group P21/c.\n tumour progression, drug resistance and metastasis) was The solid-state structure of 1 is represented in Fig. 1.\n investigated and their ability to inhibit the formation and via- Complex 1 exhibits the typical coordination geometry for\n bility of three-dimensionally cultured mammospheres ( pro- such organometallic compounds.28\u201330 The Ir\u2013Cl, Ir\u2013P and Ir\u2013C\n duced from breast CSCs) was subsequently examined. Finally, bond lengths of respectively 2.407(1), 2.259(1) and 2.070(2) \u00c5\n the mode of cell death, i.e. apoptosis or necrosis, induced by 1 are analogous to reported values for similar complexes.31,32\n and 2 was studied in detail, for instance using specific inhibi- The distance between the iridium atom and the centroid of\n tors of biomolecules involved in the two di\ufb00erent pathways.\n\n\n\n Results and discussion\n Synthesis of the organometallic complexes\n The synthesis of the ligand diphenyl(1-pyrenyl)phosphane (L)\n was reported earlier.22,27\n Cyclometalated complexes 1 and 2 were typically prepared\n by reaction of the corresponding pentamethylcyclopentadienyl\n metal dichloride dimer ([Cp*MCl2]2; M = Ir or Rh; Cp* = penta-\n methylcyclopentadienyl) with two equivalents of ligand L Fig. 1 Representation of the solid-state structure of 1. The donor\n (Scheme 1, top) and four equivalents of sodium acetate, in atoms coordinated to the metal centre are labelled. Hydrogen atoms are\n methanol at room temperature for two hours. The cationic omitted for clarity.\n\n\n\n This journal is \u00a9 the Partner Organisations 2025 Inorg. Chem. Front., 2025, 12, 2404\u20132416 | 2405\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\n\n the Cp* ring amounts to 1.873(1) \u00c5, which is also in the range\n observed for such molecules. The coordination angles varying\n from 81.60(7) to 89.86(3)\u00b0 for the \u201cthree legs\u201d of the piano-\n stool complex and from 122.56(5) to 132.84(5)\u00b0 for those invol-\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n ving the Cp* centroid (Table S2\u2020) are comparable to reported\n values for related iridium(III) complexes.30,32\n Rhodium(III) complex 2 (Fig. S1\u2020) shows comparable coordi-\n nation features, as evidenced by the published crystallographic\n data for this compound.27\n As aforementioned, substitution of the chlorido ligand by a\n DMSO molecule generates complexes 1\u00b7DMSO and 2\u00b7DMSO.\n Single crystals of these two DMSO-containing compounds were\nOpen Access Article. Published on 28 2025. Downloaded on 2026/5/12 12:45:15.\n\n\n\n\n obtained in dichloromethane/n-hexane. The crystal data and\n structure refinement parameters for the Ir(III) and Rh(III) com-\n plexes are listed in Tables S1 and S3,\u2020 respectively. Selected bond\n lengths and angles are provided in Table S2.\u2020 Representations of\n Fig. 2 (a) UV-Vis spectra of a DMSO solution of 1 recorded at 70 \u00b0C for\n their X-ray structures are depicted in Fig. S2.\u2020 Replacement of the\n 21 hours, in the range 600\u2013280 nm; insert: time evolution of the\n chlorides by DMSO molecules does not significantly a\ufb00ect the absorption at 417 nm. (b) Eyring plots of the temperature dependence of\n structures of the complexes. The coordination bond lengths and k for 1 (black triangles) and 2 (red squares) in DMSO. (c) Plots of the\n angles are comparable with those of the original chlorido com- pressure dependence of ln k for 1 in DMSO.\n plexes, except for the P\u2013M\u2013S angles which are larger than the P\u2013\n M\u2013Cl ones (viz., 97 vs. 90\u00b0; see Table S2\u2020). These higher angle\n values are most likely due to steric repulsion between the methyl\n Table 1 Summary of the kinetic, thermal, and pressure activation para-\n groups of the DMSO and the phenyls of the phosphane ligand.\n meters for the solvation of 1 and 2 in DMSO and 1 and 2 and 1\u00b7DMSO\n and 2\u00b7DMSO in H2O\n Solution behaviour of the complexes in di\ufb00erent (relevant)\n media\n \u0394H\u2021 \u0394S\u2021 (J \u0394V\u2021 (cm3\n The solution behaviour of DMSO stock solutions of the com- Compd. k (s\u22121) t1/2 a (kJ mol\u22121) K\u22121 mol\u22121) mol\u22121)\n plexes, as well as their stability upon aqueous dilution (final In DMSO\n complex concentration of 5 \u03bcM), were first investigated. These 1 2.4 \u00d7 10\u22125 160 h 115 \u00b1 2 10 \u00b1 6 n.d.b\n studies were aimed at evaluating the stability of 1 and 2 under (330 K)\n 2 15 \u00d7 10\u22125 33 h 120 \u00b1 6 39 \u00b1 19 \u22125.1 \u00b1 0.7c\n the cell culture conditions. (330 K)\n Thus, DMSO solutions of 1 and 2 in the range 3\u20136 \u00d7 10\u22125 M In H2O\n were monitored by time-resolved UV-Vis spectroscopy at 1 0.065 0.50 s 47 \u00b1 2 \u2212112 \u00b1 12 n.d.b\n (298 K)\n di\ufb00erent temperatures and pressures. The observed spectral 2 0.025 28 s 50 \u00b1 1 \u2212110 \u00b1 4 n.d.b\n changes agreed with a first-order reaction, as expected for a (298 K)\n solvolysis process. The Reactlab and Specfit software33,34 were 1\u00b7DMSO 2.7 \u00d7 10\u22123 0.5 h 72 \u00b1 2 \u221279 \u00b1 9 n.d.b\n (330 K)\n used to determine the rate constants for the two complexes. As 2\u00b7DMSO 1.3 \u00d7 10\u22123 1h 76 \u00b1 7 \u221273 \u00b1 22 n.d.b\n an example, Fig. 2a illustrates the spectral changes observed (330 K)\n for iridium complex 1 at 70 \u00b0C for 21 hours. The Eyring plots a\n Extrapolated at 310 K. b Not determined. c At 327 K.\n obtained for the variation of the values of k with the tempera-\n ture for 1 and 2 are depicted in Fig. 2b. Finally, Fig. 2c shows\n the ln k versus P plot for the variation of the rate constant for 1.\n The corresponding values for the kinetic and activation para-\n meters for the DMSO solvolysis of complexes 1 and 2, deter-\n mined from these spectroscopic data, are listed in Table 1.\n Subsequently, stability studies of freshly prepared DMSO\n solutions of the chlorido complexes 1 and 2 in aqueous and\n PBS medium were carried out as well, as some reactivity may\n be expected. Clear solubility issues under these relevant con-\n ditions were evident and Fig. 3a shows the UV-Vis spectral\n changes observed in the minute time-scale after the addition\n of 2% of a 250 \u03bcM DMSO solution of 1 to neat water.\n Fig. 3 (a) UV-Vis spectra of a 2% DMSO aqueous solution of 1 recorded\n It is clear that after an initial well-behaved first-order at 25 \u00b0C for 20 minutes, in the range 600\u2013280 nm; insert: time evolution\n process (as expected for a solvolysis reaction), the intensity of of the absorption at 417 nm. (b) Fitted33,34 concentration pro\ufb01les for\n the spectrum keeps increasing while maintaining the same parallel A \u2192 B and precipitate \u2192 B processes of the reaction indicated.\n\n\n\n 2406 | Inorg. Chem. Front., 2025, 12, 2404\u20132416 This journal is \u00a9 the Partner Organisations 2025\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\n\n isosbestic points and overall characteristics (see inset of in aqueous solution. The ESI mass spectrum of 1 and 2\n Fig. 3a). The same behaviour is observed for complex 2 under (500 \u00b5M) in H2O : DMSO (5 : 1) was recorded over the course of\n the same conditions. Fitting the full spectral changes to two 72 h at 37 \u00b0C (Fig. S5 and S7\u2020). The positive mode of the ESI\n first-order parallel processes33,34 leading to the same final mass spectra of 1 and 2 displayed distinctive signals for the\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n solvato complex (that is an A \u2192 B and precipitate \u2192 B aquated species [1-Cl + H2O + Na]+ (m/z = 754.3) and [2-Cl +\n sequence) produces the concentration profiles indicated in H2O + Na]+ (m/z = 664.3), respectively (Fig. S5 and S7\u2020) through-\n Fig. 3b, which are in excellent agreement with the monitored out the incubation period. This suggests that 1 and 2 undergo\n time-resolved spectral changes. aquation in the presence of water and the resulting aquated\n In summary, the DMSO solutions of the chlorido complexes 1 species remains stable for at least 72 h. The ESI mass spectrum\n and 2 are stable for days at room temperature with no significant of 1 and 2 (500 \u00b5M) in H2O : DMSO (5 : 1) in the presence of\n changes. However, when they are added to aqueous solutions, glutathione (500 \u00b5M) was also recorded over the course of 72 h\n they evolve within minutes to new solvato species, provided solu- at 37 \u00b0C (Fig. S6 and S8\u2020). The resultant ESI mass spectra of 1\nOpen Access Article. Published on 28 2025. Downloaded on 2026/5/12 12:45:15.\n\n\n\n\n bility is achieved. To examine this fast process, the monitoring of and 2 were dominated by signals associated to the aquated\n the UV-Vis spectral changes of 10 \u03bcM aqueous (and PBS) solu- species for 1 and 2, [1-Cl + H2O + Na]+ (m/z = 754.3) and [2-Cl +\n tions of the complexes in 20% DMSO was conducted. In these H2O + Na]+ (m/z = 664.3), respectively (Fig. S6 and S8\u2020). This\n cases, a perfect first-order fitting of the time-resolved changes is implies that the aquated species of 1 and 2 (formed in the pres-\n obtained (Fig. S3a\u2020) and the corresponding Eyring activation ence of water) are resistant to reaction with glutathione.\n parameters for the reactions can be derived (Fig. S3b\u2020). These\n data are also collected in Table 1. The values of the thermal acti- Interaction with DNA\n vation parameters are rather di\ufb00erent from those determined for The interaction of 1 and 2 with pBR322 plasmid DNA was\n the DMSO solvolysis, while \u0394H\u2021 decreases to half the value, the examined by agarose gel electrophoresis. Aliquots of di\ufb00erent\n entropy of activation becomes very negative. The data for the concentrations of the complexes, viz. from 0.1 to 20 \u00b5M, were\n chlorido-to-water substitution seem thus to fit to a clear associat- used with 15 \u00b5M DNA (in base pairs). The corresponding gel\n ive interchange mechanism, opposite to that observed for the electrophoresis images are shown in Fig. 4. For free DNA, only\n chlorido-to-DMSO substitution. Even so, the disagreement may form I (supercoiled DNA) is observed (lane 1), whereas great\n suggest that both processes are, in fact dissociatively activated, amounts of form II are detected for DNA samples treated with\n but with a large electrostriction component that produces a the highest concentrations of the complexes (lanes 2\u20139). For\n higher ordering in the more polar solvent (i.e., water) upon going instance, complex 1 induces the formation of 70\u201399% form II\n through a charge separated M\u03b4+\u22efCl\u03b4\u2212 transition state, thus (see lanes 3\u20139 in Fig. 4 top), using concentrations ranging\n leading to a more enthalpicaly favoured reaction. from 0.5 to 20 \u00b5M. For complex concentrations <0.1 \u00b5M, only\n Given the data obtained for 1 and 2, the possible aqueous form I is seen in the agarose gel (lane 2 in Fig. 4 top).\n reactivity of complexes 1-DMSO or 2-DMSO was also examined. Comparable features are observed for complex 2 (Fig. 4\n Aqueous dilution of a DMSO solution of the 1-DMSO or 2-DMSO bottom). However, lower amounts of form II are comparatively\n complexes at the 5\u201310 \u03bcM concentration level (final solution found for concentrations of 2 ranging from 1 to 15 \u00b5M (see\n containing 20% DMSO) was conducted and monitored via time- Fig. S9\u2020). Hence, complex 2 appears to be a slightly less DNA-\n resolved UV-Vis spectroscopy. Provided solubility issues are dealt damaging agent than complex 1.\n with (filtering was needed for the highest concentrations), the\n time-resolved spectral changes indicated very small, but consist- Cytotoxicity towards breast cancer cells\n ent and reproducible, changes in the hours\u2019 time-scale at room The cytotoxicity of the cyclometalated complexes 1 and 2\n temperature (Fig. S4a\u2020); these were associated to a rather small- toward bulk breast cancer cells (HMLER) and breast CSCs\n shifted solvolysis equilibrium. The temperature dependence of\n these equilibrium aquation reactions produced the kinetic and\n thermal activation parameters indicated in Table 1, as derived\n from the Eyring plots shown in Fig. S4b.\u2020 Clearly, the DMSO\n derivatives of the parent compounds undergo a partial aquation\n process that is limited by solubility issues. The values deter-\n mined for \u0394H\u2021 are larger than those obtained for the aquation\n of the chlorido ligands, agreeing with a dissociatively activated\n process from a stronger M-SO(CH3)2 bond (as expected for the\n soft nature of the metals involved). As for the \u0394S\u2021 values, these\n are much less negative than for the solvolysis of the parent chlor-\n ido complexes 1 and 2 in DMSO solution, as the above-men-\n Fig. 4 Agarose gel electrophoresis images of pBR322 plasmid DNA\n tioned electrostriction factors are at a minimum and only hydro-\n ([DNA]bp = 15 \u00b5M) incubated with decreasing concentrations\n gen-bonding interactions may be relevant. (20\u20130.1 \u00b5M) of 1 (top) and 2 (bottom) for 1 h minutes at 37 \u00b0C. Lane 1:\n Time course ESI mass spectrometry studies were performed DNA control; lanes 3\u20139: DNA and complexes at concentrations 0.1, 0.5,\n with 1 and 2 to provide insight into the solvato species formed 1, 2.5, 5, 10, 15, 20 \u00b5M.\n\n\n\n This journal is \u00a9 the Partner Organisations 2025 Inorg. Chem. Front., 2025, 12, 2404\u20132416 | 2407\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\n\n (HMLER-shEcad) cultured in monolayers was assessed using\n the colorimetric MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-\n nyltetrazolium bromide] assay. The IC50 values (concentrations\n required to reduce cell viability by 50%) were derived from\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n dose\u2013response curves (Fig. S10 and S11\u2020) and are summarised\n in Table 2.\n The cyclometalated complexes 1 and 2 displayed similar\n IC50 values towards bulk breast cancer cells and breast CSCs,\n within the sub-micromolar range. This suggests that the metal\n in the cyclometalated complexes does not a\ufb00ect their potency\n towards bulk breast cancer cells and breast CSCs. Notably the\n cyclometalated complexes 1 and 2 exhibited up to 7-fold and\nOpen Access Article. Published on 28 2025. Downloaded on 2026/5/12 12:45:15.\n\n\n\n\n 9-fold higher potency towards breast CSCs than salinomycin (a\n gold standard anti-breast CSC agent) and cisplatin (a metallo-\n drug used in certain breast cancer treatment regimens),\n respectively.35,36 To shed light on the therapeutic potential of 1\n and 2, their cytotoxicity towards non-cancerous bronchial epi-\n thelial BEAS-2B cells was determined (Fig. S12\u2020). The iridium\n (III) complex 1 displayed similar toxicity towards BEAS-2B cells\n as HMLER and HMLER-shEcad cells whereas the rhodium(III)\n complex 2 displayed significantly ( p < 0.05) lower toxicity Fig. 5 (a) Quanti\ufb01cation of mammosphere formation with HMLER-\n towards BEAS-2B cells compared to HMLER and HMLER- shEcad cells untreated and treated with 1, 2, cisplatin, or salinomycin (at\n shEcad cells (up to 3.5-fold higher IC50 value). their IC20 values, 5 days). Error bars represent standard deviations. (b)\n Representative bright-\ufb01eld images (\u00d710) of HMLER-shEcad mammo-\n spheres in the absence and presence of 1, 2, cisplatin, or salinomycin (at\n Mammosphere studies their IC20 values, 5 days).\n\n Given the promising potencies of 1 and 2 toward two-dimen-\n sionally cultured breast CSCs, their ability to inhibit the for-\n mation and to lessen the viability of three-dimensionally cul- rhodium(III) complex 2, implying that the metal influences\n tured breast CSCs was probed using established mammo- mammosphere potency, possibly as the result of the observed\n sphere-based experiments.37 The incubation of 1 and 2 within di\ufb00erence in aquation rates (see Table 1). The potency of 2\n single cell suspensions of HMLER-shEcad cells (IC20 value for towards mammospheres was comparable to the potencies\n 5 days) significantly ( p < 0.05) reduced the number and size of reported for cisplatin (IC50 value = 13.50 \u00b1 2.34 \u00b5M) and salino-\n mammospheres formed (Fig. 5). mycin (IC50 value = 18.50 \u00b1 1.50 \u00b5M) under identical con-\n Under identical conditions cisplatin and salinomycin inhib- ditions, however the potency of 1 towards mammospheres was\n ited the formation of mammospheres from single cell suspen- up to 4.5-fold greater than cisplatin and salinomycin.38\n sions of HMLER-shEcad cells to a slightly greater extent than 1 Overall, the mammosphere studies indicate that both 1 and 2\n and 2 (with respect to the number and size of mammospheres are able to e\ufb00ectively reduce mammosphere formation and\n formed) (Fig. 5). To gauge the ability of 1 and 2 to reduce the that 1 is able to reduce mammosphere viability significantly ( p\n viability of mammospheres, the colorimetric resazurin-based < 0.05) greater than cisplatin and salinomycin.\n reagent, TOX8 was used (Fig. S13 and S14\u2020). The IC50 value of\n 1 and 2 was in the micromolar range (IC50 value for 1 = 4.11 \u00b1 Mode of cell death\n 0.13 \u00b5M and IC50 value for 2 = 12.60 \u00b1 0.62 \u00b5M). Interestingly, To provide insight into the mode of breast CSC death induced\n the potency of iridium(III) complex 1 was 3-fold higher than the cyclometalated complexes 1 and 2, the Annexin\n V-propidium iodide assay was conducted. This assay probes\n the presences of phosphatidylserine residues on the exterior of\n Table 2 IC50 values of the cyclometalated complexes 1 and 2, cisplatin, cells (as sign of early- and late-stage apoptosis) and the uptake\n and salinomycin against HMLER cells, HMLER-shEcad cells, and\n of propidium iodide (as sign of late-stage apoptosis and necro-\n BEAS-2B cells\n sis).39 Upon treatment of HMLER-shEcad cells with 2 (4 \u00d7 IC50\n HMLER IC50/ HMLER-shEcad BEAS-2B IC50/ value for 72 h) there was no statistically significant change in\n Compound \u00b5M IC50/\u00b5M \u00b5M the population of apoptotic or necrotic cells (Fig. 6).\n 1 0.53 \u00b1 0.02 0.63 \u00b1 0.01 0.70 \u00b1 0.02 In contrast, incubation of HMLER-shEcad cells with 1 (4 \u00d7\n 2 0.63 \u00b1 0.04 0.79 \u00b1 0.01 2.23 \u00b1 0.07 IC50 value for 72 h) induced a significant population of cells to\n Cisplatina 2.57 \u00b1 0.02 5.65 \u00b1 0.30 13.50 \u00b1 2.34 take up propidium iodide (but not expose phosphatidylserine\n Salinomycina 11.43 \u00b1 0.42 4.23 \u00b1 0.35 18.50 \u00b1 1.50\n on the cell membrane exterior), indicative of necrosis (Fig. 6).\n a\n Reported in ref. 50, 51 and 53. As expected, treatment of HMLER-shEcad cells with cisplatin\n\n\n 2408 | Inorg. Chem. Front., 2025, 12, 2404\u20132416 This journal is \u00a9 the Partner Organisations 2025\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\n\n inhibiting caspase-dependent apoptosis in breast CSCs pro-\n motes 1-mediated cell death. Comparable increases in potency\n in the presence of z-VAD-FMK have been reported for bona fide\n necroptosis inducers.45,46 Complementary immunoblotting\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n studies showed that the expression of the necrosome com-\n ponents RIP1, RIP3, and MLKL remained largely unaltered in\n HMLER-shEcad cells treated with increasing concentrations of\n 1 (0.32\u20131.26 \u00b5M for 72 h) (Fig. S16\u2020). This suggests that 1-\n induced necroptosis is dependent on the formation of the\n necrosome complex (association of RIP1, RIP3, and MLKL) and\n not on the perturbation of the expression of its individual\n protein kinase components. Necroptosis is associated with a\nOpen Access Article. Published on 28 2025. Downloaded on 2026/5/12 12:45:15.\n\n\n\n\n number of morphological changes such as the increase in\n volume of certain organelles, overall cell volume expansion,\n and the preservation of the nuclear membrane but disinte-\n gration of the plasma membrane.47 Bright-field microscopy\n studies of HMLER-shEcad cells treated with 1 (4 \u00d7 IC50 value\n for 24 h) showed no clear cell membrane but a preserved\n nuclear membrane (Fig. 7), consistent with necroptotic cell\n death.\n In certain cases, the formation of necrosomes leads to\n downstream reactive oxygen species (ROS) generation.48\u201350 To\n determine if 1-induced necroptosis leads to ROS elevation, the\n established ROS indicator, 6-carboxy-2\u2032,7\u2032-dichlorodihydro-\n fluorescein diacetate (DCFH-DA), was used. The intracellular\n ROS levels in HMLER-shEcad cells dosed with 1 (1.5 \u00b5M or\n 3 \u00b5M) did not change significantly ( p > 0.05) over the course of\n 24 h (Fig. 7). This implies that 1-induced necroptosis is inde-\n Fig. 6 (a) FITC Annexin V-propidium iodide binding assay plots of\n pendent of ROS elevation. Another common feature of necrop-\n untreated HMLER-shEcad cells and HMLER-shEcad cells treated with 1 tosis is the hyperactivation of PARP-1, which leads to DNA\n (4 \u00d7 IC50 value for 72 h) or 2 (4 \u00d7 IC50 value for 72 h) or cisplatin (25 \u00b5M damage and the depletion of ATP and NAD levels.51,52\n for 72 h). (b) Graphical representation of the IC50 values of 1 towards Cytotoxicity studies were performed to determine whether 1-\n HMLER-shEcad cells in the absence and presence of IM-54 (10 \u03bcM),\n induced necroptosis was related to PARP-1 hyperactivation.\n necrostatin-1 (20 \u03bcM) or z-VAD-FMK (5 \u03bcM). Error bars represent stan-\n dard deviations.\n More specifically, the potency of 1 towards HMLER-shEcad\n cells was determined in the presence of well-known PARP-1\n inhibitors, veliparib (ABT-888, 10 \u03bcM) and 4-amino-1,8-\n naphthalimide (ANA, 10 \u03bcM) (Fig. 7 and S17\u2020).53,54 The IC50\n (25 \u00b5M for 72 h) induced large populations to express early- value of 1 decreased in the presence of ABT-888 (IC50 value =\n and late-stage apoptosis features (Fig. 6). 0.34 \u00b1 0.01 \u00b5M) and ANA (IC50 value = 0.21 \u00b1 0.01 \u00b5M),\n To further validate the mode of cell death induced by 1, suggesting that hyperactivation of PARP-1 was not part of\n cytotoxicity studies were carried out in the presence of necrosis necroptosis mechanism evoked by 1. The cytotoxicity data also\n inhibitors. Necrosis can occur in an unregulated, random suggests that the activity of 1 toward breast CSCs can be\n manner as well as a regulated ordered form called necroptosis enhanced by co-treating with PARP-1 inhibitors.\n which relies on the formation of necrosomes (the amalgama-\n tion of three protein kinases; RIP1, RIP3, and MLKL).40,41 Co-\n treatment of HMLER-shEcad cells with 1 and IM-54 (10 \u03bcM), Experimental\n an inhibitor of unregulated necrosis,42 led to an increase in\n potency (rather than a decrease) implying that 1 does not Materials and methods\n induce unregulated, random necrosis (Fig. 6b and S15\u2020). All compounds were prepared from commercially available\n Similar studies in the presence of necrostatin-1 (20 \u03bcM), a reagents, under a dinitrogen atmosphere using standard\n RIP1 inhibitor that attenuates necroptosis,43 led to a significant vacuum-line techniques. The solvents were obtained from a puri-\n decrease (3-fold) in the potency of 1 (Fig. 6b and S15\u2020). This fication system (Innovative Technology, Pure Solv) or dried apply-\n suggests that 1 may induce necrosome-mediated necroptotic ing standard procedures,55 and stored under dinitrogen. 1H, 13C\n cell death of breast CSCs. In the presence of z-VAD-FMK {1H},56 31P{1H}, 1H\u201313C HSQC NMR spectra were recorded at\n (10 \u03bcM), a caspase-dependent apoptosis inhibitor,44 the room temperature with 400 or 500 MHz spectrometers.\n potency of 1 increased (Fig. 6 and S15\u2020). This implies that that Chemical shifts are reported downfield from standards and the\n\n\n This journal is \u00a9 the Partner Organisations 2025 Inorg. Chem. Front., 2025, 12, 2404\u20132416 | 2409\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\n\n (\u03b75-pentamethylcyclopendadienyl)]2 (M = Ir,58 Rh59) were\n obtained applying reported procedures.\n\n Preparation of complexes 1 and 2, and their DMSO-\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n coordinated derivatives 1\u00b7DMSO and 2\u00b7DMSO\n [IrCl(\u03b75-pentamethylcyclopendadienyl)(k2C-diphenyl(1-\n pyrenyl)phosphane)] (1). Iridium dimer [IrCl(\u00b5-Cl)(\u03b75-Cp*)]2\n (79 mg, 0.10 mmol), phosphane ligand L (77 mg, 0.20 mmol)\n and sodium acetate (33 mg, 0.40 mmol) were purged under\n dinitrogen in a Schlenk flask. Subsequently, 10 mL of metha-\n nol were added and the reaction mixture was stirred for\n 24 hours under dinitrogen. Next, the solvent was removed\nOpen Access Article. Published on 28 2025. Downloaded on 2026/5/12 12:45:15.\n\n\n\n\n under reduced pressure, and the crude compound was puri-\n fied by flash chromatography (SiO2; dichloromethane). The\n isolated compound was recrystallized from CH2Cl2/hexane,\n producing pure 1 as a yellow solid with a yield of 35%\n (51.8 mg). 31P{1H} NMR (162 MHz, CDCl3): \u03b4 = +32.7 (s)\n (Fig. S18\u2020). 1H NMR (400 MHz, CDCl3): \u03b4 = 8.27 (s, 1HAr),\n 8.20\u20138.06 (m, 8HAr), 7.97 (t, J = 7.6, 1HAr), 7.51\u20137.47 (m, 3H),\n 6.98\u20136.94 (m, 2H), 1.65 (d, J = 2.0, 15H) ppm (Fig. S19 and\n S20\u2020). 13C{1H} NMR (101 MHz, CDCl3): \u03b4 = 152.2\u2013122.5 (m,\n CAr, CHAr), 94.6 (d, J = 3.4, 5C, Cp*), 8.8 (s, 5CH3, Cp*) ppm\n (Fig. S20 and S21\u2020). IR: \u02c9\u03bd = 3045, 2816, 1572, 1434, 1374, 1095,\n 1028, 866, 832, 732, 619 cm\u22121. HRMS: calcd for [M]+ 748.1632,\n found 748.1598; for [M \u2212 Cl]+ 713.1943, found 713.1934.\n [RhCl(\u03b75-pentamethylcyclopendadienyl)(k2C-diphenyl(1-\n pyrenyl)phosphane)] (2). 2 was prepared following the pro-\n cedure applied for 1 but using rhodium dimer [RhCl(\u00b5-Cl)(\u03b75-\n Cp*)]2 (63 mg, 0.10 mmol), and 77 mg (0.20 mmol) of ligand L\n and 33 mg (0.40 mmol) of sodium acetate. The resulting reac-\n tion mixture was stirred for 5 days. After evaporation of the\n solvent, the crude compound was purified flash chromato-\n Fig. 7 (a) Representative bright-\ufb01eld images (\u00d720) of untreated graphy (SiO2; cyclohexane/diethyl ether, 1 : 1). Pure 2 was\n HMLER-shEcad cells (left) and HMLER-shEcad cells treated with 1 (4 \u00d7 obtained as an orange solid with a yield of 52% (69.0 mg). 31P\n IC50 value for 24 h, right). (b) Normalised ROS activity in untreated {1H} NMR (162 MHz, CDCl3): \u03b4 = +66.4 (d, JPRh = 161.0) ppm\n HMLER-shEcad cells and HMLER-shEcad cells treated with 1 (4 \u00d7 IC50\n (Fig. S22\u2020). 1H NMR (400 MHz, CDCl3): \u03b4 = 8.40 (s, 1H),\n value for 0.5, 1, 3, 6, 16, and 24 h). Error bars represent standard devi-\n ations. (c) Graphical representation of the IC50 values of 1 towards 8.32\u20138.06 (m, 5HAr), 7.99 (t, J = 7.6, 1HAr), 7.52\u20137.49 (m, 3H),\n HMLER-shEcad cells in the absence and presence of ABT-888 (10 \u03bcM) 7.34\u20137.289 (m, 1H), 7.26\u20137.21 (m, 4H), 6.98\u20136.94 (m, 2H), 1.62\n or ANA (10 \u03bcM). Error bars represent standard deviations. (d, J = 3.2, 15H) ppm (Fig. S23 and S24\u2020). 13C{1H} NMR\n (101 MHz, CDCl3): \u03b4 = 164.5 (dd, J = 32.1, 10.2; C), 151.0 (d, J =\n 37.4, C), 136.4\u2013122.5 (m, CAr, CHAr), 100.6 (dd, J = 4.8, 3.2, 5C,\n coupling constants are given in Hz. All NMR data were analysed Cp*), 9.3 (s, 5CH3, Cp*) ppm (Fig. S24 and S25\u2020).\n using MestRe Nova (Mnova) version 14.2.1.57 The infrared [Ir(\u03b75-pentamethylcyclopendadienyl)(kS-dmso)(k2C-diphenyl\n spectra were recorded using attenuated total reflection (ATR) in (1-pyrenyl)phosphane)]PF6 (1\u00b7DMSO). Complex 1 (18 mg,\n the range 4000\u2013400 cm\u22121, and the main absorption bands are 0.024 mmol) and dimethylsulfoxide (0.1 mL, 110 mg,\n expressed in cm\u22121. High-resolution mass analyses (HRMS) were 1.41 mmol) were dissolved in 10 mL of dichloromethane and\n carried out at the Centres Cientifics i Tecn\u00f2logics de la thallium hexafluorophosphate (9.0 mg, 0.026 mmol) was sub-\n Universitat de Barcelona, with a time-of-flight instrument using sequently added. The initial yellow solution was stirred for\n electrospray ionization. UV-Vis spectroscopy was conducted on 12 h. The resulting cloudy and white solution was filtered to\n Cary 60, Agilent 8453 or Cary 100 instruments in thermally con- remove thallium chloride, and the solvent was evaporated\n trolled 1 cm path-length quartz cuvettes. Kinetic runs were con- under reduced pressure. The crude compound was recrystal-\n ducted as described previously.21 lized in dichloromethane/diethyl ether at \u221220 \u00b0C. Pure white\n and crystalline 1\u00b7DMSO was isolated by filtration and washed\n Synthesis with pentane (yield = 89%; 20 mg). 31P{1H} NMR (162 MHz,\n The ligand diphenyl(1-pyrenyl)phosphane (L) was prepared as CDCl3): \u03b4 = +24.0 (s), \u2212144.3 (hept, JPF = 713.4) ppm\n described earlier.22 The metallic dimers of formula [MCl(\u00b5-Cl) (Fig. S26\u2020). 1H NMR (400 MHz, CDCl3): \u03b4 = 8.37\u20138.30 (m,\n\n\n 2410 | Inorg. Chem. Front., 2025, 12, 2404\u20132416 This journal is \u00a9 the Partner Organisations 2025\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\n\n 5HAr), 8.27\u20138.06 (m, 6HAr), 7.75\u20137.67 (m, 3HAr), 7.44\u20137.40 (m, bu\ufb00er. pBR322 DNA was diluted as well in cacodylate bu\ufb00er to\n 1HAr), 7.33 (br, 1HAr), 6.96 (br, 2HAr), 2.51 (s, 3H), 2.16 (s, 3H), obtain a DNA concentration of 15 \u03bcM (in base pairs) and incu-\n 1.76 (d, J = 2.0, 15H) ppm (Fig. S27 and S28\u2020). 13C{1H} NMR bated with distinct concentrations of the two complexes. Two\n (101 MHz, CDCl3): \u03b4 = 152.0\u2013122.8 (m, CAr, CHAr), 101.7 (s, 5C, types of DNA-complex samples were prepared, namely (i) the\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n Cp*), 44.9 (s, CH3, dmso), 44.5 (s, CH3, dmso), 9.3 (s, 5CH3, dark controls for which the complex (at the required concen-\n Cp*) ppm (Fig. S28 and S29\u2020). IR: \u02c9\u03bd = 2964, 1568, 1472, 1436, tration) and DNA were incubated for 30 min at 37 \u00b0C and kept\n 1294, 1261, 1181, 1046, 1013, 830 (\u03bd(PF6)), 701, 556 cm\u22121. in the dark at room temperature; (ii) the irradiated samples for\n HRMS: calcd for [M \u2212 PF6]+ 791.2083, found 791.2081. which a solution of the complex (at a specific concentration)\n [Rh(\u03b75-pentamethylcyclopendadienyl)(kS-dmso)(k2C-diphe- and DNA was first incubated for 30 min at 37 \u00b0C and sub-\n nyl(1-pyrenyl)phosphane)]PF6 (2\u00b7DMSO). Complex 2 (18 mg, sequently irradiated for 30 min with blue light (\u03bb = 450 nm;\n 0.032 mmol) and dimethylsulfoxide (0.1 mL, 110 mg, 1.2 J cm\u22122). Next, all the samples were mixed with loading\n 1.41 mmol) were dissolved in 10 mL of dichloromethane and bu\ufb00er (viz. xylene cyanol 0.25% aqueous solution containing\nOpen Access Article. Published on 28 2025. Downloaded on 2026/5/12 12:45:15.\n\n\n\n\n thallium hexafluorophosphate (12.0 mg, 0.034 mmol) was sub- 30% glycerol) and loaded onto 1% agarose gels (1% in TBE\n sequently added. The initial orange solution was stirred for bu\ufb00er). The gels were run at 100 V for 1 h using a Bio-Rad\n 48 h. The resulting cloudy and yellowish solution was filtered horizontal tank connected to a Consort EV231 variable poten-\n to remove thallium chloride, and the solvent was evaporated tial power supply. Subsequently, the DNA was stained with\n under reduced pressure. The crude compound was recrystal- SYBR\u2122 Safe overnight, and the gels were imaged with a\n lized in dichloromethane/diethyl ether at \u221220 \u00b0C. Pure yellow BioRad GelDoc EZ Imager.\n and crystalline 2\u00b7DMSO was isolated by filtration and washed\n with pentane (yield = 85%; 27 mg). 31P{1H} NMR (162 MHz, Crystallography\n CDCl3): \u03b4 = +59.1 (d, J = 151.8), \u2212144.2 (hept, JPF = 713.4) ppm Data for compound 1 were collected on a Bruker APEX II\n (Fig. S30\u2020). 1H NMR (400 MHz, CDCl3): \u03b4 = 8.39\u20138.13 (m, QUAZAR di\ufb00ractometer equipped with a microfocus multilayer\n 8HAr), 8.05\u20138.00 (m, 2HAr), 7.77\u20137.65 (m, 3HAr), 7.48\u20137.42 (m, monochromator with MoK\u03b1 radiation (\u03bb = 0.71073 \u00c5). Data for\n 1HAr), 7.42\u20137.34 (m, 1HAr), 7.02 (br, 2HAr), 2.19 (s, 3H), 2.01 (s, compounds 1\u00b7DMSO and 2\u00b7DMSO were collected at BL13-\n 3H), 1.72 (d, J = 3.6, 15H) ppm (Fig. S31 and S32\u2020). 13C{1H} XALOC beamline62 of the ALBA synchrotron (\u03bb = 0.72931 \u00c5).\n NMR (101 MHz, CDCl3): \u03b4 = 156.0\u2013122.7 (m, CAr, CHAr), 106.2 Data reduction and absorption corrections were performed by\n (s, 5C, Cp*), 44.8 (s, CH3, dmso), 44.3 (s, CH3, dmso), 9.8 (s, using SAINT and SADABS, respectively.63 The structures were\n 5CH3, Cp*) ppm (Fig. S32 and S33\u2020). IR: \u02c9\u03bd = 2990, 1568, 1471, solved using SHELXT64 and refined by full-matrix least-squares\n 1436, 1385, 1309, 1292, 1184, 1046, 1099, 1013, 832 (\u03bd(PF6)), on F2 with SHELXL.65 For compounds 1\u00b7DMSO and 2\u00b7DMSO, a\n 751, 693, 567 cm\u22121. HRMS: calcd for [M-PF6-dmso]+ 623.1385, void containing only di\ufb00use electron density was analysed and\n found 623.1369. taken into account with Olex2/Solvent Mask.66 An estimated\n content of one di\ufb00use lattice CH2Cl2 molecule per formula\n UV-Vis binding studies unit was deduced for both compounds, and included in the\n The potential interaction of Calf Thymus DNA (ct-DNA) with formula. All details can be found in CCDC 2390209\u20132390211,\u2020\n complexes 1 and 2 was investigated by recording UV-Visible which contain the supplementary crystallographic data for this\n spectra in cacodylate/NaCl bu\ufb00er solutions (1 mM sodium paper.\n cacodylate, 20 mM NaCl, pH 7.3). Before the interaction with\n DNA, the stability of 25 \u03bcM solutions of 1 and 2 in 1 mM caco- Cell culture\n dylate\u201320 mM NaCl bu\ufb00er solution (containing 1% DMSO) The human mammary epithelial cell lines, HMLER and\n was first monitored over a period of 72 h. Subsequently, DNA- HMLER-shEcad were kindly donated by Prof. R. A. Weinberg\n binding experiments were performed using solutions of (Whitehead Institute, MIT). HMLER and HMLER-shEcad cells\n increasing concentrations of ct-DNA (0\u201325 \u03bcM), which were were maintained in mammary epithelial cell growth medium\n added to a 25 \u03bcM solution of the complexes in cacodylate\u2013 (MEGM) with supplements and growth factors (BPE, hydrocor-\n NaCl bu\ufb00er. The concentration of calf thymus DNA (ct-DNA) tisone, hEGF, insulin, and gentamicin/amphotericin-B). The\n was determined spectrophotometrically at 260 nm using the BEAS-2B bronchial epithelium cell line was acquired from\n nucleobase molar absorptivity of 6600 M\u22121 cm\u22121.60 Moreover, American Type Culture Collection (ATCC, Manassas, VA, USA)\n the absorbance ratio at 260 and 280 nm (A260/A280) of 1.90 indi- and cultured in RPMI 1640 medium with 2 mM L-glutamine\n cated that the DNA was su\ufb03ciently free of protein.61 supplemented with 1% penicillin and 10% fetal bovine serum.\n The cells were grown at 310 K in a humidified atmosphere con-\n Agarose gel electrophoresis taining 5% CO2.\n Electrophoretic studies were performed with pBR322 plasmid\n DNA in cacodylate bu\ufb00er (10 mM sodium cacodylate and Monolayer cytotoxicity studies\n 50 mM NaCl in milliQ water). 3 mM stock solutions of 1 and 2 Exponentially growing cells were seeded at a density of\n were prepared in DMSO. The samples at the di\ufb00erent concen- approximately 5 \u00d7 103 cells per well in 96-well flat-bottomed\n trations required for the electrophoresis (viz., 25, 15, 12.5, 10, microplates and allowed to attach for 24 h prior to addition of\n 5, 2.5, 1 and 0.5 \u00b5M) were obtained by dilution in cacodylate compounds. Various concentrations of the test compounds\n\n\n This journal is \u00a9 the Partner Organisations 2025 Inorg. Chem. Front., 2025, 12, 2404\u20132416 | 2411\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\n\n (0.0004\u2013100 \u03bcM) were added and incubated for 72 h at 37 \u00b0C binding and the FL2 channel was used to assess PI uptake.\n (total volume 200 \u03bcL). Stock solutions of the compounds were Cell populations were analysed using Floreada.io.\n prepared as 10 mM DMSO solutions and diluted using cell\n media. The final concentration of DMSO in each well was Immunoblotting analysis\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n \u22641%. After 72 h, 20 \u03bcL of MTT (4 mg mL\u22121 in PBS) was added HMLER-shEcad cells (1 \u00d7 106) were incubated with 1\n to each well and the plates incubated for an additional 4 h at (0.32\u20131.26 \u03bcM for 72 h) at 37 \u00b0C. HMLER-shEcad cells were\n 37 \u00b0C. The media/MTT mixture was eliminated and DMSO harvested and isolated as pellets. SDS-PAGE loading bu\ufb00er\n (100 \u03bcL per well) was added to dissolve the formazan precipi- (64 mM Tris-HCl ( pH 6.8), 9.6% glycerol, 2% SDS, 5%\n tates. The optical density was measured at 550 nm using a \u03b2-mercaptoethanol, 0.01% bromophenol blue) was added to\n 96-well multiscanner autoreader. Absorbance values were nor- the pellets, and this was incubated at 95 \u00b0C for 10 min. Cell\n malised to (DMSO-containing) control wells and plotted as lysates were resolved by 4\u201320% sodium dodecylsulphate polya-\n concentration of compound versus % cell viability. IC50 values cylamide gel electrophoresis (SDS-PAGE; 200 V for 25 min) fol-\nOpen Access Article. Published on 28 2025. Downloaded on 2026/5/12 12:45:15.\n\n\n\n\n were interpolated from the resulting dose dependent curves. lowed by electro transfer to polyvinylidene difluoride mem-\n The reported IC50 values are the average of three independent brane, PVDF (350 mA for 1 h). Membranes were blocked in 5%\n experiments (n = 18). (w/v) non-fat milk in PBST (PBS/0.1% Tween 20) and incubated\n with the appropriate primary antibodies (Cell Signalling\n Mammosphere formation and viability studies\n Technology). After incubation with horseradish peroxidase-\n HMLER-shEcad cells (5 \u00d7 103) were plated in ultralow-attach- conjugated secondary antibodies (Cell Signalling Technology),\n ment 96-well plates (Corning) and incubated in MEGM sup- immune complexes were detected with the ECL detection\n plemented with B27 (Invitrogen), 20 ng mL\u22121 EGF and 4 \u03bcg reagent (BioRad) and analysed using a chemiluminescence\n mL\u22121 heparin (Sigma) for 5 days. Studies were also conducted imager (Bio-Rad ChemiDoc Imaging System).\n in the presence of 1, 2, cisplatin, and salinomycin (0\u2013133 \u00b5M).\n Mammospheres treated with 1, 2, cisplatin, and salinomycin Intracellular ROS assay\n (at their respective IC20 values, 5 days) were counted and HMLER-shEcad cells (5 \u00d7 103) were seeded in each well of a\n imaged using an inverted microscope. The viability of the 96-well plate. After incubating the cells overnight, they were\n mammospheres was determined by addition of a resazurin- treated with 1 (1.5 \u00b5M or 3 \u00b5M for 0.5\u201324 h), and incubated\n based reagent, TOX8 (Sigma). After incubation for 16 h, the with 6-carboxy-2\u2032,7\u2032-dichlorodihydrofluorescein diacetate\n fluorescence of the solutions was read at 590 nm (\u03bbex = (20 \u03bcM) for 30 min. The intracellular ROS level was determined\n 560 nm). Viable mammospheres reduce the amount of the oxi- by measuring the fluorescence of the solutions in each well at\n dised TOX8 form (blue) and concurrently increase the amount 529 nm (\u03bbex = 504 nm).\n of the fluorescent TOX8 intermediate (red), indicating the\n degree of mammosphere cytotoxicity caused by the test com-\n pound. Fluorescence values were normalised to DMSO-con- Conclusions\n taining controls and plotted as concentration of test com-\n pound versus % mammosphere viability. IC50 values were Following earlier studies with ruthenium and osmium, the phos-\n interpolated from the resulting dose dependent curves. The phane ligand diphenyl(1-pyrenyl)phosphane was used to prepare\n reported IC50 values are the average of two independent experi- cycloiridated and cyclorhodiated half-sandwich complexes\n ments, each consisting of three replicates per concentration bearing a pentamethylcyclopentadienyl (Cp*) ring. The crystal\n level (n = 6). structures of the organometallic compounds show features that\n are found in other piano-stool complexes. It can be pointed out\n Annexin V-propidium iodide assay here that 1 and 2 represent uncommon examples of Cp*-con-\n HMLER-shEcad cells were incubated with and without 1 and 2 taining cyclometalated Ir(III) and Rh(III) complexes which have\n (4 \u00d7 IC50 value for 72 h) and cisplatin (25 \u00b5M for 72 h) at 37 \u00b0C. been structurally characterised by X-ray di\ufb00raction.27,31,32,67\u201370\n Cells were harvested from adherent cultures by trypsinisation. The compounds are slowly converted into the DMSO solvato\n The FITC Annexin V/dead cell apoptosis kit was used. The complexes, namely 1\u00b7DMSO and 2\u00b7DMSO, if dissolved in DMSO,\n manufacture\u2019s (Thermo Fisher) protocol was followed to carry as shown by solution studies. Moreover, 1 and 2, which are\n out this experiment. Briefly, untreated and treated cells (1 \u00d7 poorly soluble in water, are gradually converted into the aquated\n 106) were suspended in 1\u00d7 Annexin binding bu\ufb00er (100 \u00b5L) [MCp*L(H2O)]+ species, as suggested by UV-Vis studies and con-\n (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4), then firmed by mass spectrometry; as expected these new cationic\n 5 \u00b5L of FITC Annexin V and 1 \u00b5L of PI (100 \u00b5g mL\u22121) were species become more water soluble. Gel electrophoresis revealed\n added to each sample and incubated at room temperature for that 1 and 2 interact with DNA and in vitro cytotoxicity assays\n 15 min. After which more 1\u00d7 Annexin binding bu\ufb00er (400 \u00b5L) showed that they are significantly cytotoxic against tumorigenic\n was added while gently mixing. The cells were analysed using mammary epithelial HMLER (which is a genetically modified\n a FACSCanto II flow cytometer (BD Biosciences) (10 000 events cell line representative of bulk breast cancer cells) and HMLER-\n per sample were acquired) at the University of Leicester FACS shEcad (where E-cadherin was silenced, to generate breast CSC-\n Facility. The FL1 channel was used to assess Annexin V enriched populations) cells. Remarkably, rhodium(III) complex 2\n\n\n 2412 | Inorg. Chem. Front., 2025, 12, 2404\u20132416 This journal is \u00a9 the Partner Organisations 2025\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\n\n is more active against bulk breast cancer cells and breast CSCs References\n (viz., HMLER and HMLER-shEcad cells) compared to non-\n tumorigenic cells (namely BEAS-2B cells), while iridium(III) 1 WHO, Global cancer burden growing, amidst mounting\n complex 1 is toxic for both cancer and healthy cells. Subsequent need for services, https://www.who.int/news/item/01-02-\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n investigation of the ability of 1 and 2 to inhibit the formation 2024-global-cancer-burden-growing-amidst-mounting-need-\n and reduce the viability of three-dimensionally cultured breast for-services, (accessed October 18th, 2024).\n CSCs showed that 1 was thrice more e\ufb03cient than 2, in contrast 2 K. D. Miller, L. Nogueira, T. Devasia, A. B. Mariotto,\n to the monolayer cytotoxicity data (which indicated that 1 and 2 K. R. Yabro\ufb00, A. Jemal, J. Kramer and R. L. Siegel, Cancer\n had similar potency). Moreover, while 2 was as cytotoxic as cis- treatment and survivorship statistics, 2022, CA-Cancer J.\n platin and salinomycin against the mammospheres, 1 was 4.5- Clin., 2022, 72, 409\u2013436.\n fold more e\ufb00ective than cisplatin and salinomycin. In depth 3 S. Dasari and P. B. Tchounwou, Cisplatin in cancer\n mechanistic studies indicated that whereas 2 was not producing therapy: Molecular mechanisms of action,\nOpen Access Article. Published on 28 2025. Downloaded on 2026/5/12 12:45:15.\n\n\n\n\n any significant changes with respect to the population of apopto- Eur. J. Pharmacol., 2014, 740, 364\u2013378.\n tic or necrotic cells, 1 was causing necrosis. This di\ufb00erence may 4 A. Casini, A. Vessi\u00e8res and S. M. Meier-Menches, Metal-\n be explained but the faster aquation of complex 1. It was shown based Anticancer Agents Preface, The Royal Society of\n that the necroptotic cell death provoked by 1 was potentially Chemistry, 2019.\n dependent on the formation of the necrosome complex and 5 R. A. Alderden, M. D. Hall and T. W. Hambley, The discov-\n independent of intracellular ROS levels. Furthermore, hyperacti- ery and development of cisplatin, J. Chem. 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Helmin-Basa, Latest develop-\n locale=en. ments in metal complexes as anticancer agents, Coord.\n The data supporting this article have been included as part Chem. Rev., 2022, 452, 21.\n of the ESI.\u2020 9 A. K. Singh, A. Kumar, H. Singh, P. Sonawane, P. Pathak,\n Crystallographic data for compounds 1, 2, 1\u00b7DMSO and M. Grishina, J. P. Yadav, A. Verma and P. Kumar, Metal\n 2\u00b7DMSO (CCDC 2390209\u20132390211\u2020). Complexes in Cancer Treatment: Journey So Far, Chem.\n Biodivers., 2023, 20, 50.\n 10 P. Moharana, D. Ghosh and P. Paira, Drive to organoruthe-\n Con\ufb02icts of interest nium and organoiridium complexes from organoplatinum:\n Next-generation anticancer metallotherapeutics, Inorg.\n There are no conflicts to declare. Chem. Commun., 2021, 124, 18.\n 11 E. J. Anthony, E. M. Bolitho, H. E. Bridgewater,\n O. W. L. Carter, J. M. Donnelly, C. Imberti, E. C. Lant,\n Acknowledgements F. Lermyte, R. J. Needham, M. Palau, P. J. Sadler, H. Y. Shi,\n F. X. Wang, W. Y. Zhang and Z. J. Zhang, Metallodrugs are\n Financial support from the Ag\u00e8ncia de Gesti\u00f3 dels Ajuts unique: opportunities and challenges of discovery and\n Univeristaris i de Recerca ( project 2021-SGR-01107), Spanish development, Chem. Sci., 2020, 11, 31.\n Ministerio de Ciencia, Innovaci\u00f3n y Universidades ( projects 12 S. Thota, D. A. Rodrigues, D. C. Crans and E. J. Barreiro, Ru\n PID2020-115537RB-I00, PID2020-115658GB-I00 and PCI2021- (II) Compounds: Next-Generation Anticancer\n 122027-2B, MCIU/AEI/10.13039/501100011033 and European Metallotherapeutics?, J. Med. Chem., 2018, 61, 5805\u20135821.\n Union NextGenerationEU/PRTR) and the RSC (RSC Research 13 C. Sonkar, S. Sarkar and S. Mukhopadhyay, Ruthenium(II)-\n Fund grant RF19-7147) is kindly acknowledged. P. G. thanks arene complexes as anti-metastatic agents, and related\n the Catalan Institution for Research and Advanced Studies techniques, RSC Med. Chem., 2022, 13, 22\u201338.\n (ICREA). K. 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Front., 2025, 12, 2404\u20132416 This journal is \u00a9 the Partner Organisations 2025\n\f", "pages_extracted": 13, "text_length": 167549}