Half-Sandwich Ir(III) and Os(II) Complexes of Pyridyl-Mesoionic Carbenes as Potential Anticancer Agents

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Article Cite This: Organometallics XXXX, XXX, XXX−XXX pubs.acs.org/Organometallics Half-Sandwich Ir(III) and Os(II) Complexes of Pyridyl-Mesoionic Carbenes as Potential Anticancer Agents Juran Kralj,†,# Aljoša Bolje,‡,# Darija Stupin Polancě c,§ Ivana Steiner,† Tena Gržan,† Ana Tupek,† Nikolina Stojanovic,́ † Stephan Hohloch,∥ Damijana Urankar,‡ Maja Osmak,† Biprajit Sarkar,*,∥ Anamaria Brozovic,*,† and Janez Košmrlj*,‡ † Division of Molecular Biology, Ruđer Bošković Institute, HR-10000 Zagreb, Croatia Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, SI-1000 Ljubljana, Slovenia § Fidelta Ltd., HR-10000 Zagreb, Croatia ∥ Institut für Chemie und Biochemie, Freie Universität Berlin, Fabeckstraße 34-36, D-14195 Berlin, Germany Downloaded via 109.94.221.159 on October 10, 2019 at 03:22:59 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. ‡ S Supporting Information * ABSTRACT: A series of cationic chlorido arene-iridium(III) and arene-osmium(II) complexes with bidentate pyridyl functionalized mesoionic carbenes (MIC) of the 1,2,3triazol-5-ylidene type have been prepared. The variations in the ligand structures include the position of the pyridyl substituent relative to the triazolylidene ring (N-wingtip vs Cwingtip), phenyl versus ethyl substituents, and incorporation of several functional groups at the phenyl substituents. Five complexes have been characterized by X-ray structural analysis. All complexes, including osmium(II) and ruthenium(II) analogues having a pyrimidyl in place of the pyridyl group, have been studied for their cytotoxic activity on a human cervical carcinoma HeLa cell line. Two of the compounds, Ir5 and Ir9, were the most cytotoxic with IC50 values of 7.33 μM and 2.01 μM, respectively. Examination of their cytotoxic eﬀect on diﬀerent cell lines revealed that they preferentially kill cancer over normal cells. The Ir5 and Ir9 compounds arrested cells in G2 and induced a dose-dependent increase in SubG0/G1 cell population. Apoptosis, as the primary mode of cell death, was conﬁrmed by Annexin V/PI staining, detection of cleaved PARP, and caspases 3 and 7 activity upon treatment of HeLa cells with both compounds. The higher toxicity of Ir9 is probably due to its increased accumulation in the cells compared to Ir5. The role of glutathione (GSH) in the protection of cells against Ir5 and Ir9 cytotoxicity was conﬁrmed by pretreatment of cells either with buthionine sulfoximine (inhibitor of GSH synthesis) or Nacetyl-cysteine (precursor in GSH synthesis). ■ INTRODUCTION Cancer is one of the leading causes of death worldwide, treated mostly by surgery, radiotherapy, and chemotherapy.1 Despite a broad use of well-known metal-based anticancer drugs, such as cisplatin and its derivatives, two main disadvantages of chemotherapy remain a problem in successful treatment of a variety of tumor types. Chemotherapeutics are largely ineﬃcient against drug-resistant tumors and are followed by severe side eﬀects including nephrotoxicity, hepatotoxicity, ototoxicity, and cardiotoxicity.2 This urges a need for investigation of new compounds that will be highly toxic against tumor cells and nontoxic for the healthy cell population. Recently, organometallic compounds have gained interest as anticancer agent candidates, owing to their increased structural variety, diverse stereochemistry, and modular way of ligand selection compared to organic molecules. Despite possessing a carbon−metal bond, these compounds are often kinetically stable, uncharged, and relatively lipophilic. Their metal atoms can be in a low oxidation state, making them suitable for application in medicinal chemistry.3 © XXXX American Chemical Society Mesoionic carbenes (MIC) of the 1,2,3-triazol-5-ylidene type are currently highly popular ligands in organometallic chemistry.4,5 Apart from the fact that their use as a ligand class is barely 10 years old, an important reason for their popularity is their modular synthesis by Click-reaction. Thus, introducing additional heteroatom donors on such ligands and tuning of the steric and electronic properties is often easier compared to the most other classes of N-heterocyclic carbenes (NHC). Hence, metal complexes of such ligands were successfully used in a variety of homogeneous catalytic processes. Additionally, metal complexes of these ligands have been successfully investigated for their intriguing redox properties, such as electrocatalysts, and for their photochemical properties. One ﬁeld of research, where metal complexes of MICs are slowly but surely displaying promise, is their potential use in anticancer research.5,6 This is perhaps not that surprising considering the potential that metal complexes of various other NHC ligands have shown in medicinal chemistry. Recently, we Received: May 15, 2019 A DOI: 10.1021/acs.organomet.9b00327 Organometallics XXXX, XXX, XXX−XXX Article Organometallics Scheme 1. Synthesis of Os(II), Ir(III), and Ru(II) Complexes chelating pyridine at C5 (C-wingtip). Ligand precursors L1− L12 were used for the synthesis of osmium(II) (Os1−Os12) and iridium(III) half-sandwich complexes (Ir1−Ir9). Transmetalation protocol8 with Ag2O turned out to be the route of choice aﬀording the products from moderate 50% yields to higher. Additionally, in the particular case of ligand precursor L13, pyrimidyl was used as a heterocycle (N-wingtip) instead of pyridyl to construct complexes Os12 and Ru1. The complexes were characterized via 1H and 13C NMR spectroscopy and mass spectrometry. The disappearance of the signal corresponding to the triazolium C−H proton in the 1H NMR spectrum of the complexes was already a ﬁrst indication of the formation of the complexes. In the 13C NMR spectra, the peak corresponding to the metal-bound MIC-C was observed between 159.6 and 160.9 ppm for the Os(II) complexes and between 156.3 and 158.4 ppm for the Ir(III) complexes. Molecular peaks corresponding to the cations were observed for all the complexes in their HRESI mass spectra. Crystal Structures. We were successful in obtaining single crystals of compounds Os8, Os11, Ir5, Ir6, and Ir7 that were suitable for X-ray diﬀraction studies (Figure 1). The two osmium complexes and Ir6 crystallized as dichloromethane solvates. All the metal centers were coordinated in a pianostool type of coordination. The Cp* ligands were bound in an η5 way to the Ir(III) centers, and the cymene ligands were bound in an η6 fashion to the Os(II) centers. The three “legs” have shown that 1,2,3-triazolium salts, precursor for the 1,2,3triazol-5-ylidene MIC ligands, also possess anticancer activity.7 In the past years, we have been involved in the synthesis of pyridyl-substituted MIC ligands and their metal complexes, which are potent catalysts for a variety of homogeneous catalytic processes.8 An important discovery in terms of ligand synthesis was the development of methodologies for selective alkylation of pyridyl-triazoles.9 Herein, we report on the synthesis of a series of Os(II) and Ir(III) complexes, as well as one Ru(II) analogue with pyridyl-MICs. We present a comprehensive study involving 12 Os(II), 8 Ir(III) complexes, and 1 Ru(II) complex, along with their cytotoxic properties. The eﬀects of introduced ligands and metal centers on cytotoxic properties as well as their mechanisms of action in cells are discussed. ■ RESULTS AND DISCUSSION Synthesis and Characterization. The ligand precursors, triazolium salts, were synthesized following procedures that were recently reported by us.9,10 The variations in ligand precursor structures of L1−L12 include the position of the pyridyl substituent (N vs C-wingtip of the triazolylidene ring), phenyl versus ethyl substituents, and incorporation of several functional groups on the phenyl substituents (Scheme 1). Triazole rings in L1−L7 are characterized by the N1-pyridyl substituent (N-wingtip), whereas those in L8−L12 have the B DOI: 10.1021/acs.organomet.9b00327 Organometallics XXXX, XXX, XXX−XXX Article Organometallics Figure 1. Ball and stick models of Os8, Os11, Ir5, Ir6, and Ir7. Hydrogen atoms, counterions, and solvent molecules are omitted for clarity. Selected bond lengths and angles are reported in Table 1. of the piano-stool are then made up of the pyridyl-N and MICC of the chelating ligands and an additional chloride ligand. As it has been observed earlier, the bond lengths inside the triazolylidene rings point to a delocalized situation (Table 1). The metal-C(MIC) and the metal-N(pyridine) distances are all in the expected range.4c,e,f The aryl substituents on the triazolylidene rings are twisted out of plane as seen from angles close to 50° between the two rings (Table 1). Eﬀect of the Complexes on Cell Viability. This work was stimulated by recent reports on cytotoxic properties of the ligand precursors, 1,2,3-triazolium salts,7 as well as organometallic compounds of triazolylidene structure.5,6 Some triazolium salts exhibited high cell-type dependent cytotoxicity against diﬀerent tumor cells. Those did not bind double stranded DNA, but induced formation of reactive oxygen species, which further triggered cell death.7 An excellent activity proﬁle against a range of cell lines has been shown for half-sandwich Ru(II) and Os(II) organometallics having triazolylidene as a monodentate MIC ligand.6a The structures of the half-sandwich Os(II), Ir(III), and Ru(II) compounds Os1−Os12, Ir1−Ir9, and Ru1 are distinctly diﬀerent from the compounds mentioned above, possessing bidentate pyridyltriazolylidene ligand that chelates the metal in N(pyridine) ^C(MIC) mode. In Os12 and Ru1 the pyridyl dent is replaced by pyrimidyl. First, all the complexes were evaluated for their cytotoxicity potential on human cervical carcinoma (HeLa) cells, the cell model system that was previously identiﬁed as suitable for the screening of new compounds.11,12 The results of the screening are collected in Table 2. Cytotoxicity of complexes Os1, Os3−Os5, Os7, Os8, Os11, 1 Ir −Ir3, Ir8, and Ru1 could not be determined due to their poor solubility. Compound Ir9 with IC50 of 2.01 μM was the Table 1. Bond Lengths (Å), Angles, and Dihedral Angles (deg) atoms and planes Os8 Bond lengths M1−Cl1 2.40(1) M1−C1 2.02(1) M1−N4 2.15(1) C1−C2 1.38(2) C2−C3 1.45(2) C2−N1 1.36(2) N1−N2 1.32(2) N2−N3 1.33(1) N3−C1 1.35(1) N1−C16 1.45(2) N3−C8 1.46(1) C3−N4 1.38(2) M1− 1.70(1) center Bond angles C1−M1− 75.9(4) N4 C1−M1− 85.9(3) Cl1 N4−M1− 84.8(3) Cl1 N3−C1− 102.9(1) C2 Dihedral angles Tz−Py 5.9(4) Tz−Ar 48.1(5) Os11 Ir5 Ir6 Ir7 2.401(2) 2.040(6) 2.124(4) 1.398(7) 1.430(8) 1.358(7) 1.323(6) 1.343(6) 1.353(7) 1.462(7) 1.465(7) 1.385(7) 1.699(1) 2.40(1) 2.03(1) 2.13(1) 1.39(1) 1.44(1) 1.35(1) 1.32(1) 1.36(1) 1.36(1) 1.47(1) 1.44(1) 1.38(1) 1.82(1) 2.40(1) 2.01(1) 2.12(1) 1.38(1) 1.44(1) 1.35(1) 1.31(1) 1.35(1) 1.36(1) 1.47(1) 1.44(1) 1.37(1) 1.82(1) 2.39(1) 2.01(1) 2.14(1) 1.34(1) 1.47(1) 1.36(1) 1.30(1) 1.34(1) 1.38(1) 1.47(1) 1.44(1) 1.36(1) 1.83(1) 76.4(2) 76.7(3) 76.3(2) 75.9(4) 85.4(2) 88.2(2) 89.7(2) 87.9(3) 84.6(1) 85.6(2) 87.4(2) 86.8(2) 105.5(5) 102.3(6) 102.9(5) 102.0(8) 2.0(1) − 7.4(2) 44.1(2) 8.5(3) 53.0(3) 10.2(3) 56.4(3) most toxic followed by Ir5 (IC50 of 7.33 μM). Both compounds are Cp*−iridium complexes of very similar structure having C DOI: 10.1021/acs.organomet.9b00327 Organometallics XXXX, XXX, XXX−XXX Article Organometallics primary keratinocytes (Table 3). The therapeutic index was not calculated since the IC50 doses of both compounds in keratinocytes could not be determined. Cell Cycle and Cell Death Analysis. To shed light into the mode of action that underlies the antiproliferative or/and toxic activity of Ir5 and Ir9, we ﬁrst investigated their eﬀect on the cell cycle. The ﬂow cytometric analysis upon propidium iodide staining of cells treated with diﬀerent concentrations of Ir5 and Ir9 during 72 h (Figure 2A) or with 7 μM Ir5 and 2 μM Ir9 during 24−72 h (Figure 2B) was performed. It seems that both compounds tend to arrest cells in the G2 phase of the cell cycle, and push the cells further into cell death. But, it is interesting to notice that IC50 dose of Ir5 induces accumulation of cells in the S phase of cell cycle, which is not a case for IC50 of Ir9. The S phase arrest implies problems with DNA replication that triggers a “checkpoint”a cascade of signaling events that puts the phase on hold until the problem is resolved. It seems that these cells are unable to progress through S phase when the checkpoint is inhibited and often enter directly into premature mitosis.13 Further investigation is needed to understand why this eﬀect is seen only upon Ir5 treatment and not after the treatment with Ir9. The SubG0/G1 cell population, which is measured in cell cycles analysis as well, is characterized with a lower amount of DNA. The cells that entered apoptosis from the G2/M phase of the cell cycle or have lost DNA for any other reason, e.g., death by some other form of oncosis, will appear in the SubG0/G1 region. The Ir5 and Ir9 induce dose- and time-dependent increase of SubG0/G1 cell population (Figure 2A and 2B). To shed more light on the type of cell death, HeLa cells were treated with Ir5 (7 μM) or Ir9 (2 μM), following the Annexin V-FITC and PI staining 24−72 h after. The analyzed data shown in Figure 3A indicated that the majority of cells die by apoptosis, which we further conﬁrmed by detection of cleaved PARP, a well-accepted apoptotic marker,14 in the treated cells. As shown in Figure 3B, the treatment of HeLa cells with 7 μM of Ir9 induced cleavage of PARP already 24 h after the treatment. Due to the lower toxicity, the cleaved PARP was not detected upon cell treatment with the same concentration of Ir5 (7 μM). However, when the equitoxic doses of Ir5 (33 μM) and Ir9 (10 μM) were used the cleavage of PARP was detected for both compounds after 72 h treatment (Figure 3C). In addition, 72 h upon cell treatment with 7 μM and 33 μM of Ir5, and 2 μM and 10 μM of Ir9, the activity of executor caspases 3 and 7 was measured. The results showed that both compounds activated caspases 3/7, although the activation was more visible upon Ir5 treatment (Figure 3D). It is likely that Ir9 induces the activity of other caspases such as caspase 1.15 The possible involvement of autophagy and necroptosis as cell death models was checked by pretreatment of cells with baﬁlomycin A (BAF A), a known inhibitor of the late phase of autophagy,16 and with the inhibitor of necroptosis, necrostatin-1 (Nec-1).17 Afterward, the cells were treated with diﬀerent concentrations of either Ir5 or Ir9. Both inhibitors failed in protection of the cells from cell death, indicating that neither autophagy nor necroptosis are involved in cell death induced by Ir5 or Ir9 (Figure 3E). One of the possibilities for higher toxicity of Ir9 compared to Ir5 could be increased accumulation of Ir9 in the cells due to the small structural diﬀerence. In order to test this hypothesis, we treated HeLa cells with diﬀerent concentrations of both compounds during 3 h and measured the accumulation of iridium by high resolution inductively coupled plasma mass Table 2. Eﬀect of the Compounds on Viability of HeLa Cellsa compound IC50 (μM) 1 nd 33.01 ± 6.34 nd nd nd 24.42 ± 5.56 nd nd 89.41 ± 13.80 100.88 ± 11.61 nd 52.81 ± 5.97 nd nd nd 28.52 ± 8.56 7.33 ± 0.28 17.52 ± 0.64 45.02 ± 3.07 nd 2.01 ± 0.28 nd Os Os2 Os3 Os4 Os5 Os6 Os7 Os8 Os9 Os10 Os11 Os12 Ir1 Ir2 Ir3 Ir4 Ir5 Ir6 Ir7 Ir8 Ir9 Ru1 a nd = not determined due to insuﬃcient solubility. the N-wingtip ligand series, while only diﬀering in the phenyl moiety being unsubstituted in Ir5 and 2,6-diisopropyl substituted in Ir9. On the basis of the results from Table 2, compounds Ir5 and 9 Ir were selected for screening on tumor cells of diﬀerent origins and one normal cell line. These included HeLa cells, laryngeal carcinoma (HEp2) cells, large cell lung carcinoma (H460) cells, colorectal carcinoma (HCT-116) cells, ovarian cancer (MES-OV) cells, and normal primary keratinocytes. The results collected in Table 3 demonstrate similar toxicity of Table 3. Eﬀect of Ir5 and Ir9 on Viability of Diﬀerent Cell Lines cell linea Ir5 (IC50 (μM))b Ir9 (IC50 (μM))b HeLa HEp2 H460 HCT-116 MES-OV keratinocytes 7.33 ± 0.28 13.30 ± 1.20 6.00 ± 0.90 6.50 ± 0.81 7.6 ± 0.86 <100 2.01 ± 0.28 4.66 ± 0.59 3.2 ± 0.89 3.02 ± 0.17 4.08 ± 0.5 <100 a HeLa = cervical carcinoma cells; HEp2 = laryngeal carcinoma cells; H460 = large cell lung carcinoma cells; HCT-116 = colorectal carcinoma cells; MES-OV = ovarian cancer cells; Keratinocytes = normal primary keratinocytes. bThe data are shown as mean values of three experiments (±SD). both compounds on all tested tumor cell lines independent from their origin, with Ir9 being consistently more cytotoxic than Ir5. One of the most important features of a compound to be considered as potential anticancer agent is selectivity, i.e., as high as possible cytotoxicity against tumor as compared to the nontumor cells. It is noteworthy that all examined tumor cell lines proved to be considerably more sensitive to both compounds as compared to the normal cell line, normal D DOI: 10.1021/acs.organomet.9b00327 Organometallics XXXX, XXX, XXX−XXX Article Organometallics Figure 2. Eﬀects of Ir5 and Ir9 on cell cycle. HeLa cells were treated either with diﬀerent concentrations of Ir5 and Ir9 during 72 h (A) or with equitoxic doses of Ir5 (7 μM) and Ir9 (2 μM) during 24−72 h (B). A cell cycle distribution was assessed as described in the Experimental Section. Data of one from three independent experiments are presented. signal transduction, cell proliferation, and apoptosis.23 Previously, we showed importance of GSH detoxiﬁcation role in cell defense against toxicity of several compounds.24 Elucidating the mechanisms through which GSH is involved in defense against chlorido arene-iridium(III) complexes with bidentate pyridyl functionalized mesoionic carbenes (MIC) of the 1,2,3-triazol-5-ylidene type will be interesting for future investigation. spectrometry (HR ICPMS). The accumulation of both compounds in the cells increased with the time, but the content of iridium upon Ir9 was higher than the one measured upon Ir5 treatment (Figure 4). The higher level of Ir9 in the cell, compared to the level of Ir5, could be one of the reasons for its greater toxicity. The Role of Glutathione (GSH) in Cell Response to Ir5 and Ir9. The role of GSH as a cell defense mechanism is well described in the literature.18,19 Previously, we described GSH as an important factor in the protection of cell upon induction of reactive oxygen species (ROS),20,21 and upon treatment with newly synthesized12 or newly isolated compounds.22 Recently, we identiﬁed a triazolium salt of 4-(4-methoxyphenyl)-3-methyl-1-(2-picolyl)-1H-1,2,3-triazolium hexaﬂuorophosphate(V) structure, which induced ROS and showed that GSH is involved in reduction of ROS-induced toxicity.7 Here we show that pretreatment of HeLa cells with buthionine sulfoximine (BSO), a speciﬁc inhibitor of GSH synthesis, decreased cell survival upon treatment with Ir5 and Ir9 (Figure 5A). The data imply a role of GSH in cell defense against damages induced by both investigated compounds. The important role of GSH in cell-stress protection was further conﬁrmed by pretreatment of cells with N-acetyl-cysteine (NAC), a precursor of GSH synthesis. Increased GSH amount in the cell protects it against either Ir5 or Ir9 toxicity (Figure 5B). The protection role of GSH is more expressed in HeLa cells upon Ir9 treatment as compared to Ir5. In order to investigate the possibility of ROS-induced toxicity by Ir5 and Ir9, we stained cells upon treatment with ﬂuorescent dye 5(and-6)-chloromethyl-29,79-dichlorodihydroﬂuorescein diacetate, acetyl ester (CM-H2DCFDA). The data obtained indicate that neither Ir5 nor Ir9 induce ROS (Figure 5C). This was further conﬁrmed by pretreatment of cells with a well-accepted ROS scavenger tempol, which did not change survival rate of cells compared to the one treated only with compounds Ir5 and Ir9 (Figure 5D). It is known that GSH has a role in other physiological processes such as nutrient metabolism, defense by detoxiﬁcation, and regulation of cellular metabolic functions ranging from gene expression, DNA, and protein synthesis to ■ CONCLUSION We have synthesized 12 osmium(II), 9 iridium(III), and 1 ruthenium(II) chloride arene organometallic compounds that are additionally coordinated with bidentate pyridyl and pyrimidyl functionalized mesoionic carbenes (MIC) of the 1,2,3-triazol-5-ylidene type. All new compounds have been fully characterized by means of standard analytical and spectroscopic techniques. For ﬁve of them we were able to provide X-ray structural analysis. The complexes have been tested for their anticancer activity, and it is noteworthy that this is the ﬁrst report on biological activity of this type of organometallic compounds. Ir5 and Ir9 displayed the highest eﬀect on cell viability in HeLa cells. They had a similar eﬀect on diﬀerent tumor cell lines, but they were more cytotoxic against tumor cells than normal cells. Moreover, both complexes induced programmed cell death. GSH plays an important role as a protection mechanism in Ir5- and Ir9induced cell death. Organoiridium complexes seem very interesting for further investigation as potential anticancer agents due to their selective highly toxic eﬀect on diﬀerent tumor cell types. ■ EXPERIMENTAL SECTION Chemistry. The reagents and solvents were used as obtained from the commercial sources (Sigma-Aldrich, Fluka, Alfa Aesar). Ag2O25 triazolium salts L1−L13,10 complexes Os1−Os7,8b Os12,8d Ir1−Ir4,8b Ir8,8c and Ru18d were prepared as described previously. NMR spectra were measured with a Jeol ECS 400 spectrometer at 25 °C. Proton and carbon spectra were referenced to Si(CH3)4 as the internal standard. Chemical shifts are given on the δ scale (ppm). E DOI: 10.1021/acs.organomet.9b00327 Organometallics XXXX, XXX, XXX−XXX Article Organometallics Figure 3. Impact of Ir5 and Ir9 on cell death. HeLa cells were treated with 7 μM Ir5 and 2 μM Ir9 during 24−72 h. The percent of live, early apoptotic, and late apoptotic/necrotic cells are shown. The data of one from three independent experiments are presented. Heat shock was used to adjust data analysis parameters (10 min, 56 °C) (A). HeLa cells were treated 24 h with equimolar concentration (7 μM) of Ir5 and Ir9 (B) and with equitoxic concentrations, Ir5 (33 μM) and Ir9 (10 μM) during 72 h (C). Control cells were collected either at 24 (B) or 72 h (C) time points. Precision Plus Protein All Blue Prestained Protein Standard (Bio-Rad, USA) is indicated in the ﬁgure. ERK1/2 (B) and ERK2 (C) were used as loading controls. Data of one from three independent experiments are presented. HeLa cells were treated with Ir5 (7 μM and 33 μM) and Ir9 (2 μM and 10 μM). The activity of caspase 3/7 was measured after 72 h. The values are presented as fold of control. Signiﬁcance was determined between control (nontreated) and Ir5, respectively, Ir9 treated cells (*P < 0.05; **P < 0.01; ***P < 0.001). The experiment was repeated at least three times (D). HeLa cells were pretreated either with 0.5 and 1 nM BAF A or 12.5 and 25 μM Nec-1. Two hours later the cells were treated with diﬀerent concentrations of Ir5 and Ir9. Cell survival was determined 72 h later by MTT assay. Each point represents mean ± SD of at least three independent experiments. All data are expressed as the average percentage of survival values relative to the untreated control ± SD or samples treated with inhibitors alone (E). F DOI: 10.1021/acs.organomet.9b00327 Organometallics XXXX, XXX, XXX−XXX Article Organometallics Os9. From L9 (71 mg) and [Os(p-Cym)Cl2]2 (79 mg). Gray-green solid. Yield: 53% (0.106 mmol, 82 mg). 1H NMR (400 MHz, CD2Cl2) δ = 9.17 (dt, J = 5.7, 1.1 Hz, 1H, Py−H), 8.03−7.98 (m, 1H, Py−H), 7.94 (dt, J = 8.1, 1.1 Hz, 1H, Py−H), 7.77 (d, J = 9.0 Hz, 2H, Ar−H), 7.36 (ddd, J = 7.4, 5.7, 1.5 Hz, 1H, Py−H), 7.09 (d, J = 9.0 Hz, 2H, Ar−H), 5.44 (dd, J = 5.8, 1.1 Hz, 1H, Cym−CH), 5.38 (d, J = 5.9 Hz, 1H, Cym−CH), 5.28 (d, J = 5.9 Hz, 1H, Cym−CH), 4.86 (dd, J = 5.5, 1.1 Hz, 1H, Cym−CH), 4.51 (s, 3H, N−CH3), 3.87 (s, 3H, O−CH3), 2.24 (sept, J = 7.0 Hz, 1H, CH(CH3)2), 2.09 (s, 3H, Cym−CH3), 0.86 (d, J = 6.9 Hz, 3H, CH(CH3)2), 0.85 (d, J = 6.9 Hz, 3H, 21.7 (CH(CH3)2). 13C NMR (100 MHz, CD2Cl2) δ = 160.9 (ArC), 160.8 (Tz−C), 160.0, 157.0, 147.9, 147.5, 138.9, 129.7, 125.9, 125.4, 120.4, 114.9 (all Ar/Py−C), 98.2, 97.9, 81.0, 76.9, 76.8, 71.2 (all Cym−C), 55.1 (O−CH3), 38.3 (N−CH3), 30.6 (CH(CH3)2), 21.8 (CH(CH3)2), 21.0 (CH(CH3)2), 17.8, 13.06 (Cym−CH3). HRMS (ESI+) m/z calcd. for C25H28ClN4OOs+ [M]+ = 627.1566, found 627.1576. Os10. From L10 (78 mg) and [Os(p-Cym)Cl2]2 (79 mg). Yellow solid. Yield: 61% (0.122 mmol, 98 mg). 1H NMR (400 MHz, CD2Cl2) δ = 9.17 (d, J = 5.8, 1.0 Hz, 1H, Py−H), 8.11 (d, J = 8.8 Hz, 2H, Ar−H), 8.03 (dd, J = 7.9, 1.3 Hz, 1H, Py−H), 7.96 (d, J = 8.1 Hz, 1H, Py−H), 7.91 (d, J = 8.9 Hz, 2H, Ar−H), 7.38 (ddd, J = 7.3, 5.9, 1.4 Hz, 1H, Py−H), 5.43 (d, J = 5.9 Hz, 1H, Cym−CH), 5.39 (d, J = 5.8 Hz, 1H, Cym−CH), 5.32 (d, J = 5.9 Hz, 1H, Cym−CH), 4.89 (d, J = 5.6 Hz, 1H, Cym−CH), 4.55 (s, 3H, N−CH3), 2.27 (sept, J = 6.9 Hz, 1H, CH(CH3)2), 2.09 (s, 3H, Cym−CH3), 0.88 (d, J = 6.9 Hz, 3H, CH(CH3)2), 0.87 (d, J = 6.9 Hz, 3H, CH(CH3)2). 13C NMR (100 MHz, CD2Cl2) δ = 160.2 (Tz−C), 156.9, 147.7, 147.0, 140.0, 128.1, 126.5 (q, J = 4 Hz, Ar−C), 125.6, 125.4, 125.3, 120.6, (all Ar/ Py−C), 99.0, 97.5, 80.8, 77.1, 77.0, 72.0 (all Cym−C), 38.6 (N− CH3), 30.6 (CH(CH3)2), 21.9 (CH(CH3)2), 20.9 (CH(CH3)2), 17.8 (Cym−CH3). HRMS (ESI+) m/z calcd. for C25H25ClF3N4Os+ [M]+ = 665.1335, found 665.1340. Os11. From L11 (55 mg) and [Os(Cym)Cl2]2 (79 mg). Yellow solid. Yield: 69% (0.137 mmol, 95 mg). 1H NMR (400 MHz, CD2Cl2) δ = 9.23−9.17 (m, 1H, Py−H), 8.05−7.99 (m, 1H, Py−H), 7.94−7.88 (m, 1H, Py−H), 7.41−7.34 (m, 1H, Py−H), 5.95 (d, J = 5.6 Hz, 1H, Cym−CH), 5.85 (d, J = 6.1, 1H, Cym−CH), 5.78 (d, J = 5.8, 1H, Cym−CH), 5.49 (d, J = 5.8, 1H, Cym−CH), 4.78−4.57 (m, 2H, N−CH2CH3), 4.45 (s, 3H, N−CH3), 2.36 (sept, J = 6.9 Hz, 1H, CH(CH3)2), 2.21 (s, 3H, Cym−CH3), 1.66 (t, J = 7.4 Hz, 3H, N− CH2CH3), 0.98−0.90 (m, 6H, CH(CH3)2). 13C NMR (100 MHz, CD2Cl2) δ = 159.6 (Tz-5C), 157.7, 151.8, 148.7, 147.2, 139.6, 126.0, 120.8 (all Ar/Py−C), 97.7, 96.9, 81.1, 79.3, 77.1, 72.0 (all Cym−C), 49.4 (N−CH2CH3), 38.9 (N−CH3), 31.5 (CH(CH3)2), 22.3 (CH(CH3)2), 18.6 (CH(CH3)2), 15.3 (N−CH2CH3). HRMS (ESI +) m/z calcd. for C20H26ClN4Os+ [M]+ = 549.1455, found 549.1453. Iridium Complexes. Ir5. From L8 (65 mg) and [IrCp*Cl2]2 (80 mg). Bright-yellow solid. Yield: 63% (0.126 mmol, 93 mg). 1H NMR (400 MHz, CD2Cl2) δ = 8.76 (dt, J = 5.7, 1.1 Hz, 1H, Py−H), 8.13− 8.06 (m, 2H, Py−H), 8.00−7.96 (m, 2H, Ar−H), 7.67−7.61 (m, 3H, Ar−H), 7.50 (ddd, J = 6.8, 5.7, 2.2 Hz, 1H, Py−H), 4.61 (s, 3H, N− CH3), 1.42 (s, 15H, Cp*−CH3). 13C NMR (100 MHz, CD2Cl2) δ = 156.5 (Tz−C), 153.7, 148.7, 148.5, 140.2, 138.0, 131.1, 130.0, 126.3, 125.3, 121.3 (all Ar/Py−C), 91.2 (Cp*−C), 39.3 (N−CH3), 8.7 (Cp*−CH3). HRMS (ESI+) m/z calcd. for C24H27ClIrN4+ [M]+ = 599.1553, found 599.1508. Ir6. From L9 (71 mg) and [IrCp*Cl2]2 (80 mg). Bright-yellow solid. Yield: 73% (0.143 mmol, 112 mg). 1H NMR (400 MHz, CD2Cl2) δ = 8.76 (dd, J = 5.8, 0.5 Hz, 1H, Py−H), 8.08 (ddd, J = 7.9, 1.6, 0.3 Hz, 1H, Py−H), 8.01 (d, J = 8.1 Hz, 1H, Py−H), 7.88 (d, J = 8.9 Hz, 2H, Ar−H), 7.49 (ddd, J = 7.4, 5.9, 1.4 Hz, 1H, Py−H), 7.09 (d, J = 7.0 Hz, 2H, Ar−H), 4.57 (s, 3H, N−CH3), 3.88 (s, 3H, O− CH3), 1.44 (s, 15H, Cp*−CH3). 13C NMR (100 MHz, CD2Cl2) δ = 161.6 (Ar−C), 156.3 (Tz−C), 153.8, 148.8, 148.2, 140.1, 131.0, 126.7, 126.3, 122.6, 121.0, 114.9 (all Ar/Py−C), 91.2 (Cp*−CH3), 55.9 (O−CH3), 39.1 (N−CH3), 8.8 (Cp*−CH3). HRMS (ESI+) m/z calcd. for C25H29ClIrN4O+ [M]+ = 629.1659, found 629.1616. Ir7. From L10 (78 mg) and [IrCp*Cl2]2 (80 mg). Bright-yellow solid. Yield: 75% (0.15 mmol, 122 mg). 1H NMR (400 MHz, Figure 4. The accumulation of Ir5 and Ir9 in cells. HeLa cells were treated with diﬀerent concentrations of Ir5 or Ir9 for 3 h. The cells were collected, and the amount of iridium was measured by HR ICPMS. The data of three independent experiments are presented. Signiﬁcance was determined between control (nontreated) and Ir5, respectively, Ir9 treated cells (***P < 0.001). Coupling constants (J) are given in Hertz. The multiplicities are indicated as follows: s (singlet), d (doublet), t (triplet), q (quartet), sept (septet), and m (multiplet). An Agilent 6210 ESI-TOF spectrometer and Agilent 6224 time-of-ﬂight (TOF) mass spectrometer equipped with a double orthogonal electrospray source at atmospheric pressure ionization (ESI) coupled to an Agilent 1260 HLPC were used for recording HRMS spectra. X-ray data were collected with a Bruker Smart AXS. Data were collected at 140(2) K using graphite-monochromated Mo Kα radiation (Kα = 0.71073 Å). The strategy for the data collection was evaluated using the Smart software. The data were collected by the standard “omega scan techniques” and were scaled and reduced using Saint+ software. The structures were solved by direct methods using SHELXS-97 and reﬁned by full matrix least-squares with SHELXL-97, reﬁning on F2.26 General Procedure for the Preparation of Complexes Os8− Os11 and Ir5−Ir7, Ir9. The corresponding ligand precursor (2 equiv, 0.2 mmol) was mixed with basic silver(I) oxide (7 equiv, 0.7 mmol, 163 mg) and potassium chloride (20 equiv, 2 mmol, 155 mg) and was dissolved under nitrogen in dry acetonitrile (10 mL). The reaction mixture was stirred under the exclusion of light for 2 days. Afterward, the corresponding chloro-bridged metal dimer precursor (1 equiv, 0.1 mmol) was added and stirring was continued for additional 2−3 days. The remaining silver(I) oxide and silver(I) chloride that formed were ﬁltered oﬀ through a pad of Celite, and all volatiles were removed in vacuo. The crude product was then dissolved in methanol (5 mL). KPF6 (8 equiv, 0.8 mmol, 147 mg) was added, and the solution was stirred for 20 min. Slow addition of water (80 mL) resulted in precipitation of the desired complexes, which were collected by ﬁltration and air-dried. If the precipitation from water was not successful, the product was extracted with dichloromethane (3 × 20 mL). Combined organic layers were dried over NaSO4 and concentrated to ca. 5 mL volume. The product was precipitated by the addition of hexane (50 mL) and collected by ﬁltration. The complexes were obtained in moderate yields of 53% and higher. Osmium Complexes. Os8. From L8 (65 mg) and [Os(pCym)Cl2]2 (79 mg). Green solid. Yield: 65% (0.13 mmol, 96.4 mg). 1H NMR (400 MHz, CD2Cl2) δ = 9.18 (td, J = 5.7, 0.7 Hz, 1H, Py−H), 8.01 (td, J = 8.1, 0.7 Hz, 1H, Py−H), 7.96 (ddd, J = 8.1, 1.4, 0.6 Hz, 1H, Py−H), 7.86 (ddd, J = 5.5, 2.4, 1.1 Hz, 2H, Ar−H), 7.67−7.61 (m, 3H, Ar−H), 7.37 (ddd, 1H, Py−H), 5.40 (dd, J = 13.7, 5.8 Hz, 2H, Cym−CH), 5.23 (d, J = 5.6 Hz, 1H, Cym−CH), 4.84 (d, J = 5.6 Hz, 1H, Cym−CH), 4.53 (s, 3H, N−CH3), 2.24 (sept, J = 6.9 Hz, 1H, CH(CH3)2), 2.08 (s, 3H, Cym−CH3), 0.86 (d, J = 6.9 Hz, 3H, CH(CH3)2), 0.84 (d, J = 6.9 Hz, 3H, CH(CH3)2). 13C NMR (100 MHz, CD2Cl2) δ = 160.9 (Tz−C), 157.8; 148.6; 147.5, 139.7, 137.8, 131.4, 130.0, 126.3, 125.4, 121.2 (all Ar/Py−C), 99.1, 98.4, 81.9, 77.7, 77.6, 72.4 (all Cym−C), 39.2 (N−CH3), 31.4 (CH(CH3)2), 22.6 (CH(CH3)2), 21.7 (CH(CH3)2), 18.6 (Cym−CH3). HRMS (ESI+) m/z calcd. for C24H26ClN4Os+ [M]+ = 597.1461, found 597.1464. G DOI: 10.1021/acs.organomet.9b00327 Organometallics XXXX, XXX, XXX−XXX Article Organometallics Figure 5. The role of GSH level on cell survival upon treatment with Ir5 and Ir9. Eight hours after seeding cells were pretreated for 16 hours with 0.001 μg/mL BSO (A) or 24 h after the seeding with 5 mM of NAC for 2 h (B). Afterward diﬀerent concentrations of Ir5 or Ir9 were added. The cell survival was determined 72 h later by MTT assay. Each point represents the mean ± SD of at least three independent experiments. All data are expressed as the average percentage of survival values relative to an untreated control ± SD or samples treated with either BSO or NAC alone. The signiﬁcance in diﬀerences is indicated (*P < 0.05; **P < 0.01; ***P < 0.001). Twenty-four hours after seeding the cells were treated with diﬀerent concentrations of Ir5 or Ir9 during either 60 or 180 min. H2O2 treatment (0.01%, 30 min) was used as a positive control for formation of ROS (C). Each point represents the mean ± SD of two independent measurements performed in quadruplicates. Twenty-four hours after seeding cells were pretreated for 2 h with 20 μM tempol. Afterward diﬀerent concentrations of Ir5 or Ir9 were added. The cell survival was determined 72 h later by MTT assay. (D) Each point represents the mean ± SD of at least three independent experiments. All data are expressed as the average percentage of survival values relative to an untreated control ± SD or samples treated with tempol alone. CD2Cl2) δ = 8.77 (ddd, J = 5.7, 1.5, 0.7 Hz, 1H, Py−H), 8.21 (d, J = 8.3 Hz, 2H, Ar−H), 8.10 (ddd, J = 8.0, 1.4, 0.3 Hz, 1H, Py−H), 8.04 (td, J = 8.1, 1.4 Hz, 1H, Py−H), 7.91 (d, J = 8.8 Hz, 2H, Ar−H), 7.51 (ddd, J = 6.0, 1.4, 0.3 Hz, 1H, Py−H), 4.61 (s, 3H, N−CH3), 1.43 (s, 15H, Cp*−CH3). 13C NMR (100 MHz, CD2Cl2) δ = 157.2 (Tz−C), 153.8, 148.8, 148.6, 140.7, 140.2 (all Ar/Py−C), 132.9 (q, J = 33 Hz, H DOI: 10.1021/acs.organomet.9b00327 Organometallics XXXX, XXX, XXX−XXX Article Organometallics 30 μg of whole cell proteins were loaded on SDS polyacrylamide gel. Well accepted marker of apoptosis, cleaved PARP,14 was determined by using speciﬁc anti-PARP antibody (Cell Signaling Technology, USA). As internal loading controls ERK1/2 or ERK2 were used (Santa Cruz Technology, USA). In addition, the activities of caspases 3 and 7 were determined. Cells were seeded into tissue culture plates and after 1 day treated with diﬀerent concentrations of investigated compounds. Upon 72 h incubation Caspase Glo 3/7 Mix (Promega, USA) was added, and the producer’s protocol was followed. Luminometric signal was read on Fluoroskan Ascent FL (Thermo Fisher Scientiﬁc, USA) spectrometer. The possible role of autophagy and necroptosis as cell death models was checked as previously described.24 Accumulation of Iridium in the Cells. The total accumulation of iridium was measured as described previously for measurement of total cell platination21 with modiﬁcations. In short, upon treatment of cells with diﬀerent concentrations of Ir5 or Ir9 during 3 h, the cells were rinsed with ice-cold PBS and harvested into 10 mL of ice-cold PBS using a rubber policeman. After centrifugation, the cells were resuspended in PBS, an aliquot was used for determination of cell number. and the rest was digested in 70% nitric acid. Cell lysates were heated for 2 h at 75 °C, diluted in 5% nitric acid and assayed for iridium content. The amount of iridium was measured by a validated high-resolution inductively coupled plasma mass spectrometry (HR ICPMS) using the Element 2 (Thermo Finnigan, Germany). Calibration standards were prepared from single-element standard diluted in hydrochloric acid and water (1000 μg/mL; Agilent, USA). The Role of Glutathione in Cell Response to Ir5 and Ir9. The role of glutathione (GSH) in cell response to Ir5 and Ir9 was investigated by MTT assay by pretreatment of cells with speciﬁc inhibitor of GSH synthesis, buthionine sulfoximine (BSO) or precursor in GSH synthesis N-acetyl-cysteine (NAC). To examine the inﬂuence of GSH depletion on response of HeLa cells to selected compounds, 8 h after the seeding the cells were pretreated with 0.001 μg/mL of BSO. Sixteen hours later the cells were treated with diﬀerent concentrations of either Ir5 or Ir9, and the cytotoxicity eﬀect was determined 72 h later. To determine the inﬂuence of increase of GSH synthesis on cells response to selected compounds, 5 mM NAC was added 2 h before treatment of cells with diﬀerent concentrations of either Ir5 or Ir9. Generation of ROS was determined using the ﬂuorescent dye 5-(and6)-chloromethyl-29,79-dichlorodihydroﬂuorescein diacetate, acetyl ester (CM-H2DCFDA) (Life Technologies, USA) according to the previously described protocol.19 Possible role of ROS in induction of cells death was additionally checked by 2 h pretreatment with 20 μM tempol, well accepted antioxidant30 and then treated with diﬀerent concentrations of either Ir5 or Ir9. The cytotoxicity eﬀect was determined 72 h later by MTT assay. Statistical Analysis. Data were analyzed by unpaired Student’s t test and expressed as a mean ± standard error of the mean. Data were considered signiﬁcant when P values were lower than 0.05, and in the ﬁgures, these are designated as *P < 0.05, **P < 0.01, or ***P < 0.001. Experiments were performed in triplicate and repeated at least twice. Ar−C), 127.2 (q, J = 4 Hz, Ar−C), 126.4, 126.1 (all Ar/Py−C), 123.5 (q, J = 273 Hz, CF3), 121.2 (Ar/Py−C), 91.4 (Cp*−C), 39.4 (N− CH 3 ), 8.7 (Cp*−CH 3 ). HRMS (ESI+) m/z calcd. for C25H26ClF3IrN4+ [M]+ = 667.1427, found 667.1391. Ir9. From L12 (82 mg) and [Ir(Cp*)Cl2]2 (80 mg). Yellow solid. Yield: 75% (0.15 mmol, 124 mg). 1H NMR (400 MHz, CD2Cl2) δ = 8.79−8.71 (m, 1H, Py−H), 8.14−8.08 (m, 1H, Py−H), 8.08−8.04 (m, 1H, Py−H), 7.60 (t, J = 8.0 Hz, 1H, Ar−H), 7.55−7.52 (m, 1H, Py−H), 7.44−7.39 (m, 2H, Ar−H), 4.61 (s, 3H, N−CH3), 3.17 (sept, J = 6.8 Hz, 1H, CH(CH3)2), 2.35 (sept, J = 6.8 Hz, 1H, CH(CH3)2), 1.41 (s, 15H, Cp*−CH3), 1.34 (d, J = 6.8 Hz, 3H, CH(CH3)2), 1.27 (d, J = 6.8 Hz, 3H, CH(CH3)2), 1.19 (d, J = 6.8 Hz, 3H, CH(CH3)2), 0.78 (d, J = 6.8 Hz, 3H, CH(CH3)2). 13C NMR (100 MHz, CD2Cl2) δ = 158.4 (Tz−C), 153.3, 149.6, 148.7, 146.8, 146.4, 139.9, 134.4, 132.1, 126.5, 124.6, 124.4, 121.2 (all Ar−C), 91.1 (Cp*−C), 39.4 (N−CH3), 29.0 (CH(CH3)2), 27.9 (CH(CH3)2), 27.0 (CH(CH3)2), 25.3 (CH(CH3)2), 21.7 (CH(CH3)2), 21.6 (CH(CH3)2), 8.8 (Cp*− CH3). HRMS (ESI+) m/z calcd. for C30H39ClIrN4+ [M]+ = 683.2492, found 683.2464. Biology. Cell Culture. Human cervical carcinoma HeLa and laryngeal carcinoma HEp2 cells were obtained from cell culture bank (GIBCO BRL-Invitrogen, USA). Large cell lung carcinoma H460 and colorectal carcinoma HCT-116 cell lines were obtained from American Type Culture Collection (ATCC, USA). Human ovarian cancer cell line MES-OV was obtained from Prof. Sikic group (Stanford University, USA). Normal human skin keratinocyte line was obtained from the foreskin of healthy boys, aged 3−8 years. Foreskin samples were noninﬂamed and the children were free of any therapy at least 1 month before the surgery.27 The cells were obtained at the Neurochemical Laboratory, Department of Chemistry and Biochemistry, School of Medicine, University of Zagreb. All cell lines were grown as a monolayer culture in Dulbecco’s modiﬁed Eagle’s medium (DMEM; Sigma-Aldrich, USA), supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich) in a humidiﬁed atmosphere of 5% CO2 at 37 °C and were subcultured every 3−4 days. Cytotoxicity Assay. Cytotoxic activity of organometallic complexes was determined by MTT assay28 adjusted accordingly. In short, the cells were seeded into 96-well tissue culture plates. The next day diﬀerent concentrations of compounds were added to each well in quadruplicate. Upon 72 h incubation at 37 °C, the medium was aspirated and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye (Sigma-Aldrich) was added. Three hours later, formazan crystals were dissolved in DMSO, the plates were mechanically agitated for 5 min, and the optical density at 545 nm was determined on a microtiter plate reader (Awareness Technology Inc., USA). The cytotoxicity was expressed either as IC20, IC50, or IC80 (survival rate 80, 50, and 20% compared to nontreated, control cells, which survival was set at 100%). Cell Cycle and Cell Death Analysis. HeLa cells were seeded into tissue culture plates and treated with diﬀerent concentrations of compounds for the indicated time. Thereafter, both adherent and ﬂoating cells were collected, washed with PBS and ﬁxed overnight in 96% ethanol at −20 °C. Fixed cells were treated with RNase A (0.1 mg/mL, Sigma-Aldrich) for 30 min in water bath (37 °C) and afterward stained with propidium iodide (50 μg/mL, Sigma-Aldrich) for 30 min in the dark. The DNA content was analyzed on ﬂow cytometer (FACS Calibur, Becton Dickinson, USA). Data were analyzed for cell cycle distribution in FlowLogic software (Inivai Technologies, Australia) using cell cycle speciﬁc gating protocol and Watson pragmatic convolution. In order to determine the type of cell death, HeLa cells were treated with diﬀerent concentrations of investigated compounds. After speciﬁc time, both adherent and ﬂoating cells were collected, washed two times with PBS and stained with Annexin V and/or PI (propidium iodide) according to the producer’s protocol (ThermoFisher Scientiﬁc, USA). The DNA content and Annexin V ﬂuorescence were analyzed by ﬂow cytometry (FACS Calibur, Becton Dickinson). Data were analyzed with FlowLogic software (Inivai Technologies, Australia) using speciﬁc gating protocol. After determination of protein concentration by Bradford analysis,29 the ■ ASSOCIATED CONTENT S Supporting Information * The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.organomet.9b00327. Crystallographic details, 1H and 13C NMR spectra for all new compounds (PDF) Accession Codes CCDC 1044233−1044236 and 973835 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/ cif, or by emailing data_request@ccdc.cam.ac.uk, or by I DOI: 10.1021/acs.organomet.9b00327 Organometallics XXXX, XXX, XXX−XXX Article Organometallics lics: A Preliminary Investigation into the Biological Activity of “Click” Carbene Complexes. 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ORCID Stephan Hohloch: 0000-0002-5353-0801 Biprajit Sarkar: 0000-0003-4887-7277 Janez Košmrlj: 0000-0002-3533-0419 Author Contributions # JK and AB contributed equally. Notes The authors declare no competing ﬁnancial interest. ■ ACKNOWLEDGMENTS The authors acknowledge the ﬁnancial support from the Slovenian Research Agency (Research Core Funding Grant P1-0230 and Projects J1-8147 and J1-9166), the Croatian Science Foundation (HrZZ, project number IP-2016-061036), Croatian-Slovenian bilateral project BI-HR/18-19-028, and the Freie Universität Berlin. Financial support from Joint PPP-Project DAAD-ARRS BI-DE/17-19-9 funded by the DAAD through funds from the Bundesministerium fer Bildung und Forschung (BMBF) is also acknowledged. ■ REFERENCES (1) Peters, G. J.; Raymond, E. Contemporary reviews on cancer treatment. Cancer Chemother. Pharmacol. 2016, 77, 3−4. (2) Dasari, S.; Tchounwou, P. B. Cisplatin in cancer therapy: Molecular mechanisms of action. Eur. J. 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