Cytotoxicity of osmium(II) and cycloosmated half-sandwich complexes from 1-pyrenyl-containing phosphane ligands.

Open Access Article. Published on 26 May 2023. Downloaded on 5/2/2026 3:00:56 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Dalton Transactions View Article Online PAPER Cite this: Dalton Trans., 2023, 52, 8391 View Journal | View Issue Cytotoxicity of osmium(II) and cycloosmated half-sandwich complexes from 1-pyrenylcontaining phosphane ligands† Dana Josa, a,b David Aguilà,a,b Pere Fontova,c Vanessa Soto-Cerrato, Piedad Herrera-Ramírez,a Laia Rafols, a Arnald Grabulosa *a,b and Patrick Gamez *a,b,f d,e Five metal–arene complexes of formula [MX2(η6-p-cymene)(diR(1-pyrenyl)phosphane)] (M = Os or Ru, X = Cl or I, R = isopropyl or phenyl) and symbolized as MRX2 were synthesized and fully characterized, iPr Ph Ph Ph namely OsiPr Cl2 , OsI2 , OsCl2 , OsI2 and RuI2 . Furthermore, nine cyclometalated half-sandwich complexes of 6 2 formula [MX-(η -p-cymene)(k C-diR(1-pyrenyl)phosphane)] (M = Os or Ru, X = Cl or I, R = isopropyl or phenyl) or [M(η6-p-cymene)(kS-dmso)(k2C-diR(1-pyrenyl)phosphane)]PF6 (M = Os or Ru, R = isopropyl or iPr iPr Ph Ph phenyl) and symbolized as c-MRX were prepared; hence, c-OsiPr Cl , c-OsI , c-Osdmso , c-OsCl , c-OsI , cPh Ph Ph Ph Osdmso , c-RuCl , c-RuI and c-Rudmso were obtained and fully characterized. The crystal structures of ten out of the fourteen complexes were solved. All complexes exhibit notable cytotoxic properties against A549 (Lung Adenocarcinoma) human cells, with IC50 values ranging from 48 to 1.42 μM. In addition, complex c-OsiPr dmso shows remarkable toxic behaviours agains other cell lines, namely MCF7 (breast carciReceived 10th March 2023, Accepted 26th May 2023 noma), MCF10A (non-tumorigenic epithelial breast) and MDA-MB-435 (melanoma) human cells, as illus- DOI: 10.1039/d3dt00743j trated by IC50 values of 4.36, 4.71 and 2.32 μM, respectively. Finally, it has been found that OsiPr I2 aﬀects the cell cycle of A549 cells, impeding their replication (i.e., the cell cycle is blocked), whereas OsPh I2 (namely rsc.li/dalton with phenyl groups instead of isopropyl ones) does not induce this eﬀect. Introduction The search for new anticancer agents with increased eﬃciency and less unpleasant side eﬀects is a topical area of research, for instance in bioinorganic chemistry.1–4 Metal-based chemotherapeutic drugs have gained increasing interest after the discovery of cisplatin, viz. cis-diamminedichloridoplatinum(II), in 1965 by Rosenberg.5,6 In the context of the development of a Departament de Química Inorgànica i Orgànica, Facultat de Química, Secció de Química Inorgànica, Universitat de Barcelona, Martí i Franquès, 1-11, 08028 Barcelona, Spain. E-mail: arnald.grabulosa@qi.ub.es, patrick.gamez@qi.ub.es b Institute of Nanoscience and Nanotechnology (IN2UB), Universitat de Barcelona, 08028 Barcelona, Spain c Department of Chemistry, Universidad de Burgos, 09001 Burgos, Spain d Department of Pathology and Experimental Therapeutics, Faculty of Medicine and Health Sciences, Universitat de Barcelona, Campus Bellvitge, Feixa LLarga s/n, 08907 L’Hospitalet de Llobregat, Spain e Oncobell Program, Institut d’Investigació Biomèdica de Bellvitge (IDIBELL), 08908 L’Hospitalet de Llobregat, Spain f Catalan Institution for Research and Advanced Studies, Passeig Lluís Companys 23, 08010 Barcelona, Spain † Electronic supplementary information (ESI) available. CCDC 2237617–2237626. For ESI and crystallographic data in CIF or other electronic format see DOI: https://doi.org/10.1039/d3dt00743j This journal is © The Royal Society of Chemistry 2023 alternative systems, some ruthenium-containing compounds were found to be promising anticancer drug candidates,7–9 including ruthenium(II)–arene complexes,4,10,11 like [RuCl(η6fluorene)(ethylenediamine)]PF6, [RuCl(η6-5,6-dihydrophenanthrene)(ethylenediamine)]PF6 or [RuCl2(η6-p-cymene)( pta)] (i.e., RAPTA-C with pta = 1,3,5-triaza-7phosphaadamantane).12,13 Many osmium(II)–arene analogues of such organoruthenium(II) compounds have subsequently been reported,14,15 and the eﬀect of the metal exchange on the cytotoxic properties appears not to be predictable.16–18 In some cases, the Os(II) complexes are more active than their Ru (II) counterparts,19–21 whereas no diﬀerences or lower activities for the Os(II) compounds were observed with other systems.22–24 A few years ago, we started to develop a family of ruthenium (II)–arene complexes of general formula [RuX2(η6-arene)(P(1pyrenyl)R2R3)] (Scheme 1a, M = Ru), which displayed interesting cytotoxic properties, with IC50 values down to 0.4 µM.25 We also found that such Ru(II) compounds containing a phosphane ligand with a 1-pyrenyl group could undergo cyclometalation in the presence of a base, generating complexes of formula [RuX(η6-arene)(k2C-(P(1-pyrenyl)R2R3))] or [Ru(η6arene)(k2C-(P(1-pyrenyl)R2R3))(dmso)]PF6 (Scheme 1b, M = Dalton Trans., 2023, 52, 8391–8401 | 8391 View Article Online Open Access Article. Published on 26 May 2023. Downloaded on 5/2/2026 3:00:56 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Paper Scheme 1 Representations of (a) the piano-stool complexes based on a dialkyl/aryl(1-pyrenyl)phosphane ligand and (b) the cyclometalated ( phosphane)metal complexes described earlier25–27 and in the present study. Ru).26 Such cyclometalated piano-stool complexes displayed better cytotoxic activities than their non-cyclometalated analogues.27 In the present study, we investigated the eﬀect of the replacement of Ru(II) with Os(II) on the cytotoxic behaviour in this family of organometallic compounds. It is indeed generally found that substitutions reactions (e.g. aquation) of Os(II) complexes are slower than for the analogous Ru(II) complexes.28–31 Hence, we prepared a series of [OsX2(η6-pcymene)(P(1-pyrenyl)R2R3)] complexes (Scheme 1a, M = Os) with diisopropyl-(1-pyrenyl)phosphane or diphenyl-(1-pyrenyl) phosphane as monodentate phosphane ligand (R2 = R3 = isopropyl or phenyl) and chloride or iodide as anions (X = Cl or I). Moreover, the corresponding cyclometalated complexes, namely [OsX(η6-p-cymene)(k2C-(P(1-pyrenyl)R2R3))] and [Os(η6p-cymene)(k2C-(P(1-pyrenyl)R2R3))(dmso)]PF6, were synthesized as well (Scheme 1b, M = Os). In addition, the cyclometalated ruthenium(II) complexes [RuX(η6-p-cymene)(k2C-diphenyl(1pyrenyl)phosphane)] (X = Cl or I) and [Ru(η6-p-cymene)(k2Cdiphenyl(1-pyrenyl)phosphane)(dmso)]PF6 (Scheme 1b, M = Ru; R2 = R3 = phenyl) were prepared to complete the series of Ru(II) compounds described previously and for comparison purposes with the newly reported Os(II) complexes. The biological properties of the new compounds were subsequently investigated and compared with those of the previously described complexes. Dalton Transactions Fig. 1 Synthetic procedures used to prepare the non-cyclometalated chlorido (top) and iodido (bottom) osmium(II) complexes. excess of sodium iodide in technical acetone under reflux (Fig. 1, bottom). The chlorido cyclometalated complexes were synthesized by reaction of the dimeric precursors (ruthenium or osmium) with the phosphane ligand in the presence of 3 equivalents of base, namely sodium acetate, in methanol at room temperature (Fig. 2 top). The iodido cyclometalated complexes were obtained through halide exchange, using 24 equivalents of sodium iodide in acetone under reflux (Fig. 2, middle). Finally, the cationic cyclometalated complexes containing an S-coordinated DMSO molecule were prepared by reaction of the corresponding chlorido cyclometalated complexes with 10 equivalents of DMSO and 1.1 equivalent of thallium hexafluorophosphate in dichloromethane at room temperature (Fig. 2, bottom). Results and discussion Synthesis of the organometallic complexes The ligands diisopropyl(1-pyrenyl)phosphane (PPyriPr2)27 and diphenyl(1-pyrenyl)phosphane (PPyrPh2)25 were prepared as described earlier. The non-cyclometalated osmium(II) complexes were synthesized as illustrated in Fig. 1. The chlorido complexes were typically obtained by reaction of the osmium dimeric precursor [OsCl(μ-Cl)(η6-p-cymene)]2 with the phosphane ligand in dichloromethane at room temperature (Fig. 1, top). The corresponding iodido complexes were prepared through halide exchange, in the presence of an 8392 | Dalton Trans., 2023, 52, 8391–8401 Fig. 2 Synthetic procedures to prepare the chlorido cyclometalated complexes (top), iodido cyclometalated complexes (middle) and cationic cyclometalated complexes with an S-coordinated DMSO molecule (bottom). This journal is © The Royal Society of Chemistry 2023 View Article Online Open Access Article. Published on 26 May 2023. Downloaded on 5/2/2026 3:00:56 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Dalton Transactions Paper 10 osmium(II) and 4 ruthenium(II) complexes were obtained applying these synthetic procedures, which are listed in Table 1. It can be pointed out that c-OsPh could not be preI pared from c-OsPh Cl through halide exchange (Fig. 2, middle). Instead, c-OsPh I was synthesized by reaction of the dimeric precursor [OsI(μ-I)(η6-p-cymene)]2 with diphenyl(1-pyrenyl)phosphane in the presence of NaOAc. Crystal structures Single crystals could be obtained for 10 out of the 14 complexes prepared. Hence, crystal data and structure refinement iPr parameters are listed in Table S1† for complexes OsiPr Cl2 , OsI2 , ciPr iPr Ph Ph Ph OsCl and c-OsI , Table S2† for OsCl2 , OsI2 and c-OsCl , and Ph Table S3† for c-RuPh and c-RuPh Cl , c-RuI dmso . Selected bond lengths and angles are given in Tables S4–S6.† Since the solidstate structures of the non-cyclometalated and cyclometalated complexes are comparatively analogous, solely the structures iPr of OsiPr Cl2 and c-OsCl are subsequently described. A representation of the crystal structure of OsiPr Cl2 is shown in ˉ Fig. 3. OsiPr Cl2 crystallises in the triclinic space group P1 (Table S1†). OsiPr Cl2 exhibits the characteristic “three-legged piano-stool” geometry for this type of organoosmium(II) complexes. The Os–Cl bond lengths are 2.41 Å and the Os–P bond distance is 2.39 Å (Table S4†). The distance between the p-cymene ring centroid and the osmium atom is 1.70 Å. These values are comparable with those previously reported for analogous compounds.27,32–34 The coordination angles vary from 85 to 128° (Table S4†), which is within the expected range for such molecules.35,36 The solid-state structures of non-cyclometaPh Ph lated complexes OsiPr I2 , OsCl2 and OsI2 are depicted in Fig. S1, S3 and S4,† respectively. These compounds display coordination features that are analogous to those of OsiPr Cl2 (see Tables S4 and S5†). It can be noted that, as expected, the Os–I bond Table 1 List of the 14 new complexes prepared in the present study with their corresponding labelling (see Scheme 1 for the representation of their corresponding structure). The complexes for which an X-ray crystal structure could be obtained are mentioned through the corresponding ﬁgure Complex M R X X-ray structure OsiPr Cl2 OsiPr I2 c-OsiPr Cl c-OsiPr I c-OsiPr dmso OsPh Cl2 Ph OsI2 c-OsPh Cl c-OsPh I c-OsPh dmso Ph RuI2 c-RuPh Cl c-RuPh I c-RuPh dmso Os Os Os Os Os Os Os Os Os Os Ru Ru Ru Ru Isopropyl Isopropyl Isopropyl Isopropyl Isopropyl Phenyl Phenyl Phenyl Phenyl Phenyl Phenyl Phenyl Phenyl Phenyl Cl I Cl I dmso Cl I Cl I dmso I Cl I dmso Fig. 3 Fig. S1† Fig. 4 Fig. S2† n.d.a Fig. S3† Fig. S4† Fig. S5† n.d.a n.d.a n.d.a Fig. S6† Fig. S7† Fig. S8† a n.d. = not determined (as single crystals could not be obtained). This journal is © The Royal Society of Chemistry 2023 Fig. 3 Representation of the crystal structure of OsiPr Cl2 . The atoms bonded to the metal centre are labelled. Hydrogen atoms are omitted for clarity. distances are longer than the Os–Cl ones, by about 0.3 Å (Tables S4 and S5†).27 The cyclometalated complex c-OsiPr Cl crystallises in the monoclinic space group P21/c (Table S1†). A representation of its crystal structure is shown in Fig. 4. The pseudo-octahedral geometry of the Os centre of c-OsiPr Cl is strongly distorted, as the result of the cyclometalation involving the 1-pyrenyl ring. The Os–Cl, Os–P, Os–C and Os–centroid distances are 2.43, 2.32, 2.11 and 1.73 Å, respectively (Table S4†). These bond lengths are similar to those of analogous Ru(II) complexes.27,37,38 The coordination angles span from 81 to 133° (Table S4†), which is in the range of related Ru (II) complexes.26 It can be stressed here that this type of cycloosmated complexes (from a monophosphane ligand) was not found in the Cambridge Structural Database (CSD, accessed in November 2022). To the best of our knowledge, iPr compounds c-OsiPr and c-OsPh Cl , c-OsI Cl therefore represent the first examples of such cyclometalated osmium(II) complexes that have been characterized by X-ray diﬀraction. The cycloosmated compounds c-OsiPr and c-OsPh I Cl exhibit structural features comparable to those of c-OsiPr (Tables S4 Cl and S5†). Cell viability studies iPr iPr iPr The ability of the Os(II) compounds OsiPr Cl2 , OsI2 , c-OsCl , c-OsI iPr and c-Osdmso was assessed against human lung adenocarcinoma (A549) cells. Half-maximum inhibitory concen- Fig. 4 Representation of the crystal structure of c-OsiPr Cl . The atoms bonded to the metal centre are labelled. Hydrogen atoms are omitted for clarity. Dalton Trans., 2023, 52, 8391–8401 | 8393 View Article Online Open Access Article. Published on 26 May 2023. Downloaded on 5/2/2026 3:00:56 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Paper Dalton Transactions trations (IC50) were determined for all compounds after an incubation time of 24 h and using freshly prepared stock solutions of the complexes in DMSO. The IC50 values obtained are listed in Table 2. These values were compared with those reported earlier for the corresponding Ru(II) compounds, iPr iPr iPr iPr namely compounds RuiPr Cl2 , RuI2 , c-RuCl , c-RuI and c-Rudmso . After 24 h incubation, the non-cyclometalated osmium iPr complexes OsiPr Cl2 and OsI2 appear to be more active than their ruthenium counterparts (Table 2). It can be pointed out here that for the Ru(II) compounds an increase of cytotoxicity was observed with ageing solutions of RuiPr I2 ; for instance, a 7-dayold DMSO solution of RuiPr I2 gave an IC50 value of 9.5 µM (vs. 48 µM at day 0).27 Cell viability assays have been performed for iPr both OsiPr Cl2 and OsI2 using 7-day-old DMSO solutions but no cytotoxicity diﬀerences were observed compared to the freshly prepared solutions (data not shown). Finally, it can be emphasized that the low IC50 value obtained for OsiPr I2 appears to result from its perturbing eﬀect on the cell cycle, since no significant cell death was observed, unlike with the other compounds. Therefore, cell cycle studies have been carried out with this complex, which are discussed below. Regarding the cyclometalated complexes, in all cases, lower IC50 values are observed for the Ru(II) complexes, which are 2.5-to-3.2 times more active than the Os(II) ones. In a previous study with analogous Ru(II) complexes, it was observed that the complex c-RuiPr dmso was the most eﬃcient compound and it appears to be the case as well for the Os(II) complexes (see Table 2). Thus, cell viability studies were subsequently performed with c-OsiPr dmso against various cancer and healthy cell lines, namely breast adenocarcinoma (MCF7), non-tumorigenic epithelial breast (MCF10A) and melanoma (MDA-MB-435) cells. The IC50 values obtained (including those for A549 cells) are listed in Table 3, together with those found for c-RuiPr dmso , for comparison purposes. iPr The Ru(II) complex c-RuiPr dmso is more cytotoxic than c-Osdmso against all cell lines tested (Table 3). Both compounds are clearly more active than the reference compound RAPTA-C, with IC50 values in the 1.2–4.7 μM range (viz., all the complexes show high activities). It can be noted the selectivity indexes, corresponding to the ratio [IC50 MCF10A (non-cancerous cells)/IC50 MCF7 (cancerous cells)], are 0.55 and 1.08 for the Table 2 Half-maximum inhibitory concentrationsa (IC50, µM) of comiPr iPr iPr pounds OsiPr and c-OsiPr dmso and the corresCl2 , OsI2 , c-OsCl , c-OsI ponding ruthenium complexes reported earlier27 for A549 (lung adenocarcinoma) human cells, after incubation of 24 h, using freshly prepared stock solutions of the complexes Os complex OsiPr Cl2 OsiPr I2 c-OsiPr Cl c-OsiPr I c-OsiPr dmso IC50 Ru Complex 17.3 ± 4.6 1.4 ± 0.3c 6.0 ± 0.9 18.8 ± 5.0 4.3 ± 0.3 RuiPr Cl2 RuiPr I2 c-RuiPr Cl c-RuiPr I c-RuiPr dmso Complex A549 MCF7 MCF10A MDA-MB-435 c-OsiPr dmso c-RuiPr dmso 4.3 ± 0.3 1.7 ± 0.7 4.4 ± 1.0 2.7 ± 0.5 4.7 ± 0.7 1.5 ± 0.6 2.3 ± 0.2 1.2 ± 0.2 a The results are expressed as mean values ± SD out of three independent experiments. Ru(II) and Os(II) complexes, respectively. Thus, c-OsiPr dmso is less toxic towards the healthy cells than c-RuiPr . dmso Next, the eﬀect of the phosphane R groups (Fig. 1 and 2) on the cytotoxic properties was investigated. Hence, all complexes from the ligand diphenyl(1-pyrenyl)phosphane (i.e., R = Phenyl instead of isopropyl) were tested against A549 cells. The IC50 values obtained are given in Table 4. It can first be stressed that the complexes with the phenyl groups are notably less active than those with the isopropyl groups (see Tables 2 and 4). As observed with the non-cyclometalated complexes bearing isopropyl groups, the osmium(II) compounds are more cytotoxic than the ruthenium(II) ones. On the contrary, for the cyclometalated compounds, the ruthenium(II) ones are more cytotoxic, although the diﬀerences in activity is less pronounced compared with those of isopropylcontaining complexes. For instance, the highest activities are again observed for the dmso-coordinated complexes c-OsPh dmso and c-RuPh dmso with IC50 values of 6.76 and 5.17, respectively. Ph Thus, c-RuPh dmso is 1.3-times more active than c-Osdmso while it iPr is 2.5 times for c-RuiPr and c-Os . The R groups of the dmso dmso 1-pyrenyl-based phosphane ligand do aﬀect the cytotoxic properties.25 The observed toxicity variations between the R = Ph and R = iPr complexes may result from solubility diﬀerences. Ph iPr Therefore, the lipophilic character of c-OsPh Cl , c-RuCl , c-OsCl , cPh Ph iPr iPr RuiPr , c-Os , c-Ru , c-Os and c-Os was deterCl dmso dmso dmso dmso mined using the “shake-flask” procedure, which was used to calculate their partition coeﬃcients in an octan-1-ol (o)/water (w) system.39 Thus, the lipophilicity of the selected cyclometalated compounds can be expressed as the logarithm of the par- Ph Ph Ph Table 4 IC50 valuesa (µM) of compounds OsPh Cl2 , OsI2 , c-OsCl , c-OsI and c-OsPh and the corresponding ruthenium complexes in A549 dmso cells, after incubation of 24 h, using freshly prepared stock solutions of the complexes IC50b Os complex 24 ± 1 48 ± 8 2.3 ± 0.3 5.8 ± 1.9 1.7 ± 0.7 OsPh Cl2 OsPh I2 c-OsPh Cl c-OsPh I c-OsPh dmso a The results are expressed as mean values ± SD out of three independent experiments. b Values reported earlier.27 c OsiPr I2 appears to aﬀect the cell cycle. 8394 | Dalton Trans., 2023, 52, 8391–8401 iPr Table 3 IC50 valuesa (µM) of compounds c-OsiPr dmso and c-Rudmso in A549, MCF7 (breast adenocarcinoma), MCF10A (non-tumorigenic epithelial breast) and MDA-MB-435 (melanoma) human cells, after incubation of 24 h, using freshly prepared stock solutions of the complexes IC50 Ru complex IC50 37.3 ± 5.7 >10c >50 >10c 6.8 ± 2.0 RuPh Cl2 RuPh I2 c-RuPh Cl c-RuPh I c-RuPh dmso 74.7 ± 2.3b >10 >50 7.9 ± 0.7 5.2 ± 1.0 a The results are expressed as mean values ± SD out of three independent experiments. b Value reported earlier.25 c Not tested with concentrations higher than 10 µM due to solubility limitations. This journal is © The Royal Society of Chemistry 2023 View Article Online Open Access Article. Published on 26 May 2023. Downloaded on 5/2/2026 3:00:56 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Dalton Transactions tition coeﬃcients using these solvents (log Po/w), which can be estimated using eqn (1): Acs log PO=W ¼ log ð1Þ Afs Acs where Afs is the absorption value corresponding to the maximum absorption band of the compound after partition in water saturated with octan-1-ol and Acs is the absorption value after subsequent partition in octan-1-ol saturated with water. The corresponding UV-Vis spectra are shown in Fig. S68.† From these spectroscopic data (experiments carried out in triplicate), log Po/w values could be obtained for the eight complexes (Table S7†). The positive values obtained characterize a lipophilic character. Though, in all cases, the complexes bearing a R = iPr group are less lipophilic than the R = Ph ones; hence, the log Po/w values are about 15% lower for the chloride complexes and about 5% lower for the dmso complexes (Table S7†). Consequently, the comparatively higher hydrophobicity of the R = Ph complexes may explain, at least in part, their lower cytotoxicity. As mentioned above, OsiPr I2 exhibited the lowest IC50 value iPr iPr with A549 cells. However, in contrast to OsiPr Cl2 , c-OsCl , c-OsI , iPr Ph Ph iPr c-Osdmso , OsCl2 , c-Osdmso (Tables 2 and 4), OsI2 did not induce a decrease in cell viability of more than 55%, even at the highest concentrations used; actually, few dead cells were observed. This behaviour suggested that OsiPr I2 was aﬀecting the cell cycle. Therefore, cell-cycle analyses by quantifying the DNA content with flow cytometry were carried out in A549 cells by Ph incubating them for 48 h with 5 µM OsiPr I2 and with 10 µM OsI2 (negative control). The corresponding results as the percentage of cells in the G0/G1 ( pre-initiation of DNA replication), S (DNA replication), and G2/M ( post-replication, initiation of cell division/mitosis) phases of the cell cycle are listed in Table S8† and illustrated in Fig. 5 and S69.† The observed debris population was gated mainly as a subdiploid population and is related to cell death. As anticipated, OsiPr I2 clearly aﬀects the cell cycle of A549 cells. This compound significantly arrests the cycle at the G0/ Fig. 5 Cell cycle distribution of A549 cells after 48 h treatment with Ph OsiPr I2 and OsI2 in the G0/G1, S and G2/M phases, using ﬂow cytometry. The Debris state was characterised mainly as a subdiploid population related to cell death. The data are presented as mean ± standard deviation. One-way ANOVA with Dunnett post hoc analysis was used to analyse the diﬀerences between treated cells and non-treated control groups (CT). ***p < 0.001. This journal is © The Royal Society of Chemistry 2023 Paper G1 phase (68% of the cells at this phase vs. 60% for the control). Due to G0/G1 phase blockade, statistically significant lower percentages of cells in the S and G2/M phases were observed compared to the control groups (9 vs. 17% and 11 vs. 20%, respectively). Thus, DNA production is reduced in the presence of OsiPr I2 ; hence, cell division is hampered by this compound and the observed IC50 value of 1.42 μM (Table 2) is most likely not solely due to cell death (approximately 12%, debris population) but also to a significant reduction of cell division. When the isopropyl groups of the phosphane ligand are replaced by phenyl ones, the resulting complex, viz. OsPh I2 , does not aﬀect at all the cell cycle showing values like those of the control groups. These cell-cycle data again show that the R groups of the phosphane ligand influence the behaviour of the corresponding compounds towards the cells. To discard that the lack of eﬀect of OsPh I2 on cell viability is caused by a restricted entrance of the compound inside the cell, live-cell imaging with confocal microscopy was carried out. It could be noticed that, despite its low fluorescence intensity, OsPh I2 can enter the cell and is localized in all the cell cytoplasm, and mainly accumulates inside lysosomes after 3 h of treatment (Fig. S70†). In summary, all the Os(II) compounds with isopropyl groups showed higher eﬀects on cell viability than the corresponding compounds with phenyl groups. Moreover, although OsiPr I2 showed the lowest IC50 value (namely, 1.42 µM), its eﬀect is not only due to toxicity, but also to cell-cycle arrest. In contrast, the other Os(II) compounds with isopropyl groups were cytotoxic, c-OsiPr dmso being the most potent one (IC50 = 4.26 µM). Experimental General considerations All compounds were prepared under a purified dinitrogen atmosphere using standard Schlenk and vacuum-line techniques. The solvents were purified by a solvent purification system or by standard procedures40 and stored under dinitrogen. 1H, 13C{1H}, 31P{1H}, 1H–13C HSQC NMR spectra were recorded at room temperature with 400 or 500 MHz spectrometers. Chemical shifts are reported downfield from standards and the coupling constants are given in Hz. The IR spectra were recorded using Attenuated Total Reflection (ATR) and the main absorption bands are expressed in cm−1. Highresolution mass analyses (HRMS) were carried out using electrospray ionisation (ESI). The metallic dimers of formula [MX (μ-X)(η6-p-cymene)]2 (M = Ru, Os; X = Cl, I)41–45 and diphosphane ligands PPyriPr2 27 and PPyrPh2 25 were prepared following reported procedures. Synthesis Preparation of the osmium compounds [OsCl2(η6-p-cymene)(diisopropyl(1-pyrenyl)phosphane)] (OsiPr Cl2 ). A suspension of [OsCl(μ-Cl)(η6-p-cymene)]2 (79 mg, 0.10 mmol) and PPyriPr2 (96 mg, 0.30 mmol; 1.5 equiv.) in 5 mL of dichloromethane was stirred for 24 h at room temperature. The Dalton Trans., 2023, 52, 8391–8401 | 8395 View Article Online Open Access Article. Published on 26 May 2023. Downloaded on 5/2/2026 3:00:56 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Paper solvent was subsequently removed under reduced pressure and the crude product was recrystallized in dichloromethane/ hexane at −20 °C. The crystalline compound was filtered and washed with pentane. OsiPr Cl2 was obtained as a yellow solid with a yield of 83% (119 mg). 31P{1H} NMR (162 MHz, CDCl3) δ/ppm: −4.3 (s) (Fig. S12†). 1H NMR (400 MHz, CDCl3) δ/ppm: 8.79 (d, J = 9.2, 1HAr), 8.46 (t, J = 10.0, 1HAr), 8.31–8.08 (m, 7HAr), 5.57 (br, s, 1H), 5.22 (br, s, 1H), 5.02 (br, s, 1H), 4.40 (br, s, 1H), 4.00 (br, s, 1H), 3.48 (br, s, 1H), 2.89 (hept, J = 6.8, 1HAr), 1.80 (br, s, 3H), 1.67 (br, s, 3H), 1.58 (s, 3H), 1.29–1.20 (m, 9H), 0.64 (br, s, 3H) (Fig. S13 and S14†). 13C{1H} NMR (101 MHz, CDCl3) δ/ppm: 133.8–123.8 (m, CAr, CHAr), 104.8 (d, JCP = 4.8, C, p-cymene), 90.3 (s, C, p-cymene), 81.7 (s, CH, p-cymene), 78.7 (s, CH, p-cymene), 76.3 (s, 2CH, p-cymene), 30.5 (s, CH3), 23.3–17.9 (m, CH, CH3) (Fig. S14 and S15†). FTIR (ATR, neat) ν/cm−1: 3038, 2962, 2925, 2869, 1581, 1377, 1204, 1023, 851, 644. HRMS (TOF AP(+))/m/z: [M − 2Cl − H]+; calcd for C32H36OsP 643.2169. Found 643.2167. Anal. calcd for C32H37Cl2OsP: C, 53.85%; H, 5.23%. Found: C, 52.34%; H, 5.34%. [OsI2(η6-p-cymene)(diisopropyl(1-pyrenyl)phosphane)] (OsiPr I2 ). OsiPr Cl2 (101 mg, 0.14 mmol) and sodium iodide (300 mg, 2.00 mmol) were suspended in 20 mL of technical acetone. The reaction mixture was refluxed for 1 h and the resulting dark suspension was evaporated under reduced pressure. The residue was extracted with dichloromethane (3 × 10 mL) and water (10 mL). The combined organic phase was dried with anhydrous sodium sulfate, filtered and the solvent was removed under reduced pressure. The crude compound was recrystallized in dichloromethane/hexane at −20 °C. The crystalline compound was subsequently isolated and washed with pentane. OsiPr I2 was obtained as a pale orange solid with a yield of 49% (62 mg). 31P{1H} NMR (162 MHz, CDCl3) δ/ppm: −16.7 (s) (Fig. S16†). 1H NMR (400 MHz, CDCl3) δ/ppm: 8.87 (d, J = 9.5, 1HAr), 8.43 (t, J = 8.5, 1HAr), 8.31–8.08 (m, 7HAr), 5.63 (br, s, 1H), 5.14 (br, s, 1H), 5.03 (br, s, 1H), 4.48 (br, s, 1H), 4.35 (br, s, 1H), 3.61 (br, s, 1H), 3.24 (hept, J = 6.5, 1H), 1.87 (dd, J = 16.5, 7.5, 3H), 1.71 (br, s, 3H), 1.66 (t, J = 8.0, 3H), 1.50 (dd, J = 11.0, 5.0, 3H), 1.26 (br, s, 3H), 1.03 (d, J = 7.0, 3H), 0.62 (br, s, 3H) (Fig. S17 and S18†). 13C{1H} NMR (101 MHz, CDCl3) δ/ppm: 132.8–123.2 (m, CAr, CHAr), 105.0 (s, C, p-cymene), 92.4 (s, C, p-cymene), 80.9 (s, CH, p-cymene), 79.8 (s, 2CH, p-cymene), 79.3 (s, CH, p-cymene), 79.2 (s, 2CH, p-cymene), 30.5 (s, CH3), 24.4–18.7 (m, CH, CH3) (Fig. S18 and S19†). FTIR (ATR, neat) ν/ cm−1: 2953, 2925, 2866, 1582, 1457, 1375, 1285, 1203, 1107, 1027, 868, 753, 643, 610. HRMS (TOF AP(+))/m/z: [M − I]+; calcd for C32H37IOsP 771.1293. Found 771.1276. Anal. calcd for C32H37I2OsP: C, 42.86%; H, 4.16%. Found: C, 42.88%; H, 4.17%. [OsCl-(η6-p-cymene)(k2C-diisopropyl(1-pyrenyl)phosphane)] (c6 OsiPr Cl ). A suspension of [OsCl(μ-Cl)(η -p-cymene)]2 (128 mg, 0.16 mmol), PPyriPr2 (233 mg, 0.73 mmol) and sodium acetate (77 mg, 0.94 mmol) in 40 mL of methanol was stirred for 24 h at room temperature. The solvent was removed under reduced pressure and the residue was extracted with dichloromethane (3 × 10 mL) and water (10 mL). The combined organic phase was dried with anhydrous sodium sulfate, filtered and the 8396 | Dalton Trans., 2023, 52, 8391–8401 Dalton Transactions solvent was removed under reduced pressure. The crude product was recrystallized in dichloromethane/hexane at −20 °C. The crystalline compound was filtered and washed with pentane. c-OsiPr Cl was obtained as a dark yellow solid with a yield of 75% (163 mg). 31P{1H} NMR (162 MHz, CDCl3) δ/ ppm: +39.0 (s) (Fig. S20†). 1H NMR (400 MHz, CDCl3) δ/ppm: 8.81 (s, 1HAr), 8.11–7.92 (m, 7HAr), 6.14 (d, J = 5.6, 1H), 6.11 (d, J = 6.0, 1H), 5.35 (d, J = 5.6, 1H), 5.05 (d, J = 5.6, 1H), 3.15–3.09 (m, 1H), 3.00–2.96 (m, 1H), 2.73 (hept, J = 7.2, 1H), 2.11 (s, 3H), 1.56 (dd, J = 14.8, 7.2, 3H), 1.41 (dd, J = 15.6, 7.2, 3H), 1.19 (d, J = 6.8, 3H), 1.11 (d, J = 7.2, 3H), 1.09–1.01 (m, 6H) (Fig. S21 and S22†). 13C{1H} NMR (101 MHz, CDCl3) δ/ppm: 153.0–122.5 (m, CAr, CHAr), 100.8 (s, C, p-cymene), 88.4 (s, CH, p-cymene), 87.1 (s, C, p-cymene), 83.7 (s, CH, p-cymene), 80.1 (s, CH, p-cymene), 78.5 (s, CH, p-cymene), 30.6 (s, CH), 28.8 (d, JCP = 31.7, CH), 25.3 (d, JCP = 30.3, CH), 23.7 (s, CH3), 22.8 (s, CH3), 21.6 (s, CH3), 19.8 (s, CH3), 19.3 (s, CH3), 19.0 (s, CH3), 18.0 (s, CH3) (Fig. S22 and S23†). FTIR (ATR, neat) ν/cm−1: 2921, 2852, 1566, 1457, 1303, 1033, 868, 755, 662. HRMS (TOF AP(+))/m/z: [M − Cl]+; calcd for C32H36OsP 643.2170. Found 643.2169. Anal. calcd for C32H36ClOsP: C, 56.75%; H, 5.36% Found: C, 55.82%; H, 6.80%. [OsI-(η6-p-cymene)(k2C-diisopropyl(1-pyrenyl)phosphane)] (ciPr OsiPr I ). Complex c-OsCl (163 mg, 0.24 mmol) and sodium iodide (899 mg, 6.00 mmol) were suspended in 40 mL of technical acetone. The resulting reaction mixture was refluxed for 12 h. The dark suspension was evaporated under reduced pressure and the residue was extracted with dichloromethane (3 × 10 mL) and water (10 mL). The combined organic phase was dried with anhydrous sodium sulfate, filtered and the solvent was removed under reduced pressure. The crude compound was first purified by column chromatography (silica, dichloromethane). The eluent from the fraction corresponding to the desired compound was evaporated under reduced pressure. The remaining solid was subsequently recrystallized in dichloromethane/hexane at −20 °C. The pure crystalline compound was isolated by filtration and washed with pentane. c-OsiPr I was obtained as a pale brown solid with a yield of 24% (44 mg). 31P{1H} NMR (202 MHz, CDCl3) δ/ppm: +34.6 (s) (Fig. S24†). 1H NMR (500 MHz, CDCl3) δ/ppm: 8.64 (s, 1HAr), 8.09–7.94 (m, 7HAr), 5.98 (d, J = 6.0, 1H), 5.93 (d, J = 6.0, 1H), 5.52 (d, J = 5.5, 1H), 5.23 (d, J = 5.5, 1H), 3.32 (br, 1H), 2.99–2.92 (m, 2H), 2.21 (s, 3H), 1.61 (dd, J = 13.0, 7.0, 3H), 1.49 (dd, J = 15.0, 7.0, 3H), 1.27 (d, J = 7.0, 3H), 1.09 (d, J = 7.0, 3H), 1.02 (dd, J = 13.5, 7.0, 3H), 0.90 (dd, J = 12.5, 7.0, 3H) (Fig. S25 and S26†). 13C{1H} NMR (125 MHz, CDCl3) δ/ppm: 151.9–122.8 (m, CAr, CHAr), 102.3 (s, C, p-cymene), 88.3 (s, C, p-cymene), 87.3 (s, CH, p-cymene), 81.9 (s, CH, p-cymene), 80.9 (s, CH, p-cymene), 79.8 (s, CH, p-cymene), 30.9 (s, CH), 30.0 (d, JCP = 28.4, CH), 29.3 (d, JCP = 31.1, CH), 23.9 (s, CH3), 23.2 (s, CH3), 22.8 (s, CH3), 19.8 (s, CH3), 19.7 (s, CH3), 19.1 (s, CH3), 18.4 (s, CH3) (Fig. S26 and S27†). FTIR (ATR, neat) ν/cm−1: 3033, 2958, 2922, 2866, 1566, 1461, 1439, 1301, 1174, 1028, 855, 842, 738, 607. HRMS (TOF AP(+))/m/z: [M − I]+; calcd for C32H36OsP 643.2170. Found 643.2155. Anal. calcd for C32H36IOsP: C, 50.00%; H, 4.72% Found: C, 48.24%; H, 4.71%. This journal is © The Royal Society of Chemistry 2023 View Article Online Open Access Article. Published on 26 May 2023. Downloaded on 5/2/2026 3:00:56 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Dalton Transactions [Os(η6-p-cymene)(kS-dmso)(k2C-diisopropyl(1-pyrenyl)phosphane)] iPr PF6 (c-OsiPr dmso ). Complex c-OsCl (73 mg, 0.11 mmol) and dimethylsulfoxide (0.1 mL, 110 mg, 1.41 mmol) were dissolved in 15 mL of dichloromethane and thallium hexafluorophosphate (45 mg, 0.13 mmol) was added. The initially transparent reaction mixture was stirred for 12 h, giving a cloudy solution. The thallium chloride obtained was removed by filtration using a filter paper and the solvent was evaporated under reduced pressure. The crude compound was recrystallized in dichloromethane/diethyl ether at −20 °C. The resulting crystalline product was isolated by filtration and washed with pentane. c-OsiPr dmso was obtained as a pale-yellow solid with a yield of 45% (39 mg). 31P{1H} NMR (162 MHz, CDCl3) δ/ppm: +43.5 (s), −144.0 (hept, JPF = 713.1) (Fig. S28†). 1H NMR (400 MHz, CDCl3) δ/ppm: 8.59 (s, 1HAr), 8.35 (d, J = 6.8, 1HAr), 8.21–8.05 (m, 6HAr), 6.66 (d, J = 6.4, 1H), 6.61 (d, J = 6.0, 1H), 5.93 (s, br, 1H), 5.61 (s, br, 1H), 3.49 (s, 3H), 3.34–2.28 (m, 1H), 3.14 (hept, J = 6.8, 1H), 2.65–2.59 (m, 1H), 2.40 (s, 3H), 1.75 (s, 3H), 1.68 (dd, J = 16.0, 6.8, 3H), 1.47 (dd, J = 17.2, 6.4, 3H), 1.43 (d, J = 7.2, 3H), 1.33 (dd, J = 13.2, 6.4, 3H), 1.12 (d, J = 6.8, 3H), 0.25 (dd, J = 16.4, 6.8, 3H) (Fig. S29 and S30†). 13C{1H} NMR (101 MHz, CDCl3) δ/ppm: 151.3–122.7 (m, CAr, CHAr), 88.3 (s, CH, p-cymene), 85.4 (s, CH, p-cymene), 54.7 (s, CH3), 46.2 (s, CH3), 31.2 (s, CH), 31.0 (d, JCP = 32.7, CH), 25.9 (d, JCP = 32.2, CH), 24.7 (s, CH3), 21.4 (s, CH3), 19.3 (s, 2CH3), 19.1 (s, CH3), 18.6 (s, CH3), 18.4 (d, JCP = 5.4, CH3) (Fig. S30 and S31†). FTIR (ATR, neat) ν/cm−1: 2970, 1442, 1385, 1289, 1106, 1010, 843 (ν(PF6)), 741, 668. HRMS (TOF AP(+))/m/z: [M − PF6]+; calcd for C32H36OsP 721.2309. Found 721.2294. [OsCl2(η6-p-cymene)(diphenyl(1-pyrenyl)phosphane)] (OsPh Cl2 ). iPr The procedure used to prepare OsCl2 was followed but using [OsCl(μ-Cl)(η6-p-cymene)]2 (56 mg, 0.07 mmol) and PPyrPh2 (70 mg, 0.18 mmol). OsPh Cl2 was obtained as an orange-brown solid with a yield of 73% (80 mg). 31P{1H} NMR (162 MHz, CDCl3) δ/ppm: −4.8 (s) (Fig. S32†). 1H NMR (400 MHz, CDCl3) δ/ppm: 8.91 (d, J = 9.2, 1HAr), 8.31–8.27 (m, 2HAr), 8.20 (d, J = 9.2, 1HAr), 8.13–8.08 (m, 4HAr), 7.66 (br, 4H), 7.57 (dd, J = 10.8, 8.0, 1HAr), 7.40–7.36 (br, 2HAr), 7.29 (br, 6HAr), 5.43 (br, s, 2H), 4.71 (br, s, 2H), 3.00 (hept, J = 7.2, 1H), 1.74 (br, s, 3H), 1.25 (d, J = 6.8, 6H) (Fig. S33 and S34†). 13C{1H} NMR (125 MHz, CDCl3) δ/ppm: 136.1–123.9 (m, CAr, CHAr), 107.5 (d, JCP = 6.7, C, p-cymene), 90.8 (s, C, p-cymene), 30.4 (s, CH), 22.3 (s, 2CH3), 18.4 (s, CH3) (Fig. S34 and S35†). FTIR (ATR, neat) ν/ cm−1: 3052, 2956, 1581, 1434, 1386, 1158, 1092, 1027, 856, 740, 690, 636, 607. HRMS (TOF AP(+))/m/z: [M − 2Cl − H]+; calcd for C38H32OsP 711.1856. Found 711.1853. Anal. calcd for C38H33Cl2OsP: C, 58.38%; H, 4.25% Found: C, 58.22%; H, 4.27%. [OsI2(η6-p-cymene)(diphenyl(1-pyrenyl)phosphane)] (OsPh I2 ). The Ph procedure used to prepare OsiPr I2 was followed but using OsCl2 (79 mg, 0.10 mmol) and sodium iodide (210 mg, 1.40 mmol). OsPh I2 was obtained as an orange solid with a yield of 75% (72 mg). 31P{1H} NMR (162 MHz, CDCl3) δ/ppm: −20.2 (s) (Fig. S36†). 1H NMR (400 MHz, CDCl3) δ/ppm: 8.80 (br, s, 1HAr), 8.28 (d, J = 7.6, 1HAr), 8.24 (d, J = 8.0, 1HAr), 8.19 (d, J = 8.8, 1HAr), 8.13–8.01 (m, 5HAr), 7.70 (br, 5HAr), 7.33 (br, 5HAr), This journal is © The Royal Society of Chemistry 2023 Paper 5.58 (br, 2H), 5.08–4.50 (br, 2H), 3.43 (hept, J = 6.8, 1H), 1.78 (s, 3H), 1.23 (d, J = 6.8, 6H) (Fig. S37 and S38†). 13C{1H} NMR (101 MHz, CDCl3) δ/ppm: 133.7–123.8 (m, CAr, CHAr), 106.7 (d, JCP = 6.3, C, p-cymene), 93.2 (s, C, p-cymene), 31.9 (s, 2CH3), 31.7 (s, CH), 18.9 (s, CH3) (Fig. S38 and S39†). FTIR (ATR, neat) ν/cm−1: 3037, 2957, 2865, 1582, 1433, 1370, 1207, 1181, 1090, 1022, 851, 752, 689, 631. HRMS (TOF AP(+))/m/z: [M − I]+; calcd for C38H33IOsP 839.0979. Found 839.0976. Anal. calcd for C38H33I2OsP: C, 47.31%; H, 3.45% Found: C, 46.99%; H, 3.78%. [OsCl-(η6-p-cymene)(k2C-diphenyl(1-pyrenyl)phosphane)] (c-OsPh Cl ). The procedure used to prepare c-OsiPr Cl was applied but using [OsCl(μ-Cl)(η6-p-cymene)]2 (126 mg, 0.16 mmol), PPyrPh2 (286 mg, 0.74 mmol) and sodium acetate (77 mg, 0.94 mmol). c-OsiPr Cl was obtained as a dark yellow solid with a yield of 97% (231 mg). 31P{1H} NMR (202 MHz, CDCl3) δ/ppm: +28.5 (s) (Fig. S40†). 1H NMR (500 MHz, CDCl3) δ/ppm: 8.84 (s, 1HAr), 8.18–8.05 (m, 9HAr), 7.98 (t, J = 7.5, 1HAr), 7.73–7.41 (m, 4HAr), 7.34–7.32 (m, 3HAr), 7.22–7.19 (m, 2HAr), 5.90 (d, J = 6.0, 1H), 5.81 (d, J = 5.5, 1H), 4.77 (d, J = 5.5, 1H), 4.55 (d, J = 5.5, 1H), 2.35 (hept, J = 7.0, 1H), 2.11 (s, 3H), 1.03 (d, J = 7.0, 3H), 0.73 (d, J = 7.0, 3H) (Fig. S41 and S42†). 13C{1H} NMR (125 MHz, CDCl3) δ/ppm: 152.8–122.9 (m, CAr, CHAr), 98.4 (s, C, p-cymene), 91.3 (s, C, p-cymene), 86.3 (d, JCP = 3.4, CH, p-cymene), 84.9 (d, JCP = 5.3, CH, p-cymene), 84.7 (d, JCP = 5.9, CH, p-cymene), 78.5 (d, JCP = 4.5, CH, p-cymene), 30.0 (s, CH), 22.9 (s, CH3), 22.7 (s, CH3), 18.0 (s, CH3) (Fig. S42 and S43†). FTIR (ATR, neat) ν/cm−1: 3035, 2956, 1581, 1480, 1433, 1381, 1305, 1181, 1095, 1028, 846, 752, 601. HRMS (TOF AP(+))/m/z: [M − Cl]+; calcd for C38H32OsP 711.1857. Found 711.1847. Anal. calcd for C38H32ClOsP: C, 61.24%; H, 4.33% Found: C, 61.69%; H, 4.35%. [OsI-(η6-p-cymene)(k2C-diphenyl(1-pyrenyl)phosphane)] (c-OsPh I ). A suspension of [OsI(μ-I)(η6-p-cymene)]2 (88 mg, 0.08 mmol), PPyrPh2 (88 mg, 0.23 mmol) and sodium acetate (50 mg, 0.61 mmol) in 25 mL of methanol was stirred for 12 h at room temperature. The solvent was removed under reduced pressure and the residue was extracted with dichloromethane (3 × 10 mL) and water (10 mL). The combined organic phase was dried with anhydrous sodium sulfate, filtered and the filtrate was evaporated under reduced pressure. The crude was recrystallized in dichloromethane/diethyl ether at −20 °C. The pure crystalline compound was isolated by filtration and washed with pentane. c-OsPh I was obtained as an olive-green solid with a yield of 46% (61 mg). 31P{1H} NMR (202 MHz, CDCl3) δ/ppm: +24.1 (s) (Fig. S44†). 1H NMR (500 MHz, CDCl3) δ/ppm: 8.70 (s, 1HAr), 8.18–7.96 (m, 9HAr), 7.45–7.40 (m, 4HAr), 7.30–7.26 (m, 3HAr), 7.18–7.14 (m, 2HAr), 5.70 (d, J = 6.0, 1H), 5.60 (d, J = 7.2, 1H), 4.93 (d, J = 6.0, 1H), 4.58 (d, J = 5.6, 1H), 2.64 (hept, J = 7.2, 1H), 2.27 (s, 3H), 1.13 (d, J = 6.8, 3H), 0.66 (d, J = 7.2, 3H) (Fig. S45 and S46†). 13C{1H} NMR (125 MHz, CDCl3) δ/ppm: 153.2–122.7 (m, CAr, CHAr), 101.2 (s, C, p-cymene), 90.1 (s, C, p-cymene), 86.4 (d, JCP = 5.1, CH, p-cymene), 86.0 (d, JCP = 3.6, CH, p-cymene), 82.9 (d, JCP = 5.6, CH, p-cymene), 78.4 (d, JCP = 4.1, CH, p-cymene), 30.5 (s, CH), 23.1 (s, CH3), 22.3 (s, CH3), 19.0 (s, CH3) (Fig. S46 and S47†). FTIR (ATR, neat) ν/cm−1: Dalton Trans., 2023, 52, 8391–8401 | 8397 View Article Online Open Access Article. Published on 26 May 2023. Downloaded on 5/2/2026 3:00:56 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Paper 3038, 2956, 2919, 2865, 1566, 1459, 1433, 1381, 1304, 1180, 1094, 1030, 846, 754, 693, 619. HRMS (TOF AP(+))/m/z: [M − I]+; calcd for C38H32OsP 711.1857. Found 711.1848. Anal. calcd for C38H32IOsP: C, 54.54%; H, 3.85% Found: C, 54.99%; H, 4.01%. [Os(η6-p-cymene)(kS-dmso)(k2C-diphenyl(1-pyrenyl)phosphane)] iPr PF6 (c-OsPh dmso ). The procedure used to prepare c-Osdmso was folPh lowed but using c-OsCl (101 mg, 0.14 mmol), dimethylsulfoxide (0.1 mL, 110 mg, 1.41 mmol) and thallium hexafluorophosphate (65 mg, 0.19 mmol). The reaction mixture was stirred for 2.5 h at room temperature. c-OsPh dmso was obtained as a pale-green solid with a yield of 68% (89 mg). 31P {1H} NMR (162 MHz, CDCl3) δ/ppm: +28.1 (s), −144.2 (hept, JPF = 714.4) (Fig. S48†). 1H NMR (400 MHz, CDCl3) δ/ppm: 8.77 (s, 1HAr), 8.51 (d, J = 7.6, 1HAr), 8.32–8.13 (m, 7HAr), 7.88 (m, 1HAr), 7.64–7.63 (m, 3HAr), 7.45–7.33 (m, 4HAr), 6.48 (d, J = 5.6, 1H), 5.99 (d, J = 6.0, 1H), 5.73 (d, J = 6.0, 1H), 4.57 (d, J = 6.4, 1H), 3.44 (s, 3H), 2.87 (hept, J = 6.8, 1H), 2.51 (s, 3H), 1.76 (s, 3H), 1.24 (d, J = 7.2, 3H), 0.95 (d, J = 6.8, 3H) (Fig. S49 and S50†). 13C{1H} NMR (125 MHz, CDCl3) δ/ppm: 143.3–125.2 (m, CAr, CHAr), 119.4 (s, C, p-cymene), 106.7 (s, C, p-cymene), 93.4 (s, CH, p-cymene), 90.4 (d, JCP = 3.4, CH, p-cymene), 86.3 (d, JCP = 5.3, CH, p-cymene), 81.5 (d, JCP = 5.9, CH, p-cymene), 52.8 (s, CH3), 41.2 (s, CH3), 30.5 (s, CH), 23.7 (s, CH3), 20.4 (s, CH3), 19.0 (s, CH3) (Fig. S50 and S51†). FTIR (ATR, neat) ν/cm−1: 2972, 1568, 1472, 1437, 1387, 1292, 1183, 1097, 1013, 843 (ν(PF6)), 694, 606. HRMS (TOF AP(+))/m/z: [M − PF6]+; calcd for C40H38OPOsS 789.1996. Found 789.1979. Preparation of the ruthenium compounds [RuI2(η6-p-cymene)(diphenyl(1-pyrenyl)phosphane)] (RuPh I2 ). The Ph procedure used to prepare OsiPr I2 was followed but using RuCl2 (450 mg, 0.65 mmol) and sodium iodide (1500 mg, 10.00 mmol). RuPh I2 was obtained as a dark brown solid with a yield of 65% (369 mg). 31P{1H} NMR (162 MHz, CDCl3) δ/ppm: +23.1 (s) (Fig. S52†). 1H NMR (400 MHz, CDCl3) δ/ppm: 8.61 (br, s, 1HAr), 8.28 (d, J = 7.6, 1HAr), 8.23–8.04 (m, 7HAr), 7.95 (d, J = 9.6, 1HAr), 7.73 (br, s, 4HAr), 7.35 (br, 5HAr), 5.43 (br, s, 2H), 4.71 (br, s, 2H), 3.53 (hept, J = 6.8, 1H), 1.79 (s, 3H), 1.23 (br, s, 6H) (Fig. S53 and S54†). 13C{1H} NMR (101 MHz, CDCl3) δ/ ppm: 135.2–123.7 (m, CAr, CHAr), 112.8 (d, JCP = 6.1, C, p-cymene), 100.8 (s, C, p-cymene), 88.6 (d, JCP = 4.5, CH, p-cymene), 88.1 (br, s, CH, p-cymene), 32.1 (s, CH), 22.8 (br, s, 2CH3), 19.1 (s, CH3) (Fig. S54 and S55†). FTIR (ATR, neat) ν/ cm−1: 3048, 2957, 2917, 2861, 1470, 1430, 1374, 1087, 852, 687, 635. HRMS (TOF AP(+))/m/z: [M − I]+; calcd for C38H33IPRu 749.0408. Found 749.0413. Anal. calcd for C38H33I2PRu: C, 52.13%; H, 3.80% Found: C, 51.80%; H, 3.92%. [RuCl-(η6-p-cymene)(k2C-diphenyl(1-pyrenyl)phosphane)] (c-RuPh Cl ). A suspension of [RuCl(μ-Cl)(η6-p-cymene)]2 (643 mg, 1.05 mmol), PPyrPh2 (870 mg, 2.25 mmol) and sodium acetate (492 mg, 6.00 mmol) in 160 mL of methanol was stirred for 4 h at room temperature. The solvent was removed under reduced pressure and the residue was extracted with dichloromethane (3 × 10 mL) and water (10 mL). The combined organic phase was dried with anhydrous sodium sulfate and the filtrate was evaporated under reduced pressure. The crude 8398 | Dalton Trans., 2023, 52, 8391–8401 Dalton Transactions was recrystallized in dichloromethane/diethyl ether at −20 °C. The resulting crystalline material was isolated by filtration and washed with pentane. c-RuPh Cl was obtained as an orange solid with a yield of 57% (787 mg). 31P{1H} NMR (162 MHz, CDCl3) δ/ppm: +66.4 (s) (Fig. S56†). 1H NMR (400 MHz, CDCl3) δ/ppm: 8.94 (s, 1HAr), 8.18–7.97 (m, 9HAr), 7.45–7.42 (m, 3HAr), 7.36–7.29 (m, 3HAr), 7.23–7.18 (m, 2HAr), 5.97 (d, J = 7.2, 1H), 5.95 (d, J = 7.2, 1H), 4.65 (d, J = 6.0, 1H), 4.60 (d, J = 6.0, 1H), 2.51 (hept, J = 6.8, 1H), 1.98 (s, 3H), 1.07 (d, J = 6.8, 3H), 0.75 (d, J = 7.2, 3H) (Fig. S57 and S58†). 13C{1H} NMR (101 MHz, CDCl3) δ/ppm: 168.3–122.7 (m, CAr, CHAr), 108.9 (s, C, p-cymene), 99.2 (s, C, p-cymene), 95.6 (d, JCP = 4.1, CH, p-cymene), 93.1 (d, JCP = 4.9, CH, p-cymene), 91.9 (d, JCP = 5.5, CH, p-cymene), 87.2 (d, JCP = 4.6, CH, p-cymene), 30.4 (s, CH), 22.62 (s, CH3), 22.56 (s, CH3), 18.4 (s, CH3) (Fig. S58 and S59†). FTIR (ATR, neat) ν/cm−1: 3065, 2955, 2920, 1570, 1432, 1304, 801, 652, 600. HRMS (TOF AP(+))/m/z: [M − Cl]+; calcd for C38H32PRu 621.1285. Found 621.1298. Anal. calcd for C38H32ClPRu: C, 69.56%; H, 4.92% Found: C, 67.38%; H, 4.87%. [RuI-(η6-p-cymene)(k2C-diphenyl(1-pyrenyl)phosphane)] (c-RuPh I ). The procedure used to prepare c-OsPh I was followed but using [RuI(μ-I)(η6-p-cymene)]2 (254 mg, 0.26 mmol), PPyrPh2 (290 mg, 0.75 mmol) and sodium acetate (164 mg, 2.00 mmol); the reaction mixture was stirred for 24 h at room temperature. c-RuPh I was obtained as a brown solid with a yield of 74% (287 mg). 31P{1H} NMR (162 MHz, CDCl3) δ/ppm: +65.0 (s) (Fig. S60†). 1H NMR (400 MHz, CDCl3) δ/ppm: 8.78 (s, 1HAr), 8.18–7.96 (m, 9HAr), 7.45–7.40 (m, 3HAr), 7.30–7.26 (m, 3HAr), 7.15–7.10 (m, 2HAr), 5.77 (d, J = 5.6, 1H), 5.74 (d, J = 6.0, 1H), 4.88 (d, J = 6.4, 1H), 4.66 (d, J = 6.0, 1H), 2.78 (hept, J = 6.8, 1H), 2.15 (s, 3H), 1.15 (d, J = 6.8, 3H), 0.70 (d, J = 7.2, 3H) (Fig. S61 and S62†). 13C{1H} NMR (101 MHz, CDCl3) δ/ppm: 164.2–122.7 (m, CAr, CHAr), 111.6 (s, C, p-cymene), 99.2 (s, C, p-cymene), 95.1 (d, JCP = 4.0, CH, p-cymene), 93.1 (d, JCP = 5.2, CH, p-cymene), 91.4 (d, JCP = 5.3, CH, p-cymene), 86.7 (d, JCP = 4.6, CH, p-cymene), 31.0 (s, CH), 23.0 (s, CH3), 22.4 (s, CH3), 19.3 (s, CH3) (Fig. S62 and S63†). FTIR (ATR, neat) ν/cm−1: 3045, 2954, 2866, 1570, 1468, 1433, 1385, 1302, 1173, 1091, 1027, 846, 754, 631, 604. HRMS (TOF AP(+))/m/z: [M − I]+; calcd for C38H32PRu 621.1285. Found 621.1298. Anal. calcd for C38H32IPRu: C, 61.05%; H, 4.31% Found: C, 61.26%; H, 3.70%. [Ru(η6-p-cymene)(kS-dmso)(k2C-diphenyl(1-pyrenyl)phosphane)] iPr PF6 (c-RuPh dmso ). The procedure used to prepare c-Osdmso was folPh lowed but using c-RuCl (336 mg, 0.51 mmol), dimethylsulfoxide (0.35 mL, 385 mg, 4.94 mmol) and thallium hexafluorophosphate (196 mg, 0.56 mmol). c-RuPh dmso was obtained as a yellow solid with a yield of 64% (274 mg). 31P{1H} NMR (162 MHz, CDCl3) δ/ppm: +67.3 (s), −144.0 (hept, JPF = 713.4) (Fig. S64†). 1H NMR (400 MHz, CDCl3) δ/ppm: 8.79 (s, 1HAr), 8.52 (d, J = 6.8, 1HAr), 8.31–8.13 (m, 8HAr), 7.65 (m, 3HAr), 7.47–7.36 (m, 3HAr), 7.06 (br, 2HAr), 6.60 (d, J = 5.2, 1H), 6.16 (d, J = 6.0, 1H), 5.81 (d, J = 6.0, 1H), 4.62 (d, J = 6.0, 1H), 3.27 (s, 3H), 2.91 (hept, J = 6.8, 1H), 2.40 (s, 3H), 1.50 (s, 3H), 1.19 (d, J = 6.8, 3H), 0.45 (d, J = 6.8, 3H) (Fig. S65 and S66†). 13C{1H} NMR (125 MHz, CDCl3) δ/ppm: 157.7–125.3 (m, CAr, CHAr), This journal is © The Royal Society of Chemistry 2023 View Article Online Open Access Article. Published on 26 May 2023. Downloaded on 5/2/2026 3:00:56 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Dalton Transactions 122.5 (s, C, p-cymene), 113.0 (s, C, p-cymene), 100.4 (s, CH, p-cymene), 99.3 (s, CH, p-cymene), 95.6 (d, JCP = 5.0, CH, p-cymene), 89.2 (s, CH, p-cymene), 52.3 (s, CH3), 44.7 (s, CH3), 31.0 (s, CH), 23.8 (s, CH3), 20.6 (s, CH3), 19.2 (s, CH3) (Fig. S66 and S67†). FTIR (ATR, neat) ν/cm−1: 1570, 1479, 1436, 1314, 1184, 1094, 1014, 843 (ν(PF6)), 697, 620. HRMS (TOF AP(+))/m/z: [M − PF6]+; calcd for C40H38OPRuS 699.1424. Found 699.1434. X-ray crystallography iPr Ph Ph Ph Ph Data for compounds OsiPr Cl2 , OsI2 , OsCl2 , OsI2 , c-OsCl , c-RuCl Ph and c-RuI were collected on a Bruker APEX II QUAZAR diﬀractometer equipped with a microfocus multilayer monochromator with Mo Kα radiation (λ = 0.71073 Å). Data for comiPr pounds c-OsiPr and c-RuPh Cl , c-OsI dmso were collected at BL1346 XALOC beamline of the ALBA synchrotron (λ = 0.72931 Å). Data reduction and absorption corrections were performed by using SAINT and SADABS, respectively.47 The structures were solved using SHELXT48 and refined by full-matrix least-squares on F2 with SHELXL.49 For compound c-RuPh dmso , a void containing only diﬀuse electron density was analysed and taken into account with Olex2/Solvent Mask.50 An estimated content of two diﬀuse lattice CH2Cl2 molecules per formula unit were deduced, and included in the formula. All details can be found in CCDC 2237617–2237626,† which contain the supplementary crystallographic data for this paper. Cell viability assays To screen the eﬀect of all the Os(II) compounds on cell viability, the A549 human cell line (lung adenocarcinoma) was chosen. In some experiments, the MCF7 (breast adenocarcinoma), MCF10A (non-tumorigenic epithelial breast) and MDA-MB-435 (melanoma) human cell lines were used as well. For these experiments, the diﬀerent cell lines were cultured in a 96-well plate (1 × 105 cells per well) for 24 h with their proper medium at 37 °C and 5% CO2. A549, was cultured in Dulbecco’s modified Eagle’s medium (DMEM, Biological Industries, Beit Haemek, Israel), MCF7, MCF10A and MDA-MB-435 were maintained in DMEM/Ham’s F12 [1 : 1]. All mediums were supplemented with 100 U mL−1 penicillin, 100 μg mL−1 streptomycin, and 2 mM glutamine. Moreover, A549, MCF7 and MDA-MB-435 were supplemented with 10% fetal bovine serum, whereas MCF10A was supplemented with 5% horse serum, 10 µg mL−1 insulin, 100 ng mL−1 cholera toxin, 500 ng mL−1 hydrocortisone and 20 ng mL−1 epidermal growth factor. The next day, the cells were treated with freshly prepared complex solutions at the chosen concentration range for 24 h. Subsequently, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution (MTT) of 5 mg mL−1 was added and incubation was carried out for 2 h at 37 °C. The formazan crystals formed were dissolved in 100 µL of DMSO and the absorbance at 570 nm was recorded using a multiwell plate reader (Multiskan FC, Thermo Fisher Scientific Inc). The estimated half-inhibitory concentration (IC50) for each compound was calculated using GraphPad Prism v8.0.1 software (Graph-Pad, San Diego, CA, USA). This journal is © The Royal Society of Chemistry 2023 Paper Lipophilicity The lipophilicity of selected compounds was quantified by calculating the partition coeﬃcients in an octan-1-ol/water system using the “shake-flask” method. The complexes were suspended in milliQ water saturated with octan-1-ol. After sonicating them for 1 h at 298 K, the suspensions were shaken for 24 h using an orbital-shaker at a rate of 120 rpm. The samples were subsequently filtered with a 0.2 μm Puradisc FP 30 mm Cellulose Acetate Syringe Filter (Whatman). Some aliquots (of 4 mL) of the filtrates (viz., fs samples) were reserved (for the UV-Vis measurements). Other aliquots of 4 mL were poured onto 4 mL of octan1-ol saturated with milliQ water. The resulting mixtures were shaken for 24 h at 298 K. The samples were then centrifuged, and the organic phases were isolated (viz., cs samples). UV-Vis spectra were recorded for both the fs and cs samples (Fig. S68†). The observed diﬀerences between the MLCT absorptions of the two types of samples, namely Afs and Acs (see ESI†), were used to calculate the log Po/w values applying eqn (1). The data obtained after measurements in triplicate are listed in Table S7.† Cell cytometry A549 cells were seeded in a 6-well plate (2 × 105 cells per well) and incubated for 24 h at °C. Then, the cells were treated with the diﬀerent Os(II) complexes. After 48 h of incubation, the cells were harvested and fixed with a cold 70% ethanol solution and were kept at −20 °C for at least 3 h following the MUSE™ Cell cycle kit (EMD Millipore, Burlington, MA, USA) manufacturer’s instructions. Afterwards, the cells were incubated with the MUSE Cell cycle reagent for 30 min and examined using the flow cytometry MUSE Cell Analyzer, to characterise the diﬀerent populations (G0/G1, S and G2/M) depending on their DNA content. One-way ANOVA with Dunnett post hoc analysis was used to analyse the observed diﬀerences. Confocal microscopy To localize OsPh I2 inside the cells, A549 cells were cultured in 8-well sterile-Slide (Ibidi, Gräfelfing, Germany) (3 × 104 cells per well). After 24 h of incubation, the cells were treated with Ph 50 µM OsPh I2 for 3 h. The high concentration of OsI2 used is due to the low fluorescence emission exhibited by this complex. To visualize the Os(II) compound, it was excited at 405 nm with a laser and the emission between 413 and 488 nm was recorded. To localize the lysosomes in the cell, Lysotracker Red (Molecular Probes, OR, USA) at a concentration of 15 nM was pre-incubated for 30 min. The images were obtained using a Carl Zeiss LSM 880 spectral confocal laser scanning microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) and processed with ZEN 2 blue edition software (Zeiss). Representative images from three independent experiments are shown in Fig. S70.† Conclusions The present study was carried out to compare the cytotoxic properties of osmium(II) arene complexes from hindered Dalton Trans., 2023, 52, 8391–8401 | 8399 View Article Online Open Access Article. Published on 26 May 2023. Downloaded on 5/2/2026 3:00:56 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Paper monophosphane ligands with those of their ruthenium(II) counterparts. We have indeed previously shown that such [RuX2(η6-arene)(diR(1-pyrenyl)phosphane)] exhibit interesting cytotoxic behaviours as well as chemical properties, since they can undergo cyclometalation under basic conditions, leading to organometallic compounds with distinct biological activities (compared with the parent, non-cyclometalated complexes). The eﬀect of the metal centre, namely osmium(II) vs. ruthenium(II), on the properties of this family of half-sandwich complexes was investigated. Hence, 10 osmium(II) complexes were prepared from two diﬀerent diR(1-pyrenyl)phosphane ligands (R = isopropyl or phenyl), halides (X = Cl or I) and DMSO (X = dmso for the cyclometalated complexes). The X-ray structures of 7 of these osmium(II) complexes were obtained; it can be pointed out here that, to the best of our knowledge, solid-state structures of cycloosmated half-sandwich complexes have not been reported so far. To be able to perform a complete comparison with all ruthenium(II) analogues, 4 ruthenium(II) complexes were also prepared; the other 6 compounds to complete the series were reported earlier. The crystal structures of 3 of these new ruthenium(II) complexes were obtained. The cytotoxic behaviours of the two related metallic series revealed some interesting features. The toxicity of the noncyclometalated osmium(II) compounds is higher than the corresponding ruthenium(II) complexes, with IC50 values down to 1.42 µM for A549 cells (while the lowest value is 24 µM for ruthenium). Notably, the osmium(II) complexes do not undergo rapid cyclometalation in DMSO contrary to the ruthenium(II) ones, indicating that the reactivity of Os(II) is slower than that of Ru(II), as one would have expected it.30 Indeed, as shown in a previous study, as soon as they are dissolved in DMSO, such ruthenium(II) complexes progressively convert into cyclometalated c-RuRX species through a multi-step process.27 Therefore, the cytotoxic activity of “pure” ruthenium (II) compounds of the type RuRX2 cannot be determined and it appears that the intermediate species are not very active. For the OsRX2 complexes, their cytotoxic properties can be determined since their multi-step conversion towards the formation of c-OsRX is significantly slower. Regarding the cyclometalated complexes, while the ruthenium ones are very active in various cell lines (IC50 values between 2.61 and 1.19 µM), the osmium ones are comparatively less toxic (IC50 values between 4.36 and 2.32 µM); the cycloosmated compounds are twice less active but are still quite cytotoxic with IC50 < 5 µM. Again, the observed diﬀerence may be ascribed to the distinct activity of ruthenium compared with osmium, the latter being usually more inert than the former. It can also be stressed that cellcycle studies have shown that the highest activity, viz. lowest IC50 value, exhibited by OsiPr I2 with A549 cells is not solely due to its cytotoxicity but also to its ability to arrest the cell cycle, which is an interesting property regarding the possibility to stop tumour growth. It has been shown as well that the nonactivity exhibited by OsPh I2 does not arise from its inability to enter A549 cells, therefore illustrating the importance of the R group of the diR(1-pyrenyl)phosphane ligand, namely phenyl 8400 | Dalton Trans., 2023, 52, 8391–8401 Dalton Transactions vs. isopropyl, properties. regarding the corresponding biological Conﬂicts of interest There are no conflicts to declare. Acknowledgements Financial support from the Spanish Ministerio de Ciencia e Innovación (Projects PID2020-115537RB-I00, PID2020115658GB-I00 and RED2018-102471-T; MCIN/AEI/10.13039/ 501100011033), the RSC (RSC Research Fund grant RF19-7147) and the AGAUR (Project 2021-SGR-01107) is kindly acknowledged. P. G. thanks the Institució Catalana de Recerca i Estudis Avançats (ICREA). This research used resources of the ALBA synchrotron. The corresponding crystallographic measurements were performed with the collaboration of ALBA staﬀ at BL13-XALOC beamline. 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