Highly effective Ru(II) and Os(II) half-sandwich complexes induce cytotoxicity in cancer cells through combined mitochondrial and endoplasmic reticulum stress.

{"full_text": " European Journal of Medicinal Chemistry 297 (2025) 117970\n\n\n Contents lists available at ScienceDirect\n\n\n European Journal of Medicinal Chemistry\n journal homepage: www.elsevier.com/locate/ejmech\n\n\nResearch paper\n\nHighly effective Ru(II) and Os(II) half-sandwich complexes induce\ncytotoxicity in cancer cells through combined mitochondrial and\nendoplasmic reticulum stress\nJan Hos\u030cek a,* , Kamila Petrz\u030celova\u0301 b, Renata He\u0301z\u030cova\u0301 a , Nicol Strakova\u0301 a , Simona Kajabova\u0301 a ,\nIvan Nemec b, Pavl\u00edna S\u030cimec\u030ckova\u0301 a , Kater\u030cina Pe\u030cnc\u030c\u00edkova\u0301 a , Josef Mas\u030cek a , Ja\u0301n Monco\u013e c ,\nPavel S\u030ctarha b,**\na\n Department of Pharmacology and Toxicology, Veterinary Research Institute, Hudcova 296/70, Brno, 62100, Czech Republic\nb\n Department of Inorganic Chemistry, Faculty of Science, Palacky University Olomouc, 17. listopadu 12, 77146, Olomouc, Czech Republic\nc\n Department of Inorganic Chemistry, Faculty of Chemical and Food Technology, Slovak University of Technology in Bratislava, Bratislava, SK-81237, Slovakia\n\n\n\n\nA R T I C L E I N F O A B S T R A C T\n\nKeywords: A series of ruthenium(II) and osmium(II) half-sandwich complexes was synthesized and characterized for its\nRuthenium potential as a new class of anticancer agents. The complexes feature polycyclic aromatic hydrocarbon (PAH)-\nOsmium substituted Schiff bases and were rationally designed to combine the redox-modulating MoA of half-sandwich\nAntiproliferative activity\n Ru, Rh, Os and Ir complexes, connected with their ability to induce the formation of various reactive oxygen\nStress gene expression\n species (ROS), with the ability of PAH-substituents to target and disrupt DNA. The complexes [Ru(\u03b76-pcym)Cl(L)]\nEndoplasmic reticulum\nMitochondria PF6 (1\u20134) and [Os(\u03b76-pcym)Cl(L)]PF6 (5\u20138) were stable in aqueous environments, in contrast to the rapid\n degradation observed for the co-studied rhodium(III) (9\u201312) and iridium(III) (13\u201316) [M(\u03b75-Cp*)Cl(L)]PF6\n complexes; L = ethane-1,2-diamine-based Schiff bases (L1\u2013L4) bearing two terminal PAH substituents 2-naphtyl\n (for L1), 9-anthracenyl (for L2), 9-phenanthrenyl (L3) or 1-pyrenyl (L4); pcym = 1-methyl-4-(propan-2-yl)ben\u00ad\n zene (p-cymene), Cp* = pentamethylcyclopentadienyl. Biological testing demonstrated that 1\u20138 possess signif\u00ad\n icant antiproliferative activity against various lung cancer cell lines, including those resistant to cisplatin, with\n Os(II) complex 5 showing the highest cytotoxicity. Treatment with these complexes led to the activation of stress-\n related gene pathways, including unconventional endoplasmic reticulum stress, apoptotic signalling, and mito\u00ad\n chondrial membrane depolarization. Activation of p21/GADD45A pathway indicates DNA-damage response, as\n well. Notably, these complexes did not induce significant inflammatory responses, a notable advantage over\n cisplatin. The results highlight the potential of Ru and Os half-sandwich complexes as alternative metallodrugs,\n capable of overcoming platinum resistance and minimizing inflammatory side effects. This study suggests that\n these compounds could serve as a promising class of anticancer agents for future clinical development.\n\n\n\n\n1. Introduction conventional anti-cancer therapies are not completely effective against\n lung cancer and new types of drugs need to be developed.\n Lung cancer represents one of the most dangerous cancer types. Two Chemotherapy remains an integral part of lung cancer treatment,\nmain types of lung cancer can be distinguished - small cell lung cancer usually in combination with surgery and radiotherapy [6]. Cisplatin, a\n(SCLC) and non-small cell lung cancer (NSCLC), with SCLC being more platinum-based metallodrug, is commonly used in clinical practice\naggressive and typically diagnosed at a later stage. [1,2]. In terms of against a wide range of solid tumours, including lung cancer, especially\nincidence, lung cancer is one of the most frequently newly diagnosed as part of combination therapy [7,8]. However, the development of\ncancers (3rd in incidence) and the most common cause of cancer deaths resistance to such cisplatin-based chemotherapy is the problem for\n(over 20 % of all cancer-related deaths) [3\u20135]. It is therefore evident that oncological patients, in addition to negative side effects (e.g.,\n\n\n\n * Corresponding author. Department of Pharmacology and Toxicology, Veterinary Research Institute, Hudcova 296/70, Brno, 621 00, Czech Republic.\n ** Corresponding author. Department of Inorganic Chemistry, Faculty of Science, Palack\u00fd University Olomouc, 17. listopadu 12, Olomouc, 77146, Czech Republic.\n E-mail addresses: jan.hosek@vri.cz (J. Hos\u030cek), pavel.starha@upol.cz (P. S\u030ctarha).\n\nhttps://doi.org/10.1016/j.ejmech.2025.117970\nReceived 23 May 2025; Received in revised form 10 July 2025; Accepted 11 July 2025\nAvailable online 11 July 2025\n0223-5234/\u00a9 2025 Elsevier Masson SAS. All rights are reserved, including those for text and data mining, AI training, and similar technologies.\n\fJ. Hos\u030cek et al. European Journal of Medicinal Chemistry 297 (2025) 117970\n\n\nnephrotoxicity or myelosuppression). were checked by elemental analysis and 1H NMR. Compound L3 is new\n With the discovery of the antineoplastic effects of cisplatin in 1965, and is reported here for the first time.\ncompounds of other d-block metals (including cis-[Ru(NH3)4(OH)Cl]Cl; Complexes [Ru(\u03b76-pcym)Cl(L)]PF6 (1\u20134), [Os(\u03b76-pcym)Cl(L)]PF6\nFig. 1) were also studied for their biological activity [9]. The biological (5\u20138), [Rh(\u03b75-Cp*)Cl(L)]PF6 (9\u201312) and [Ir(\u03b75-Cp*)Cl(L)]PF6 (13\u201316)\npotential of non-platinum complexes, including those of ruthenium (Fig. 2 and Supporting Information, Fig. S2) were synthesized using\n(Ru), was further explored [10], and over the years, several Ru com\u00ad microwave-assisted methods, following typical protocols for this type of\nplexes have entered clinical trials for the treatment of various types of Ru(II)/Os(II) arene and Rh(III)/Ir(III) arenyl complexes. The starting\ncancer, including lung cancer, in human cancer patients [11\u201314]. These compounds were the corresponding dimers [M\u2032(\u03bc-Cl)(\u03b76-pcym)Cl]2 (M\u2019\nachievements make Ru one of the most promising d-block metals in the = Ru or Os) and [M\u2019\u2019(\u03bc-Cl)(\u03b75-Cp*)Cl]2 (M\u2019 = Rh or Ir). Complex 4 has\nfield of anticancer drug development. Importantly, its success was fol\u00ad been previously reported in the literature [28], while other complexes\nlowed by other group 8 (Os; see Fig. 1 for [Os(\u03b76-pcym)Cl2(pta)]) [15] (1\u20133, 5\u201316) are reported here for the first time. Formerly reported\nand group 9 (Rh, Ir) [15,16] platinum metals offering similar chemistry complex [Ru(\u03b76-pcym)Cl(en)]PF6 (0) was involved in this study for\nand structural types; pcym = 1-methyl-4-(propan-2-yl)benzene comparative purposes [21].\n(p-cymene), pta = 1,3,5-triaza-7-phosphaadamantane [14,17]. The complexes were characterized by CHN elemental analysis, mass\n These metals generally offer various biologically promising struc\u00ad spectrometry (MS), nuclear magnetic resonance (NMR), and Fourier\ntural types, including half-sandwich one [14,17\u201320]. Half-sandwich Transform Infrared Spectroscopy (FT-IR) (see Experimental section).\ncomplexes consist of a \u03b7-coordinated arene (for Ru and Os) or arenyl The formation of the [M\u2032(\u03b76-pcym)Cl(L)]+ (M\u2019 = Ru for 1\u20134 and Os for\n(for Rh and Ir) ligand, along with three others coordination sites, usually 5\u20138) and [M\u2019\u2019(\u03b75-Cp*)Cl(L)]+ (M\u2019\u2019 = Rh for 9\u201312 and Ir for 13\u201316)\noccupied by one bidentate and one monodentate ligand. While the complex cations was clearly proved by ESI+ MS through the detection of\nmonodentate ligand (e.g., halogenido or pta) can ensure the hydrolytic dominant peaks corresponding to the [M\u2019Cl(L)(pcym)]+ and [M\u2019\u2019Cl\nactivation of such complexes, the bidentate ligand is primarily respon\u00ad (Cp*)(L)]+ species, assigned based on their m/z values and characteristic\nsible for modulating the biological activity. Furthermore, although not isotopic patterns (Supporting Information, Fig. S3\u2013S18); ESI+ refers to\nyet fully elucidated, the mechanism of action (MoA) of anticancer electrospray ionization in positive ionization mode. In addition to these\nhalf-sandwich Ru, Os, Rh and Ir complexes appears to differ from the pseudomolecular peaks of complex cations of the prepared coordination\nconventional anticancer metallodrug cisplatin and its Pt-based ana\u00ad compounds 1\u201316, fragment of general compositions {[M\u2032(L)(pcym)]\u2013\nlogues [14,18,20]. Their MoA is thought to be multifaceted, involving H}+ and {[M\u2019\u2019(Cp*)(L)]\u2013H}+ were detected in the mass spectra of the\nenhanced production of reactive oxygen species (ROS), interactions with studied compounds.\nproteins, and disruption of mitochondrial function. Unlike cisplatin, The purity of complexes 1\u20138 was checked by HPLC and the results\nDNA does not appear to be the main target of Ru, Os, Rh, and Ir indicate acceptable purity (96.2\u201398.4 %) for the following chemical and\nhalf-sandwich complexes. biological studies (Supporting Information, Fig. S19).\n Based on these assumptions, we chose in this work one of the most\nbasic bidentate ligands - ethylene-1,2-diamine (en) - which was used in 2.2. NMR characterization\npioneer papers reporting among the first on anticancer activity of Ru and\nOs half-sandwich complexes, such as [Ru(\u03b76-bip)Cl(en)]PF6 (RM175; The 1H NMR spectra of the free ligands L1\u2013L4 (measured in CDCl3)\nFig. 1) and its Os analogue [Os(\u03b76-bip)Cl(en)]PF6 (AFAP51) [21,22]. We showed the characteristic \u2013CH\u2013 Schiff base group resonance as a singlet\nthen modified en through a Schiff condensation with DNA-targeting at 8.46\u20139.50 ppm (Supporting Information, Fig. S20\u2013S23). This reso\u00ad\nsubstituents from a family of polycyclic aromatic hydrocarbons nance was accompanied by a complex aromatic region with numerous\n(PAHs), namely 2-naphtyl (for L1), 9-anthracenyl (for L2), 9-phenan\u00ad multiplets in the 7.12\u20138.92 ppm range assignable to the PAH sub\u00ad\nthrenyl (for L3) or 1-pyrenyl (for L4). We hypothesized that this chem\u00ad stituents, and by a singlet belonging to aliphatic ethylene protons\nical modification would result in strong intercalative interaction with (4.08\u20134.52 ppm). In the case of L2, residual signals of free anthracene-9-\nDNA [23], while the ability of complexes bearing such ligand to carbaldehyde (e.g., 11.53 ppm for \u2013CHO) were detected in CDCl3, even\ngenerate high ROS populations will be not reduced. Overall, this com\u00ad after purification by several recrystallizations. The same behaviour was\nbined biological effect could lead to high antiproliferative activity and observed in DMSO\u2011d6, whereas in deuterated benzene (C6D6), only the\nthe ability of such rationally designed coordination compounds to signals of L2 were detected. This implied that L2 is partially unstable in\novercome acquired resistance of tumour cells. CDCl3 and DMSO\u2011d6, but it can be used for the preparation of complexes,\n since it was prepared in adequate purity, as proved by 1H NMR in C6D6\n2. Results and discussion (Supporting Information, Fig. S21).\n The discussion of the NMR results is divided into two parts. First, Ru\n2.1. Synthesis and general properties and Os complexes 1\u20138 will be discussed. Their 1H NMR spectra were\n measured in DMSO\u2011d6 and contained all the expected resonances with\n Compounds L1\u2013L4 (Fig. 2 and Supporting Information, Fig. S1) were appropriate integral intensities (Supporting Information, Fig. S24\u2013S27).\nprepared by a standard Schiff base condensation of en and the appro\u00ad The singlet corresponding to the \u2013CH\u2013 Schiff base group was detected at\npriate aldehydes of PAHs. Compounds L1, L2, and L4 were formerly lower fields (9.37\u201310.24 ppm) as compared with free ligands. Impor\u00ad\nreported in the literature [24\u201327], and their composition and purity tantly, no changes were observed for this characteristic resonance (0\u201324\n h), demonstrating that no cleavage of the used en-based ligands L1\u2013L4\n occurred and that the Ru and Os complexes 1\u20138 are stable in the used\n solvent (DMSO\u2011d6). As a consequence of the coordination of L1\u2013L4 to\n the metal centres, the ethylene hydrogens showed as two broad signals\n at 3.63\u20134.45 ppm in the spectra of 1\u20138. The presence of pcym was\n proved by its characteristic set of resonances, for example by two dou\u00ad\n blets (or broad signals) at 4.05\u20135.79 ppm (aromatic hydrogens of pcym),\n or by a singlet at 1.60\u20132.39 ppm (methyl of pcym).\n In contrast to Ru and Os complexes 1\u20138, their Rh and Ir analogues\n 9\u201316 were unstable in the used solvents (CDCl3, DMSO\u2011d6). Specifically,\n gradual cleavage of the Schiff base bond was observed, resulting in the\n Fig. 1. Pioneer anticancer Ru and Os complexes. release of the PAH substituents, as proved by detection of the signals of\n\n 2\n\fJ. Hos\u030cek et al. European Journal of Medicinal Chemistry 297 (2025) 117970\n\n\n\n\n Fig. 2. General structural formulas of the studied complexes 1\u201316, given together with their polycyclic aromatic substituents.\n\n\nfree PAH carbaldehydes, while the characteristic singlet of the \u2013CH\u2013 Information, Fig. S28), but their hydrolytic stability was poor (see\nSchiff base group gradually disappeared in 1H NMR spectra of Rh and Ir below). The spectra of other Rh and Ir complexes (10\u201312 and 14\u201316)\ncomplexes 9\u201316 (Supporting Information, Fig. S28\u2013S31). Only the L1- contained the resonances of the degradation products (e.g. singlets of\ncontaining complexes 9 and 13 were stable enough to obtain their 1H carbaldehydes or more Cp* resonances) even in the very fresh solutions\nNMR spectrum before the beginning of degradation (Supporting (t > 5 min; Supporting Information, Fig. S29\u2013S31).\n\n\n\n\nFig. 3. The molecular structures of the complex cations in compounds 1 (A), 3 (B) and 4 (C), and in fragments 9\u2019 (D) and 13\u2019 (E). The PF\u22126 anions and hydrogen\natoms were omitted for clarity. The colour code used is as follows: light yellow (carbon), green (chlorine), dark teal (metal atom) and light blue (nitrogen). (For\ninterpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)\n\n 3\n\fJ. Hos\u030cek et al. European Journal of Medicinal Chemistry 297 (2025) 117970\n\n\n Given the pronounced instability of Rh and Ir complexes 9\u201316, they initial complexes were transformed in 5 h (for 9) and in 20 h (for 13).\nwere excluded from the following chemical analysis (CHN analysis, FTIR After these times, two sets of resonances were detected in the spectra\nspectroscopy), as well as from the stability studies in the presence of instead of the initial Schiff bond resinance. One was clearly assignable to\nwater and biological assays. the released 2-naphthaldehyde (\u03b4 = 9.99 ppm for both 9 and 13), while\n the second one (\u03b4 = 8.98 ppm for 9 and 9.10 ppm for 13) most likely\n2.3. Crystal structures corresponded to fragments [M(\u03b75-Cp*)Cl(L1\u2032)]+ (9\u2032, 13\u2032) with only one\n 2-naphthaldehyde substituent (L1\u2032). Similar fragmentation was detected\n We successfully prepared single crystals of Ru compounds 1, 3 and 4, by a single-crystal X-ray analysis of 13\u2019 (see above; Fig. 3).\nwhich were suitable for single-crystal X-ray diffraction (Supporting In\u00ad Analogical 2-naphthyl-bearing Ru (1) and Os (5) complexes were\nformation, Tables S1 and S2). The experiments confirmed the antici\u00ad markedly more stable than their Rh (9) and Ir (13) congeners. Complex\npated composition of these compounds, revealing complex cations 1 released less than 1 % of its PAH substituent in 50 % DMSO\u2011d6/50 %\npaired with PF\u22126 anions. The complex cations consist of arene ligand, one D2O with PBS after 1 h of standing at r.t. (Fig. 4 and Supporting Infor\u00ad\nbidentate Schiff base, and one chlorido ligand within the inner coordi\u00ad mation, Fig. S34). A similar extent of PAH release was observed by 1H\nnation sphere, forming a pseudo-octahedral piano-stool arrangement NMR in the presence of excess of NADH coenzyme (5 molar equiv.),\n(Fig. 3A\u2013C). The longest metal-ligand bond lengths observed are the which was used as a model highly-concentrated intracellular biomole\u00ad\nRu\u2212 Cl bonds, which measure approximately 2.40 \u00c5 in all three com\u00ad cule (Supporting Information, Fig. S34). For this mixture of 1 with\nplexes. In contrast, the Ru\u2212 N bonds are shorter and more variable, NADH, only negligible NADH oxidation (ca. 4 % of NADH) was detected\nranging between 2.09 and 2.13 \u00c5. All metal\u2013ligand bond lengths are by 1H NMR. In contrast with 9 and 13, no new set of signals assignable to\nsummarized in Supporting Information, Table S3. the fragments with only one 2-naphthaldehyde substituent (L1\u2032) was\n During the crystallization of compounds 9 and 13, we isolated found in the 1H NMR spectra of 1 or its mixture with excess NADH. Os\ncrystals that contained partially (for 9) or fully (for 13) cleaved Schiff complex 5 was the most stable of the L1-bearing complexes 1, 5, 9 and\nbase ligands. In the Rh complex 9, the Schiff base ligand in the resulting 13, because 5 did not undergo any changes when dissolved alone or in a\nstructure of [Rh(\u03b75-Cp*)Cl(en)]PF6 (9\u2032) was fully cleaved with both the mixture with excess NADH (5 molar equiv.) for 24 h (50 % DMSO\u2011d6/50\nnaphthyl moieties released and leaving ethylene-1,2-diamine (en) to act % D2O with PBS; Fig. 4 and Supporting Information, Fig. S35). Only\nas the N,N\u2032-chelating ligand (Fig. 3D). Regarding the Ir complex 13, the negligible NADH oxidation (ca. 2 % of NADH) was observed for the\nresulting structure [Ir(\u03b75-Cp*)Cl(L1\u2032)]PF6 (13\u2032) contains a partially mixture of 5 with an excess of NADH, which was not accompanied by the\ncleaved Schiff base ligand N-(naphthalen-2-ylmethylidene)ethane-1,2- release of the 2-naphthaldehyde substituent of 5.\ndiamine with only one 2-naphtyl substituent (L1\u2032; Fig. 3E). Both com\u00ad Similarly to the L1-containing complexes 1 and 5, their analogues\nplexes 9\u2032 and 13\u2032 adopt a pseudo-octahedral piano-stool arrangement, 2\u20134 and 6\u20138 with more extended PAH substituent (in L2\u2013L4) were found\nsimilar to those observed in compounds 1, 3 and 4. The [Rh(\u03b75-Cp*)Cl to be adequately stable in the used DMSO/D2O mixture of solvents after\n(en)]+ cation of 9\u2032 was formerly reported in the literature [29]. 1 h of standing at r.t. (Fig. 4). This finding of sufficient stability allowed\n subsequent biological experiments to be performed on compounds 1\u20138.\n2.4. Aqueous chemistry In contrast, complexes 9\u201316 were not suitable for the planned biological\n experiments due to low stability in DMSO (section 2.2) or its mixture\n Ru and Os complexes 1\u20138 and 2-naphthyl-bearing compounds 9 (Rh) with water (this section).\nand 13 (Ir), which showed acceptable stability in CDCl3 and DMSO\u2011d6\n(see above), were investigated by 1H NMR for their stability in a water- 2.5. In vitro cytotoxicity on 2D and 3D cell models\ncontaining mixture of solvents (50 % DMSO\u2011d6/50 % D2O with PBS);\nPBS = phosphate-buffered saline (pH 7.4), added to mimic physiological To evaluate the therapeutic potential against lung cancer, a\nconditions (pH, chloride ions). screening cytotoxicity assay was conducted on several cell lines. In\n Regarding Rh and Ir complexes 9 and 13, the results revealed that addition to lung cancer-derived cell lines (the epithelial cell line A549,\nboth the complexes underwent rapid degradation associated with a the adenocarcinoma cell line MOR, and its cisplatin-resistant variant\nrelease of the PAH substituent (Fig. 4 and Supporting Information, MOR/CPR), the assay also included the foetal lung tissue cell line MRC-5\nFig. S32 and S33). The characteristic resonances of free 2-naphthalde\u00ad and human peripheral blood mononuclear cells (PBMCs) (Table 1).\nhyde were detected (e.g., the \u2013CHO resonance at 9.99 ppm). The Generally, Os(II) complexes 5\u20138 showed higher cytotoxic effect than\n their Ru(II) analogues 1\u20134, as it was previously demonstrated on other\n complexes and cell lines (e.g., Refs. [30\u201332]). The most cytotoxic was Os\n (II) complex 5, which showed the highest potential across all cell lines.\n This compound together with its Ru(II) analogue 1 were selected for\n further analysis to reveal mechanisms of action.\n It is worth noting that all tested complexes were more active against\n cisplatin-resistance MOR/CPR cell line than against cisplatin-sensitive\n MOR. This indicates the ability of these molecules to bypass several\n drug resistances. All complexes were also more cytotoxic than cisplatin\n against all used cell lines. On the other hand, no selectivity towards\n cancerous cell lines was observed.\n A previous investigation reported on biological and catalytic activity\n of similar Ru compounds derived from two different en-based Schiff\n bases, including complex 4 with L4 (the second one is N,N\u2032-bis{(E)-[4-(L-\n methylethyl)phenyl]methylene}ethane-1,2-diamine (L5)) [28]. Both\n compounds were tested for in vitro antitumor activity against human\nFig. 4. Extent of the release of polycyclic aromatic substituents (PAHs) from\nthe studied complexes, as determined by 1H NMR in 50 % DMSO\u2011d6/50 % D2O breast adenocarcinoma (MCF7) and human hepatocellular carcinoma\nwith phosphate-buffered saline (PBS, pH 7.4). An extent of the PAH release was (HepG2) cell lines. The compound [Ru(\u03b76-pcym)Cl(L5)]PF6 showed only\ncalculated from an integral intensity of the \u2013CH\u2013 Schiff bond resonance (i.e. low in vitro cytotoxicity on these human cell lines (IC50 = 74.9 and\nbound PAH substituent in complexes) and the \u2013CHO resonance of a free car\u00ad 115.5 \u03bcM, respectively), whereas 4 was inactive on the human cell lines\nbaldehyde (i.e. released PAH substituent). used in the concentration range tested (IC50 > 200 \u03bcM). However, it is\n\n 4\n\fJ. Hos\u030cek et al. European Journal of Medicinal Chemistry 297 (2025) 117970\n\n\nTable 1 incubation. These results indicate that tested complexes 1 and 5 trigger\nThe results (IC50; given as mean \u00b1 SE and 95 % confidence interval [\u03bcM]) of in different type of cell death than apoptosis or necrosis.\nvitro testing of antiproliferative activity of compounds 1\u20138 against human lung\ncarcinomas (A549, MOR), human cisplatin-resistant lung carcinoma (MOR/ 2.7. Mitochondrial membrane potential and mitochondria visualisation\nCPR), human foetal lung fibroblasts (MRC-5), and human peripheral blood\nmononuclear cells (PBMCs); CCK-8 assay, 72 h exposure time. Cisplatin (CDDP)\n Although only low induction of apoptosis was observed in cells after\nand complex [Ru(\u03b76-pcym)Cl(en)]PF6 (0) were used as the reference drugs.\n incubation with complexes 1 and 5, the change of mitochondrial\n Compd. Cell line membrane potential (\u0394\u03c8m) was evaluated as it could be connected with\n A549 MOR MOR/CPR PBMCs MRC-5 early stages of apoptosis [36].\n 1 6.0 \u00b1 1.1 3.1 \u00b1 1.2 2.1 \u00b1 1.1 1.7 \u00b1 1.1 6.2 \u00b1 1.1 Complex 5, as well as cisplatin, was able to significantly disrupt the\n (4.7\u20137.8) (2.2\u20134.6) (1.6\u20132.7) (1.5\u20132.0) (5.5\u20137.2) \u0394\u03c8m after 24 h incubation with 7.1 % and 7.4 % of cells with depolarised\n 2 3.3 \u00b1 1.2 4.2 \u00b1 1.0 3.0 \u00b1 1.1 1.1 \u00b1 1.1 1.4 \u00b1 1.2 mitochondrial membrane, respectively, whereas complex 1 had only\n (2.3\u20134.8) (3.9\u20134.5) (2.5\u20133.6) (1.0\u20131.3) (0.9\u20132.0) minor effect (2.3 % of cells with depolarised mitochondrial membrane)\n 3 3.9 \u00b1 1.1 5.1 \u00b1 1.1 2.0 \u00b1 1.0 1.1 \u00b1 1.2 1.9 \u00b1 1.1\n (3.1\u20134.9) (4.2\u20136.3) (1.9\u20132.2) (0.8\u20131.6) (1.7\u20132.2)\n (Fig. 7A). This effect was eliminated after prolonged (72 h) incubation\n 4 3.0 \u00b1 1.2 3.6 \u00b1 1.1 2.6 \u00b1 1.1 0.5 \u00b1 1.1 0.7 \u00b1 1.2 with tested compounds (Supporting Information, Fig. S37). Our results\n (2.1\u20134.1) (2.9\u20134.8) (2.2\u20133.0) (0.4\u20130.6) (0.5\u20131.3) indicate a greater effect of Os(II) complex 5 on mitochondrial function\n 5 1.2 \u00b1 1.2 1.7 \u00b1 1.1 0.5 \u00b1 1.1 0.6 \u00b1 1.0 1.6 \u00b1 1.1 than Ru(II) compound 1, as earlier reported for different pair of Ru and\n (0.9\u20131.6) (1.3\u20132.1) (0.4\u20130.6) (0.5\u20130.7) (1.4\u20131.8)\n Os complexes [32].\n 6 3.4 \u00b1 1.1 5.2 \u00b1 1.0 2.9 \u00b1 1.0 0.2 \u00b1 1.2 2.8 \u00b1 1.1\n (2.5\u20134.4) (4.7\u20135.7) (2.7\u20133.1) (0.1\u20130.3) (2.4\u20133.9) It is in the accordance with previous studies, where similar half-\n 7 1.5 \u00b1 1.2 2.8 \u00b1 1.1 2.0 \u00b1 1.1 1.3 \u00b1 1.2 0.8 \u00b1 1.0 sandwich Ru(II) complexes have demonstrated a remarkable ability to\n (1.1\u20132.2) (2.4\u20133.2) (1.7\u20132.8) (1.0\u20131.8) (0.7\u20130.9) depolarize mitochondrial membranes. For instance, a specific Ru(II)\n 8 2.2 \u00b1 1.2 3.8 \u00b1 1.1 2.2 \u00b1 1.1 0.5 \u00b1 1.2 0.8 \u00b1 1.0 complex exhibited an eightfold increase in mitochondrial depolarization\n (1.5\u20133.2) (3.2\u20135.1) (1.6\u20132.9) (0.3\u20130.6) (0.7\u20130.8)\n 0 >10 >10 Not tested Not tested Not tested\n in A549 lung cancer cells within just 1 h treatment. This rapid depo\u00ad\n CDDP 10.4 \u00b1 1.1 6.3 \u00b1 1.1 >20 8.6 \u00b1 1.0 2.6 \u00b1 1.1 larization indicates a potent mechanism by which these complexes\n (8.6\u201312.4) (5.4\u20137.6) (7.7\u20139.5) (2.3\u20133.0) induce mitochondrial dysfunction [37].\n Further studies have shown that Ru(II) complexes can induce\n apoptosis through mitochondrial pathways. The Ru(II) complex HB324,\nworth noting that in this experiment only 24 h incubation was used (in\n for example, activates the pro-apoptotic protein Harakiri, which inhibits\nthe comparison with 72 h used in our study). Complex [Ru(\u03b76-pcym)Cl\n anti-apoptotic proteins Bcl-2 and BCL-xL, leading to mitochondrial outer\n(en)]PF6 (0), which was formerly reported to be cytotoxic in various\n membrane permeabilization and subsequent cell death [38]. Addition\u00ad\nhuman cancer cell lines (e.g. A2780 ovarian carcinoma) [28], was\n ally, half-sandwich Ru(II) complexes have been observed to impair\ninvolved in this study for comparative purposes. 0 did not show any\n mitochondrial function by reducing both mitochondrial membrane po\u00ad\ncytotoxicity in A549 and MOR cells (Table 1), which indicates that the\n tential and respiration, further contributing to their cytotoxic effects on\npresence of PAH substituents in highly cytotoxic compounds 1\u20138 is\n cancer cells [39].\nresponsible for the cytotoxic effect.\n The effect of Os(II) complexes is less described, but several studies\n Based on obtained results, complex 5, as the most potent compound,\n have confirmed their role in \u0394\u03c8m disruption. For instance, Os(II) arene\nand its ruthenium analogue 1 were selected to further in vitro analyses.\n complexes with azopyridine ligands have demonstrated potent anti\u00ad\nTo verify the anticancer potential of selected complexes 1 and 5, their\n cancer activity in A549 human non-small cell lung cancer cells, caused,\neffect on 3D cell culture model was analyzed. This model is closer to real\n at least, by the change of \u0394\u03c8m [40].\ntumour and fill the gap between common 2D cell cultures and animal in\n Tested complexes 1 and 5 were also able to increase the mitochon\u00ad\nvivo experiments. The ability of complexes 1 and 5 to cause cell death in\n drial mass in cells (Fig. 7B and C). This behaviour could be understood as\n3D spheroids formed from A549 cell was analyzed (Fig. 5 and Sup\u00ad\n compensation mechanisms of disrupted mitochondrial functions. Ac\u00ad\nporting Information, Fig. S36). Obtained results demonstrated similar\n cording to our best knowledge, this effect was observed for Ru(II) and Os\npotential of tested complexes to induce cell death as cisplatin (CDDP).\n (II) complexes for the first time. However, high content of mitochondria\n in cells has related to cisplatin resistance [41]. On the other hand, high\n2.6. Cell cycle and apoptosis evaluation mitochondria mass increased sensitivity to pemetrexed treatment [41]\n and elevated production of ROS, contributing to oxidative stress and\n Modification of cell cycle and induction of apoptosis is a common enhancing the cytotoxic effects of the drug [42].\nmechanism of action of cytostatics. Tested complexes 1 and 5 did not\nsignificantly affect cell cycle (Fig. 6). Only minor non-significant G1 2.8. Stress-related gene expression\narrest was visible after treatment with these compounds, apart from\ncisplatin, which caused significant G2/M arrest. Several studies To reveal the potential mode of action of tested complexes, their\ndemonstrated that the ability of Ru(II) and Os(II) complexes to affect cell effect on expression of stress-related genes was evaluated. The effect of\ncycle strongly depends on type of ligand and cell line. However, it is complexes 1 and 5 was described on cancerous (A549; Fig. 8) and non-\npossible to summarize that most of studied Ru and Os complexes tend to cancerous (differentiated HepaRG; Fig. 9) cells, which mimic the pri\u00ad\ncause G1/G0 arrest [33,34]. mary hepatocytes responsible for elimination of xenobiotics from a\n On the other hand, it can be supposed that 1 and 5 were able to body.\ninduce apoptosis after 24 h incubation and this effect was dissipated In most cases, Ru(II) complex 1 affected gene expression more\nafter prolonged (72 h) incubation (Fig. 6). The ability to induce effectively than its Os(II) analogue 5 in A549 cells. Both the studied\napoptosis has been described for many Ru(II) and Os(II) complexes, but complexes appear to target cellular organelles and proteins, eliciting\nthere is not clear structure-activity relationship [34,35]. The level of multi-faceted stress responses.\napoptotic cells was relatively low after 24 h incubation \u2013 11.9 % for Consistent with possible proteotoxic and organelle stress, complexes\nCDDP, 16.6 % for 1, and 12.8 % for 5, when high concentration of both 1 and 5 strongly engage the unfolded protein response (UPR)/endo\u00ad\ntested complexes was used (3 \u00d7 IC50). The number of necrotic cells was plasmic reticulum (ER) stress, both in A549 and HepaRG cells. On the\nlow, as well \u2013 0.8\u20131.4 % after 24 h and 1.7\u20132.2 % after 72 h. The only other hand, DDIT3 expression was not affected in HepaRG cells treated\nexception was CDDP which caused necrosis in 11 % of cell after 72 h by cisplatin. Complexes 1 and 5 upregulate DDIT3 (aka CHOP), a\n\n 5\n\fJ. Hos\u030cek et al. European Journal of Medicinal Chemistry 297 (2025) 117970\n\n\n\n\nFig. 5. 3D spheroids from A549 cells were treated with complex 1 and 5 and cisplatin (CDDP) at concentrations corresponding to the IC50 values obtained from 2D\nexperiments (i.e., 6 \u03bcM for 1, 1.2 \u03bcM for 5, and 10 \u03bcM for cisplatin). After 72 h of incubation, the spheroids were stained with a Live/Dead Cell Imaging Kit to\ndistinguish live (green) and dead (red) cells, and confocal microscopy was used to capture the images (magnification 10 \u00d7 ). Cisplatin (CDDP) was used as the\nreference drug. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)\n\n\nsentinel of unresolved ER stress-induced apoptosis, and ATF3, an stress occurs. This could engage p53 \u2013 indeed many Ru-based complexes\nadaptive stress-response transcription factor, in a concentration- can activate p53 pathways, though often less potently than cisplatin\ndepended manner. A striking finding is that 1 and 5 treatment down- [43]. In prior comparative analyses, a Ru(II) organometallic compound\nregulates HSPA5 (BiP/GRP78), the master ER chaperone. HSPA5 nor\u00ad RDC11 induced p53-dependent genes to a lesser degree than cisplatin,\nmally helps refold misfolded proteins and suppress UPR sensor gene which aligns with a more moderate DNA damage profile. Instead,\nactivation; its suppression by HSPA5 depletion would amplify ER stress RDC11 favoured other stress responses (oxidative and ER stress) over the\nsignalling. In cells exposed to complexes 1 and 5, GDF15 is indeed classical p53 response [43]. Thus, complexes 1 and 5 likely trigger p53\nupregulated alongside DDIT3 and ATF3, reinforcing the notion that and cell-cycle arrest as a secondary consequence of DNA damage, while\nthese compounds elicit an ER stress response akin to HSPA5 inhibition. their primary cytotoxic signals come from proteotoxic and metabolic\nThe outcome of severe UPR activation is apoptotic commitment, as stress. Apart from cisplatin that induces a broader and more intense set\nDDIT3 drives pro-apoptotic genes and inhibits Bcl-2 family survival of DNA damage response genes than formerly reported Ru(II) and Os(II)\nproteins. In line with this, global transcriptomic analyses have shown complexes. In this context, adding a 2-naphtyl PAH substituent to the\nthat a structurally different Ru(II) compound (an octahedral polypyridyl structures of 1 and 5 was hypothesized to increase their ability to\ncomplex) preferentially activates ER stress genes compared to cisplatin intercalate DNA, as reported for similar Ru(II) complexes derived from\n[43]. In other words, this seems to be the first report on half-sandwich polycyclic aromatic diamines, which showed a dramatic increase of\nRu(II) and Os(II) complexes aiming, at least partially, their anticancer DNA intercalation and binding [44]. Thus 1 and 5 may partially act by\nactivity to ER. wedging into DNA and inducing damage or transcriptional dysregula\u00ad\n Tested complexes 1 and 5 also activate DNA damage signalling, tion. However, unlike cisplatin, they are not expected to primarily create\nalthough to a lesser extent than cisplatin. Treated cells show increased fixed crosslinks; instead, they might form monofunctional adducts or\np21 and GADD45A, indicating that some DNA damage or replication associate with DNA transiently, evoking different DNA damage\n\n 6\n\fJ. Hos\u030cek et al. European Journal of Medicinal Chemistry 297 (2025) 117970\n\n\n\n\nFig. 6. The effect of 1 and 5 on cell death (A, C) and on the cell cycle (B, D) detected in the A549 cell line. The cells were incubated with the test compounds at the\nIC50, half-IC50, and triple-IC50 concentrations (i.e., 3.0, 6.0, and 18.0 \u03bcM for 1; 0.6, 1.2, and 3.6 \u03bcM for 5). Cisplatin (CDDP) at a concentration of 10 \u03bcM served as the\npositive control. The analysis was performed after 24 h (A, B) and 72 h (C, D) of exposure. The data are shown as the mean \u00b1 SEM; * indicates statistical significance\n(p < 0.05) compared with the DMF group; ** indicates statistical significance (p < 0.01) compared with the DMF group; *** indicates statistical significance (p <\n0.001) compared with the DMF group; **** indicates statistical significance (p < 0.0001) compared with the DMF group. The quadrants of the dot plots for cell\napoptosis/death were as follows: early apoptosis, lower right quadrant (LR); late apoptosis, upper right quadrant (UR); and necrotic/dead cells, upper left quad\u00ad\nrant (UL).\n\n\nsignalling dynamics. not in HepaRG cells, a cytochrome P450 enzyme not appreciably\n Importantly, complexes 1 and 5 do not significantly induce IL-6 or IL- induced by cisplatin. On the other hand, in HepaRG cells, the CYP3A4\n8 in cancerous A549 cells, unlike cisplatin, that robustly induces IL-6 gene expression was elevated (this gene is not present in A549 cells [46].\nand IL-8 transcripts (and protein secretion) as part of an acute CYP1A1 is a classic target of the aryl hydrocarbon receptor (AhR), a\nresponse (Fig. 8) [45]. The gene expression data show no upregulation ligand-activated transcription factor that senses polyaromatic xenobi\u00ad\nof these inflammatory cytokines with 1 and 5 treatment. This suggests otics. The naphthyl-containing ligand in 1 and 5 is a polyaromatic\nminimal activation of NF-\u03baB or senescence-associated secretory pro\u00ad structure that may act as an AhR ligand or be metabolized into one.\ngrams by 1 and 5. The absence of an IL-6/8 response could reflect a lack Induction of CYP1A1 strongly suggests AhR activation, as AhR binding\nof robust DNA damage-associated pattern recognition (e.g. no cytosolic leads to transcription of CYP1A1 as part of the detoxification response\nDNA to trigger innate immune sensors) or a more direct apoptotic [47]. Thus, the cell may recognize 1 and 5 as a xenobiotic stress,\nelimination of cells before they mount an inflammatory response. In engaging chemical detox pathways. Cisplatin, being an inorganic com\u00ad\npharmacological terms, this is potentially beneficial \u2013 inducing cancer plex without an aromatic moiety, does not trigger this pathway. This\ncell death without stimulating pro-inflammatory cytokines might reduce difference highlights the pharmacological role of the naphthyl ligand: it\npro-tumour inflammation or paracrine survival loops that cytokines like essentially flags 1 and 5 as an AhR-active xenobiotic, adding another\nIL-6 can provide. It underscores a mechanistic difference: cisplatin\u2019s layer to its mechanism (one that cisplatin lacks).\ndamage response crosses into immune signalling, whereas 1 and 5 The oxidative stress indicated as elevated expression of HMOX1 and\ncaused stress response is more contained to the tumour cell\u2019s internal HSP70 genes was observed only in HepaRG cells treated by Os(II)\nstress pathways. On the other hand, the situation is opposite in complex 5. However, minute elevation of HMOX1 was also observed for\nnon-cancerous HepaRG cells - 1 and 5 induced IL-6/8 expression, Ru(II) complex 1 in A549 cells.\nwhereas cisplatin not. The absence of IL-6/8 might also suggest that Overall, the gene expression signature for complexes 1 and 5 points\ncomplexes 1 and 5 do not drive cells into a senescent state but rather to ER stress-mediated apoptosis as a central mechanism, whereas for\npush them directly to apoptosis. cisplatin the UPR is more of a secondary, downstream consequence.\n Uniquely, complexes 1 and 5 upregulate CYP1A1 in A549 cells, but From a molecular pharmacology standpoint, these differences could\n\n\n 7\n\fJ. Hos\u030cek et al. European Journal of Medicinal Chemistry 297 (2025) 117970\n\n\n\n\nFig. 7. The effect of 1 and 5 on mitochondrial membrane depolarization in the A549 cell line (A). The cells were incubated with the test compounds at the IC50\nconcentrations (i.e., 6 \u03bcM for 1 and 1.2 \u03bcM for 5). Cisplatin (CDDP) at a concentration of 10 \u03bcM and carbonyl cyanide chlorophenylhydrazone (cccp; 50 \u03bcM) were\nused as a control for impaired mitochondrial membrane potential. The analysis was performed after 24 h of exposure by TMRM staining and confocal microscopy.\nThe effect of 1 and 5 on mitochondrial cellular mass in A549 cells (B \u2013 C). After 72 h incubation of cells with the test compounds at the IC50 concentrations and\ncisplatin (CDDP) at a concentration of 10 \u03bcM, they were stained by MitoTracker and observed either under confocal microscopy (magnification 63 \u00d7 for wide view,\ndigital zoom for detail) (B) or by flow cytometry (C). The data are shown as the mean \u00b1 SEM; * indicates statistical significance (p < 0.05) compared with the DMF\ngroup; *** indicates statistical significance (p < 0.001) compared with the DMF group; **** indicates statistical significance (p < 0.0001) compared with the\nDMF group.\n\n\ntranslate to distinct anticancer profiles, including e.g. efficacy in tu\u00ad resistance. Toxicity-wise, the available data hint at a favourable profile\nmours with dysfunctional p53 or resistance towards conventional anti\u00ad but warrant careful evaluation: the lack of significant myelosuppression\ncancer drugs (such as cisplatin), since they kill via alternate stresses. or nephrotoxicity in analogous Ru(III) trials (e.g. KP1339) is encour\u00ad\n aging [11] and the new Ru(II) complexes may similarly spare these\n systems. However, their recognition by AhR and induction of liver en\u00ad\n2.9. In vivo outlook zymes in vitro suggest that hepatic metabolism and potential hepato\u00ad\n toxicity will be key considerations. The induction of CYP3A4 in\n Based on their physicochemical stability and potent in vitro profile, hepatocytes (as seen with Os(II) analogues) and inflammatory cytokines\ntested Ru(II) and Os(II) PAH-Schiff base complexes hold considerable in normal cells indicates that normal tissues might mount a detoxifica\u00ad\ntherapeutic promise. Their ability to overcome cisplatin resistance in tion or stress response to these agents. Thus, while anticancer efficacy\nvitro by engaging alternative cytotoxic pathways (ER stress and pro\u00ad may be high, a careful balance between tumoricidal action and host\nteotoxic) could translate into efficacy in tumours that are refractory to toxicity must be managed. Overall, the PAH-bearing Ru(II)\nconventional platinum therapy. Importantly, the minimal pro- half-sandwich complexes emerge as promising anticancer candidates\ninflammatory cytokine response in tumour cells suggests they might that could complement or surpass existing Ru(III) drugs. They appear\nprovoke less collateral tissue inflammation or immunosuppressive side- capable of bypassing classic resistance mechanisms and killing cancer\neffects in vivo, potentially improving tolerability. In preclinical animal cells via nontraditional routes (ER stress and metabolic collapse) while\nmodels, we would expect these complexes to induce robust apoptosis potentially reducing certain side effects. Continued in vivo studies and\nwithin tumours through mitochondrial/ER stress mechanisms, leading early-phase trials will be crucial to confirm their therapeutic potential,\nto tumour regressions even in cases with p53 dysfunction or platinum\n\n 8\n\fJ. Hos\u030cek et al. European Journal of Medicinal Chemistry 297 (2025) 117970\n\n\n\n\nFig. 8. Changes in the gene expression caused by complexes 1 and 5, and cisplatin (CDDP) in A549 cancer cells. A549 cells were incubated with tested compounds at\nthe IC50, half-IC50, and double-IC50 concentrations (i.e., 3.0, 6.0, and 12.0 \u03bcM for 1; 0.6, 1.2, and 2.4 \u03bcM for 5) or DMF (0.1 % v/v) only for 5 (A\u2013E) and 24 h (F\u2013J).\nThe gene expression was evaluated by q-RT-PCR. The data are shown as the mean \u00b1 SEM; * indicates statistical significance (p < 0.05) compared with the DMF\ngroup; ** indicates statistical significance (p < 0.01) compared with the DMF group; *** indicates statistical significance (p < 0.001) compared with the DMF group;\n**** indicates statistical significance (p < 0.0001) compared with the DMF group.\n\n\n 9\n\fJ. Hos\u030cek et al. European Journal of Medicinal Chemistry 297 (2025) 117970\n\n\n\n\nFig. 9. Changes in the gene expression caused by complexes 1 and 5, and cisplatin (CDDP). HepaRG cells were incubated with tested compounds at indicated\nconcentrations or DMF (0.1 % v/v) only for 5 (A\u2013E) and 24 h (F\u2013J). The gene expression was evaluated by q-RT-PCR. The data are shown as the mean \u00b1 SEM; *\nindicates statistical significance (p < 0.05) compared with the DMF group; ** indicates statistical significance (p < 0.01) compared with the DMF group; *** indicates\nstatistical significance (p < 0.001) compared with the DMF group; **** indicates statistical significance (p < 0.0001) compared with the DMF group.\n\n\n\n\n 10\n\fJ. Hos\u030cek et al. European Journal of Medicinal Chemistry 297 (2025) 117970\n\n\noptimize dosing (considering possible metabolism by AhR-induced en\u00ad Vitrogel was obtained from InvivoGen. A Live/Dead Cell Imaging Kit\nzymes), and determine whether their novel mode of action indeed and LIVE/DEAD Fixable Violet Dead Cell Stain Kit were obtained from\ntranslates into improved outcomes with acceptable toxicity in whole Invitrogen (Eugene, OR, USA). An ApoflowEx FITC kit was produced by\norganisms. If their in vitro profile holds true in vivo, these Exbio (Prague, Czech Republic). Cytotoxicity Detection KitPLUS and\nPAH-functionalized Ru and Os complexes could significantly broaden Universal Probe Library probes were obtained from Roche (Mannheim,\nthe metallotherapeutic arsenal against cancer. Germany). TaqMan\u2122 Gene Expression Assays were produced by\n Thermo Fisher Scientific (Waltham, MA, USA); the Kapa Probe Fast One-\n3. Conclusion Step reaction mixture was purchased from Kapa Biosystems Pty (Cape\n Town, South Africa); and reference gene qPCR assays were obtained\n The research presented in this study explores the development and from Generi Biotech (Hradec Kra\u0301love\u0301, Czech Republic).\ncharacterization of novel ruthenium(II) and osmium(II) half-sandwich The starting dinuclear complexes [Ru(\u03bc-Cl)(\u03b76-pcym)Cl]2 [48,49],\ncomplexes with polycyclic aromatic hydrocarbons (PAH)-substituted [Rh(\u03bc-Cl)(\u03b75-Cp*)Cl]2 [49,50], [Os(\u03bc-Cl)(\u03b76-pcym)Cl]2 [51,52], and [Ir\nSchiff bases. The novel complexes exhibit high antiproliferative activity (\u03bc-Cl)(\u03b75-Cp*)Cl]2 [49,50] have been previously reported in the litera\u00ad\nagainst lung cancer cells, including a cisplatin-resistant line. The ture and were prepared using the Monowave 300 (Anton Paar) micro\u00ad\nincorporation of PAH groups in the Schiff bases leads to enhanced DNA wave reactor. Complex [Ru(\u03b76-pcym)Cl(en)]PF6 (0) was prepared as\nintercalation, contributing to the potent antiproliferative effects formerly reported in the literature [21].\nobserved. Notably, these complexes induced cell death via mechanisms\nthat diverge from traditional DNA cross-linking, involving cellular stress 4.2. Synthesis of organic compounds L1\u2013L4\nresponses, oxidative damage, and mitochondrial dysfunction.\n The cellular studies revealed that complexes 1 and 5 caused Ethane-1,2-diamine (1 mmol) was added to a suspension of a stoi\u00ad\napoptosis primarily through ER stress pathways, including the upregu\u00ad chiometric amount (2 mmol) of the aldehyde of the corresponding\nlation of stress-related genes like DDIT3 and ATF3. The compounds were polycyclic aromatic hydrocarbon in 10 mL of methanol. The reaction\nalso observed to induce mitochondrial membrane depolarization, a mixture is acidified with four drops of acetic acid and then heated in a\nhallmark of early apoptosis, and increase mitochondrial mass, possibly microwave reactor at 60 \u25e6 C for 3 min. The resulting organic compounds\nas a compensatory mechanism. These findings suggest that the Ru and (L1\u2013L4) were filtered off, washed with cold methanol, and dried at 50\nOs complexes engage multiple cellular stress pathways, including pro\u00ad \u25e6\n C.\nteotoxicity, oxidative stress, and mitochondrial dysfunction, contrib\u00ad N,N\u2032-bis [(E)-naphthalen-2-ylmethylidene]ethane-1,2-diamine (L1):\nuting to their unconventional anticancer efficacy. In comparison with Yield: 85 %. CHN analysis (%): calcd. for C24H20N2: C, 85.7; H, 6.0; N,\ncisplatin, which primarily activates DNA damage response pathways, 8.3 %; found: C, 86.1; H, 6.2; N, 8.2 %. 1H NMR (400 MHz, CDCl3, 298 K,\nthe Ru and Os complexes display a distinct mode of action. They do not ppm): \u03b4 8.46 (s, 2H), 7.97 (m, 4H), 7.84 (m, 6H), 7.49 (m, 4H), 4.08 (s,\nsignificantly activate inflammatory cytokines in cancer cells, an 4H).\nadvantage that could reduce pro-tumour inflammation, making these N,N\u2032-bis [(E)-anthracen-9-ylmethylidene]ethane-1,2-diamine (L2):\ncomplexes a promising alternative to cisplatin. Moreover, the potential Yield: 80 %. CHN analysis (%): calcd. for C32H24N2: C, 88.0; H, 5.5; N,\nfor overcoming platinum resistance by targeting alternative stress 6.4 %; found: C, 87.7; H, 5.4; N, 6.5 %. 1H NMR (400 MHz, C6D6, 298 K,\npathways provides an exciting avenue for the development of more ppm): \u03b4 9.22 (s, 2H), 8.67 (d, J = 8.6 Hz, 4H), 8.13 (s, 2H), 7.72 (d, J =\neffective anticancer drugs. These findings pave the way for further 8.2 Hz, 4H), 6.99 (m, 4H), 4.21 (s, 4H).\npreclinical and clinical investigations aimed at developing these com\u00ad N,N\u2032-bis [(E)-phenanthren-9-ylmethylidene]ethane-1,2-diamine\nplexes as viable alternatives to current chemotherapy options. The (L3): Yield: 90 %. CHN analysis (%): calcd. for C32H24N2: C, 88.0; H, 5.5;\nability to bypass cisplatin resistance while minimizing inflammation N, 6.4 %; found: C, 87.9; H, 5.3; N, 6.2 %. 1H NMR (400 MHz, CDCl3,\ncould enhance the therapeutic outcomes for patients suffering from 298 K, ppm): \u03b4 8.99 (s, 2H), 8.92 (d, J = 8.2 Hz, 2H), 8.67 (m, 4H), 8.08\nresistant forms of cancer, particularly lung cancer. (s, 2H), 7.88 (d, J = 7.8 Hz, 2H), 7.68 (d, J = 7.6 Hz, 2H), 7.59 (q, J =\n 8.3 Hz, 4H), 7.40 (t, J = 7.6 Hz, 2H), 4.26 (s, 4H).\n4. Experimental section N,N\u2032-bis [(E)-pyren-1-ylmethylidene]ethane-1,2-diamine (L4): Yield:\n 85 %. CHN analysis (%): calcd. for C36H24N2: C, 89.2; H, 5.0; N, 5.8 %;\n4.1. Materials found: C, 89.0; H, 5.3; N, 5.6 %. 1H NMR (400 MHz, CDCl3, 298 K, ppm):\n \u03b4 9.33 (s, 2H), 8.68 (d, J = 9.4 Hz, 2H), 8.52 (d, J = 8.2 Hz, 2H), 8.16 (d,\n Chemicals (RuCl3\u22c5xH2O, RhCl3\u22c5xH2O, OsCl3\u22c5xH2O, IrCl3\u22c5xH2O, J = 8.2 Hz, 4H), 8.01 (m, 8H), 7.79 (d, J = 9.4 Hz, 2H), 4.36 (s, 4H).\n1,2,3,4,5-pentamethylcyclopenta-1,3-diene (HCp*), 4-methyl-1-(1-\nmethylethyl)-1,3-cyclohexadiene (\u03b1-terpinen), NH4PF6, KPF6, MgSO4, 4.3. Synthesis of complexes\nethane-1,2-diamin, naphthalene-2-carbaldehyde, anthracene-9-\ncarbaldehyde, phenanthrene-9-carbaldehyde, pyrene-1-carbaldehyde, 0.05 mmol of dimers [M(\u03bc-Cl)(\u03b76-pcym)Cl]2 (M = Ru for 1\u20134 or Os\ncisplatin, phosphate-buffered saline (PBS; powder), \u03b2-nicotinamide for 5\u20138) or [M(\u03bc-Cl)(\u03b75-Cp*)Cl]2 (M = Rh for 9\u201312 or Ir for 13\u201316) were\nadenine dinucleotide reduced disodium salt (NADH), \u03b2-nicotinamide suspended in MeOH (5 mL) in a microwave reaction vial, and 0.15 mmol\nadenine dinucleotide hydrate (NAD+)), solvents (methanol, diethyl of en-based Schiff base L1 (for 1, 5, 9 and 13), L2 (for 2, 6, 10 and 14),\nether, dichloromethane (DCM), n-hexane, acetic acid) and deuterated L3 (for 3, 7, 11 and 15) or L4 (for 4, 8, 12 and 16) was added. The\nNMR solvents (CDCl3, C6D6, DMSO\u2011d6, D2O, DMF-d7) were supplied by reaction mixtures were heated in a microwave reactor (1 min, 100 \u25e6 C),\nMerck Life Science (Prague, Czech Republic), VWR International cooled to room temperature (r.t.), and filtered. After that, NH4PF6 or\n(Str\u030c\u00edbrna\u0301 Skalice, Czech Republic), Lach-Ner (Neratovice, Czech Re\u00ad KPF6 (0.25 mmol) was added to these solutions and these mixtures were\npublic) and Chemstar Czech Republic (Plzen\u030c, Czech Republic). The stirred (r.t., 15 min). A portion of the MeOH was evaporated under a\nchemicals and solvents were used as received. stream of nitrogen until complexes 1\u201316 precipitated. The products\n Cell culture media and their supplements [foetal bovine serum (FBS), were removed and re-dissolved in 3 mL of DCM. An equal volume of\nantibiotics, non-essential amino acids (NEAA)] were purchase from water was added to this solution and the resulting mixture was shaken\nBiosera (Nuaille, France), anti-microbial agent Normocin was obtained thoroughly. After separation of the DCM phase, this solution was dried\nfrom InvivoGen (San Diego, CA, USA). Collagenase I and Histopaque\u00ae by the addition of MgSO4 and after filtration the filtrate was concen\u00ad\n1077 were purchased from Merck (Darmstadt, Germany). A Cell trated (nitrogen gas) to ca. 1 mL volume. Subsequent addition of n-\nCounting Kit 8 (CCK-8) was obtained from Abcam (Cambridge, UK). hexane (ca. 5 mL) led to the formation of the solid product (1\u201316).\n\n 11\n\fJ. Hos\u030cek et al. European Journal of Medicinal Chemistry 297 (2025) 117970\n\n\nProducts were washed (3 \u00d7 2 mL of cold diethyl ether) and dried 2.9 %. 1H NMR (400 MHz, DMSO\u2011d6, 298 K, ppm): \u03b4 9.78 (s, 2H), 9.04\novernight at 50 \u25e6 C. Complexes are stable at r.t. and do not require special (d, J = 8.2 Hz, 2H), 8.97 (d, J = 8.2 Hz, 2H), 8.87 (s, 2H), 8.38 (m, 2H),\nstorage conditions. 8.04 (d, J = 8.0 Hz, 2H), 7.85 (m, 8H), 4.34 (bs, 4H), 4.05 (m, 4H), 2.34\n Alternatively, 0.05 mmol of a dimer was suspended in 2 mL of MeOH (bs, 1H), 1.80 (s, 3H), 0.78 (d, J = 6.9 Hz, 6H). ESI+ MS (MeOH, m/z):\nin a microwave reaction vial, and 0.105 mmol of appropriate en-based 761.2 (761.3 calcd. for {[Os(L3)(pcym)]\u2013H+}+; 5 %), 797.2 (797.2\nSchiff base was added. The mixtures were heated in a microwave calcd. for [OsCl(L3)(pcym)]+; 100 %).\nreactor (1 min ramp to 100 \u25e6 C, 2 min isothermal hold, and cooling to 45 [Os(\u03b76-pcym)Cl(L4)]PF6 (8): Yield: 70 %. CHN analysis (%): calcd.\n\u25e6\n C). After that, NH4PF6 (0.25 mmol) was added to these solutions and for C46H38N2ClF6OsP: C, 58.8; H, 3.9; N, 2.8 %; found: C, 58.9; H, 3.7; N,\nthese mixtures were stirred (r.t., 10 min). The resulting yellow solids 2.5 %. 1H NMR (400 MHz, DMSO\u2011d6, 298 K, ppm): \u03b4 10.12 (s), 10.06 (s),\nwere filtered and washed three times with 1 mL of MeOH, 1 mL of cold 9.18 (d, J = 8.2 Hz), 9.08 (d, J = 7.8 Hz), 8.66 (d, J = 9.0 Hz), 8.40 (m),\ndiethyl ether, and dried in a desiccator under reduced pressure. 5.79 (d, J = 5.5 Hz), 5.46 (d, J = 5.5 Hz), 5.31 (d, J = 5.5 Hz), 5.04 (d, J\n [Ru(\u03b76-pcym)Cl(L1)]PF6 (1): Yield: 85 %. CHN analysis (%): calcd. = 5.5 Hz), 4.65 (bs), 4.41 (bs), 4.28 (bs), 4.13 (bs), 4.00 (bs), 3.66 (bs),\nfor C34H34N2ClF6PRu: C, 54.3; H, 4.6; N, 3.7 %; found: C, 54.1; H, 4.3; N, 2.78 (sep, J = 6.7 Hz), 3.18 (s), 1.97 (s), 1.23 (d, J = 7.0 Hz), 0.88 (d, J =\n3.6 %. 1H NMR (400 MHz, DMSO\u2011d6, 298 K, ppm): \u03b4 9.37 (s, 2H), 8.77 6.7 Hz). ESI+ MS (MeOH, m/z): 809.3 (809.3 calcd. for {[Os(L4)\n(s, 2H), 8.48 (d, J = 8.6 Hz, 2H), 8.21 (d, J = 8.6 Hz, 2H), 8.10 (m, 4H), (pcym)]\u2013H+}+; 20 %), 845.2 (845.2 calcd. for [OsCl(L4)(pcym)]+; 100\n7.72 (m, 4H), 4.89 (d, J = 6.1 Hz, 2H), 4.68 (d, J = 6.1 Hz, 2H), 4.07 (m, %).\n2H), 3.74 (m, 2H), 2.63 (sep, J = 6.9 Hz, 1H), 2.07 (s, 3H), 1.04 (d, J = [Rh(\u03b75-Cp*)Cl(L1)]PF6 (9): Yield: 95 %. CHN analysis (%): calcd. for\n6.9 Hz, 6H). ESI+ MS (MeOH, m/z): 571.3 (571.2 calcd. for {[Ru(L1) C34H35N2ClF6PRh: C, 54.1; H, 4.7; N, 3.7 %; found: C 54.0; H 4.5; N, 3.3\n(pcym)]\u2013H+}+; 65 %), 607.2 (607.1 calcd. for [RuCl(L1)(pcym)]+; 100 %. 1H NMR (400 MHz, DMSO\u2011d6, 298 K, ppm): \u03b4 9.29 (s, 1H), 9.06 (s,\n%). 1H), 8.90 (s, 1H), 8.71 (dd, J = 8.6, 1.7 Hz, 1H), 8.57 (s, 1H), 8.20\u20138.06\n [Ru(\u03b76-pcym)Cl(L2)]PF6 (2): Yield: 75 %. CHN analysis (%): calcd. (m, 6H), 7.98 (dd, J = 8.7, 1.7 Hz, 1H), 7.77\u20137.66 (m, 4H), 4.29\u20134.12\nfor C42H38N2ClF6PRu: C, 59.2; H, 4.5; N, 3.3 %; found: C, 59.1; H, 4.2; N, (m, 3H), 3.90\u20133.80 (m, 1H), 1.52 (s, 15H). ESI+ MS (MeOH, m/z): 573.1\n2.9 %. 1H NMR (400 MHz, DMSO\u2011d6, 298 K, ppm): \u03b4 10.14 (s, 2H), 8.93 (573.2 calcd. for {[Rh(Cp*)(L1)]\u2013H+}+; 100 %), 609.1 (609.1 calcd. for\n(bs, 2H), 8.47 (d, J = 8.6 Hz, 1H), 8.37 (d, J = 8.6 Hz, 1H), 8.22 (m, 6H), [RhCl(Cp*)(L1)]+; 70 %).\n7.65 (m, 8H), 4.35 (bs, 4H), 4.11 (d, J = 6.3 Hz, 1H), 3.63 (d, J = 6.3 Hz, [Rh(\u03b75-Cp*)Cl(L2)]PF6 (10): ESI+ MS (MeOH, m/z): 673.3 (673.2\n1H), 2.29 (d, J = 5.9 Hz, 1H), 1.97 (d, J = 5.9 Hz, 1H), 1.86/1.49 (2 \u00d7 calcd. for {[Rh(Cp*)(L2)]\u2013H+}+; 40 %), 709.2 (709.2 calcd. for [RhCl\nsep, J = 6.8 Hz, 1H), 1.60/1.23 (2 \u00d7 s, 3H), 0.58/\u20130.06 (2 \u00d7 d, J = 6.6 (Cp*)(L2)]+; 100 %).\nHz, 6H). ESI+ MS (MeOH, m/z): 671.1 (671.2 calcd. for {[Ru(L2) [Rh(\u03b75-Cp*)Cl(L3)]PF6 (11): ESI+ MS (MeOH, m/z): 673.3 (673.2\n(pcym)]\u2013H+}+; 5 %), 707.1 (707.2 calcd. for [RuCl(L2)(pcym)]+; 100 calcd. for {[Rh(Cp*)(L3)]\u2013H+}+; 40 %), 709.2 (709.2 calcd. for [RhCl\n%). (Cp*)(L3)]+; 100 %).\n [Ru(\u03b76-pcym)Cl(L3)]PF6 (3): Yield: 70 %. CHN analysis (%): calcd. [Rh(\u03b75-Cp*)Cl(L4)]PF6 (12): ESI+ MS (MeOH, m/z): 721.2 (721.2\nfor C42H38N2ClF6PRu: C, 59.2; H, 4.5; N, 3.3 %; found: C, 58.8; H, 4.4; N, calcd. for {[Rh(Cp*)(L4)]\u2013H+}+; 15 %), 757.2 (757.2 calcd. for [RhCl\n3.0 %. 1H NMR (400 MHz, DMSO\u2011d6, 298 K, ppm): \u03b4 9.78 (s, 2H), 9.01 (Cp*)(L4)]+; 100 %).\n(m, 6H), 8.45 (bs, 2H), 8.07 (d, J = 7.8 Hz, 2H), 7.87 (m, 8H), 4.34 (bs, [Ir(\u03b75-Cp*)Cl(L1)]PF6 (13): Yield: 90 %. CHN analysis (%): calcd. for\n4H), 4.24 (bs, 2H), 3.91 (bs, 2H), 2.39 (sep, J = 7.0 Hz, 1H), 1.76 (s, 3H), C34H35N2ClF6IrP: C, 48.4; H, 4.2; N, 3.3 %; found: C, 47.8; H, 4.0; N, 3.4\n0.78 (d, J = 6.9 Hz, 6H). ESI+ MS (MeOH, m/z): 671.1 (671.2 calcd. for %. 1H NMR (400 MHz, DMSO\u2011d6, 298 K, ppm): \u03b4 9.38 (s, 1H), 8.99 (d, J\n{[Ru(L3)(pcym)]\u2013H+}+; 5 %), 707.2 (707.2 calcd. for [RuCl(L3) = 14.5, 3.8 Hz, 2H), 8.58 (s, 1H), 8.58 (s, 1H), 8.20\u20138.04 (m, 6H), 7.98\n(pcym)]+; 100 %). (dd, J = 8.6, 1.8 Hz, 1H), 7.77\u20137.65 (m, 4H), 4.29\u20134.20 (m, 2H),\n [Ru(\u03b76-pcym)Cl(L4)]PF6 (4): Yield: 80 %. CHN analysis (%): calcd. 4.11\u20133.92 (m, 2H), 1.48 (s, 15H). ESI+ MS (MeOH, m/z): 663.2 (663.2\nfor C46H38N2ClF6PRu: C, 61.4; H, 4.3; N, 3.1 %; found: C, 61.1; H, 4.0; N, calcd. for {[Ir(Cp*)(L1)]\u2013H+}+; 45 %), 699.1 (699.2 calcd. for [IrCl\n2.9 %. 1H NMR (400 MHz, DMSO\u2011d6, 298 K, ppm): \u03b4 10.12 (s, 2H), 9.20 (Cp*)(L1)]+; 100 %).\n(d, J = 8.0 Hz, 2H), 8.71 (d, J = 9.2 Hz, 2H), 8.45 (m, 12H), 8.20 (t, J = [Ir(\u03b75-Cp*)Cl(L2)]PF6 (14): ESI+ MS (MeOH, m/z): 763.4 (763.3\n9.2 Hz, 2H), 4.40 (m, 4H), 4.10 (bs, 2H), 4.00 (m, 2H), 2.51 (s, 1H), 1.91 calcd. for {[Ir(Cp*)(L2)]\u2013H+}+; 20 %), 799.3 (799.2 calcd. for [IrCl\n(s, 3H), 0.85 (d, J = 6.9 Hz, 6H). ESI+ MS (MeOH, m/z): 719.1 (719.3 (Cp*)(L2)]+; 100 %).\ncalcd. for {[Ru(L4)(pcym)]\u2013H+}+; 15 %), 755.2 (755.2 calcd. for [RuCl [Ir(\u03b75-Cp*)Cl(L3)]PF6 (15): ESI+ MS (MeOH, m/z): 763.4 (763.3\n(L4)(pcym)]+; 100 %). calcd. for {[Ir(Cp*)(L3)]\u2013H+}+; 10 %), 799.3 (799.2 calcd. for [IrCl\n [Os(\u03b76-pcym)Cl(L1)]PF6 (5): Yield: 90 %. CHN analysis (%): calcd. (Cp*)(L3)]+; 100 %).\nfor C34H34N2ClF6OsP: C, 48.5; H, 4.1; N, 3.3 %; found: C, 48.2; H, 4.5; N, [Ir(\u03b75-Cp*)Cl(L4)]PF6 (16): ESI+ MS (MeOH, m/z): 811.4 (811.3\n3.0 %. 1H NMR (400 MHz, DMSO\u2011d6, 298 K, ppm): \u03b4 9.38 (s, 2H), 8.69 calcd. for {[Ir(Cp*)(L4)]\u2013H+}+; 30 %), 847.2 (847.2 calcd. for [IrCl\n(s, 2H), 8.35 (d, J = 8.6 Hz, 2H), 8.17 (d, J = 8.6 Hz, 2H), 8.08 (d, J = (Cp*)(L4)]+; 100 %).\n8.2 Hz, 4H), 7.71 (m, 4H), 5.14 (d, J = 5.6 Hz, 2H), 4.83 (d, J = 5.6 Hz,\n2H), 4.05 (bs, 2H), 3.86 (bs, 2H), 2.54 (sep, J = 6.9 Hz, 1H), 2.08 (s, 3H), 4.4. General methods\n1.04 (d, J = 6.9 Hz, 6H). ESI+ MS (MeOH, m/z): 661.2 (661.2 calcd. for\n{[Os(L1)(pcym)]\u2013H+}+; 85 %), 697.1 (697.1 calcd. for [OsCl(L1) Electrospray ionization mass spectrometry (ESI-MS; methanol solu\u00ad\n(pcym)]+; 100 %). tions) was carried out with an LCQ Fleet ion trap spectrometer (Thermo\n [Os(\u03b76-pcym)Cl(L2)]PF6 (6): Yield: 75 %. CHN analysis (%): calcd. Scientific; QualBrowser software, version 2.0.7) in the positive (ESI+)\nfor C42H38N2ClF6OsO: C, 53.6; H, 4.1; N, 3.0 %; found: C, 53.7; H, 3.9; N, ionization mode. Elemental analysis was performed by a Flash 2000\n2.8 %. 1H NMR (400 MHz, DMSO\u2011d6, 298 K, ppm): \u03b4 10.24 (s, 2H), 8.92 CHNS Elemental Analyser (Thermo Scientific).\n 1\n(bs, 2H), 8.40 (d, J = 8.6 Hz, 2H), 8.27 (m, 4H), 8.16 (t, J = 8.8 Hz, 2H), H NMR spectroscopy and 1H\u20131H COSY experiments were recorded\n7.64 (m, 8H), 4.45 (m, 5H), 3.96 (d, J = 9.4 Hz, 1H), 2.20 (d, J = 9.4 Hz, using CDCl3 or DMSO\u2011d6 solutions at 298 K on a Varian-400 spec\u00ad\n1H), 1.88/1.34 (2 \u00d7 bs, 1H), 1.80 (d, J = 14.8 Hz, 1H), 0.61 (m, 6H), trometer (400 MHz); COSY = correlation spectroscopy. 1H spectra were\n\u2212 0.03 (bs, 3H). ESI+ MS (MeOH, m/z): 761.2 (761.2 calcd. for {[Os(L2) calibrated against the residual signals of the used solvent (1H at 7.26\n(pcym)]\u2013H+}+; 25 %), 797.2 (797.2 calcd. for [OsCl(L2)(pcym)]+; 100 ppm for CDCl3, 7.16 for C6D6 and 2.50 ppm for DMSO\u2011d6). The splitting\n%). of proton resonances in the reported 1H spectra is defined as s = singlet,\n [Os(\u03b76-pcym)Cl(L3)]PF6 (7): Yield: 85 %. CHN analysis (%): calcd. d = doublet, t = triplet, m = multiplet and bs = broad signal.\nfor C42H38N2ClF6OsP: C, 53.6; H, 4.1; N, 3.0 %; found: C, 53.9; H, 3.7; N, Reversed-phase high-performance liquid chromatography (RP-\n\n 12\n\fJ. Hos\u030cek et al. European Journal of Medicinal Chemistry 297 (2025) 117970\n\n\nHPLC) coupled to ESI + MS was performed on the UHPLC-MS device antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin), and\n(Dionex/Thermo Fisher Scientific) equipped with an Acclaim 120 (C18 antimicrobial agent Normocin. The cells were kept at 37 \u25e6 C in a hu\u00ad\nstationary phase; 5 \u03bcm pore size, 120 \u00c5, 2.1 \u00d7 50 mm). The mixture of midified atmosphere containing 5 % CO2. MOR/CPR cells were culti\u00ad\nMeCN (A) and 0.1 % formic acid in H2O (B) was used as the mobile phase vated in the presence of 1 \u03bcg/mL CDDP, as recommended by the\nat the gradients of 20 % A (t = 0 min), 80 % A (t = 15 min), 80 % A (t = supplier. Cells were usually passaged twice a week, and their viability\n20 min), 20 % A (t = 21 min) and 20 % A (t = 30 min) over a 30 min was checked by vital staining with trypan blue.\nperiod (0.4 mL min\u2212 1 flow rate). The detection wavelength was 254 nm. Human peripheral blood mononuclear cells (PBMCs) were isolated\n from the buffy coat prepared from whole blood of healthy volunteers at\n4.5. Crystallography the Department of Transfusion & Tissue Medicine of the University\n Hospital Brno. The buffy coat was mixed with PBS (NaCl, 137 mM; KCl,\n Monocrystals suitable for a single-crystal X-ray analysis were pre\u00ad 2.7 mM; Na2HPO4, 10 mM; KH2PO4, 1.8 mM; pH 7.4) at a ratio of 1:1 and\npared for Ru complexes 1, 2 and 4. Furthermore, several crystalline subsequently transferred into a cuvette containing Histopaque\u00ae 1077\nproducts obtained from the solutions of the Rh and Ir complexes (mother with half the volume of the buffy coat/PBS mixture. After centrifugation\nliquors, re-crystallized products) were tested and we were able to (at 500 g/30 min/r.t.), the layer containing the PBMCs was aspirated,\ndetermine the crystal structures of the complexes with the cleaved Schiff transferred to a new cuvette, and washed twice with cold PBS (followed\nbase ligands 9\u2032 and 13\u2019. by centrifugation at 500 g/10 min/4 \u25e6 C). The washed cells were resus\u00ad\n Data collection for single crystals of 1, 3, and 4 was performed using pended in complete RPMI 1640 medium [containing 10 % FBS, antibi\u00ad\na Stoe StadiVari equipped with a Pilatus3R 300K hybrid pixed array otics (100 U/mL penicillin and 100 mg/mL streptomycin), and\ndetector (Dectris) and a microfocused X-ray source Xenocs Genics 3D Cu Normocin] and counted.\nHF (Cu K\u03b1). All the crystal structures were solved using SHELXT pro\u00ad\ngram [53] and refined by the full matrix least-squares procedure with 4.8. In vitro 2D and 3D cytotoxicity testing\nSHELXL (version 2019/2) [54] in OLEX2 (version 1.5) [55]. The\nmulti-scan absorption corrections were applied using the program Stoe The effect of the tested compounds on cell viability was evaluated\nLANA [56]. The molecular structures and packing diagram were drawn using a Cell Counting Kit 8 (CCK8) according to the manufacturer\u2019s\nwith MERCURY [57]. The crystal structure of compound 4 was previ\u00ad manual. The test compounds were dissolved in DMF (the concentration\nously published [28], but the completeness of the collected diffraction of DMF did not exceed 0.1 % v/v in the presence of cells) and CDDP was\ndata was low, and the experiment was performed at ambient tempera\u00ad used as the positive control. Floating PBMCs were seeded at a concen\u00ad\nture, compared to 150 K in the redetermination presented here. The tration of 5 \u00d7 104 cells/well in a 96-well plate. Adherent A549, MOR,\npcym ligand in structure 4 is disordered in two positions with occupancy MOR/CPR, and MRC-5 cells were seeded into 96-well plate at a density\nfactors of 0.61 (1) and 0.39 (1). The disordered pcym ligand was of 1 \u00d7 104 cells/well and allowed to adhere overnight. The next day, the\nmodeled using same fragment restraints and rigid body (RIGU) re\u00ad cultivation medium was changed (MOR/CPR cell obtained medium\nstraints. CCDC deposition numbers: 2450152 (for 1), 2450153 (for 3) without CDDP), and the tested complexes were added 2 h later. Cell\nand 2450154 (for 4). viability was measured 72 h later, and the IC50 values (the concentra\u00ad\n The data collection for the complexes with the cleaved Schiff base tions of the tested compounds that caused a 50 % decrease of metabolic\nligands 9\u2032 and 13\u2032 was done using an XtaLAB Synergy-I diffractometer active cells in comparison with the number of untreated cells) were\nwith a HyPix3000 hybrid pixel array detector and microfocused calculated according to four-parameter logistic (4 PL) analysis,\nPhotonJet-I X-ray source (Cu K\u03b1). The data integration and multi-scan excluding outstanding values (ROUT algorithms, Q = 5 %) in Prism 7.05\nabsorption corrections were applied using the program CrysAlisPro software (GraphPad Software, Inc., San Diego, CA, USA).\n1.171.40.82a [58]. The crystal structures of 9\u2032 and 13\u2032 were solved and To evaluate the anti-cancer potential of the tested complexes in a 3D\nrefined using the same programs as in the case of 1, 3, and 4. The quality cell model, which is more closely related to real cancer than 2D models\nof the crystals of was very poor leading to unsatisfactorily diffraction [61], spheroids formed from A549 cells were used. A549 cells resus\u00ad\ndata and high R-values. Therefore, both crystal structures were sub\u00ad pended in serum-free DMEM were seeded at a density of 16 \u00d7 103\nmitted to CSD as database communications [59,60]. For 9\u2019, the [Rh cells/well into a 96-well U-shaped bottom microtitre plate with a low\n(\u03b75-Cp*)Cl(en)]+ cation was formerly reported [29]. cell binding surface (Thermo Scientific, Roskilde, Denmark). After 4\n days, the medium was exchanged for medium containing 10 % FBS, and\n4.6. Stability studies the spheroids were cultivated for another 3 days. Then, the grown\n spheroids (diameter of 0.5\u20131.0 mm) were transferred to a 96-well plate\n The appropriate amount of the complexes for 1 mM solutions in 50 % with a flat bottom, which was covered with Vitrogel matrix, forming a\nDMSO\u2011d6/50 % D2O (v/v) was dissolved in DMSO\u2011d6 (250 \u03bcL) and 250 wide U-shaped bed. During the transfer, the medium was exchanged.\n\u03bcL of the phosphate-buffered saline (PBS) solution in D2O was added Complexes 1 and 5 and CDDP at concentrations corresponding to their\n(DMSO\u2011d6 was added due to low solubility of the complexes in water). IC50 values obtained from 2D experiments (i.e., 6 \u03bcM for 1, 1.2 \u03bcM for 5,\n1\n H NMR spectra were recorded at various time points (t = 0\u201324 h). The and 10 \u03bcM for CDDP) were added after 2 h of acclimatization to the\nobtained spectra were calibrated against the residual signal of DMSO\u2011d6 spheroids in a CO2 incubator. After 3 days of incubation, the spheroids\n(2.50 ppm). Analogical experiments were performed with an addition of were stained with a Live/Dead Cell Imaging Kit containing calcein AM\nan excess of NADH (5 molar equiv.), which was dissolved in 250 \u03bcL of and BOBO-3 iodide dyes to distinguish live and dead cells. After 30 min\nthe PBS solution in D2O prior its addition to the complexes dissolved in of incubation in the dark at room temperature, the shape of the spher\u00ad\n250 \u03bcL of DMSO\u2011d6. oids and the amount and distribution of dye were analyzed via confocal\n microscopy (Leica TSP SP 8). At least 3 spheroids were analyzed for each\n4.7. Cell culture compound.\n\n The cytotoxic effects of the tested complexes were evaluated on the 4.9. Cell cycle analysis\nlung cell lines A549 and MOR, the cisplatin-resistant MOR/CPR, and\nMRC-5 (all obtained from ECACC, Salisbury, UK). MOR cells were Flow cytometry was used to evaluate the number of cells in the\ncultured in RPMI 1640 medium, A549 cells in DMEM High glucose particular phases of the cell cycle. The protocol was described previously\nmedium, and MRC-5 cell in EMEM supplemented by 1 % NEAA; all [62]. The cells were seeded at 6 well plate at a concentration of 3 \u00d7 105\nmedia were supplemented with 10 % fetal bovine serum (FBS), cells/well and treated complexes 1 and 5, CDDP, and DMF only for 24 h\n\n 13\n\fJ. Hos\u030cek et al. European Journal of Medicinal Chemistry 297 (2025) 117970\n\n\nand 72 h. For 24 h, the concentrations corresponding to their \u03bcM) was used as a control for impaired mitochondrial membrane po\u00ad\n3 \u00d7 and 1 \u00d7 IC50 values obtained for 72 h incubation were used to tential. Unstained cells and cells treated with carbocyanine iodide were\nachieve strong cellular response. On the other hand, for 72 h incubation, used as controls for proper setting of flow cytometry analysis. A mini\u00ad\nthe concentrations corresponding to their 0.5 \u00d7 and 1 \u00d7 IC50 values mum of 1.5 \u00d7 104 live cells excluding doublets and debris were sub\u00ad\nobtained for 72 h incubation were used to evaluate the effect of level of jected to analysis. TMRM fluorescence was detected using the\ntested compounds. The cells were washed in cold PBS and fixed by appropriate excitation/emission settings (Ex/Em: 548/574 nm). TMRM\nlow-speed vortexing in 70 % (v/v) cold ethanol at 4 \u25e6 C overnight. Next, negative cells were gated as cells with \u0394\u03a8m, which is a typical feature of\ncells were stained by 100 \u03bcL Vindel solution (propidium iodide, RNase) early apoptosis. The data were evaluated by CellStream Analysis 1.2.55\nfor 20 min at RT and analyzed by flow cytometer Amnis CellStream software. Experiments were performed in duplicate and in three inde\u00ad\n(Luminex, USA). A minimum of 1.5 \u00d7 104 events were collected per pendent repetitions.\nsample for evaluation of the percentage of cells in G1, S, and G2/M\nphases of the cell cycle. The data were evaluated by CellStream Analysis\n 4.12. Detection of amount and size of mitochondria\n1.2.55 software. Experiments were performed in duplicate and in three\nindependent repetitions.\n The amount and size of mitochondria were measured after staining\n cells with MitoTracker Green FM (Invitrogen, USA, cat. No. M46750).\n4.10. Assessment of cell death\n MitoTracker Green FM is a cell-permeant reagent that labels mito\u00ad\n chondria in live cells. The cells were incubated with MitoTracker Green,\n The assessment of cell death and apoptosis was determined by flow\n allowing the dye to passively diffuse across the plasma membrane and\ncytometry in the human lung cancer cell line A549. The staining pro\u00ad\n accumulate in active mitochondria. Confocal microscopy was used to\ntocol has been described previously [62] with minor modifications. The\n determine the localization and size of mitochondria. Meanwhile, flow\ntreated cells were stained with Annexin V Dyomics 647 dye (Exbio,\n cytometry analysis was employed to quantify the mitochondrial content.\nCzech Republic; dilution 5 \u03bcL in 100 \u03bcL Annexin Binding Buffer) and\n Briefly, A549 cells (2 \u00d7 105 cells/well) were seeded during the log\u00ad\nLIVE/DEAD Fixable Violet Dead Cell Stain Kit (ThermoFisher Scientific,\n arithmic growth phase into a 10 % FBS medium on IBIDI chamber slides\nUSA; dilution 1 \u03bcL in 100 \u03bcL Annexin Binding Buffer). After 15 min\n (cat. No. 80826) for confocal microscopy analysis. Similarly, A549 cells\nincubation at 37 \u25e6 C in the dark, samples were centrifuged at 500\u00d7g for 5\n (1.4 \u00d7 105 cells/well) were plated on 6-well plates for flow cytometry\nmin at room temperature. Detection was performed using Amnis Cell\u00ad\n and allowed them to adhere overnight at 37 \u25e6 C with 5 % CO2. The next\nStream (Luminex, USA) flow cytometer. Fluorescence was recorded\n day, the culture medium was replaced, and the tested compounds were\nusing the appropriate filter sets for Annexin V Dyomics 642 (Ex/Em:\n added 2 h later at concentrations based on their IC50 values for 72 h.\n642/702 nm) and Live/Dead Violet (Ex/Em: 405/450 nm). Unstained\n After the 72 h incubation, the culture medium was gently removed.\nand single-stained controls were used to adjust compensation and define\n MitoTracker Green FM solution diluted in medium (200 nM) was added\nthe gating strategy. A minimum of 2 \u00d7 104 events excluding doublets\n to the cells and incubated at 37 \u25e6 C for 30 min. In the end, the cells were\nand debris were subjected to analysis.\n imaged under a Leica SP8 confocal fluorescence microscope.\n Analysis of cell death and apoptosis was performed using double\n For flow cytometry, double staining was used to detect viability and\nstaining on dot plots, with Annexin V Dyomics 642 on the x-axis and\n mitochondrial content. First, cells were detached from the 6-well plate\nLive/Dead Violet on the y-axis. The dot plots were divided into four\n with trypsin (Gibco, USA, cat. No. 15400054), washed, and stained with\nquadrants. Live cells (live cells/Annexin V negative; low left quadrant;\n the LIVE/DEAD Fixable Violet Dead Cell Stain Kit (ThermoFisher Sci\u00ad\nLL), early apoptotic cells (live cells/Annexin V positive; low right\n entific, USA, cat. No. L34964) for 15 min at 37 \u25e6 C. The cells were then\nquadrant; LR), late apoptotic cells (dead cells/Annexin V positive; upper\n washed, stained with Mitotracker Green FM for 40 min at 37 \u25e6 C, washed\nright quadrant; UR), and necrotic/dead cells (dead cells/Annexin V\n again, resuspended in 100 \u03bcL PBS, and finally analyzed using the Amnis\nnegative; upper left quadrant; UL) were distinguished after double\n CellStream flow cytometer (Luminex, USA). Fluorescence was recorded\nstaining. The data were evaluated by CellStream Analysis 1.2.55 soft\u00ad\n with the appropriate filter sets for MitoTracker Green FM (Ex/Em: 488/\nware. Experiments were performed in three independent repetitions.\n 528 nm) and Live/Dead Violet (Ex/Em: 405/450 nm). Unstained con\u00ad\n trols were used to establish the gating strategy. A minimum of 2 \u00d7 104\n4.11. Mitochondrial membrane potential studies\n events, excluding doublets and debris, were analyzed.\n\n Changes in mitochondrial membrane potential (\u0394\u03a8m) after treat\u00ad\nment by complexes 1 and 5, CDDP, and DMF were assessed using the 4.13. Expression of stress related genes\ncationic dye tetramethylrhodamine methyl ester (TMRM) after 24\nhours. This dye is permeable to cells that accumulate in active mito\u00ad Changes in the gene expression of cellular stress markers were\nchondria. Reduced fluorescence indicates mitochondrial depolarization, determined by quantitative real-time PCR (qRT\u2013PCR) on human liver\nwhich is a typical feature of early apoptosis. A549 cells were seeded into HepaRG\u2122 (Biopredic, Rennes, France) and A549 cells. HepaRG\u2122 were\na 6-well cultivation plate at a concentration of 3 \u00d7 105 cells/well and differentiated in 24-well plates as described previously [63], with minor\nallowed to adhere overnight. The cultivation medium was changed after changes. After differentiation, the cells were exposed to 1, 5, or CDDP in\n24 h and cells were treated. Following 24 h treatment, cells were medium without the differentiating agent (2 % DMSO) for 5 or 24 h.\ncollected and resuspended in culture medium containing TMRM at a Cytotoxicity was tested after 24 h with the Cytotoxicity Detection\nfinal concentration of 100 nM. Cells were incubated with the dye at 37 KitPLUS, according to the manufacturer\u2019s instructions. A549 cell were\n\u25e6\n C for 30 min in the dark to allow adequate dye accumulation in seeded as described above (chapter 4.8). Total RNA was isolated, and\npolarized mitochondria. After incubation, cells were centrifuged at 500 g qRT\u2012PCR was performed with primers, Universal Probe Library probes\nfor 5 min at room temperature and samples were washed once with pre- or TaqMan\u2122 Gene Expression Assays, as described recently [63], with\nwarmed PBS buffer to remove excess dye and stained by LIVE/DEAD minor changes. The amplifications were carried out in 10 \u03bcL of Kapa\nFixable Violet Dead Cell Stain Kit (ThermoFisher Scientific, USA) at a Probe Fast One-Step reaction mixture containing 1 \u03bcL of the sample.\ndilution of 1:1000. After staining of viable and dead cells (15 min, 37 Reference gene qPCR assays were used for the housekeeping genes\n\u25e6\n C), the samples were washed, centrifuged at 500 g for 5 min at room human \u03b22-microglobulin (B2M; #3030) and hydroxymethylbilane syn\u00ad\ntemperature and resuspended in PBS for analysis. Flow cytometry thase (HMBS; #3032). The Cp values of these two genes were averaged\nanalysis was performed using the flow cytometer Amnis CellStream to serve as a reference, and the changes in relative gene expression were\n(Luminex, USA). Carbonyl cyanide chlorophenylhydrazone (cccp; 50 calculated based on the comparative threshold cycle method [64].\n\n 14\n\fJ. Hos\u030cek et al. European Journal of Medicinal Chemistry 297 (2025) 117970\n\n\nCRediT authorship contribution statement WHO classification of lung tumors: impact of advances since 2015, J. Thorac.\n Oncol. 17 (2022) 362\u2013387, https://doi.org/10.1016/j.jtho.2021.11.003.\n [6] M. Wang, R.S. Herbst, C. Boshoff, Toward personalized treatment approaches for\n Jan Hos\u030cek: Writing \u2013 review & editing, Writing \u2013 original draft, non-small-cell lung cancer, Nat. 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