Novel organo-osmium(ii) proteosynthesis inhibitors active against human ovarian cancer cells reduce gonad tumor growth inCaenorhabditis elegans

{"full_text": " INORGANIC CHEMISTRY\n FRONTIERS\n\n View Article Online\n RESEARCH ARTICLE View Journal | View Issue\n\n\n\n\n Novel organo-osmium(II) proteosynthesis\n Cite this: Inorg. Chem. Front., 2021,\n inhibitors active against human ovarian cancer\n 8, 141 cells reduce gonad tumor growth in\n Caenorhabditis elegans\u2020\nPublished on 27 October 2020. Downloaded on 5/12/2026 12:52:24 PM.\n\n\n\n\n Enrique Ortega, a Francisco J. Ballester, a Alba Hern\u00e1ndez-Garc\u00eda, a\n Samanta Hern\u00e1ndez-Garc\u00eda, b M. Alejandra Guerrero-Rubio,b Delia Bautista,c\n M. Dolores Santana, *a Fernando Gand\u00eda-Herrero*b and Jos\u00e9 Ruiz *a\n\n This work reports the synthesis and characterization of some novel osmium(II) complexes with potential\n as anticancer drugs tested in vitro and in vivo. The complexes have a structure [(\u03b76-p-cym)Os(C^N)(X)]0/+,\n where the C^N ligand is deprotonated 2-phenylpyridine ( ppy) or 4-(2-pyridin)benzaldehyde ( ppy-CHO)\n and X is chloride, pyridine ( py) or the pyridine derivative 4-NMe2-py. The in vitro cytotoxic studies\n showed that complexes [(\u03b76-p-cym)Os(C^N)(4-NMe2-py)]+ (C^N = ppy 2a and ppy-CHO 5a) exerted\n e\ufb00ective antiproliferative activity towards both cisplatin-sensitive ovarian cancer cells (A2780) and cispla-\n tin-resistant cells (A2780cis). The mechanism underlying the antiproliferative e\ufb00ects in vitro was studied\n showing a reduction of proteosynthesis up to 58% and an increase of apoptosis modulated by caspase-3.\n Model animal Caenorhabditis elegans was used to estimate the e\ufb00ects of 2a and 5a and the in-house\n 4-NMe2-py Ru(II) analogue 5b in vivo. Compounds 2a, 5a and 5b were able to reduce tumor growth up\n 32.2%, 19% and 30%, respectively in the tumoral strain JK1466 and presented low toxicity in both tumoral\n and wild-type strains. The mechanistic study using reporter gene expression showed that 2a, 5a and 5b\n were able to maintain the reactive oxygen species (ROS) levels in the animals by increased expression of\n the mitochondrial superoxide dismutase 3 (SOD-3), an indication that they were able to regulate oxidative\n stress genes speci\ufb01cally. Interestingly the three complexes showed a similar mechanism of action,\n suggesting that the identity of the metal ion does not matter and the e\ufb00ect is more related to the whole\n Received 31st December 2019, structure of the complex. Worthy of note, cisplatin treatment produced elevated ROS levels in the animals\n Accepted 26th October 2020\n and induced the expression of glutathione transferase 4 (GST-4) suggesting di\ufb00erent mechanisms of\n DOI: 10.1039/c9qi01704f action for the two complexes. Altogether the results showed that osmium(II) complexes can be potential\n rsc.li/frontiers-inorganic candidates in the search for novel chemotherapeutic drugs.\n\n\n\n Introduction synthesis rates compared to their normal counterparts.1 As the\n translation process integrates all the oncogenic signals, this\n Targeting inherently unique biological activities in cancer cells biological property of cancer cells might be exploited to prefer-\n might improve therapeutic activity and prevent the develop- entially eliminate these cells.2\n ment of drug resistance. In order to accommodate tumor In the field of medicinal inorganic chemistry, the DNA\n growth demands, most cancer cells exhibit elevated protein damaging agent CDDP (cisplatin) has succeeded as a clinical\n drug for epithelial ovarian cancer chemotherapy. However,\n a\n severe toxicity and resistance phenomena often develop, which\n Departamento de Qu\u00edmica Inorg\u00e1nica, Universidad de Murcia, and Institute for Bio-\n subsequently leads to treatment failure.3 Hence, significant\n Health Research of Murcia (IMIB-Arrixaca), E-30071 Murcia, Spain.\n E-mail: dsl@um.es, jruiz@um.es e\ufb00orts have been made in medicinal chemistry to identify\n b\n Departamento de Bioqu\u00edmica y Biolog\u00eda Molecular A, Unidad Docente de Biolog\u00eda, novel metal-based agents that overcome CDDP major draw-\n Facultad de Veterinaria, Universidad de Murcia, E-30071 Murcia, Spain. backs.4 In particular, ruthenium and osmium-based com-\n E-mail: fgandia@um.es pounds o\ufb00er promise as anticancer candidates as they exhibit\n c\n SAI, Universidad de Murcia, E-30071 Murcia, Spain\n improved therapeutic activity and selectivity in some cancer\n \u2020 Electronic supplementary information (ESI) available: Experimental details,\n synthesis, cell cycle profiles. CCDC 1968966-1968968. For ESI and crystallo- cells through distinct mechanisms of action (MoA), thus pre-\n graphic data in CIF or other electronic format see DOI: 10.1039/c9qi01704f venting resistance to readily develop.5\u201321\n\n\n This journal is \u00a9 the Partner Organisations 2021 Inorg. Chem. Front., 2021, 8, 141\u2013155 | 141\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\n\n In this sense, a series of octahedral metal complexes have ease of maintenance and the small body size. It takes three\n been reported to target the protein synthesis process, such as days for an embryo to reach adulthood and reproduce. A single\n the type [Ru(N^O)(N^N)2]+ described by Glazer,22 two in-house hermaphrodite has the ability to produce approximately 300\n families of compounds of the type [Ru(C^N)( phen)2]+ and o\ufb00spring, which enables high-throughput analyses. C. elegans\n [Ir(C^N)2( phen)]+ (C^N = C, N chelating ligand; phen = 1,10- is less complex than the mammalian system, while still\n phenanthroline)7,23 and very recently, a half-sandwich ruthe- sharing high genetic homology (60\u201380%).30 Using RNAi\n nium-based complex [(\u03b76-p-cym)Ru(C^N)(X)]0/+ with a higher technology, the knockdown of gld-1 gene avoided the germ\n cytotoxic profile than CDDP against ovarian cancer cells.24 cells to exit from mitosis and therefore induced cell prolifer-\n A Re(I) polypyridyl complex [Re(CO)3(dmphen)( p-tol-ICN)]+ ation throughout the gonad, forming a germline tumor that is\n (dmphen = 2,9-dimethyl-1,10-phenanthroline; p-tol-ICN = para- lethal to the animal.31,32 Once the tumor has been induced,\n tolyl isonitrile) developed by Wilson has been also shown to C. elegans could be used to study evolutionary conserved sig-\n inhibit translation.25 Although there is a growing amount of naling pathways in response to drug exposure.32,33 To the best\n research on metal-based anticancer candidates, the vast of our knowledge, this is the first time a tumoral strain of\n majority failed to enter clinical trials due to systemic toxicity C. elegans has been used to evaluate the e\ufb03ciency of a metallo-\nPublished on 27 October 2020. Downloaded on 5/12/2026 12:52:24 PM.\n\n\n\n\n issues and only a few complexes, i.e., (N)KP1339 ([Na][trans- chemotherapeutic agent. An in-house ruthenium translation\n RuCl4(Ind)2] (also known as IT-139; Ind = 1H-indazole) and the inhibitor 5b has also included in this study for comparison\n Ru(II) polypyridyl photosensitizer TLD1433, are at present in purposes. Our results provide insights into the potential com-\n phase I/II clinical trials.26\u201328 Thus, there is an urgent need for bination of both in vitro cell-based assays and in vivo screening\n establishing in vivo models for cancer research that allow cost- in C. elegans for the identification of new organometallic anti-\n e\ufb00ective identification and validation of new chemotherapeu- cancer agents.\n tic candidates.\n Based on some of the previous discovery on ruthenium\n protein synthesis inhibitors,24 we developed a new series of Results and discussion\n Os(II) arene complexes of the type [(\u03b76-p-cym)Os(C^N)(X)]0/+\n ( p-cym = para-cymene; C^N = ppy or ppy-CHO; X = Cl, py or Synthesis and characterization of complexes\n 4-NMe2-py) (Chart 1), the ppy-CHO chelating ligand being The new osmium compounds synthesized for this study are\n selected as a handle for further functionalization.29 Chloride depicted in Chart 1. The Os(II) dimer [(\u03b76-p-cym)OsCl2]2 has\n was replaced by py or 4-NMe2-py which could modulate the been used as a precursor for the preparation of neutral com-\n anticancer activity of the complexes. We hypothesized that plexes 1a and 3a, treating it with the corresponding proligand\n these novel osmium complexes could act as proteosynthesis ppy or ppy-CHO in the presence of sodium acetate and in\n inhibitors in ovarian cancer cells and a series of in vitro experi- methanol (Scheme S1; ESI\u2020). Complexes 1a and 3a were used\n ments were conducted to elucidate the main cytotoxic MoA in the synthesis of the cationic compound 2a, and complexes\n involved. Furthermore, in the present study, the small nema- 4a and 5a, respectively, in which the chloride ligand was dis-\n tode Caenorhabditis elegans (C. elegans) was chosen for the placed with silver triflate and py (4a) or 4-NMe2-py (2a and 5a)\n development of an in vivo model for cancer disease. were added subsequently (Scheme S2; ESI\u2020). The new com-\n Characteristics that have contributed to its success include the plexes 1a\u20135a gave satisfactory elemental analysis results and\n genetic manipulability, the well-characterized genome, the were also characterised by ESI-HRMS, NMR (including COSY,\n and HSQC) and UV-vis spectroscopy (Fig. S1\u2013S35, ESI\u2020), and\n X-ray di\ufb00raction studies. Their purity was confirmed also by\n HPLC-MS. The 1H and 13C NMR data are consistent with the\n proposed structures of the complexes with the Os atom co-\n ordinated to the p-cymene ligand \u03b76 in a pseudo-octahedral\n piano stool geometry, with some of the resonances of the \u03b76-p-\n cymene protons being resolved to distinct peaks as one would\n expect for molecules with C1 symmetry proving that the\n rotation of the arene is restricted.\n The positive ion ESI-MS spectra of 1a and 3a displayed the\n corresponding [M \u2212 Cl]+ peak with the expected isotopic distri-\n bution pattern, whereas the ESI-MS spectra of 2a, 4a and 5a\n displayed [M]+ peaks. Reversible hydrolysis of the Ru\u2013Cl bond\n is relatively rapid in the MeOD-d4/D2O 2 : 1 mixture of 3a as\n observed by 1H NMR (Fig. S43 and S44, ESI\u2020). No hydrolysis\n was observed for 2a, 4a and 5a (Fig. S45, ESI\u2020) after 24 h under\n Chart 1 New osmium(II) complexes 1a\u20135a studied in this work and\n the same conditions than those used for 3a. In addition, the\n some in-house analogous ruthenium(II) complexes 1b and 5b (ref. 34 HPLC chromatograms (Fig. S39\u2013S42, ESI\u2020) of 4a and 5a in\n and 24, respectively). Roswell Park Memorial Institute (RPMI) cell culture medium\n\n\n 142 | Inorg. Chem. Front., 2021, 8, 141\u2013155 This journal is \u00a9 the Partner Organisations 2021\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\n\n remained unaltered after 24 h. The stability of complexes 1a Table 1 IC50 values [\u00b5M] for tested arene metal complexes and CDDP\n and 2a was also checked by UV-VIS (Fig. S38. ESI\u2020). The UV-Vis after 48 ha\n absorption spectra of the solutions of complexes in acetonitrile\n Chinese Selectivity\n at 5 \u00d7 10\u22125 M have been obtained. As shown in Fig. S36 in the Complexes A2780 A2780cisR hamster ovary factorb\n ESI,\u2020 all complexes have intense bands between 200 and\n 1a 23 \u00b1 3 18 \u00b1 4 55 \u00b1 5 2.4\n 350 nm due to ligand-to-ligand charge transfer (LLCT) tran-\n 2a 0.16 \u00b1 0.01 0.13 \u00b1 0.03 (0.8) 4.3 \u00b1 0.8 26.9\n sitions, with extinction coe\ufb03cients between 13 000\u201340 000 M\u22121 3a 13.0 \u00b1 0.7 10.4 \u00b1 0.2 (0.8) 63 \u00b1 3 4.8\n cm\u22121, the highest value found for 5a. In addition, between 350 4a 15.1 \u00b1 1.3 15.8 \u00b1 0.3 (1.0) >100 >6.6\n [46.6% \u00b1 2.1]\n and 550 nm the spectra show a band due to metal\u2013ligand\n 5a 2.8 \u00b1 0.2 6.7 \u00b1 0.5 (2.4) 41 \u00b1 3 14.6\n charge transfer transitions, MLCT, with extinction coe\ufb03cients 5bc 2.1 \u00b1 0.2 4.6 \u00b1 0.2 (2.2) 14.6 \u00b1 0.3 7.0\n around 3000 M\u22121 cm\u22121. Noteworthy, complex 5a exhibited an CDDP 2.0 \u00b1 0.1 22 \u00b1 1 (11) 6.3 \u00b1 0.5 3.2\n intense emission around 400 nm when excited at 330 nm a\n Cell viability was determined by the MTT assay after 48 h treatment\n (Fig. S37, ESI\u2020). Complex 5a contains simultaneously the C^N and IC50 values were calculated as described in the ESI.\u2020 Each value\n ligand with the CHO group and the 4-NMe2-py ligand, probably represents the mean \u00b1 SD of two independent experiments (n = 4).\n Resistance factor, defined as IC50(resistant A2780cisR)/IC50(sensitive\nPublished on 27 October 2020. Downloaded on 5/12/2026 12:52:24 PM.\n\n\n\n\n a charge transfer between both groups is responsible for the A2780), is given in parentheses. The term \u201c>100\u201d indicates that no IC50\n intense emission observed. Crystallographic data and refine- value reached up to 100 \u00b5M and the respective percentage of the\n ment parameters for all complexes are collected in Table S1 in inhibitory concentration value is given between brackets. b Selectivity\n factor defined as IC50 (normal Chinese hamster ovarian cells)/IC50\n the ESI,\u2020 and selected bond distances and angles are listed in (tumoral ovarian cells A2780). c Source of IC50 values is ref. 24.\n Table S2 in the ESI.\u2020 The structures of complexes 3a, 4a and 5a\n were unambiguously confirmed by the X-ray crystallographic\n study, confirming the anticipated molecular structure (Fig. 1).\n low resistance factors and being remarkably less toxic than\n As shown, the 2-phenylpyridine-type ligand assumes a bidentate CDDP in normal Chinese hamster ovary cells. Compared to\n chelate coordination mode (\u03ba2-C,N), occupying two coordination\n the previously reported 4-NMe2-py Ru(II) analogue, namely\n positions in all complexes. The Os cations of 4a and 5a crystal-\n 5b,24 the Os(II) complex analogue 5a showed a 2-fold increase\n lized with one CF3SO3\u2212 anion. C\u2013H\u22efO interactions were in the selectivity factor under the same experimental con-\n observed in 3a and 5a, yielding a chain parallel to axis 101\n ditions. Furthermore, 2a achieved a 10-fold increase in cyto-\n (Fig. S46, S47, and Tables S3, S4, ESI\u2020) in the solid state.\n toxicity with respect to their aldehyde containing analogues,\n 5a and 5b, and indeed exhibited higher selectivity over normal\n Antiproliferative activity of the osmium complexes\n cells. The low cytotoxicity of 1a and 3a is in agreement with\n The development of these complexes was motivated by our the low biological activity previously reported for the analogue\n recently reported cationic Ru(II) complexes [(\u03b76-p-cym)Ru(C^N) neutral ruthenium complex 1b.34\n (L)]+ with L = py and 4-NMe2-py (5b in Chart 1), which act as\n protein synthesis inhibitors in ovarian cancer cells.24 The cyto- E\ufb00ects on cellular morphology\n toxicity of the Os(II) complexes was evaluated in a panel of\n The A2780 cell line displays endothelial-like morphology, with\n human ovarian cancer cells which includes cells of the epi-\n characteristic oval and spindle-shape morphologies aggregated\n thelial ovarian carcinoma A2780 and its CDDP-resistant\n in incipient islets. Treatment with 5 \u00b5M of complex 5a induced\n ovarian cancer cell line A2780cisR, as well as non-tumorigenic\n observable changes in cell morphology such as cell shrinkage\n Chinese hamster ovary cells. For comparison, the cytotoxic\n and the increment of refringent cells, indicative of cell death\n e\ufb00ects of CDDP were also determined (Table 1).\n induction (Fig. S48, ESI\u2020). In order to prove that these metal-\n Complexes 2a, 5a and its Ru analogue 5b exhibited greater\n based agents are able to promote tumoral cell death, flow cyto-\n activity than complexes 1a, 3a and 4a, with IC50 values in the\n metry and fluorescent-based assays were performed as follows.\n low micromolar range, indicating that the 4-NMe2-py ligand\n significantly improved the antiproliferative e\ufb00ect. All the com- Morphological analysis by flow cytometry\n plexes were active against CDDP-resistant cells, thus displaying\n In order to confirm the morphological changes observed by\n microscopy, a flow cytometry analysis of the A2780 cell popu-\n lation was performed. As observed in Fig. S49 in ESI,\u2020 the\n reduction in cell size detected in forward light scatter (FSC) and\n the concomitant nuclear condensation, revealed by the transient\n increase in the side scatter (SCC), were consistent with apoptosis\n being promoted by complexes 2a and 5a. Therefore, additional\n flow cytometry experiments were conducted to verify it.\n\n Depolarization of the mitochondrial membrane potential\n Fig. 1 Molecular structures of osmium cations with atom numbering\n schemes for 3a (left), 4a (centre) and 5a (right) (50% thermal ellipsoids). Using flow cytometry after 24 h treatment with the osmium\n Selected bond lengths and angles for 3a\u20135a are shown in the ESI.\u2020 complex, changes in mitochondrial membrane potential\n\n\n This journal is \u00a9 the Partner Organisations 2021 Inorg. Chem. Front., 2021, 8, 141\u2013155 | 143\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\n\n (MMP) of A2780 cells were measured by the retention of rhoda- suggesting that the CHO group had an impact on the overall\n mine-123 dye (Fig. S50, ESI\u2020). We observed that 5a caused a structure in terms of cell death induction.\n dose-dependent decrease in the fluorescence intensity of the\n dye, which revealed a loss of MMP. This alteration in mito- Cell cycle analysis\n chondria energetics led us to think that apoptosis might be The e\ufb00ect of the osmium complex on the cell cycle progression\n triggered as a consequence. was examined using PI staining. Histogram profiling revealed\n that A2780 cells treated with 2a and 5a significantly accumu-\n Apoptosis induction by osmium complexes lated in the G1 phase in contrast to the clinical drug CDDP,\n To check the mechanism by which these osmium complexes which produces S and G2/M phase arrest (Fig. 2 and Fig. S52,\n induced cell death in cancer cells, dual Annexin-FLUOS/propi- ESI\u2020). Particularly, complex 2a produced a characteristic subG1\n dium iodide (PI) staining was performed. Treated cells showed spike (marked with an arrow in Fig. 2), an indicative of DNA\n positive staining for both Annexin-V and Annexin-V/PI quad- fragmentation probably derived from the apoptosis induction.\n rants, suggesting the induction of early and late apoptosis In order to evaluate the e\ufb00ect of the compounds in the pro-\n rather than necrosis after 24 h treatment. Moreover, we gression of the cell cycle, A2780 cells were synchronized to G0/\nPublished on 27 October 2020. Downloaded on 5/12/2026 12:52:24 PM.\n\n\n\n\n studied the influence of e\ufb00ector caspase-3 in 2a and 5a- G1 by serum starvation. Cells were then treated with the\n mediated apoptosis by pretreating cells with the caspase-3 complex for 6 or 24 h and switched to serum-containing\n inhibitor (NSCI, 5 \u00b5M) for 1 h. Pre-exposure of caspase inhibi- media to re-enter the cell cycle. As shown in Fig. S53 in the\n tor prevented complex-induced apoptosis as shown in Fig. 2 ESI,\u2020 synchronous cells treated with 5a for 6 h produced a\n and Fig. S51 in the ESI,\u2020 indicating the activation of caspase- mild G1-phase arrest in the cell population. However, 24 h\n dependent apoptosis. Compared to their aldehyde-containing treatment caused extensive G1 phase accumulation that was\n counterpart, 5a, 2a induced apoptosis to a greater extent, not reversed by compound removal. This suggests that block-\n age of G1/S transition by 2 \u00b5M of 5a was irreversible for at least\n 24 h with cells being unable to re-enter the cell cycle.\n\n Protein synthesis inhibition by osmium complexes\n Recently, the in-house 4-NMe2-py Ru(II) analogue 5b was\n demonstrated to exert its MoA via protein synthesis inhi-\n bition.24 This result led us to investigate the osmium com-\n plexes, 2a and 5a, as possible protein translation inhibitors in\n cancer cells. By using O-propargyl puromycin (OPP), a modi-\n fied translation inhibitor aminonucleoside that is incorporated\n into nascent peptides within cells, we tracked de novo protein\n synthesis within cancer cells by measuring the levels of OPP\n incorporation following treatment. This is achieved by the\n alkylation of an Alexa Fluor 488 azide fluorescent tag in OPP-\n labeled nascent proteins using click-chemistry. The quantifi-\n cation of the fluorescently labeled peptides was measured by\n whole-fluorescent readings, flow cytometry and confocal laser\n microscopy imaging.\n The A2780 cells were treated for 12 h, a su\ufb03ciently long\n period for protein synthesis to occur, at two di\ufb00erent concen-\n trations of either 2a, 5a or cycloheximide (CHX), which was\n used as a positive control. Fluorescence measurements indi-\n cated that 10 \u00b5M of osmium complexes were e\ufb00ectively\n capable of inhibiting up to 58 to 48% of the global protein syn-\n thesis in cancer cells (Fig. S54 ESI\u2020). In addition, flow cytome-\n try analysis confirmed that, as well as CHX, both 2a and 5a\n caused inhibition of protein synthesis (Fig. S55, ESI\u2020), which\n Fig. 2 (A) Flow cytometry analysis of apoptosis/necrosis induction in\n is consistent with both cell cycle arrest in the G1 phase and\n A2780 cells after 24 h treatment with either 2a, 5a alone (5 \u00b5M) or sub- caspase 3 activation as CHX induces caspase-dependent apop-\n sequent to pretreatment with caspase 3 inhibitor NSCI (5 \u00b5M) for 1 h. tosis.24 In order to corroborate the obtained results, we exam-\n CDDP (2 \u00b5M) was used as a positive control. Data presented as mean \u00b1 ined the e\ufb00ects of the present complexes in living cells under\n SD from two independent experiments (n = 2). (B) Cell cycle analysis of\n confocal microscopy. Following OPP-labeling with fluorescent\n A2780 cells after indicated treatments for 24 h measured by propidium\n iodide intensity in the FL2-A channel; subG1 phase population is\n Alexa Fluor 488 azide, confocal images showed evidence for a\n depicted by an arrow. Representative histograms from two independent decrease in total protein synthesis in both metal-based agents\n experiments (n = 2). and CHX treatments as a result of the observed reduction in\n\n\n 144 | Inorg. Chem. Front., 2021, 8, 141\u2013155 This journal is \u00a9 the Partner Organisations 2021\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\n\n pounds as antitumoral agents were measured in the C. elegans\n mutant strain JK1466. The commonly used chemotherapeutic\n agent CDDP was used as control. In C. elegans gld-1(q485)\n mutants (JK1466 strain), the germ cells fail to exit from\n mitosis and continue to proliferate throughout the gonad,\n forming a germline tumor that is lethal to the animal.32 The\n gonad area in these animals is swelled and enlarged by tumor\n development,35 as shown in Fig. 4 B\u2013E, in comparison with\n the normal gonad of the wild-type animals (Fig. 4A). The size\n of the tumors in the animals treated with the metal complexes\n was smaller in comparison with non-treated animals or the\n animals treated with 0.1% DMSO (Fig. 5).\n Measurements of the tumor area showed that complex 5a\n was able to reduce the size of the tumors between a 16.8 and\nPublished on 27 October 2020. Downloaded on 5/12/2026 12:52:24 PM.\n\n\n\n\n 19.2% when treated with 10 and 100 \u00b5M of the compound\n respectively, suggesting the potential of the novel compound\n as a chemotherapeutic agent. A Ru(II) complex, 5b, analog of\n 5a, was also tested to elucidate the e\ufb00ect of the complexes\u2019\n metal atom in tumor reduction in vivo. The treatment with 5b\n was also e\ufb00ective and reduced the tumor size in a range\n between 10.9 and 30.6% (Fig. 5 and 4, Table S5 and Fig. S57\u2020).\n Worthy of note, complex 2a seems the most e\ufb00ective of the\n tested compounds towards tumor reduction, as it was active at\n the lowest concentration used (0.1 \u00b5M), at this concentration\n the tumor growth was reduced by 14.6%, while at the\n\n\n\n\n Fig. 3 Detection of protein synthesis on A2780 cells after 12 h treat-\n ment with 2a, 5a (10 \u00b5M) or cycloheximide (500 \u00b5M) using Click-iT Alexa\n Fluor-OPP by confocal microscopy. Nuclear Mask was used for co-\n staining cell nuclei. Scale bar = 25 \u00b5m.\n\n\n\n green fluorescence (Fig. 3). Overall, the results indicated that\n these complexes share proteosynthesis inhibition as their\n main MoA, although other mechanisms might be under\n operation.\n\n Oxidative stress by complex 5a in cancer cells\n Previous studies with the Ru analogue 5b ruled out the induc-\n tion of oxidative stress as a main mechanism of action.24\n Nonetheless, the influence of ROS signaling in the complex-\n mediated proteosynthesis inhibition was assayed using the\n cell permeant reagent 2\u2032,7\u2032-dichlorofluorescein diacetate\n (DCFH-DA) after 2 h drug-exposure. N-Acetylcysteine was used\n for protection studies as a well-known ROS scavenger whereas\n hydrogen peroxide (H2O2) was used as positive control for ROS\n generation. However, no evidence of canonical ROS generation\n was found in vitro after treatment with 5a (Fig. S56 ESI\u2020),\n which suggests that the osmium complex did not act as a\n redox impairing agent within cells. Fig. 4 E\ufb00ect on tumor growth of the metal complexes. Representative\n images taken at 20\u00d7 of: (A) adult C. elegans wild-type gonad (normal\n E\ufb00ect of metal complexes on tumor growth C. elegans gonad), (B\u2013E) JK1466 tumoral strain gonads; (B) control animal gonadal\n tumor, (C) 100 \u00b5M CDDP, (D) 0.1% DMSO (control) treated animal, (E)\n Due to the high selectivity factor achieved by complexes 2a and 100 \u00b5M complex 5a in 0.1% DMSO, in all the cases the tumor is outlined\n 5a, the e\ufb00ects of these novel synthetized organometallic com- in blue. Scale bar: 100 \u03bcm.\n\n\n\n This journal is \u00a9 the Partner Organisations 2021 Inorg. Chem. Front., 2021, 8, 141\u2013155 | 145\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\nPublished on 27 October 2020. Downloaded on 5/12/2026 12:52:24 PM.\n\n\n\n\n Fig. 5 E\ufb00ect of the metal complexes on tumor growth. Tumor size\n evaluation for the tumoral strain JK1466 treated with di\ufb00erent concen-\n trations of the metal complexes (MEAN \u00b1 SEM), n \u2265 10, two indepen-\n dents trials were measured in each case, ** signi\ufb01cant at p \u2264 0.05 by\n ANOVA test.\n\n\n\n optimum concentration (1 \u00b5M), the tumor size was reduced by\n 32.2% (Fig. 5, Table S5\u2020). Furthermore, the widely used treat-\n ment, CDDP, was also able to reduce the size of the tumors to\n 28.1 and 48.9% at 10 and 100 \u00b5M, indicating that the\n employed tumoral strain is an adequate model to screen\n potential antitumoral drugs. This is the first time that a\n Fig. 6 Toxicity e\ufb00ect of the metal complexes. Lifespan histograms and\n tumoral strain of C. elegans was used to evaluate the e\ufb03ciency\n survival curves for the strains N2 and JK1466 treated with di\ufb00erent con-\n of a chemotherapeutic agent. centrations of the metal complexes. A, B, E, F are mean lifespan data and\n C, D, G, H are lifespan curves. Data are presented as mean \u00b1 SE, ** sig-\n Toxicity e\ufb00ect of complex 2a, 5a, 5b and CDDP on wild-type ni\ufb01cant at p \u2264 0.05.\n and tumoral strain JK1466\n Once the positive e\ufb00ects on tumor reduction in vivo were estab-\n lished, the e\ufb00ects on lifespan in the tumoral animals and in that the compound did not only act as tumor reduction agent\n wild type ones were measured. Thus, the toxicity of cisplatin but also could modulate transcription factors responsible for\n and complex 5a was evaluated in the animal C. elegans, which the increased longevity of wild-type worms. Indeed, Garc\u00eda-\n is an admitted model to study the toxicity of molecules. As Rodr\u00edguez and coauthors36 reported that a dose of 60 \u00b5g mL\u22121\n shown in Fig. 6, Table S6,\u2020 overall, in the range of concen- (200 \u00b5M) was able to modulate the gene expression in wild-\n trations used, none of the compounds, complex 5a and CDDP, type C. elegans strain. Their RNA microarray results showed\n were toxic as they did not reduce the animal\u2019s lifespan below that among the genes upregulated by CDDP exposure were\n the mean lifespan of the non-treated animals (control worms). apoptosis related genes and stress response genes, indicating\n On the contrary, the tested concentrations were able to signifi- that CDDP was able to confer oxidative stress resistance to the\n cantly prolong the mean lifespan for both tumoral and wild- animals by activating DAF-16/FOXO (insulin signaling\n type worms. The C. elegans treated with complex 5a showed an pathway) and SKN-1/Nrf2 (redox active pathway) downstream\n increase on lifespan of 16.9% in JK1466 animals and an genes like gst-4. These results suggest that the lifespan exten-\n increase of 16.5% in wild type worms. Complex 5b had a more sion shown in our experiments may be dependent on the\n moderate e\ufb00ect towards the lifespan of both the wild-type and insulin signaling pathway and the redox active pathway and its\n the tumoral strains, nevertheless at 100 \u03bcM was able to extend transcription factors DAF-16 and SKN-1 respectively, protecting\n the JK1466 animals\u2019 lifespan by 11.7% and the wild-type by tumoral animal\u2019s lifespan while reducing tumor growth.\n 4.4% using 10 \u03bcM (Table S6 and Fig. S58\u2020). On the other hand,\n complex 2a increased the lifespan by 6.3 and 13.5% of both E\ufb00ect of complexes 2a, 5a, 5b, and CDDP on ROS production\n strains treated with 1 \u00b5M of the complex, however higher con- in C. elegans\n centrations of the compound were toxic for the wild-type As cell-based assays did not reveal oxidative stress in cancer\n animals and reduced the lifespan (Table S6, Fig. S59\u2020). CDDP cells, we decided to check the e\ufb00ects of complex 5a on oxi-\n treatment also prolonged the animal\u2019s lifespan by 18% and dative stress resistance and intracellular ROS levels in\n 16.1% in both strains, respectively. These results indicated C. elegans. Reactive oxygen species were measured in vivo by\n\n\n 146 | Inorg. Chem. Front., 2021, 8, 141\u2013155 This journal is \u00a9 the Partner Organisations 2021\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\n\n using the cell permeant reagent DCFH-DA. As shown in shock proteins of the nematode seems to be a\ufb00ected by CDDP\n Fig. S60 and S61 in the ESI,\u2020 CDDP produced an increase in treatment.36\n the ROS levels 4.5 times (Fig. S60C, ESI\u2020) compared with the In the strain CF1553 superoxide dismutase 3 (SOD-3) is\n water control, an indication that CDDP produced reactive fused with GFP. The protein expression is shown in the head\n oxygen species in vivo. The positive control used was juglone, (Fig. 7, Fig. S66\u2020), the tail and the vulva of the animals. SOD-3\n which exerted oxidative stress in vivo as shown in Fig. S60B in is one of the two mitochondrial superoxide dismutase in\n the ESI.\u2020 C. elegans and a well known target of DAF-16.40 Mutant\n Meanwhile the same concentration of complex 5a did not animals treated with 100 \u03bcM of either 2a, 5a or 5b showed an\n increase the ROS levels in vivo. These results suggested increase of fluorescence as shown in Fig. 8, an indication of an\n di\ufb00erent mechanism of action of the two compounds. On the overexpression of SOD-3. The SOD-3 expression increase was\n other hand, 2a increased slightly the ROS production in vivo, the same for the two complexes, 1.8-fold higher than the\n suggesting that the compound is more reactive than 5a control with no significant di\ufb00erence ( p \u2264 0.05) between them.\n (Fig. S61 and S66\u2020). Meanwhile CDDP did not alter the expression of superoxide\n A number of studies have shown multiple e\ufb00ects of CDDP dismutase. Several reports had linked the expression and\nPublished on 27 October 2020. Downloaded on 5/12/2026 12:52:24 PM.\n\n\n\n\n inside the cells, the induction of DNA damage by forming\n platinum-DNA adducts being critical. CDDP also generates cel-\n lular damage by increasing the ROS levels and mitochondrial\n dysfunction by producing mtDNA adducts and electron chain\n imbalance. These e\ufb00ects activate di\ufb00erent pathways such as\n p53, p38 or JKN leading to cell death by apoptosis.37\u201339 In con-\n trast, complexes 5a and 5b did not increase the ROS pro-\n duction in comparison with the DMSO treated animals an\n indication that the mechanism of action of this compound is\n not mediated by free radicals as observed with CDDP. Worthy\n of note, 2a increased slightly the ROS production in vivo but\n not as much as CDDP, thus the MoA is clearly not mediated by\n ROS. Therefore, the downstream targets of the DAF-16/FOXO\n pathway and the redox active pathway (Nrf2/SKN-1) were\n assayed to estimate the underlying mechanism of action of 2a,\n 5a and 5b.\n The targets chosen were the heat shock protein 16.2\n (HSP-16.2), the mitochondrial superoxide dismutase (SOD-3)\n Fig. 7 E\ufb00ect of the metal complexes in SOD-3 expression.\n and the glutathione synthetase (GST-4), as they are involved in\n Representative images of: (A) C. elegans strain CF1553 bright \ufb01eld, (B)\n the longevity pathways and the resistance to oxidative stress. positive control (treated with juglone), (C) animals treated with 100 \u03bcM\n CDDP, (D) worms treated with 100 \u03bcM complex 5a. Scale bar: 50 \u03bcm.\n E\ufb00ect of metal complexes on C. elegans HSP-16.2, SOD-3 and\n GST-4 expression\n The e\ufb00ect of the metal complexes in the animal\u2019s resistance to\n oxidative stress was measured with the mutant strains TJ375,\n CF1553 and CL2166. The TJ375 strain contains the heat shock\n protein 16.2 (HSP::16.2) fused with the green fluorescent\n protein (GFP). No fluorescence increase was shown (Fig. S63\n ESI\u2020) when the worms were exposed directly to the compounds\n 5a, 5b or CDDP, indicating that the tested compounds did not\n increase the levels of HSP-16.2 protein as observed with\n juglone. However, the compound 2a was able to promote the\n expression of HSP-16.2, when the animals were exposed to\n 100 \u00b5M of the complex (Fig. S63 and S66\u2020), this e\ufb00ect may be\n a protection mechanism against the ROS produced by 2a.\n Also, in the set of experiments to estimate whether the com-\n pounds were able to protect against oxidative stress produced\n with juglone, the results showed that none of the complexes\n Fig. 8 E\ufb00ect of the metal complexes in SOD-3::GFP expression.\n were able to enhance the animals oxidative stress through the\n Integrated \ufb02uorescence of the CF1553 C. elegans strain, treated with the\n accumulation of HSP-16.2::GFP (Fig. S62, ESI\u2020). These results metal complexes (100 \u03bcM) or with juglone (20 \u03bcM) (MEAN \u00b1 SEM), n \u2265\n agree with the microarray results of Garc\u00eda-Rodr\u00edguez and 10, two independents trials were measured in each case, ** signi\ufb01cant at\n coauthors that showed that none of genes encoding the heat p \u2264 0.05 by ANOVA test.\n\n\n\n This journal is \u00a9 the Partner Organisations 2021 Inorg. Chem. Front., 2021, 8, 141\u2013155 | 147\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\n\n activity of human SOD3 to tumor response to chemotherapeu- reduced the tumor size by 30%, more than 5a using the same\n tic agents, as SOD3 is usually downregulated in tumor concentration in cells, and the IC50 for 5b was slightly lower\n cells.41\u201344 Sharma et al. reported a reduction in breast cancer than that of 5a, being 2.1 and 2.8 \u03bcM respectively. Similar to\n cell produced by metformin treatment which upregulated the 5a, complex 5b improved the lifespan of both strains but the\n expression and activity of SOD.45 In the same line Mira and e\ufb00ects were moderate in comparison with the e\ufb00ect showed by\n coauthors reported that an enhanced SOD3 expression 5a, nevertheless the complex was not toxic for the animals.\n improved the tumor response to the chemotherapeutic agent The other analog of 5a -complex 2a- reduced the tumor size by\n doxorubicin.46 In summary the tumor reduction e\ufb00ects in the 32.3% using 1 \u00b5M of the complex, being the compound most\n C. elegans tumor model and the positive e\ufb00ects on the lifespan e\ufb00ective among those tested. Furthermore, at the same con-\n of both the tumor and the wild-type strain when treated with centration, it was able to increase the wild-type worms\u2019 life up\n 2a, 5a and 5b may be related to the overexpression of SOD-3 to 13.5%, however at higher concentrations the lifespan was\n which is a canonical target of the transcription factor DAF-16 reduced by 7% an indication of toxicity. This compound\n and the insulin signaling pathway, both involved in longevity, increased slightly the ROS production as well as the expression\n tumor suppression and apoptosis.47 of HSP-16.2 suggesting that the compound is more reactive\nPublished on 27 October 2020. Downloaded on 5/12/2026 12:52:24 PM.\n\n\n\n\n As demonstrated in cells, complexes 5a and 5b caused mito- than the other analogs. Interestingly, the mechanism of action\n chondrial dysfunction, which in C. elegans could cause the seems to be the same for the three complexes suggesting that\n overexpression of SOD-3 mediated by DAF-16, to maintain the it is more related to the complete structure of the complex\n redox homeostasis of the cells, aiding the animal\u2019s than the center metal atom. Worthy of note, the absence of the\n survival.48,49 Thus SOD-3 may be more involved in maintaining aldehyde group seems to increase the reactivity of the complex\n or incrementing the C. elegans lifespan than in reducing the although it also increases the toxicity in vivo. Overall the\n tumor size. This could be a signal that the mitochondrial dys- mechanism underlying the e\ufb00ects of complexes 2a, 5a and 5b\n function showed in cells was also occurring in the nematodes. and on C. elegans wads dissimilar to the mechanism of CDDP,\n Finally, the strain CL2166 contains a glutathione-s-transfer- while CDDP reduced the tumor growth by ROS production \u2013\n ase (GST-4) fused with GFP. This enzyme is a canonical target which may be caused by DNA-platinum adducts, as mentioned\n of the redox active pathway (SKN-1/Nrf2). SKN-1 regulates the above\u2013 and GST-4 activation, the e\ufb00ects of complexes 2a, 5a\n stress-induced gst-4 transcription in the presence of redox and 5b on C. elegans lifespan and tumor reduction are related\n active compounds.36 As reported before,36 the results (Fig. S64 to SOD-3 overexpression and the insulin signaling pathway\n and S65, ESI\u2020) showed an increase in the GST-4 expression by activation. This is the first time that a tumoral strain of\n CDDP, however 2a, 5a and 5b did not, rea\ufb03rming that the C. elegans has been used to evaluate the e\ufb03ciency of a che-\n mechanism of action of the two metal complexes is di\ufb00erent, motherapeutic agent, allowing cost-e\ufb00ective identification and\n and based on DAF-16 and the insulin signaling pathway in the validation of new anticancer drug candidates.\n case of the metal complex 5a.\n\n Experimental\n Conclusions Chemical synthesis\n\n Our work demonstrates that the osmium(II)-arene complexes The purity of all compounds determined by C, H, N and S\n [(\u03b76-p-cym)Os(C^N)(4-NMe2-py)]+ (C^N = ppy and ppy-CHO) act combustion elemental analysis and HPLC was \u226595%.\n as proteosynthesis inhibitors in A2780 ovarian cancer cells (at\n 10 \u00b5M up to 58% of the global protein synthesis), but not as Synthetic procedure of complex 1a [Os(\u03b76-p-cymene)( ppy)Cl]\n redox impairing agents within cells, achieving low cytotoxicity Sodium acetate (0.4 mmol, 33 mg) and 2-phenylpyridine\n towards healthy ovarian cells (SF = 14.6 and 26.9). Complex 5a (0.2 mmol, 31 mg) were dissolved in 5 ml of dry methanol\n is also able to reduce 19% the size of the tumors in C. elegans (MeOH). It was stirred for 5 minutes then the osmium dimer\n gld-1(q485) mutants (JK1466 strain). In addition, in the used [(\u03b76-p-cymene)OsCl2]2 (0.1 mmol, 79 mg) and 5 ml of MeOH\n concentration range complex 5a was not toxic to the animals were added. The solution was stirred at room temperature\n and indeed it induces an extension of 17% in the C. elegans\u2019s under nitrogen for 24 h in the dark. After that time, the solu-\n lifespan both in the tumoral organisms and also in the wild tion was filtered, concentrated and purified by flash chromato-\n type ones. No e\ufb00ects in intracellular ROS levels were observed graphy in alumina using as eluent a mixture 9/1 (v/v) of di-\n in the expression of heat shock proteins in the animal when chloromethane/acetonitrile. The orange band was collected,\n studying the C. elegans mutant strain TJ375 (hsp-16.2/GFP the solvent was removed under reduced pressure and the solid\n reporter) in response to complex 5a treatment, showing that was dissolved in dichloromethane and precipitated with\n none of genes encoding the hsps of the nematode seems to be hexane, obtaining an intense orange solid (0.12 mmol, yield:\n a\ufb00ected by Os(II) treatment. An analog of complex 5a, which 60%). ESI-MS ( pos ion mode, CHCl3): m/z = 480.1382 [M \u2212\n only di\ufb00ers with it in the metal atom-Ru(II) was used instead of Cl]+. 1H NMR (400 MHz, CD3CN, 25 \u00b0C) \u03b4 ( ppm): 9.19 (ddd, J =\n Os(II) to elucidate the importance of the complex\u2019 metal atom 5.8, 1.6, 0.8 Hz, 1H8), 8.01 (ddd, J = 7.5, 1.3, 0.6 Hz, 1H4), 7.91\n in the e\ufb00ects produced on the animal model. Complex 5b (ddd, J = 8.2, 1.2, 0.6 Hz, 1H5), 7.78\u20137.71 (m, 2H6+1), 7.13\u20137.09\n\n\n 148 | Inorg. Chem. Front., 2021, 8, 141\u2013155 This journal is \u00a9 the Partner Organisations 2021\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\n\n (m, 1H7), 7.09\u20137.05 (m, 1H3), 6.98 (ddd, J = 7.7, 7.2, 1.3 Hz, 194.46, 168.26(q), 165.82(q), 157.29, 151.44(q), 142.75, 138.57,\n 1H2), 5.67 (dd, J = 5.5, 1.1 Hz, 1H13), 5.59 (d, J = 5.5, 1H10), 137.38(q), 124.84 (2C), 123.37, 121.00, 96.71(q), 91.93(q),\n 5.48 (dd, J = 5.7, 1.1 Hz, 1H11), 5.20 (dd, J = 5.4, 1.2 Hz, 1H12), 83.04, 81.08, 76.43, 72.21, 31.91, 23.02, 21.97, 18.75. UV-Vis\n 2.24 (sept, J = 6.9 Hz, 1H14), 2.10 (s, 3H9), 0.90 (d, J = 6.9 Hz, (CH3CN, \u03bb(nm), (\u03b5 (M\u22121 cm\u22121)): 262(18 898), 305 (24 916), 417\n 3H15), 0.77 (d, J = 6.9 Hz, 3H16). 13C NMR (100 MHz, CD3CN) \u03b4 (3564). IR (KBr, cm\u22121): \u03bd(CO) = 1681. Decomposition point =\n ( ppm): 168.1(q), 167.3(q), 156.8, 145.6(q), 140.5, 138.2, 130.5, 224 \u00b0C. Anal. Calcd for C22H22ClNOOs (542.10): C, 48.74; H,\n 124.7, 123.4, 123.0, 119.6, 95.9(q), 91.0(q), 82.7, 80.8, 76.1, 4.09; N, 2.58; found: C, 48.31; H, 3.99; N, 2.45 (%).\n 71.8, 31.9, 23.0, 22.0, 18.7. UV-Vis (CH3CN, \u03bb(nm), (\u03b5 (M\u22121\n cm\u22121)): 234 (17 240), 254 (17 688), 295 (11 572), 346 (3332), 390 Synthetic procedure of complex 4a [Os(\u03b76-p-cymene)\n (3005). Melting point = 197 \u00b0C. Anal. Calcd for C21H22ClNOs ( ppy-CHO)( py)][CF3SO3]\n (514.10): C, 49.06; H, 4.31; N, 2.72; found: C, 49.08; H, 4.26; N, Complex 3a (0.1 mmol, 54.2 mg) and silver triflate (AgSO3CF3,\n 2.64 (%). 0.1 mmol, 25.7 mg) were dissolved in 5 ml of MeOH and\n stirred for 2 hours in the dark. After this time, a white precipi-\n Synthetic procedure of complex 2a [Os(\u03b76-p-cymene) tate of the silver chloride was observed as a byproduct. It was\n ( ppy)( py)][CF3SO3]\nPublished on 27 October 2020. Downloaded on 5/12/2026 12:52:24 PM.\n\n\n\n\n filtered and pyridine (0.1 mmol, 8.1 \u00b5l) was added to the fil-\n Complex 1a (0.1 mmol, 51.4 mg) and silver triflate (AgSO3CF3, trate. The solution was stirred for 2 hours. After this time, a\n 0.1 mmol, 25.7 mg) were dissolved in 5 ml of MeOH and yellowish solution was observed. The solution was dried and,\n stirred for 2 hours in the dark. After this time, a white precipi- subsequently, extraction was performed with DCM (3 \u00d7 5 ml).\n tate of the silver chloride was observed as a byproduct. It was A yellow solid was precipitated with hexane, 35.4 mg\n filtered and 4-(dimethylamino)pyridine (4-NMe2-py) (0.048 mmol, yield 48%).\n (0.1 mmol, 8.1 \u00b5l) was added to the filtrate. The solution was ESI-MS ( pos ion mode, DMSO): m/z = 587.1726 [M]+. 1H\n stirred for 2 hours. After this time, a yellowish solution was NMR (300 MHz, CD3CN, 25 \u00b0C) \u03b4 ( ppm): 10.16 (s, 1H8), 9.50\n observed. The solution was dried and, subsequently, extraction (d, J = 5.8 Hz, 1H7), 8.77 (d, J = 1.4 Hz, 1H1), 8.43\u20138.40 (m,\n was performed with DCM (3 \u00d7 5 ml). A yellow solid was precipi- 2H9), 8.02 (d, J = 7.3 Hz, 1H4), 7.95 (ddd, J = 7.3, 7.3, 1.4 Hz,\n tated with hexane, 48.7 mg (0.065 mmol, yield 65%). 1H5), 7.86 (d, J = 8.1 Hz, 1H3), 7.68 (tt, J = 7.7, 1.5 Hz, 1H11),\n ESI-MS ( pos ion mode, CHCl3): m/z = 602.2232 [M]+. 1H 7.62 (dd, J = 8.0, 1.6 Hz, 1H2), 7.43 (ddd, J = 7.3, 5.8, 1.6 Hz,\n NMR (300 MHz, CD3CN, 25 \u00b0C) \u03b4 ( ppm): 9.43 (ddd, J = 5.9, 1.4, 1H6), 7.21\u20137.11 (m, 2H10), 6.10 (d, J = 5.7 Hz, 1H15), 6.00\u20135.90\n 0.9 Hz, 1H8), 8.22 (dd, J = 7.5, 1.3 Hz, 1H4), 7.92\u20137.80 (m, (m, 2H13+14), 5.52 (d, J = 5.5 Hz, 1H16), 2.25\u20132.17 (m, 1H17),\n 2H1+3), 7.80\u20137.74 (m, 2H9), 7.70 (dd, J = 7.7, 1.3 Hz, 1H5), 1.72 (s, 3H12), 0.81 (d, J = 7.0 Hz, 3H18), 0.72 (d, J = 7.0 Hz,\n 7.31\u20137.20 (m, 2H2+6), 7.09 (ddd, J = 7.8, 7.2, 1.3 Hz, 1H7), 3H19). 13C NMR (75 MHz, CD3CN) \u03b4 ( ppm): 194.17, 166.52(q),\n 6.24\u20136.16 (m, 2H10), 5.92 (dd, J = 5.6, 1.0 Hz, 1H16), 5.85 (m, 165.10(q), 157.56, 154.78 (2C), 151.72(q), 142.36, 140.22,\n 2H13+15), 5.40 (d, J = 5.5 Hz, 1H14), 2.86 (s, 6H11), 2.13 (sept, J = 139.70, 138.63(q), 126.87, 126.39 (2C), 125.40, 125.25, 121.81,\n 6.9 Hz, 1H17), 1.75 (s, 3H12), 0.78 (d, J = 7.0 Hz, 3H18), 0.72 (d, J 99.91(q), 92.18(q), 84.57, 84.25, 82.90, 72.79, 31.87, 23.01,\n = 6.9 Hz, 3H19). NMR (75 MHz, CD3CN) \u03b4 ( ppm): 167.9(q), 22.31, 17.69. UV-Vis (CH3CN, \u03bb(nm), (\u03b5 (M\u22121 cm\u22121)): 230\n 166.2(q), 157.0, 155.0(q), 152.7, 146.0(q), 140.4, 139.5, 131.9, (13 000), 297(16 782), 381(2722). IR (Nujol, cm\u22121): \u03bd(CO) =\n 125.2, 124.7, 124.4, 120.2, 108.6, 98.9(q), 90.9(q), 83.9, 83.4, 1682. Decomposition point = 220 \u00b0C. Anal. Calcd for\n 82.2, 71.9, 39.4, 32.0, 23.0, 22.5, 18.0. UV-Vis (CH3CN, \u03bb(nm), C28H27F3N2O4SOs (734.82): C, 45.77; H, 3.70; N, 3.81; S, 4.36;\n (\u03b5 (M\u22121 cm\u22121)): 245 (18 502), 286 (28 804), 378 (3822). found: C, 45.12; H, 3.58; N, 3.68; S, 4.22 (%).\n Decomposition point = 209 \u00b0C. Anal. Calcd for\n C29H32F3N3O3SOs (749.88): C, 46.45; H, 4.30; N, 5.60; S, 4.28; Synthetic procedure of complex 5a [Os(\u03b76-p-cymene)\n found: C, 46.28; H, 4.36; N, 5.51; S, 4.20 (%). ( ppy-CHO)(4-NMe2-py)][CF3SO3]\n The procedure used was similar to that described for 4a,\n Synthetic procedure of complex 3a [Os(\u03b76-p-cymene) except for the use of 4-(dimethylamino)pyridine (4-NMe2-py).\n ( ppy-CHO)Cl] Yellow solid. Yield: 43%. ESI-MS ( pos ion mode, DMSO): m/z =\n The procedure used was similar to that described for 1a, 630.2166 [M]+. 1H NMR (300 MHz, CD3CN, 25 \u00b0C) \u03b4 ( ppm):\n except for the use of 4-(2-pyridyl)-benzaldehyde ( ppy-CHO). 10.16 (s, 1H8), 9.50 (ddd, J = 5.8, 1.5, 0.8 Hz, 1H7), 8.75 (d, J =\n Orange solid. Yield: 42%. ESI-MS ( pos ion mode, DMSO): m/z 1.4 Hz, 1H1), 8.01 (ddd, J = 8.2, 1.5, 0.7 Hz, 1H4), 7.92 (ddd, J =\n = 508.1287 [M \u2212 Cl]+. m/z = 561.1311 [M + NH4]+. 1H NMR 7.3, 5.8, 1.5 Hz, 1H5), 7.86 (d, J = 8.1 Hz, 1H3), 7.80\u20137.74 (m,\n (300 MHz, CD3CN, 25 \u00b0C) \u03b4 ( ppm): 10.07 (s, 1H8), 9.27 (ddd, J 2H9), 7.59 (dd, J = 8.1, 1.6 Hz, 1H2), 7.40 (ddd, J = 7.3, 5.8, 1.5\n = 5.8, 1.6, 0.8 Hz, 1H7), 8.51 (d, J = 1.6 Hz, 1H1), 8.05 (d, J = 8.1, Hz, 1H6), 6.24\u20136.17 (m, 2H10), 6.02 (d, J = 5.6 Hz, 1H15),\n 1H5), 7.91 (d, J = 8.0 Hz, 1H3), 7.84 (ddd, J = 8.1, 7.4, 1.5 Hz, 5.95\u20135.85 (m, 2H13+14), 5.50 (d, J = 5.6 Hz, 1H16), 2.86 (s, 6H11)\n 1H4), 7.50 (dd, J = 8.0, 1.7 Hz, 1H2), 7.23 (ddd, J = 7.3, 5.8, 1.4 2.13 (sept, 1H17), 1.77 (s, 3H12), 0.78 (d, J = 6.9 Hz, 3H18), 0.71\n Hz, 1H6), 5.77 (d, J = 5.4 Hz, 1H10), 5.68 (dd, J = 5.4, 1.0 Hz, (d, J = 6.9 Hz, 3H19). 13C NMR (75 MHz, CD3CN) \u03b4 ( ppm):\n 1H12), 5.54 (d, J = 5.4 Hz, 1H13), 5.29 (d, J = 5.4 Hz, 1H11), 2.26 194.31, 166.43(q), 166.09(q), 157.50, 155.01(q), 152.78 (2C),\n (sept, J = 6.9 Hz, 1H14), 2.14 (s, 3H9), 0.91 (d, J = 6.9 Hz, 3H15), 151.66(q), 142.38, 139.87(q), 138.36(q), 126.06, 125.30, 124.84,\n 0.77 (d, J = 6.9 Hz, 3H16). 13C NMR (75 MHz, CD3CN) \u03b4 ( ppm): 121.57, 108.72, 99.78(q), 91.74(q), 84.13, 83.65, 82.54, 72.24,\n\n\n This journal is \u00a9 the Partner Organisations 2021 Inorg. Chem. Front., 2021, 8, 141\u2013155 | 149\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\n\n 39.35 (2C), 31.97, 23.10, 22.46, 18.02. UV-Vis (CH3CN, \u03bb(nm), (\u03b5 appropriate density for each cell line. The cell lines were con-\n (M\u22121 cm\u22121)): 297(39 648), 418(3152). IR (Nujol, cm\u22121): \u03bd(CO) = firmed to be mycoplasma-free using the Hoechst DNA staining\n 1681. Decomposition point = 229 \u00b0C. Anal. Calcd for method.52\n C30H32F3N3O4SOs (777.89): C, 46.32; H, 4.15; N, 5.40; S, 4.12; The maximum % of DMSO used in cell experiments was 0.4\n found: C, 46.07; H, 3.98; N, 5.19; S, 3.98 (%). (except for cisplatin, water diluted) and the measurements\n were corrected with a control containing the same amount of\n X-Ray structures of compounds 3a\u20135a DMSO.\n Crystals suitable for X-ray di\ufb00raction of complexes 3a, 4a and\n 5a were obtained from acetonitrile/hexane (1 : 6) and mounted In vitro antiproliferative inhibition assays\n in inert oil on a Mitegen Micromount and transferred to the Cell viability was determined using a 3-(4,5-dimethylthiazo-2-\n di\ufb00ractometer. Intensities were registered at low temperature yl)-2,5-diphenyl-tetrazolium bromide (MTT)-based vitality\n on a Bruker D8QUEST di\ufb00ractometer using monochromated assay upon exposure of the compounds. In brief, cells were cul-\n Mo K\u03b1 radiation (\u03bb = 0.71073 \u00c5) in \u03a6 and \u03c9 scan modes. tured in 96-well plates (5000 cells per well) in complete\n Absorption corrections were based on multi-scans ( program medium and incubated for 24 h. Serial dilutions of chemical\nPublished on 27 October 2020. Downloaded on 5/12/2026 12:52:24 PM.\n\n\n\n\n SADABS).50 The structures were solved by dual methods complexes were added in a range of final concentrations from\n (SHELXT 2014/5) and refined anisotropically using 0 to 100 \u03bcM in 100 \u03bcL per well and incubated for 48 h. The\n SHELXL-2018.51 All non-hydrogen positions were refined with medium was removed and 100 \u00b5L of MTT solution (1 mg\n anisotropic temperature factors. Hydrogen atoms were mL\u22121) was added. After incubation of the cells for 4 h at 310 K,\n included using rigid methyl groups or a riding model. the MTT solution was removed by suction and 100 \u00b5L of\n Graphics were drawn with Olex2. A summary of crystal data DMSO was added to solubilize the purple formazan crystals\n collection and refinement parameters for all compounds are formed in active mitochondria. The absorbance was measured\n given in ESI.\u2020 at 570 nm using a microplate reader (FLUOstar Omega) and\n the IC50 values were calculated based on the inhibitory rate\n UV-VIS stability studies of compounds 1a and 2a curves using the equation:\n The stability of the complexes 1a and 2a was analyzed by Imax\n recording the absorption spectra a t = 0 and after 48 h of incu- I\u00bc \u0012 \u0013\n IC50 n\n bation at 37 \u00b0C in a PerkinElmer Lambda 750S spectrometer. 1\u00fe\n C\n The complexes were dissolved in DMSO to a final concen-\n tration of 5 \u00d7 10\u22125 M. where I represents the percentage inhibition of viability\n observed, Imax is the maximal inhibitory e\ufb00ect, IC50 is the con-\n HPLC-MS stability studies of compounds 3a\u20135a centration that inhibits 50% of maximal growth, C is the con-\n The stability of Os(II) complexes was analyzed using a centration of the compound and n is the slope of the semi-log-\n RP-HPLC/MS TOF 6220 equipped with a double binary pump arithmic dose\u2013response sigmoidal curves. The non-linear\n (model G1312A), degasser, autosampler (model G1329A), fitting was calculated using SigmaPlot 14.0 software. All com-\n diode array detector (model G1315D) and mass detector in pounds were tested at least in two independent studies (n = 4\n series Agilent Technologies 1200. Chromatographic analyses per concentration replicate).\n were carried out on a Brisa C18 column (200 mm \u00d7 4.6 mm,\n 5 \u03bcm particle size). The mobile phase was a mixture of (A) Morphological analysis of A2780 cells\n H2O/HCOOH 0.1% and (B) acetonitrile/HCOOH 0.1%. The To evaluate cell morphology, the A2780 cancer cells were\n flow rate was 0.6 mL min\u22121 in a linear gradient starting with seeded in 6-well plates at 3 \u00d7 105 cells per well and incubated\n 10% B at 0\u201314 min, reaching 90% B at 14.1\u201318 min, and 10% overnight. The following day, cells were treated with either the\n B at 18.1\u201320 min. Chromatograms were recorded at 280 nm. complex or cisplatin and morphological changes were assessed\n The HPLC system was controlled using ChemStation software over a span of 24 h by phase contrast microscopy (NIKON\n (MASS HUNTER). Samples were dissolved in DMSO (1 mg Eclipse TE 2000U microscope). After treatment, cells were col-\n mL\u22121 final concentration). lected by trypsinization and subjected to flow cytometry\n (Beckman CoulterEpics XL), where at least 10 000 events per\n Cell lines and culture sample were recorded and analyzed by plotting both light and\n Human ovarian carcinoma cell lines, A2780 and A2780cisR, forward scatter in Flowing Software version 2.5.1. Two inde-\n were grown in RPMI-1640 supplemented with 10% fetal bovine pendent measurements of the mitochondrial membrane\n serum (FBS) and 2 mM L-glutamine whereas non-tumorigenic potential were performed.\n Chinese hamster ovary cells were grown in FK-12 medium sup-\n plemented with 10% FBS and 2 mM L-glutamine. The acquired Mitochondrial membrane potential was determined by the\n resistance of A2780cisR cells was maintained by supplement- retention of rhodamine-123 dye\n ing the medium with 1 \u03bcM cisplatin every second passage. The A2780 cells were seeded onto 12-well plates (2 \u00d7 105 cells\n Cells were cultured in a humidified incubator at 310 K in a 5% per well) and incubated overnight. Cisplatin or the tested\n CO2 atmosphere and subcultured every 3\u20134 days with an complex was added to the cells for 24 h. After treatment, cells\n\n\n 150 | Inorg. Chem. Front., 2021, 8, 141\u2013155 This journal is \u00a9 the Partner Organisations 2021\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\n\n were incubated with rhodamine-123 (1 \u00b5M) for 15 min at screening flow cytometer (LSR Fortessa X-20) following the\n 310 K in the dark and then washed with PBS. Retention of the described protocol and adding a trypsinization step to allow\n fluorescent dye was determined by flow cytometry in the FL1-H cell capture using a cytometer. Prior to the assay, fluorescence\n channel. Two independent experiments were performed (n = 2 intensities of both Alexa Fluor 488 and NuclearMask stain\n per replicate) yielding similar results. were measured using a ClarioStar\u2122 microplate reader. The\n fluorescence intensity ratio between Alexa Fluor 488 ( protein\n Apoptosis and necrosis quantification assay synthesis) and nuclear staining (cell viability) was used as an\n The dual Annexin V-FLUOS/PI staining assay (ROCHE, Sigma- indicator for actual protein synthesis. Two independent experi-\n Aldrich) was used for the detection of apoptotic and necrotic ments were performed (n = 3 per replicate).\n cell populations. Briefly, cells were treated as indicated above\n and collected cells were resuspended in 185 \u03bcL of binding ROS generation\n bu\ufb00er. Then, 5 \u00b5L of Annexin-V-FLUOS and 10 \u00b5L of PI were\n added and the resuspended cell solution was left at room The ability of the metal complexes for reactive oxygen species\n temperature in the dark for 15 min. Protection assays were per- generation (ROS) was evaluated using 2\u2032,7\u2032-dichlorodihydro-\n fluorescein diacetate (DCFH-DA) probe following previously\nPublished on 27 October 2020. Downloaded on 5/12/2026 12:52:24 PM.\n\n\n\n\n formed by pretreating A2780 cells for 1 h with the caspase 3\n inhibitor NSCI (5 \u03bcM). Cells were analyzed by flow cytometry described protocols.5,53 Briefly, after A2780 cells have been\n (Beckman CoulterEpics XL) and a total of 10 000 events were allowed to attach the cell surface of 96-well plates, cells were\n acquired in each sample, registering at 620 and 525 nm for PI stained with 10 \u00b5M DCFH-DA for 30 min. The solution was\n and Annexin V, respectively, \u03bbexc = 488 nm. Data were analyzed then removed, and treatments were added to the cells at\n using Flowing Software version 2.5.1. Two independent assays di\ufb00erent concentrations for 2 or 24 h. The fluorescence of the\n were performed (n = 2 per replicate) yielding similar results. dichlorofluorescein (DCF) product was measured using a\n Data were analyzed using Flowing Software version 2.5.1. FLUOstar Omega spectrophotometer. Two independent experi-\n ments were performed (n = 4 per concentration replicate).\n Cell cycle analysis\n The A2780 cancer cells were seeded into 12-well plates at a C. elegans strains and culture conditions\n density of 2 \u00d7 105 cells per well overnight. Treatment with com- The C. elegans wild-type strain N2, JK1466 (gld-1(q485)/dpy-5\n plexes or cisplatin at indicated concentrations was performed (e61) unc-13(e51)), CF1553 (muIs84 [( pAD76) sod-3p::GFP + rol-6\n for 24 h. Cells were collected by trypsinization and fixed in ice- (su1006)]), CL2166 (dvIs19 [( pAF15)gst-4p::GFP::NLS] III) and\n cold ethanol 70% in PBS for 1 h. After fixative removal, a stain- TJ375 (gpIs1 [hsp-16.2p::GFP]) strains were obtained from the\n ing solution with 40 \u00b5g mL\u22121 propidium iodide and 1 \u00b5g mL\u22121 Caenorhabditis Genetic Center (CGC, St Paul, MN, USA),\n RNase was added for 30 min and the samples were analyzed which is supported by the National Institutes of Health \u2013\n using a Beckman CoulterEpics cytometer (\u03bbexc = 488 nm and O\ufb03ce of Research Infrastructure Programs (P40 OD010440).\n \u03bbem = 630). For cell cycle reversibility tests, cells were previously The strains were maintained at 20 \u00b0C in solid nematode\n synchronized by serum starvation to the G1 phase during 24 h growth medium (NGM)54 and the experiments were performed\n and then treated with complex 4a for either 6 or 24 h. After in liquid S medium with animals age-synchronized.54\n drug exposure period, cells were returned to serum-containing Escherichia coli OP50 was used as a food source to N2, CF1553,\n cell media for another 24 h to re-enter cell cycle progression. CL2166 and TJ375 strains and E. coli HT115 gld-1 was used in\n The analysis was performed from two independent experiences order to feed the JK1466 strain. E. coli was grown overnight in\n (n = 2 per replicate) and histograms were plotted using Luria\u2013Bertani (LB) medium at 37 \u00b0C and was concentrated 10\u00d7\n Flowing Software version 2.5.1. in sterile M9 bu\ufb00er.\n Protein synthesis inhibition assay\n Nascent protein synthesis was assayed using the Click-iT Plus C. elegans tumor induction via gene knockdown with RNAi\n O-propargyl-puromycin (OPP) Protein Synthesis Assay Kit RNAi feeding was used to ensure all worms had been silenced\n (Invitrogen\u2122) according to the manufacturer\u2019s instructions. in gld-1. So, JK1466 strain and wild type strain used to propa-\n Briefly, A2780 cells were incubated at 1 \u00d7 104 cells per well in gate the tumoral phenotype had silenced gld-1 gen. The E. coli\n 96-well black plates for 24 h and treated with the osmium com- strain HT115 (DE3) with the homologous DNA sequence for\n plexes or cycloheximide for 12 hours. Then 20 \u03bcM Click-iT OPP the gld-1 (T23G11.3) gene inside the vector L4440 ( pPD129.36)\n reagent for 30 min was applied, fixed with 3.7% formaldehyde was obtained from Source BioScience (sourcebioscience.com)\n in PBS, permeabilized with 0.5% Triton X-100 and stained from the library \u201cRNAi Library (Ahringer)\u201d. The RNAi feeding\n with the Click-iT Plus OPP reaction cocktail containing Alexa was done following the standard protocol.31 Briefly, the HT115\n Fluor 488 picolyl azide as instructed by the manufacturer. strain was cultured in LB supplemented with 30 \u00b5g mL\u22121 of\n Cells were then washed, counterstained with NuclearMask carbenicillin overnight at 37 \u00b0C, and the cells were then\n blue stain and imaged by confocal fluorescence microscopy. induced with 1 mM of IPTG (isopropyl \u03b2-D-1-thiogalactopyrano-\n Alternatively, quantification of OPP labeling was performed in side) at 37 \u00b0C for one hour. Cultures were centrifuged to\n 96 well-plates (80 000 cells per well) using a high-throughput remove LB and concentrated to 10\u00d7 with M9 bu\ufb00er.\n\n\n This journal is \u00a9 the Partner Organisations 2021 Inorg. Chem. Front., 2021, 8, 141\u2013155 | 151\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\n\n E\ufb00ect of CDDP, complexes 2a, 5a and 5b on tumor was used for N2 (wild-type) worms avoiding IPTG and the\n proliferation plates were seeded with concentrated OP50.\n The gld-1(q485) mutation causes a defect in oocyte develop-\n ment that results in the growth of germline tumors that fill the Statistical analysis of in vivo assays\n somatic gonad, eventually leading to the animal\u2019s death.32 The Mathematical analysis of the obtained data was performed\n e\ufb00ects of complexes 2a, 5a and 5b, and CDDP on gld-1(q485) using the on-line application for survival analysis OASIS 2,58\n germline tumors were estimated using age-synchronized with the parameters Kaplan\u2013Meier estimator, Boschloo\u2019s Test,\n JK1466 worms. The animals were maintained on S medium Kolmogorov\u2013Smirnov Test and Survival Time F-Test.\n with HT115 gld-1 bacteria as the food source and di\ufb00erent con-\n centrations of complexes 2a, 5a, 5b and CDDP (0.1, 1, 10 and E\ufb00ect of the metal complexes in ROS production in vivo\n 100 \u00b5M) were added when the worms reach the L4 stage The measurement of ROS generated by the metal complexes\n (48 hours at 20 \u00b0C). Control worms were prepared with water was measured with the 2\u2032,7\u2032-dichlorodihydrofluorescein diace-\n for the CDDP assays and with 0.1% DMSO; for the complex 2a, tate (DCFH-DA) fluorescent probe. Age synchronized N2\n 5a and 5b assays, DMSO in the samples was maintained con- worms were treated with 100 \u03bcM of the metal complexes at\nPublished on 27 October 2020. Downloaded on 5/12/2026 12:52:24 PM.\n\n\n\n\n stant at 0.1%. On the 4th day of adulthood, animals were 20 \u00b0C in liquid S medium supplemented with E. coli; after\n washed with M9 bu\ufb00er and mounted onto glass slides contain- 48 hours the worms were washed three times with M9 and\n ing 10 mM sodium azide to anesthetize them. Bright-field (BF) juglone 20 \u03bcM was used as the positive control. Then the\n images were taken using the 20\u00d7 lens in a Leica DM 2500 LED worms were incubated for one hour in S medium containing\n microscope fitted with a Leica DFC550 camera (Leica 10 \u03bcM of DCFH-DA at 37 \u00b0C in the dark. Worms were washed\n Microsystems, Wetzlar, Germany). Analyses of the gonad size again with M9 bu\ufb00er and mounted onto glass slides contain-\n was performed with the ImageJ software.55 ing 10 mM sodium azide to reduce their mobility.\n Fluorescence of 8\u201310 worms was measured for each condition.\n Tumor size evaluation Images of fluorescence were taken at constant exposure times\n The obtained bright-field images were used to measure the using the 10\u00d7 lens and the I3 filter cube (Excitation Band Pass\n area of each tumorous gonad by using ImageJ software.55 450\u2013490 nm, dichromatic mirror 510 nm) in a Leica DM 2500\n Gonad sizes were measured from the loop region to the proxi- LED microscope fitted with a Leica DFC550 camera (Leica\n mal region, including uterus area when it was filled with Microsystems, Wetzlar, Germany) with incident light beam. To\n tumorous cells. Significance of the obtained data was obtained quantify GFP fluorescence, images were analyzed using ImageJ\n using the ANOVA test with a significance level of 0.5. software (NIH).59 Each raw image was split in RGB channels\n and the mean pixel density was measured only in the green\n Survival assays channel, the significance of the data was analyzed by the one-\n Animal lifespan was determined using a platform for the auto- way ANOVA test.\n matic control of the organism C. elegans in the lifespan\n machine.55 The mechanical device was built56 to follow and E\ufb00ect of metal complexes on HSP-16.2p expression\n analyze C. elegans life cycle, by taking images of the worms- The experiments with the strain TJ375 were performed as\n settled in analysis plates- every hour and estimating the life- reported by Guerrero-Rubio et al.,57 TJ375 (hsp-16.2p::GFP)\n span using a mathematical software. After 48 hours in liquid worms were grown in liquid S medium at 20 \u00b0C and the\n media, the JK1466 or the N2 worms were centrifuged at 2000 g expression of HSP-16.2 was measured by observing the fluo-\n and washed with M9 bu\ufb00er three times. 30\u201340 worms were rescence of the fused green fluorescent protein (GFP). TJ375\n then transferred to 35 mm analysis plates, containing 8 mL of L4 age-synchronized larvae were treated for 24 hours with 0.1,\n NGM agar, supplemented with 30 \u03bcg mL\u22121 of nystatin, 30 \u03bcg 1, 10, 100 \u00b5M of each compound or juglone (20 \u03bcM) as positive\n mL\u22121 of carbenicillin and the tested compounds in the same control at 20 \u00b0C, to induce oxidative stress. After 24 h of induc-\n dose as in the liquid media. FUdR (2\u2032-deoxy-5-fluorouridine) at tion, worms were washed with M9, photographed and analyzed\n 10 \u03bcg mL\u22121 was used to avoid progeny and IPTG was added to as above. Other sets of experiments were proposed to estimate\n a final concentration of 1 mM to induce E. coli HT115 gld-1 the potential of the compounds to generate oxidative stress re-\n bacterial cells. Plates were seeded with 100 \u03bcL of E. coli HT115 sistance. For these experiments L1 larvae were treated for\n gld-1 from an overnight culture in LB at 37 \u00b0C; the culture was 48 hours with 100 \u00b5M of each compound, then the worms\n concentrated 10\u00d7 in a sterile M9 bu\ufb00er and left to induce over- were washed and transferred to fresh S medium containing\n night at 20 \u00b0C. All the experiment plates were prepared in juglone 20 \u00b5M. After 24 hours of exposition to juglone worms\n triplicate. Plates were closed and incubated for 20 minutes at where washed and visualized under a fluorescence microscope\n 20 \u00b0C. Closed lid plates were loaded into the scanners of the using 40\u00d7 lens with the I3 filter cube. The images included the\n lifespan machine. The machine acquired an image of each anterior part of the worms from the back of the pharynx, ten\n loaded plate every hour for the duration of the experiment and or more individual worms were photographed in each inde-\n the analysis detected the time of the death for each worm. The pendent trial. The image acquisition and analysis were per-\n experiments were set at 25 \u00b0C for 25 days. The same procedure formed as above.\n\n\n 152 | Inorg. Chem. Front., 2021, 8, 141\u2013155 This journal is \u00a9 the Partner Organisations 2021\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\n\n E\ufb00ect of complexes 2a, 5a and 5b and CDDP on SOD-3 expression 5 E. Ortega, J. G. Yellol, M. Rothemund, F. J. Ballester,\n Synchronous cohorts of the CF1553 strain were treated with V. Rodr\u00edguez, G. Yellol, C. Janiak, R. Schobert and J. Ruiz, A\n 100 \u03bcM of each drug or their vehicle for 48 hours at 20 \u00b0C in S new C,N-cyclometalated osmium(II) arene anticancer\n medium, juglone 20 \u03bcM was used as the positive control. Then sca\ufb00old with a handle for functionalization and antioxida-\n worms were washed with M9 and visualized under a fluo- tive properties, Chem. Commun., 2018, 54, 11120\u201311123.\n rescence microscope at 20\u00d7 lens with the I3 filter cube. The 6 J. M. Hearn, I. Romero-Canel\u00f3n, A. F. Munro, Y. Fu,\n image acquisition and analysis were performed as above. A. M. Pizarro, M. J. Garnett, U. McDermott, N. O. Carragher\n While the SOD-3 is accumulated in the head, the tail and the and P. J. Sadler, Potent organo-osmium compound shifts\n vulva of the animals, only the head fluorescence was used to metabolism in epithelial ovarian cancer cells, Proc. Natl.\n measure the SOD-3 accumulation. Acad. Sci. U. S. A., 2015, 112, E3800\u2013E3805.\n 7 (a) V. Novohradsky, J. Yellol, O. Stuchlikova, M. D. Santana,\n E\ufb00ect of complexes 2a, 5a and 5b and CDDP on GST-4p H. Kostrhunova, G. Yellol, J. Kasparkova, D. Bautista,\n expression J. Ruiz and V. Brabec, Organoruthenium Complexes with\n C^N Ligands are Highly Potent Cytotoxic Agents that Act by\n Age synchronized animals of the CL2166 strain were treated\nPublished on 27 October 2020. Downloaded on 5/12/2026 12:52:24 PM.\n\n\n\n\n a New Mechanism of Action, Chem. \u2013 Eur. J., 2017, 23,\n with 100 \u03bcM of each drug or their vehicle for 48 hours at 20 \u00b0C\n 15294\u201315299; (b) F. Ballester, E. Ortega-Forte, D. Bautista,\n in S medium, juglone 20 \u03bcM was used as the positive control.\n M. D. Santana and J. Ruiz, Ru(II) photosensitizers compe-\n Then worms were washed with M9 and visualized under a fluo-\n tent for hypoxic cancers by green light activation, Chem.\n rescence microscope at 20\u00d7 lens with the I3 filter cube. The\n Commun., 2020, 56, 10301\u201310304.\n image acquisition and analysis were performed as above.\n 8 F. E. Poynton, S. A. Bright, S. Blasco, D. C. Williams,\n J. M. Kelly and T. Gunnlaugsson, The development of\n ruthenium(II) polypyridyl complexes and conjugates for\n Con\ufb02icts of interest in vitro cellular and in vivo applications, Chem. Soc. Rev.,\n There are no conflicts to declare. 2017, 46, 7706\u20137756.\n 9 J. P. C. Coverdale, H. E. Bridgewater, J.-I. Song, N. A. Smith,\n N. P. E. Barry, I. Bagley, P. J. Sadler and I. Romero-Canel\u00f3n,\n In vivo selectivity and localization of reactive oxygen\n Acknowledgements species (ROS) induction by osmium anticancer complexes\n This work was supported by the Spanish Ministerio de Ciencia that circumvent platinum-resistance, J. Med. Chem., 2018,\n e Innovaci\u00f3n (MCI/AEI) and FEDER funds (Projects RTI2018- 61, 9246\u20139255.\n 096891-B-I00, AGL2017-86526 and MultiMetDrugs network 10 J. P. C. Coverdale, I. Romero-Canel\u00f3n, C. Sanchez-Cano,\n RED2018-102471-T) and Fundaci\u00f3n S\u00e9neca-CARM (Projects G. J. Clarkson, A. Habtemariam, M. Wills and P. J. Sadler,\n 20857/PI/18 and 19893/GERM/15). F. B. thanks Fundaci\u00f3n Asymmetric transfer hydrogenation by synthetic catalysts in\n S\u00e9neca-CARM (Project 20277/FPI/17). M. A. G.-R. holds a con- cancer cells, Nat. Chem., 2018, 10, 347\u2013354.\n tract financed by MEC-FEDER (Spain). S. H.-G. holds a contract 11 J. Yellol, S. A. P\u00e9rez, A. Buceta, G. Yellol, A. Donaire,\n financed by Fundaci\u00f3n S\u00e9neca (Spain). E.O thanks AECC P. Szumlas, P. J. Bednarski, G. Makhloufi, C. Janiak,\n (PRDMU19003ORTE). A. Espinosa and J. Ruiz, Novel C,N-Cyclometalated\n The authors are grateful to Julie Ahringer\u2019s group at The Benzimidazole Ruthenium(II) and Iridium(III) Complexes\n Wellcome CRC Institute, University of Cambridge (Cambridge, as Antitumor and Antiangiogenic Agents: A Structure\u2013\n UK) for the kind donation of the RNAi strain used in this Activity Relationship Study, J. Med. Chem., 2015, 58,\n work. 7310\u20137327.\n 12 M. J. Chow, M. Alfiean, G. Pastorin, C. Gaiddon and\n W. H. Ang, Apoptosis-independent organoruthenium anti-\n Notes and references cancer complexes that overcome multidrug resistance: self-\n assembly and phenotypic screening strategies, Chem. Sci.,\n 1 D. Ruggero and P. P. Pandolfi, Does the ribosome translate 2017, 8, 3641\u20133649.\n cancer?, Nat. Rev. Cancer, 2003, 3, 179\u2013192. 13 A. Weiss, R. H. Berndsen, M. Dubois, C. M\u00fcller, R. Schibli,\n 2 M. Bhat, N. Robichaud, L. Hulea, N. Sonenberg, J. Pelletier A. W. Gri\ufb03oen, P. J. Dyson and P. 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