Increasing the π-Expansive Ligands in Ruthenium(II) Polypyridyl Complexes: Synthesis, Characterization, and Biological Evaluation for Photodynamic Therapy Applications.

{"full_text": " pubs.acs.org/IC Article\n\n\n\n Increasing the \u03c0\u2011Expansive Ligands in Ruthenium(II) Polypyridyl\n Complexes: Synthesis, Characterization, and Biological Evaluation\n for Photodynamic Therapy Applications\n Maria Dalla Pozza, Pierre Mesdom, Ahmad Abdullrahman, Tayler D. Prieto Otoya, Philippe Arnoux,\n C\u00e9line Frochot, Germain Niogret, Bruno Saubam\u00e9a, Pierre Burckel, James P. Hall, Marcel Hollenstein,\n Christine J. Cardin, and Gilles Gasser*\n Cite This: Inorg. Chem. 2023, 62, 18510\u221218523 Read Online\nSee https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.\n\n\n\n\n ACCESS Metrics & More Article Recommendations *\n s\u0131 Supporting Information\n Downloaded via MOSCOW STATE UNIV on May 12, 2026 at 11:21:22 (UTC).\n\n\n\n\n ABSTRACT: Lack of selectivity is one of the main issues with\n currently used chemotherapies, causing damage not only to altered\n cells but also to healthy cells. Over the last decades, photodynamic\n therapy (PDT) has increased as a promising therapeutic tool due to\n its potential to treat diseases like cancer or bacterial infections with\n a high spatiotemporal control. Ruthenium(II) polypyridyl com-\n pounds are gaining attention for their application as photo-\n sensitizers (PSs) since they are generally nontoxic in dark\n conditions, while they show remarkable toxicity after light\n irradiation. In this work, four Ru(II) polypyridyl compounds with\n sterically expansive ligands were studied as PDT agents. The Ru(II)\n complexes were synthesized using an alternative route to those\n described in the literature, which resulted in an improvement of the synthesis yields. Solid-state structures of compounds\n [Ru(DIP)2phen]Cl2 and [Ru(dppz)2phen](PF6)2 have also been obtained. It is well-known that compound [Ru(dppz)(phen)2]Cl2\n binds to DNA by intercalation. Therefore, we used [Ru(dppz)2phen]Cl2 as a model for DNA interaction studies, showing that it\n stabilized two different sequences of duplex DNA. Most of the synthesized Ru(II) derivatives showed very promising singlet oxygen\n quantum yields, together with noteworthy photocytotoxic properties against two different cancer cell lines, with IC50 in the micro- or\n even nanomolar range (0.06\u22127 \u03bcM). Confocal microscopy studies showed that [Ru(DIP)2phen]Cl2 and [Ru(DIP)2TAP]Cl2\n accumulate preferentially in mitochondria, while no mitochondrial internalization was observed for the other compounds. Although\n [Ru(dppn)2phen](PF6)2 did not accumulate in mitochondria, it interestingly triggered an impairment in mitochondrial respiration\n after light irradiation. Among others, [Ru(dppn)2phen](PF6)2 stands out for its very good IC50 values, correlated with a very high\n singlet oxygen quantum yield and mitochondrial respiration disruption.\n\n\n \u25a0 INTRODUCTION\n Photodynamic therapy (PDT) is a well-established medical\n superoxide radical (O2\u2212\u2022), hydroxyl radical (HO\u2022), and\n hydrogen peroxide (H2O2). An ideal PS is characterized by\n technique used for the treatment of localized diseases and is a the ability to absorb light in the therapeutic window (600\u2212900\n valuable supplement or alternative to chemotherapy, radio- nm), an appropriate energy of the triplet state, and a lifetime of\n therapy, or immunotherapy for the treatment of some forms of the triplet state long enough to allow the production of ROS.\n cancer. PDT was developed to decrease the well-known side The most interesting feature of PDT is the spatiotemporal\n effects of chemotherapy, which very often lacks selectivity.1 control of drug activation, which makes it possible to have a\n PDT is based on the use of light, a photosensitizer (PS), and\n specific target, decreasing the severe side effects caused by the\n oxygen. The PS is ideally nontoxic in dark conditions and\n becomes toxic once irradiated with light at a desired diffusion of a toxic drug in the entire body.2,3\n wavelength. After being excited with light at a specific\n wavelength, the PS* often undergoes intersystem crossing Received: August 4, 2023\n (ISC), leading to a triplet excited state T1. From this excited Revised: October 10, 2023\n state, energy can be transferred to the surrounding Accepted: October 13, 2023\n biomolecules (PDT type I) or directly to molecular oxygen Published: November 1, 2023\n in its ground state (3O2) (PDT type II) to produce reactive\n oxygen species (ROS), such as singlet oxygen (1O2),\n\n \u00a9 2023 American Chemical Society https://doi.org/10.1021/acs.inorgchem.3c02606\n 18510 Inorg. Chem. 2023, 62, 18510\u221218523\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 1. Chemical structures of the synthesized compounds.\n\nScheme 1. Synthetic Scheme for the Four Compounds Synthesized in This Work\n\n\n\n\n Ruthenium(II) polypyridyl compounds have been widely complex [Ru(dppz)(phen)2]Cl2 has been extensively studied\nstudied in the last decades due to their promising photo- by Barton et al. for its high binding affinity to DNA and its\ndynamic properties.4\u221211 In fact, the possibility to tune the \u201clight-switch\u201d properties once intercalated into the double helix\nelectronic configuration of the metal complexes by changing of DNA.18,19,21 Taking into account the need for increasing the\nthe ligands, their outstanding values of 1O2 quantum yield after affinity of Ru(II)-based PSs for biological targets and with the\nlight activation, and their ability to interact with biological aim to improve the 1O2 production of these compounds, in\ntargets like DNA or proteins make this class of compounds this work, we planned to design Ru(II) derivatives with\nvery versatile for different applications. Remarkably, by additional \u03c0-extended ligands such as the benzo[i]dipyrido-\nmodulating the choice of the ligands, it is possible to obtain [3,2-a:2\u2032,3\u2032-c]phenazine (dppn), the dipyrido[3,2-a:2\u2032,3\u2032-c]-\na red shift in the absorption spectra, an increased cellular phenazine (dppz), and the 4,7-diphenyl-1,10-phenanthroline\nuptake, better targeting properties, and improved ROS (DIP) (Figure 1).\nproduction, responsible for cell death.12\u221214 In 2017, the These ligands were chosen for their ability to give long-lived\n 3\nRu(II)-based compound TLD1433 designed by McFarland et \u03c0\u2212\u03c0* excited states to the Ru(II) polypyridyl compounds\nal. entered clinical trials for bladder cancer, increasing the upon coordination. Furthermore, we were interested in\ninterest in this versatile class of compounds.15,16 studying the effect of the coordination of two bulky ligands\n Heteroleptic Ru(II) compounds with one bulky intercalating in the PDT activity of the resulting Ru(II) complex. While\nligand have largely been reported in the literature for their [Ru(DIP)2phen]Cl2 (1) and [Ru(dppz)2phen](PF6)2 (3)\nphotodynamic and DNA intercalating properties.17\u221220 The were already described in the literature,22,23 to the best of our\n 18511 https://doi.org/10.1021/acs.inorgchem.3c02606\n Inorg. Chem. 2023, 62, 18510\u221218523\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 2. Solid-state structures of cations of 1 (a) and 2 (b) obtained by the X-ray diffraction of single crystals. Ru atoms are in magenta, N atoms\nare in blue, and C atoms are in gray. H atoms, solvent atoms, counterions, and disordered atoms in the structure are omitted for clarity.\n\nknowledge, the other Ru(II) derivatives [Ru(DIP)2TAP]Cl2 (Scheme 1). The syntheses of [Ru(dppz)2phen](PF6)2\n(TAP = 1,4,5,8-tetraazaphenanthrene) (2) and [Ru- (compound 3) and [Ru(dppn)2phen](PF6)2 (compound 4)\n(dppn)2phen](PF6)2 (4) are not described. The synthesis of were more problematic, mainly due to solubility issues of the\nthe compounds from the precursor [Ru(LL)Cl2] with the dppz precursors. We first tried to synthesize the compounds starting\nor dppn ligands reported in the literature was giving low yield from the Ru(LL)Cl2 precursor, followed by coordination with\nand the purification was quite tedious.23 Giorgi and phen, as previously reported in the literature,23 but tedious\ncollaborators recently reported a different synthetic strategy purification steps were necessary to obtain the desired\nfor a compound similar to 4, namely, [Ru(dppn)2(dmbpy)]- compounds in low yields. One of the main problems related\n(PF6)2 (dmbpy = 4,4\u2032-dimethyl-2,2-bipyridine), where the to the synthesis was the insolubility of the precursor\nsolubility issues related to the [Ru(LL)Cl2] intermediate were [Ru(LL)Cl2], which did not allow for characterization or\novercome by a different synthesis.24 In this article, we propose purification. Therefore, we envisioned a different synthetic\na different synthetic strategy, starting from the precursor route starting from the coordination of two phendione ligands\n[Ru(phendione)2Cl2] (phendione = 1,10-phenanthroline-5,6- to the precursor [Ru(DMSO)4Cl2] in anhydrous DMF to\ndione), inspired by the work of Leveque et al.25 With the obtain the corresponding disubstituted Ru(II) dichloro\ndescribed synthetic route, the solubility issues were overcome compound [Ru(phendione)2Cl2], followed by the coordina-\nas well, the synthesis was straightforward, and the purification tion of the phen ligand in EtOH/H2O (1/1, v/v). As expected,\nby column chromatography was remarkably not needed. the intermediate could be isolated by precipitation as the PF6\u2212\nStability in dimethyl sulfoxide (DMSO) and CH3CN after salt and directly used in the following step without the need for\nlight irradiation was confirmed by 1H NMR and UV\u2212vis any chromatographic purification. Finally, the intermediate\nspectroscopies. The presence of the polypyridyl ligands allows [Ru(phendione)2phen](PF6)2 was reacted with an excess of o-\nthe possibility of intercalation in the DNA structure. Therefore, phenylenediamine in CH3CN/EtOH (1/3, v/v) to obtain final\nthe interaction of compound 3 with two different duplex DNA compound 3 or 4 after precipitation with NH 4 PF 6 .\nsequences was investigated. The (photo)cytotoxic effect Remarkably, there was no need to purify the product by\nagainst different cancer cells at different wavelengths was column chromatography, but just by filtration and washing\nassessed. The viability results highlighted a very promising with water, obtaining yields of 82 and 55% for compounds 3\nphototoxic activity of most of the tested compounds, and 4, respectively (Scheme 1). This modified synthetic\ncorrelated with very good 1O2 quantum yields. Therefore, we pathway allowed us to improve the yields described in the\nwent more in-depth to study the internalization of the most literature and therefore made it easier to study this attractive\npromising compounds in nuclei and mitochondria, and we\n class of compounds.\nchecked their effect on the mitochondrial respiration.\n X-ray Crystallography. Single crystals of 1 and 3 were\n\n\u25a0 RESULTS AND DISCUSSION\n Synthesis and Characterization. The Ru(II) derivatives\n obtained by dissolving the compounds in CH3CN, followed by\n the slow addition of diethyl ether to allow solvent diffusion.\n The structures of the cations of the compounds are shown in\nwere synthesized by using different strategies. All of them were Figure 2. As expected, both complexes are constructed around\nprepared starting from [Ru(DMSO)4Cl2], which has already the RuN6 core, where the metal center is characterized by an\nbeen reported in the literature as a useful precursor for this octahedral geometry. No substantial differences in terms of\nkind of compounds.26 The complexes bearing two DIP ligands, bond lengths or angles were observed compared to the already\n1 and 2, were obtained by coordination of the two DIP ligands described Ru(II) trispolypyridyl complexes.27 Interestingly,\nwith the metal center in anhydrous N,N-dimethylformamide both compounds show \u03c0\u2212\u03c0 stacking (Figures SI18 and SI19).\n(DMF), followed by coordination with the phen or TAP ligand On the one hand, the phen ligand of 1 seems to be intercalated\nto obtain the final compound after purification by silica column between the two DIP ligands of proximal molecules. On the\nchromatography, with yields of 73 and 42%, respectively other hand, the intercalation of the dppz ligands of 3 is much\n 18512 https://doi.org/10.1021/acs.inorgchem.3c02606\n Inorg. Chem. 2023, 62, 18510\u221218523\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nmore pronounced, which was not surprising since the aromatic for drugs, as it highly affects the cellular uptake by passive\nstructure of the dppz ligand is longer than the DIP, allowing a diffusion.30 We employed the \u201cshake-flask\u201d method31 to\nbetter \u03c0\u2212\u03c0 interaction (Figure SI19). The steric hindrance measure the Log P values of 1\u22124 (Table 1). As expected,\ncaused by the phenyl substituents of the DIP ligand probably the compounds showed Log P values between 1 and 1.5,\ncontributes to the decrease in the \u03c0\u2212\u03c0 stacking among the DIP indicating that the compounds are hydrophobic. Moreover, we\nligands too. can observe that all of the compounds have similar Log P\n Absorption and Emission Properties of the Ru(II)- values.\nComplexes. The UV\u2212vis absorption spectra in CH3CN were (Photo)stability Studies. In some cases, PSs can undergo\nthen recorded and are shown in Figure 3. The maximum degradation after light irradiation, causing the release of toxic\n moieties.32 To evaluate the photostability of complexes 1\u22124,\n the evolution with time of their absorption properties was\n monitored through UV\u2212visible spectroscopy during continu-\n ous irradiation at 540 nm (9 J/cm2). Very interestingly, the\n absorption spectra of all of the synthesized Ru(II) derivatives\n in CH3CN remained unchanged after 40 min of light\n irradiation, demonstrating that no photodegradation occurred\n (Figure SI22). The decrease in the absorption intensity\n observed in the UV\u2212vis spectra of compound 4 was attributed\n to the presence of aggregation phenomena, which have\n previously been reported in the literature for similar\n compounds.33,34 CH3CN was used since the poor solubility\n of the compounds in water did not allow us to obtain a suitable\n concentration for the UV\u2212vis analysis. Next, the stability of the\nFigure 3. UV\u2212vis spectra of compounds 1\u22124 in CH3CN. The complexes in DMSO was investigated. For this purpose, 1H\nsolutions have been prepared in CH3CN and used at the following NMR spectra of compounds 1\u22124 were recorded over a period\nconcentrations: 10.4 \u03bcM (1), 14.8 \u03bcM (2), 15.5 \u03bcM (3), and 12.5 \u03bcM of 48 h (Figures SI23\u2212SI26). Overall, the compounds showed\n(4). remarkable stability since no changes in the spectra were\n observed.\n Singlet Oxygen Quantum Yield. The PS, after excitation\nabsorption wavelength of a PS is a very important parameter with light, is excited to an unstable singlet state S1, which can\nsince it determines the wavelength to be used for its release the excess energy by luminescence or by nonradiative\nphotoactivation. In this sense, the use of long-wavelength relaxation. The most important electronic transition involved\nlight (500\u2212900) is generally preferred since it has deeper tissue in PDT is the intersystem crossing (ISC), which leads to an\npenetration, which is required for a PDT treatment to be excited triplet state, T1, that presents a longer lifetime than the\neffective, especially in in vivo and clinical settings. 28 excited singlet state S1. The excited T1 state can decay by\nCompounds 1 and 2 displayed two bands in the UV range radiative relaxation, giving phosphorescence or interacting with\n(273 and 276 nm), corresponding to the spin-allowed \u03c0\u2212\u03c0* other species present in the biological environment. This, in\ntransitions. The shoulder around 320 nm is ascribed to the turn, generates ROS, responsible for the activation of different\n1\n MLCT and 1LLCT.29 The broad band between 350 and 510 cell death pathways. Another mechanism involves molecular\nnm corresponds to the metal-to-ligand charge transfer oxygen (3O2) that is excited by energy transfer from excited T1,\n(1MLCT transition). Compounds 3 presented an intense leading to the formation of 1O2. 1O2 presents a very short\nintraligand \u03c0\u2212\u03c0* transition centered at 280 nm, while the one lifetime (<0.04\u22123 \u03bcs) and a very high reactivity, allowing a\nof compound 4 is shifted at 290\u2212340 nm. Moreover, for 4, this spatiotemporal control of its area of action (0.01\u22120.155\ntransition gives a double-humped absorption at 389 and 410 \u03bcm).12,35 In this context, it is clear that the PS efficacy of\nnm. A broad 1MLCT band is centered at 435 nm.24 triggering the formation of 1O2 species after excitation by light\n 1\u22123 are also characterized by an emission band after is very important for the efficiency of the PS in PDT. The\nexcitation at 450 nm in CH3CN, while 4 does not emit in these measurement of the 1O2 quantum yield is of main importance\nconditions, according to that reported by Giorgi et al. for to evaluate if a PS can exploit the light energy to convert 3O2 to\nsimilar compounds (Table 1 and Figure SI20).24 the reactive singlet oxygen species O2 (1\u0394g).2,3,12 Therefore,\n Distribution Coefficient (Log P Values). The octanol\u2212 we evaluated the photophysical properties of the compounds\nwater partition coefficient (Log P) is an important parameter and their 1O2 quantum yield.\n 1\u22123 present similar emission spectra after excitation at 450\nTable 1. Absorption Maxima, Emission Maxima, and Log P nm, with an emission band at around 600 nm for 1 and 3 and\nValues of the Ruthenium Complexes 682 nm for 2 (Figure SI20). On the contrary, compound 4\n absorption maxima \u03bb emission maxima \u03bb\n does not emit at all, with a luminescence quantum yield of 0\ncompound (nm)a (nm)a log Pb (Table 2).\n 1 222, 273, 442 608 1.5 \u00b1 0.3 In this context, the 1O2 quantum yield of the four Ru\n 2 222, 276, 435 682 1.6 \u00b1 0.3 complexes was measured in aerated CH3CN using [Ru-\n 3 222, 275, 363, 435 612 1.0 \u00b1 0.2 (bpy)3]Cl2 as the standard. To our delight, compounds 1\u22123\n 4 222, 242, 261, 325, 435 nd 1.3 \u00b1 0.1 showed moderate to high 1O2 quantum yields, with values\n ranging from 0.36 to 0.73. Remarkably, compound 4 showed a\n 1\na\n UV\u2212Vis and emission spectra were recorded in CH 3 CN. O2 quantum yield of 0.73 (Figure SI22). The lack of\nb\n Experimentally determined using the shake-flask method. luminescence of compound 4 together with a high 1O2\n 18513 https://doi.org/10.1021/acs.inorgchem.3c02606\n Inorg. Chem. 2023, 62, 18510\u221218523\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nTable 2. Photophysical Properties of Compounds 1\u22124 in colocalization in nuclei or mitochondria was observed by the\nCH3CN studies reported below. Sequences were chosen based on their\n 1\n differences in terms of base steps to evaluate how the\n luminescence luminescence O2 quantum\n compound quantum yield lifetime (ns) yield interaction is influenced by changing the sequence. We\n1 0.06 179 0.60\n selected two sequences of dsDNA d(CCGGTACCGG)2\n2 0.08 371 0.36\n (TA) and d(CCGGCGCCGG)2 (CG), and for each sequence,\n3 0.16 188 0.37\n we used a DNA:Ru ratio of 1:1 or 1:2. After increasing the\n4 0.00 n.d. 0.73 temperature from 10 to 80 \u00b0C with 5 \u00b0C increments, we\n[Ru(bpy)3] 0.08 159 0.57 observed shifts of the melting transitions to higher temper-\n Cl2 atures (Figure 4). In particular, both sequences were stabilized\n by the addition of the compound. Interestingly, for the TA\nquantum yield suggests that most of the energy of the T1 sequence, we can observe that the DNA is more stabilized with\nexcited state of 4* is used to produce 1O2. a 1:1 ratio, while adding complex equivalents leads to a\n DNA Thermal Denaturation and Photocleavage decreased stabilizing effect (Figure 4). Overall, the results\nStudies. The polypyridyl Ru(II) compounds have been indicate that there is a preference in terms of step for the\nintensively studied for their application as DNA intercala- interaction and that the presence of two bulky ligands instead\ntors.36\u221238 In this work, we focused our interest in under- of only one probably does not change the DNA binding.\nstanding if the synthesized compounds are able to bind the Overall, these results confirm the stabilizing effect of the\nduplex DNA and how this interaction can influence the Ru(II) derivatives bearing dppz ligands. This study is a\nstability of the DNA duplex. As previously demonstrated by preliminary step in the investigation of the interaction of 3 with\nCardin and co-workers, the intercalation of a polypyridyl metal different DNA sequences, and a more detailed investigation is\ncomplex on the duplex DNA depends not only on the metal needed to unravel their interaction and determine any\nderivative but also on the different sequences and the different sequence selectivity and/or specificity of binding and\nsteps of the DNA sequence. In particular, they observed by stabilization.\ncrystal structure analysis that the dppz ligand of the compound After obtaining the DNA stabilizing results, we also tried to\n\u039b-[Ru(phen)2(dppz)]Cl2 intercalates symmetrically and per- investigate the ability of the compounds to cleave the DNA\npendicularly from the minor groove of the d- after light activation. Unfortunately, as observed for the\n(CCGGTACCGG)2 duplex at the central TA/TA step.39 stability studies by melting curves, the compounds precipitated\n[Ru(phen)2(dppz)]Cl2 was described as a stabilizing agent for once the DNA in the duplex, single strand, G-quadruplex, or\nthe duplex structures of RNA and DNA.40 In this work, we plasmid form were added to the solution. Therefore, it was not\nstudied the effect on the thermal stability of dsDNA after possible to obtain any reliable result (data not shown). We\nruthenium interaction, using the CD thermal denaturation hypothesized that the strong interaction of the compound with\nmethod to determine the difference of melting point after the DNA might cause an unbalance of DNA charges that leads to\naddition of 1 or 2 equiv of compound 3 to different DNA the precipitation of the DNA in the presence of the Ru(II)\nsequences. Unfortunately, it was not possible to evaluate the complexes.\nDNA interaction of the other compounds since precipitation (Photo)toxicity Studies. To evaluate the biological\noccurred even with a 1:1 ratio of DNA:compound. This activity of the four compounds, we performed a two-\nappeared only when the compound was added to the DNA dimensional (2D) in vitro viability assay against two cancerous\nsolution and not in the absence of DNA. Since 3 was the only cell lines CT26 (mice colon adenocarcinoma) and HT29\ncompound that could be solubilized to the concentrations (human colorectal adenocarcinoma) and one noncancerous\nnecessary for the experiment, it was taken as a model even if no cell line RPE-1 (eye-pigmented retinal epithelium). Their\n\n\n\n\nFigure 4. dsDNA melting curve with a DNA:compound ratio of 1:0 (blue line), 1:1 (orange line), and 1:2 (gray line). Concn of dsDNA was 40\n\u03bcM, and the concentration of 3 was 40 or 80 \u03bcM. Melting curves were recorded from 10 to 80 \u00b0C, holding time 1 min, 1 \u00b0C/min, and recorded CD\neach 5 \u00b0C change in temperature. Each graph is a representative of at least two independent experiments.\n\n 18514 https://doi.org/10.1021/acs.inorgchem.3c02606\n Inorg. Chem. 2023, 62, 18510\u221218523\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nTable 3. (Photo)toxicity IC50 (\u03bcM) Values toward CT26, HT29, and RPE-1 at 540 nm (9 J/cm2), 595 nm (3.4 J/cm2), and 620\nnm (6.7 J/cm2)a\n 540 nm 595 nm 620 nm\n compound IC50 dark IC50 light PI IC50 dark IC50 light PI IC50 dark IC50 light PI\n CT26\n 1 >100 0.09 \u00b1 0.05 1111 >100 8\u00b11 13 >100 10 \u00b1 1 10\n 2 >100 2.8 \u00b1 0.1 36 >100 23 \u00b1 3 4 >100 37 \u00b1 16 3\n 3 >100 7\u00b16 14 >100 >100 1 >100 >100\n 4 >100 0.06 \u00b1 0.02 1666 >100 1.9 \u00b1 0.7 53 >100 0.75 \u00b1 0.03 133\n PpIX >100 0.12 \u00b1 0.04 833 >100 8.8 \u00b1 0.3 11 >100 0.51 \u00b1 0.06 196\n HT29\n 1 >100 1.6 \u00b1 0.1 63 >100 3.2 \u00b1 0.7 31 >100 32.5 \u00b1 0.7 3\n 2 >100 1.5 \u00b1 0.5 67 >100 20 \u00b1 15 5 >100 31 \u00b1 1 3\n 3 >100 >100 >100 >100 >100 >100\n 4 >100 0.18 \u00b1 0.08 556 >100 0.4 \u00b1 0.2 250 >100 2.5 \u00b1 0.4 40\n PpIX >100 1.1 \u00b1 0.6 91 >100 0.7 \u00b1 0.5 143 >100 2.9 \u00b1 0.6 34\n RPE-1\n 1 >100 0.9 \u00b1 0.2 111 >100 7.8 \u00b1 0.4 13 >100 17 \u00b1 3 6\n 2 >100 1.4 \u00b1 0.3 71 >100 19 \u00b1 4 5 >100 13 \u00b1 2 8\n 3 >100 9\u00b14 11 >100 >100 >100 >100\n 4 >100 0.18 \u00b1 0.02 556 >100 1.3 \u00b1 0.4 77 >100 0.3 \u00b1 0.2 333\n PpIX >100 0.5 \u00b1 0.2 200 >100 0.9 \u00b1 0.1 111 >100 0.26 \u00b1 0.08 385\na\n The reported values were obtained as the mean of three independent experiments.\n\n\n\n\nFigure 5. Subcellular localization of compounds 1 (a), 2 (b), and 3 (c) by confocal microscopy. CT26 cells were imaged live following incubation\nwith the compounds (10 \u03bcM) for 4 h and then with Hoechst 33342 and MitoTracker Deep Red for 10 min. In each picture, the top left panel is the\nbright field image, the top right panel is the MitoTracker signal (yellow), the bottom left panel is the compound signal (magenta), and the bottom\nright panel is the merge of fluorescent channels.\n\ncytotoxicity in the dark and upon light irradiation at different penetration.28 For this reason, we tested the compounds\u2019\nwavelengths (i.e., 540, 595, and 620 nm) was investigated phototoxicity also after excitation at 595 and 620 nm (3.4 and\nusing a fluorometric cell viability assay. In all of the 6.7 J/cm2, respectively). Viability results showed that the\nexperiments, protoporphyrin IX (PpIX) was used as a positive compounds were generally less phototoxic when irradiated\ncontrol. Since it is known that UV light can cause cell death,41 with longer wavelengths than after irradiation at 540 nm with\nit is important to mention that the light dose was carefully no toxicity at all for compound 3. This could be explained by\noptimized in order to have a cell survival of nontreated cells of the better luminescence quantum yield of compound 3, with a\nat least 95% after light irradiation. After adjustment of the lifetime of 188 ns, and a low 1O2 quantum yield as compared\nirradiation time, the phototoxicity of the compounds was to compound 4 and [Ru(bpy)3]Cl2, which was used as the\nevaluated in different cell lines. Very promisingly, all of the standard. Overall, these results could indicate that 3 dissipates\ntested compounds were nontoxic in dark conditions up to 100 energy by luminescence, which loses its PDT activity.\n\u03bcM and were very toxic after light irradiation at 540 nm, with However, encouraging results have been obtained with\nIC50 values in the range of 0.06\u22127 \u03bcM. Since one of the main compound 4, displaying very low IC50 values at all of the\nissues of PDT is low light tissue penetration, there is much studied wavelengths. Importantly, this compound showed\ninterest in finding PSs able to cause a phototoxic effect after similar or even better IC50 values compared to those of the\nactivation with longer wavelengths, allowing for a deeper tissue control (PpIX). Concerning 1 and 2, the IC50 values showed\n 18515 https://doi.org/10.1021/acs.inorgchem.3c02606\n Inorg. Chem. 2023, 62, 18510\u221218523\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 6. Whole cell and mitochondrial accumulation of compounds 1 and 4 following overnight incubation of CT26 cells with compounds 1 or 4\n(1 \u03bcM) assessed by Ru quantification using high-resolution ICP-MS. (a) Whole cell accumulation. (b) Fraction of the whole cellular content\naccumulated in mitochondria.\n\nbetter activity as compared to 3. Anyway, the phototoxic likely because they affect redox homeostasis within mitochon-\nactivity decreased drastically after irradiation at 620 nm. The dria and thus cellular energy production.6 Interestingly, 1 and\ndifferent activities between 1 and 2 compared to that of 3 2 were found to significantly and similarly accumulate in\ncould be explained by the luminescence quantum yield and the mitochondria (Figure 5a,b), with Pearson coefficient values of\n1\n O2 quantum yield. As we suggested, the poor activity of 0.56 and 0.65, respectively, between compounds and\ncompounds 2 and 3 could be the consequence of the low 1O2 MitoTracker fluorescence signals. Of note, the compounds\nquantum yield. However, the luminescence quantum yield of 2 presented no toxicity in the dark, indicating that they affect\nis much lower than 3, suggesting that the excited triplet state mitochondria only after light irradiation. Previous studies\ndirectly dissipates energy by producing ROS. 1 is characterized showed accumulation in mitochondria of similar Ru(II)\nby a low emission but a very good 1O2 production, which once compounds with DIP ligands,6 confirming what we observed\nagain can explain the better activity of 1 and 2 as compared to in the present study for 1 and 2. By contrast, the luminescence\nthat of 3. Interestingly, no activity of compound 3 was of compound 3 did not show any overlap with MitoTracker\nobserved after irradiation at 540 nm on HT29 cells, indicating (Pearson coefficient value of \u22120.15), suggesting that its\nthat compound 3 is more toxic against CT26 cells than HT29. phototoxicity upon light excitation does not involve its\nHowever, there was no selectivity toward cancer cells rather accumulation in mitochondria (Figure 5c). Moreover, the\nthan healthy cells. intracellular fluorescence signal from compound 3 was very\n Overall, all of the Ru(II) derivatives displayed higher toxicity low, likely because this compound is weakly internalized after 4\nafter excitation at 540 nm, while lower phototoxic effect was h of treatment, as revealed by the ICP-MS analysis (Figure\nobtained with 595 and 620 nm. The compounds do not have SI39).\nany selectivity against cancer cells if compared to non- To have a better understanding of the subcellular local-\ncancerous cells (Table 3). Remarkably, 4 displayed a very ization of the nonluminescent compound 4, we used ICP-MS\npromising IC50 at all irradiation wavelengths, with no toxicity on purified mitochondria. Compound 1 was also included in\nin the dark and, therefore, high phototoxicity index (PI). this study as a positive control. Interestingly, almost 40% of\n Cellular Internalization by Confocal Microscopy and internalized compound 1 localized in mitochondria (Figure\nInductively Coupled Plasma Mass Spectrometry (ICP- 6b), in line with what we observed by confocal microscopy. By\nMS). A drug\u2019s activity and efficacy rely on drug cellular uptake. contrast, less than 5% of compound 4 accumulated in\nMoreover, the accumulation in different organelles could lead mitochondria, despite a higher cellular uptake compared to 1\nto a variation in drug activity.42 Therefore, we decided to (Figures 6 and SI40). It is interesting to notice that\ninvestigate the internalization of the tested compounds using compounds 1 and 2, which bear the DIP ligands, accumulate\nconfocal microscopy, particularly focusing on nuclear and in mitochondria, while 3 and 4, which have dppz or dppn\nmitochondrial accumulation. Compounds 1\u22123 were easily ligands, do not significantly localize in these organelles.\nvisualized following excitation at 448 nm, whereas the dppn Metabolic Studies of Mitochondria Respiration.\nderivative 4 was not included in this study since it did not According to our results on mitochondrial internalization of\nshow any luminescence after excitation. Nuclear and 1 and 2, we decided to compare the impact of our best\nmitochondrial accumulation was assessed using colabeling compounds (1 and 4), on the cellular respiration, using a\nwith the DNA nuclear stain Hoechst 33342 and the live Seahorse XF analyzer. Mitochondria are cellular organelles\nmitochondria stain MitoTracker Deep Red. As shown in responsible for the adenosine triphosphate (ATP) production\nFigure 5, none of the compounds showed detectable by oxidative phosphorylation. They also have important roles\naccumulation in the nucleus, as confirmed by the negative in several metabolic pathways, in apoptosis and programmed\nvalues of Pearson coefficients between the Hoechst signal and cell death, and in ROS homeostasis.45,46 In cancer cells,\ncompounds 1\u22123 (\u22120.27, \u22120.35, and \u22120.17, respectively). mitochondrial function is essential and plays a central role in\n Mitochondria are pivotal organelles in cell apoptosis. In the cell viability.47 The Seahorse assay was used to investigate\nparticular, oxidative stress can trigger the mitochondria- the effect of the tested compounds on the mitochondrial\ndependent cell death signaling pathway.43 Some Ru(II) respiration and to elucidate if the accumulation of our\npolypyridyl compounds can selectively target mitochondria, compounds in the mitochondria could influence the cellular\nas previously reported by Chao and co-workers.44 However, respiration. Cells were treated for 4 h with the compounds\nalthough these compounds showed high phototoxicity after using their IC50 and IC25 concentrations and then irradiated at\nlight irradiation, they also had important toxicity in the dark, 540 nm for 40 min. After the culture medium was degassed for\n 18516 https://doi.org/10.1021/acs.inorgchem.3c02606\n Inorg. Chem. 2023, 62, 18510\u221218523\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 7. (a) Mito stress test of the OCR profile in CT26 cells after 4 h of treatment and 40 min of irradiation. Oligomycin (inhibitor of ATP\nsynthase), FCCP (uncoupling agent), antimycin A (complex III inhibitor), and rotenone (complex I inhibitor) were sequentially added. The OCR\nprofile of compound 4 (IC50 light and dark) and control light is highlighted in the graph. The blue bar indicates the time spot at which we\nregistered the bar graph below. (b) Bar graph of the OCR profile of each compound after the injection of FCCP (t = 40 min).\n\n1 h in a non-CO2 incubator, the cells were treated with compounds can trigger mitochondrial depolarization inde-\nsequential injections of specific inhibitors of the electron pendently of their cellular sublocalization.49,50\ntransport chain. First, oligomycin was added to inhibit the ATP\nsynthase, FCCP (carbonyl cyanide 4-(trifluoromethoxy)-\nphenylhydrazone) was then added as an uncoupling agent\n \u25a0 CONCLUSIONS\n In summary, in this work, we report on the synthesis,\nthat induces maximal oxygen consumption rate (OCR), and a\n characterization, and biological evaluation of four Ru(II)\ncombination of rotenone/antimycin A was injected to block\n polypyridyl compounds. An alternative synthetic method to\nthe electron transport chain to stop the mitochondrial O2 obtain Ru(II) derivatives bearing two dppz or dppn ligands in\nconsumption. The Mito stress test was performed following good yields is described. The four compounds displayed\nthe Agilent protocol.48 Our Seahorse results showed that excellent stability in DMSO over 48 h and in CH3CN after\ndespite the fact that compound 1 showed mitochondrial light irradiation at 540 nm for 40 min. Very promising\ninternalization by confocal microscopy studies, almost no effect phototoxicity properties against different cancer cell lines were\non the cellular respiration was observed at IC25 and IC50 observed. In particular, compound 4 stands out for its\nconcentrations. On the contrary, we observed a severe photoactivity in the micro- or even nanomolar range at all of\nimpairment of mitochondrial respiration in samples treated the tested wavelengths, making this compound very interesting\nwith 4 together with irradiation at 540 nm for 40 min, while no for PDT applications. The interaction with different sequences\neffect was observed in cells kept in the dark condition. of DNA has been studied, taking as model compound 3.\nCompound 4 clearly affects not only the ATP-linked Interestingly, a DNA stabilizing effect was observed, which is\nrespiration but also the spare capacity (Figure 7a). In Figure consistent with the one proposed for the largely studied\n7b, we can clearly see that the respiration capacity is compound [Ru(phen)2(dppz)]Cl2. In addition, the internal-\ndiminished by the administration of compound 4. Overall, 4 ization of these Ru(II) derivatives in organelles containing\nis the most effective, inducing a decrease in respiration higher DNA, i.e., nuclei and mitochondria, was investigated. None of\nthan that of the positive control PpIX. It is worth mentioning the compounds entered the nuclei, but compounds 1 and 2\nthat PpIX impaired mitochondrial respiration either in the dark targeted the mitochondria. Moreover, to study the mechanism\nor light conditions, while 1 and 4 showed values similar to the of action of the best-performing compounds 1 and 4, we\nnegative control in the dark. Our results suggested that the evaluated their ability to affect the cellular respiration. Very\ndysregulation of mitochondrial respiration is one of the cell interestingly, compound 4 affected the mitochondrial respira-\ndeath mechanisms driven by compound 4. This could be due tion only after light exposure, while no consistent effect was\nto depolarization of the mitochondrial membrane. In fact, observed in the dark condition. Overall, this work sheds light\nprevious studies have shown that Ru(II) polypyridyl on the promising phototoxic activity of the tested compounds\n 18517 https://doi.org/10.1021/acs.inorgchem.3c02606\n Inorg. Chem. 2023, 62, 18510\u221218523\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nand their interaction with important biological targets such as determine their relative concentrations in each phase. The measure-\nmitochondria and DNA. ments were repeated three times for each complex.\n Photostability. The photostability of the tested compounds was\n\n\u25a0 MATERIALS AND METHODS\nAll chemicals were purchased from commercial sources and used\n evaluated by irradiation at 540 nm in 96-well plates with an Atlas\n Photonics LUMOS BIO irradiator during time intervals from 0 to 40\n min. UV\u2212vis spectra were recorded using a BioTek Cytation 5. The\nwithout further purification. If necessary, solvents were dried over solutions were prepared in air-saturated CH3CN at 100 \u03bcM and read\nmolecular sieves. The Ru(II) precursors Ru(DMSO)4Cl2 and after different times of irradiation (i.e., 0, 10, 20, 30, 40 min).\nRu(DIP)2Cl2 and Ru(phendione)2Cl2 were synthesized following Singlet Oxygen and Luminescence Quantum Yield. All\npreviously reported procedures.51,52 The cell culture media and spectra were measured using 4-sided quartz cells. The absorption\nreagents were purchased from Fisher Scientific. Thin-layer chroma- values of the references and samples at the excitation wavelength were\ntography (TLC) was performed using silica gel 60 F-254 (Merck) adjusted to 0.2. All emission spectra were normalized to the same\nplates with the detection of spots by exposure to UV light. Eluent absorbance for the purpose of comparison. [Ru(bpy)3]Cl2 in\nmixtures are expressed as volume-to-volume (v/v) ratios. 1H- and 13C acetonitrile was chosen as the standard for both luminescence and\n 1\nNMR spectra were measured on Bruker Avance III HD 400 MHz or O2 quantum yield determination. The luminescence quantum yield\nBruker Avance Neo 500 MHz spectrometers. The deuterated solvent of [Ru(bpy)3]Cl2 in CH3CN is evaluated at 0.077.55 The 1O2\nsignal was used as an internal standard. The chemical shifts \u03b4 are quantum yield of [Ru(bpy)3]Cl2 in aerated CH3CN is evaluated at\nreported in parts per million (ppm) relative to tetramethylsilane 0.57.56 Time-resolved experiments were performed using for\n(TMS) or signals from the residual protons of deuterated solvents. excitation: a pulsed laser diode emitting at 407 nm (LDH-P-C-\nThe following abbreviations were used to designate multiplicities: s = 400M, fwhm <70 ps, 1 MHz) coupled with a driver PDL 800-D (both\nsinglet, d = doublet, t = triplet, m = multiplet, and dd = double\u2212 PicoQuant GmbH, BERLIN, Germany) and for detection: an\ndoublet. ESI experiments were carried out using a 6470 Triple Quad avalanche photodiode SPCM-AQR-15 (EG & G, VAUDREUIL,\ninstrument (Agilent Technologies). HPLC analysis was performed Canada) coupled with a 650 nm long-wave pass filter as the detection\nusing two Agilent G1361 1260 Prep Pumps and an Agilent G7115A system. The acquisition was performed by a PicoHarp 300 module\n1260 DAD WR detector equipped with an Agilent Pursuit XRs 5C18 with a 4 channel router PHR-800 (both PicoQuant GmbH, BERLIN,\n(100 \u00c5, C18 5 \u03bcm 250 \u00d7 4.6 mm) column. The flow rate was 1 mL/ Germany). The luminescence decays were recorded using the single\nmin with the following gradients: 0\u22123 min: isocratic 95% A (5% B); photon counting method. Data were collected up to 500 counts\n3\u221217 min: linear gradient from 95% A (5% B) to 0% A (100% B); accumulated in the maximum channel and analyzed using time\n17\u221223 min: isocratic 0% A (100% B). Chromatograms were detected correlated single photon counting (TCSPC) software Fluofit\nat 250 nm. The solvents (HPLC grade) were Millipore water (solvent (PicoQuant GmbH, BERLIN, Germany) based on iterative\nA) and acetonitrile (solvent B). Only for compound 1, we used 0.1% reconvolution using a Levensberg\u2212Marquardt algorithm, enabling\nv/v of trifluoroacetic acid in A and B. The samples were dissolved in the obtention of multiexponential profiles (mainly one or two\nCH3CN and filtered through a 0.2 mm membrane filter before the exponentials in our cases).\ninjection. The absorption spectra were measured with an Agilent Cary Solution Preparation for Annealing. Initial stock solutions of\nUV\u2212visible Multicell Peltier spectrophotometer. The luminescence the ruthenium complexes were made in EtOH, and oligonucleotides\nspectrum was measured with a Fluorolog FL3-222 spectrofluorimeter were made in water. The concentration of the solutions was checked\n(Horiba Jobin Yvon, Palaiseau, France) equipped with a 450 W xenon using the extinction coefficient of 20,000 M\u22121 cm\u22121 at 450 nm for the\narc lamp, with a thermostatically controlled cell holder compartment Ru compound, and the Eurogentec-provided extinction coefficients at\n(25 \u00b0C), a UV\u2212visible photomultiplier tube R928 (HAMAMATSU 260 nm, calculated using the nearest-neighbor model, were used for\nJapan), and an InGaAs infrared detector cooled by liquid nitrogen DNA. The stocks were then diluted with buffer and combined to form\n(DSS-IGA 020L Electro-Optical System Inc., Phoenixville, PA). The solutions with either 1:1 or 1:2 ratio of DNA strands to ruthenium\nexcitation beam is separated by a SPEX dual network monochromator complex. Annealing of the oligonucleotides, both with and without\n(1200 lines/mm blased at 330 nm). The luminescence was measured ruthenium complex present, was carried out by incubating the\nby the UV\u2212visible detector via the SPEX dual network emission buffered solution at 90 \u00b0C for 5 min and then allowing it to cool to\nmonochromator (1200 lines/mm blased at 500 nm). 1 O 2 room temperature.\nluminescence was measured with an InGaAs infrared detector CD Thermal Denaturation. CD melting experiments were\n(800\u22121550 nm) via the dual network emission monochromator carried out using a Chirascan V100 with a temperature-controlled\nSPEX (600 lines/mm blase\u0301 at 1 \u03bcm). The cell culture medium and four-cell changer. Samples were prepared at a concentration of 40 \u03bcM\nDNA plasmid were purchased from Thermo Fisher. DNA sequences of DNA and either 1 or 2 mol equiv of the ruthenium complex. The\nfor thermal studies were provided by Eurogentec. buffer consisted of 50 mM sodium cacodylate at pH 7. Absorption\n X-ray Crystal Structures. Single crystals were grown in CH3CN/ was recorded at 260 nm at 5 \u00b0C intervals between 10 and 80 \u00b0C, with\nEt2O at 18 \u00b0C. A suitable crystal was selected and mounted in a loop a temperature change rate of 1 \u00b0C/min in a 0.1 cm path length quartz\non a Synergy diffractometer. The crystals were kept at 100 K during cuvette. Melting curves were generated from these data.\nthe data collection. Using Olex2,53 the structures were solved with the Cell Culture. CT26 cells were cultured in DMEM media (Gibco,\nSHELXT54 structure solution program using intrinsic phasing and Life Technologies), HT29 cells were cultured in McCoy 5, and RPE-1\nrefined with the SHELXL54 refinement package using least-squares cells were cultured in DMEM/F-12 (Gibco) supplemented with 10%\nminimization. Full details are included in the Supporting Information. of fetal bovine serum. All cell lines were complemented with 100 U/\n Stability Studies in DMSO. The stability in DMSO-d6 at 37 \u00b0C mL of penicillin\u2212streptomycin mixture (Gibco) and maintained in a\nwas assessed by 1H NMR for 48 h. Spectra were recorded at time humidified atmosphere at 37 \u00b0C and 5% of CO2.\nzero, 1, 6, 12, 24, and 48 h. 2D (Photo)toxicity Assay. (Photo)toxicity of compounds 1\u22124\n Measurement of Octanol\u2212Water Partition Coefficient and PpIX in dark and light conditions was carried out by a\n(Log P). The log P values were determined using the shake-flask fluorometric cell viability assay using Resazurin (ACROS Organics).\nmethod. 1 mL of octanol was presaturated with 1 mL of phosphate- Cells were seeded in triplicate in 96-well plates at a density of 4 \u00d7 103\nbuffered saline (PBS) by overnight incubation with shaking of a cells/well (for CT26) or 6 \u00d7 103 cells/well (for HT29 and RPE-1) in\nbiphasic mixture of the two at room temperature. 5 \u03bcL of a 10 mM 100 \u03bcL of culture media. After 24 h of incubation at 37 \u00b0C with 5%\nDMSO stock solution of each compound was added into the biphasic CO2, cells were treated with increasing concentrations of the\nsolution of PBS/octanol to give a final concentration of 50 \u03bcM. The ruthenium complexes. Dilutions were prepared from a 10 mM stock\nmixture was shaken for 24 h using an automated shaker and allowed solution in DMSO, which was further diluted to different\nto stand for 2 h. Aliquots from the octanol phase and the aqueous concentrations (0.01\u2212100 \u03bcM) in the cell media. After 4 h of\nphase were extracted and analyzed using the UV\u2212vis detector to incubation with the tested compounds, the medium was replaced with\n\n 18518 https://doi.org/10.1021/acs.inorgchem.3c02606\n Inorg. Chem. 2023, 62, 18510\u221218523\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nfresh medium, and the cells were irradiated for a variable time Gen5 software and by utilizing the cell count and the data were\ndepending on the wavelength (40 min at 540 nm with an irradiance of normalized against the same cell number.\n9 J/cm2 and 1 h at 595 and 620 nm with an irradiance of 3.4 J/cm2 Synthetic Procedures. Ru(DMSO)4Cl2. Ru(DMSO)4Cl2 was\nand 6.7 J/cm2, respectively). Cells were incubated for up to 48 h. synthesized following an adapted literature procedure.57 Spectro-\nThen, 100 \u03bcL of complete medium containing resazurin (0.2 mg/mL scopic data were in agreement with the literature.57\n 1\nfinal concentration) was added. After incubating for 4 h at 37 \u00b0C, the H NMR (400 MHz, deuterium oxide) \u03b4 3.48 (s, 1H), 3.48 (s,\nfluorescence signal of the resorufin product was read (ex: 540 nm em: 2H), 3.45 (s, 2H), 3.41 (s, 1H), 3.37 (s, 3H).\n590 nm) in a BioTek Cytation 5 fluorimeter. IC50 values were then [Ru(DIP)2Cl2]. This precursor was synthesized according to a\ncalculated using GraphPad Prism software 9. The XY analysis with literature procedure. Ru(DIP)2Cl2 was synthesized following an\nthree replicate values in side-by-side subcolumns was chosen. Inserted adapted literature procedure.58 Spectroscopic data were in agreement\nraw data obtained from a BioTek Cytation 5 fluorimeter were treated with the literature.58\n 1\nas follows: X values were transformed into the logarithm, and data H NMR (400 MHz, CH3CN-d3) \u03b4 10.51 (d, J = 5.3 Hz, 2H), 8.17\nwere normalized to the lowest Y value. Data were then analyzed with (d, J = 9.4 Hz, 2H), 8.08 (d, J = 5.4 Hz, 2H), 8.02 (d, J = 9.4 Hz, 4H),\n\u201cnonlinear regression\u201d (curve fit) and then \u201clog(inhibitor) vs. 7.81 (d, J = 7.4 Hz, 5H), 7.70 (t, J = 7.4 Hz, 5H), 7.67\u22127.58 (m, 4H),\nnormalized response\u201d. 7.52 (d, J = 1.8 Hz, 12H), 7.25 (d, J = 5.5 Hz, 2H).\n Subcellular Localization by Confocal Microscopy. CT26 cells [Ru(DIP)2(phen)]Cl2. This compound was synthesized according to\n(3 \u00d7 104 cells/well) were seeded in 35 mm culture dishes with a 20 a literature procedure. Ru(DIP)2Cl2 was synthesized following an\nmm diameter glass coverslip bottom and incubated for 48 h. The cell adapted literature procedure.59 Spectroscopic data were in agreement\nmedium was then replaced by fresh medium containing 10 \u03bcM of 1\u2212 with the literature59 (385 mg, 73%).\n3. After incubation for 4 h in the dark at 37 \u00b0C with 5% CO2, cells Crystal data for C60H40Cl2N6O3Ru (M = 1064.95 g/mol):\nwere washed with PBS to remove the compound not internalized in orthorhombic, space group Pcca (no. 54), a = 20.2027(4) \u00c5, b =\nthe cells. Cells were stained with Hoechst 33342 (1 \u03bcg/mL) and 22.3997(6) \u00c5, c = 21.7393(4) \u00c5, V = 9837.8(4) \u00c53, Z = 8, T = 100 K,\nMitoTracker Deep Red (MTDR, 100 nM) at 37 \u00b0C for 10 min. Live \u03bc(Cu K\u03b1) = 4.013 mm\u22121, Dcalcd = 1.438 g/cm3, 60181 reflections\ncells were imaged in an SP8 confocal laser scanning microscope measured (7.16\u00b0 \u2264 2\u0398 \u2264 152.84\u00b0), 10029 unique (Rint = 0.0796,\n(Leica Microsyste\u0300mes, Nanterre, France) equipped with a 63x x 1.40 Rsigma = 0.0502), which were used in all calculations. The final R1 was\nplan apochromat objective. The excitation/emission wavelengths were 0.0703 (I > 2\u03c3(I)), and wR2 was 0.2196 (all data). The coordinates\n405/420\u2212450 nm (Hoechst), 448/600\u2212650 nm (1\u22123), and 638/ have been deposited in the Cambridge Crystallographic Data Centre,\n660\u2212700 nm (MTDR). Laser intensities were kept as low as possible no. 2287795. 1H NMR (400 MHz, DMSO-d6) \u03b4 8.84 (dd, J = 8.3, 1.2\nto avoid any phototoxicity. To quantify the amount of colocalization Hz, 2H), 8.44 (s, 2H), 8.35 (d, J = 5.5 Hz, 2H), 8.26 (s, 4H), 8.25 (d,\nbetween each compound and Hoechst or MTDR, Pearson coefficients J = 1.3 Hz, 2H), 8.19 (d, J = 5.5 Hz, 2H), 7.88 (dd, J = 8.2, 5.3 Hz,\nwere calculated by using the coloc2 plugin in ImageJ software. 2H), 7.82 (d, J = 5.5 Hz, 2H), 7.76 (d, J = 5.5 Hz, 2H), 7.69\u22127.60\n Sample Preparation for Cellular Fractionation. CT26 cells (m, 20H). 13C NMR (101 MHz, CH3CN-d3) \u03b4: 153.96, 153.52,\nwere seeded at a density of 2 \u00d7 106 in 10 cm plates and incubated for 153.38, 149.96, 149.93, 149.54, 149.43, 148.82, 137.82, 136.66,\n24 h at 37 \u00b0C with 5% of CO2. Cells were treated with 1 \u03bcM 132.01, 130.77, 130.73, 130.55, 130.04, 129.87, 129.06, 127.00,\nconcentrations of compounds 1 and 4. After overnight incubation, 126.94. HRMS (ESI) m/z: [M]2+ calcd for C60H40N6Ru 473.1174.\ncells were washed 2\u00d7 with 5 mL of cold PBS, collected, counted, and Found 473.1161; (error: 0.2 ppm) IR (neat) \u03bdmax: 3053, 1621,1556,\nthe Sigma Mitochondria Isolation Kit protocol (MITOISO2) was 1415, 848 cm\u22121. Anal. calcd For C60H50Cl2N6O5Ru\u00b75H2O: C 65.10,\nfollowed for isolation. The pellet was dried and stored at room H 4.55, N 7.59. Found: C 64.71, H 4.28, N 7.51. HPLC: TR = 15.791\ntemperature. ICP-MS samples were prepared by digestion using 70% min.\nnitric acid (100 \u03bcL for the mitochondria, 600 \u03bcL for nuclei, 60 \u00b0C 1,4,5,8-Tetraazaphenanthrene (TAP). The TAP ligand was\novernight). Samples were then further diluted 1:300 for nuclei and synthesized by the procedure reported in the literature.60 Spectro-\n1:100 for mitochondria (2% HCl solution in MQ water). Finally, the scopic data were in agreement with the literature.60\n 1\nruthenium concentration in the solution was analyzed using a high- H NMR (400 MHz, DMSO-d6) \u03b4 9.23 (d, J = 2.0 Hz, 1H), 9.20\nresolution ICP-MS of an Agilent 7900 quadrupole ICP-MS (d, J = 2.0 Hz, 1H), 8.37 (s, 1H). 13C NMR (101 MHz, DMSO-d6) \u03b4:\ninstrument at the Institut de Physique du Globe de Paris, France. 147.36, 146.14, 143.95, 140.68, 132.00.\nTo calculate the relative amount of metal present in the mitochondria, [Ru(DIP)2(TAP)]Cl2. Ru(DIP)2Cl2 (200 mg, 0.2 mmol) and 1,4,5,8-\n10% of total cell suspension was taken out from each replicate before tetraazaphenanthrene (43.5 mg, 0.2 mmol, 1 equiv) were dissolved in\nfractionation. Then, the amount of metal present in the total cells was DMF (20 mL) and stirred at reflux for 8 h under a N2 atmosphere.\ncompared to the metal in the isolated mitochondrial fraction by The solvent was then removed in vacuum. The residue was purified by\nnormalizing the initial cell number. silica gel column chromatography (CHCl3/CH3OH = 3:1) to obtain\n Mito Stress Test. One \u00d7104 CT26 cells/well was seeded in a an orange powder (104 mg, 42%).\n 1\nSeahorse XF Cell 96-well culture microplate using 80 \u03bcL of F12K H NMR (500 MHz, CH3CN-d3) \u03b4 9.01 (d, J = 2.8 Hz, 1H, Hb),\nmedium supplemented with 10% FBS and incubated for 24 h at 37 \u00b0C 8.63 (s, 1H, Hc), 8.41 (d, J = 2.8 Hz, 1H, Ha), 8.27 (d, J = 5.5 Hz, 1H,\nwith 5% CO2. The day after, the medium was replaced by an equal H1), 8.23 (d, J = 5.5 Hz, 1H, H6), 8.22 (d, J = 0.9 Hz, 2H, H3,4), 7.70\nvolume of compound dissolved in culture media (compound 1, IC25 = (d, J = 5.5 Hz, 1H, H2), 7.68\u22127.58 (m, 11H). 13C NMR (126 MHz,\n0.025 \u03bcM, IC50 = 0.09 \u03bcM; compound 4, IC25 = 0.06 \u03bcM, IC50 = 0.02 CH3CN-d3) \u03b4: 154.26, 153.49, 150.96, 150.29, 149.61, 149.01,\n\u03bcM and PpIX, IC25 = 0.004 \u03bcM, IC50 = 0.01 \u03bcM). After 4 h of 148.96, 146.46, 144.17, 136.56, 136.53, 133.77, 130.80, 130.75,\ntreatment, media was removed, and the treated cells were washed very 130.15, 130.10, 127.15, 127.08. HRMS (ESI) m/z: [M + H]+ calcd\ncarefully with the Seahorse XF medium three times. Cells were for C60H44N8RuH 979.2805. Found 979.2765; [M]2+ found 474.11.\nirradiated for 40 min at 540 nm. Finally, the plates were incubated at IR (neat) \u03bdmax: 3053, 1660, 1621,1557, 1416, 859 cm\u22121. Anal. calcd\n37 \u00b0C for 1 h in a non-CO2 incubator. The Mito stress assay was run For C58H52Cl2F12N8O7Ru\u00b77H2O: C 60.84, H 4.58, N 9.79. Found: C\nin an Agilent Seahorse XFe96 instrument at 37 \u00b0C using multiple 60.78, H 4.21, N 9.84. HPLC: TR = 11.302 min.\ninhibitors, i.e., ATP synthase inhibitor (oligomycin, 1 \u03bcM), proton [RuCl2(phendione)2]. The product was synthesized following a\ngradient, mitochondrial membrane potential collapsing agent (FCCP, reported protocol.52 Spectroscopic data were in agreement with the\n1 \u03bcM), and mitochondrial respiratory complex I and III inhibitors literature.52\n 1\n(rotenone, 1 \u03bcM and antimycin A, 1 \u03bcM, respectively). At the end of H NMR (400 MHz, DMSO-d6) \u03b4 10.11 (d, J = 5.2 Hz, 1H), 8.48\nthe run, the cells were fixed using a 4% p-formaldehyde solution and (d, J = 7.6 Hz, 1H), 8.10 (d, J = 7.6 Hz, 1H), 8.07\u22127.97 (m, 1H),\nstained with Hoechst 33342. Each well was imaged in a Cytation 5 7.77 (d, J = 5.3 Hz, 1H), 7.43\u22127.27 (m, 1H).\nCell Imaging Multimode Reader, BioTek using a 10X objective lens. [Ru(phendione)2(phen)](PF6)2. The product was synthesized\nFinally, the number of cells from each image was calculated by using following a literature procedure.61\n\n 18519 https://doi.org/10.1021/acs.inorgchem.3c02606\n Inorg. Chem. 2023, 62, 18510\u221218523\n\fInorganic Chemistry pubs.acs.org/IC Article\n 1\n H NMR (400 MHz, DMSO-d6) \u03b4 10.12 (d, J = 5.7 Hz, 2H), 8.48\n(d, J = 7.3 Hz, 2H), 8.10 (d, J = 7.9 Hz, 2H), 8.06\u22127.96 (m, 2H),\n7.77 (d, J = 4.9 Hz, 2H), 7.41\u22127.29 (m, 2H). 13C NMR (101 MHz,\n \u25a0\n *\n ASSOCIATED CONTENT\n s\u0131 Supporting Information\n\nCH3CN-d3) \u03b4 208.87 (d, J = 31.1 Hz), 176.00 (d, J = 54.4 Hz), The Supporting Information is available free of charge at\n158.02, 157.31, 154.20, 148.40, 138.81, 137.55, 137.31, 132.33, https://pubs.acs.org/doi/10.1021/acs.inorgchem.3c02606\n131.84, 129.79, 129.26, 127.13. 1\n H and 13C NMR spectra, HPLC chromatogram, and\n [Ru(dppz)2phen](PF6)2. [Ru(phendione)2phen](PF6)2 (90 mg, HRMS (Figures SI1\u2212SI16); crystal data collection,\n0.09 mmol) and o-phenylenediamine (40 mg, 0.36 mmol, 4 equiv)\n refinements, and results (Tables SI1 and SI2); structure\nwere added to a mixture of degassed CH3CN/EtOH (20 mL, 1:3, v/\nv) and stirred at 80 \u00b0C for 20 h under a N2 atmosphere. The reaction of the compounds with proton enumeration (Figure\nmixture was concentrated to ca. 10 mL and cooled to room SI17); crystal structure showing \u03c0\u2212\u03c0 stacking inter-\ntemperature, and then an aqueous saturated NH4PF6 solution (15 actions between the ligands (Figures SI18 and SI19);\nmL) was added. A red precipitate was formed, and the mixture was luminescence spectra (Figure SI20); luminescence\nkept at 4 \u00b0C overnight to allow the complete precipitation. The red decays (Figure SI21 and Table SI3); luminescence\nproduct was collected by vacuum filtration, washed with distilled lifetime (Table SI3); singlet oxygen emission spectra\nwater and ice-cold ethanol, and dried with diethyl ether. The product (Figure SI22); photobleaching studies by UV (Figure\nwas then redissolved in CH3CN and dried in vacuo (aspect: red SI23); stability studies by 1H NMR in DMSO of\npowder, 88 mg, 82%). The PF6\u2212 counterion was exchanged by using compounds 1\u22124 at 37 \u00b0C (Figures SI24\u2212SI27); IC50\nAmberlite IRA402 chloride form resin. The compound was dissolved plots of the compounds at different wavelengths\nin 1 mL of MeCN, and 5 mL of MeOH was added. The resin was\n (Figures SI28\u2212SI39); cellular uptake graphs (Figure\nadded and mixed by rotation for 5 h. After cotton filtration, the\ncompound was precipitated in pentane to remove grease traces from SI40); and CD melting profile of (CCGGCGCCGG)2\nthe resin and dried under vacuum. and d(CCGGTACCGG)2 with 3 (1:0, 1:1, and 1:3\n Crystal data for C192H112F48N40P8Ru4 (M = 4542.24 g/mol): ratio) (Figures SI41\u2212SI46) (PDF)\nmonoclinic, space group P21/n (no. 14), a = 21.8071(12) \u00c5, b =\n30.1739(17) \u00c5, c = 28.475(2) \u00c5, \u03b2 = 103.157(7)\u00b0, V = 18245(2) \u00c53,\nZ = 4, T = 293(2) K, \u03bc(Mo K\u03b1) = 0.512 mm\u22121, Dcalcd = 1.654 g/cm3,\n603650, reflections measured (3.99 \u2264 2\u0398 \u2264 46.514\u00b0), 26192 unique\n \u25a0 AUTHOR INFORMATION\n Corresponding Author\n(Rint = 0.4362, Rsigma = 0.0989), which were used in all calculations. Gilles Gasser \u2212 Chimie ParisTech, PSL University, CNRS,\nThe final R1 was 0.1178 (I > 2\u03c3(I)), and wR2 was 0.3693 (all data). Institute of Chemistry for Life and Health, Paris 75005,\nThe coordinates have been deposited in the Cambridge Crystallo- France; orcid.org/0000-0002-4244-5097;\ngraphic Data Centre (CCDC), deposition number no. 2277028. 1H Email: gilles.gasser@chimieparistech.psl.eu,\nNMR (400 MHz, CH3CN-d3) \u03b4 9.67 (ddd, J = 8.3, 5.4, 1.3 Hz, 4H, www.gassergroup.com\nH3 or 4), 8.65 (dd, J = 8.3, 1.3 Hz, 2H, Hc), 8.52\u22128.46 (m, 4H,\nH7 or 10), 8.31 (dd, J = 5.4, 1.3 Hz, 2H, H1), 8.29 (s, 2H, Hd), 8.22 Authors\n(dd, J = 5.3, 1.2 Hz, 2H, Ha), 8.17\u22128.13 (m, 4H, H8 or 9), 8.11 (dd, J Maria Dalla Pozza \u2212 Chimie ParisTech, PSL University,\n= 5.4, 1.3 Hz, 2H, H6), 7.83 (dd, J = 8.2, 5.4 Hz, 2H, H2), 7.81 (ddd, J CNRS, Institute of Chemistry for Life and Health, Paris\n= 12.8, 8.3, 5.4 Hz, 4H, H5), 7.68 (dd, J = 8.3, 5.3 Hz, 2H, Hb). 13C\n 75005, France\nNMR (101 MHz, CH3CN-d3-d3) \u03b4: 155.46, 155.21, 154.23, 151.85,\n151.77, 148.78, 143.79, 141.06, 138.13, 134.66, 134.61, 133.56, Pierre Mesdom \u2212 Chimie ParisTech, PSL University, CNRS,\n132.14, 131.88, 130.65, 129.14, 128.31, 128.25, 126.98. HRMS (ESI) Institute of Chemistry for Life and Health, Paris 75005,\nm/z: [M]2+ calcd for C48H28N10Ru 423.07. Found 423.07. IR (neat) France\n\u03bdmax: 3097, 1617, 1546, 1421, 1358, 843 cm\u22121. HPLC: TR = 10.734 Ahmad Abdullrahman \u2212 Department of Pharmacy, Chemistry\nmin. and Pharmacy Building, University of Reading, Berkshire\n [Ru(dppn)2phen](PF6)2. [Ru(phendione)2phen](PF6)2 (113 mg, RG6 6AD, U.K.\n0.11 mmol, 1 equiv) and 2,3-diaminonaphthalene (72 mg, 0.46 mmol, Tayler D. Prieto Otoya \u2212 Department of Chemistry,\n4 equiv) were suspended in degassed CH3CN/EtOH (25 mL, 1:3, v/ University of Reading, Reading RG6 6AD, U.K.\nv) and stirred at 80 \u00b0C for 20 h under a N2 atmosphere. The reaction Philippe Arnoux \u2212 Universit\u00e9 de Lorraine, CNRS, LRGP,\nmixture was concentrated in vacuo to ca. 10 mL and cooled to room Nancy F-54000, France\ntemperature, followed by the addition of an aqueous saturated\n C\u00e9line Frochot \u2212 Universit\u00e9 de Lorraine, CNRS, LRGP,\nNH4PF6 solution (15 mL). The formation of a dark red precipitate\nwas observed. The mixture was kept at 4 \u00b0C overnight to allow\n Nancy F-54000, France; orcid.org/0000-0002-7659-\ncomplete precipitation. The brown product was collected by vacuum 3864\nfiltration, washed with distilled water and ice-cold ethanol, and dried Germain Niogret \u2212 Institut Pasteur, Universit\u00e9 Paris Cit\u00e9,\nwith diethyl ether. The product redissolved in acetonitrile and dried in CNRS UMR3523, Departement of Structural Biology and\nvacuo (aspect: dark red powder, 106 mg, 55%). Chemistry, Laboratory for Bioorganic Chemistry of Nucleic\n 1\n H NMR (400 MHz, CH3CN-d3) \u03b4 9.68 (ddd, J = 9.5, 8.2, 1.4 Hz, Acids, Paris 75015, France\n2H, H3 or 6), 9.15 (s, 2H, H7 or 12), 8.66 (dd, J = 8.3, 1.3 Hz, 1H, Hc), Bruno Saubam\u00e9a \u2212 Universit\u00e9 Paris Cit\u00e9, INSERM, CNRS, P-\n8.42\u22128.35 (m, 2H, H8 or 11), 8.33 (dd, J = 5.4, 1.3 Hz, 1H, H1 or 4), MIM, Plateforme d\u2019Imagerie Cellulaire et Mol\u00e9culaire\n8.30 (s, 1H, Hd), 8.27 (dd, J = 5.3, 1.3 Hz, 1H, Ha), 8.08 (dd, J = 5.4, (PICMO), Paris F-75006, France\n1.3 Hz, 1H, H1 or 4), 7.84 (dd, J = 8.2, 5.5 Hz, 1H, H2 or 5), 7.81\u22127.74 Pierre Burckel \u2212 Universit\u00e9 de Paris, Institut de physique du\n(m, 3H, H9 or 10), 7.71 (dd, J = 8.3, 5.3 Hz, 1H, Hb). 13C NMR (126\n globe de Paris, CNRS, Paris F-75005, France\nMHz, CH3CN-d3) \u03b4: 155.56, 155.26, 154.29, 152.49, 152.40, 148.78,\n141.94, 139.73, 138.19, 136.27, 134.79, 134.72, 132.26, 132.18, James P. Hall \u2212 Department of Pharmacy, Chemistry and\n129.64, 129.34, 129.16, 128.50, 128.46, 127.03. HRMS (ESI) m/z: Pharmacy Building, University of Reading, Berkshire RG6\n[M]2+ calcd for C60H40N6Ru 473.0922. Found 473.0926; (error: 0.2 6AD, U.K.; orcid.org/0000-0003-3716-4378\nppm) IR (neat) \u03bdmax: 3088, 1632,1515, 1419, 1357, 842 cm\u22121. Anal. Marcel Hollenstein \u2212 Institut Pasteur, Universit\u00e9 Paris Cit\u00e9,\ncalcd for C56H32F12N10P2Ru\u00b74H2O: C 51.42, H 3.08, N 10.71. Found: CNRS UMR3523, Departement of Structural Biology and\nC 51.18, H 2.88, N 10.16. HPLC: TR = 11.689 min. Chemistry, Laboratory for Bioorganic Chemistry of Nucleic\n 18520 https://doi.org/10.1021/acs.inorgchem.3c02606\n Inorg. Chem. 2023, 62, 18510\u221218523\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n Acids, Paris 75015, France; orcid.org/0000-0003-0263- Two-Photon Photodynamic Anticancer Agents. Biomaterials 2015, 56,\n 9206 140\u2212153.\n Christine J. Cardin \u2212 Department of Chemistry, University of (7) Li, G.; Sun, L.; Ji, L.; Chao, H. Ruthenium(II) Complexes with\n Reading, Reading RG6 6AD, U.K.; orcid.org/0000-0002- Dppz: From Molecular Photoswitch to Biological Applications. Dalton\n Trans. 2016, 45 (34), 13261\u221213276.\n 2556-9995\n (8) Tang, S.-J.; Wang, M.; Yang, R.; Liu, M.; Li, Q.; Gao, F. More-Is-\nComplete contact information is available at: Better Strategy for Constructing Homoligand Polypyridyl Ruthenium\nhttps://pubs.acs.org/10.1021/acs.inorgchem.3c02606 Complexes as Photosensitizers for Infrared Two-Photon Photo-\n dynamic Therapy. Inorg. Chem. 2023, 62 (21), 8210\u22128218.\nNotes (9) Wachter, E.; Heidary, D. K.; Howerton, B. S.; Parkin, S.; Glazer,\n E. C. Light-Activated Ruthenium Complexes Photobind DNA and\nThe authors declare no competing financial interest.\n Are Cytotoxic in the Photodynamic Therapy Window W. Chem.\n\n\u25a0 ACKNOWLEDGMENTS\nThe authors are grateful for financial support from the\n Commun. 2012, 48, 9649\u22129651.\n (10) Karges, J.; Kuang, S.; Maschietto, F.; Blacque, O.; Ciofini, I.;\n Chao, H.; Gasser, G. Rationally Designed Ruthenium Complexes for\nEuropean Union\u2019s Horizon 2020 research and innovation 1- and 2-Photon Photodynamic Therapy. Nat. Commun. 2020, 11 (1),\n No. 3262.\nprogramme (Marie Sk\u0142odowska-Curie grant agreement No. (11) Karges, J.; Heinemann, F.; Jakubaszek, M.; Maschietto, F.;\n861381), the ERC Consolidator Grant PhotoMedMet to G.G. Subecz, C.; Dotou, M.; Vinck, R.; Blacque, O.; Tharaud, M.; Goud,\n(GA 681679), and the program \u201cInvestissements d\u2019 Avenir\u201d \u0301\n B.; Vin\u0303uelas Zahlnos, E.; Spingler, B.; Ciofini, I.; Gasser, G. Rationally\nlaunched by the French Government and implemented by the Designed Long-Wavelength Absorbing Ru(II) Polypyridyl Complexes\nANR with the reference ANR-10-IDEX-0001-02 PSL (G.G.). as Photosensitizers for Photodynamic Therapy. J. Am. Chem. Soc.\nThis work was carried out with the support of Diamond Light 2020, 142 (14), 6578\u22126587.\nSource, instrument B23 (proposal number SM30390). Part of (12) Heinemann, F.; Karges, J.; Gasser, G. Critical Overview of the\nthe ICP-MS analyses was supported by IPGP multidisciplinary Use of Ru(II) Polypyridyl Complexes as Photosensitizers in One-\nprogram PARI and by Paris-IdF region SESAME Grant no. Photon and Two-Photon Photodynamic Therapy. Acc. Chem. Res.\n12015908. 2017, 50, 2727\u22122736.\n (13) Mari, C.; Pierroz, V.; Rubbiani, R.; Patra, M.; Hess, J.; Spingler,\n\n\u25a0 ABBREVIATIONS\nPDT, photodynamic therapy; PS, photosensitizer; DIP, 4,7-\n B.; Oehninger, L.; Schur, J.; Ott, I.; Salassa, L.; Ferrari, S.; Gasser, G.\n DNA Intercalating RuII Polypyridyl Complexes as Effective Photo-\n sensitizers in Photodynamic Therapy. Chem. - Eur. J. 2014, 20 (44),\ndiphenyl-1,10-phenanthroline; dppn, benzo[i]dipyrido[3,2- 14421\u221214436.\na:2\u2032,3\u2032-c]phenazine; dppz, dipyrido[3,2-a:2\u2032,3\u2032-c]phenazine; (14) Gandosio, A.; Purkait, K.; Gasser, G. Recent Approaches\n towards the Development of Ru(II) Polypyridyl Complexes for\nTAP, 1,4,5,8-tetraazaphenanthrene; phen, 1,10-phenanthro-\n Anticancer Photodynamic Therapy. Chimia 2021, 75 (10), 845\u2212855.\nline; phendione, 1,10-phenanthroline-5,6-dione; MLCT, (15) Chamberlain, S.; Cole, H. 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ChemBioChem 2020, 21,\n2966\u22122973.\n\n\n\n\n 18523 https://doi.org/10.1021/acs.inorgchem.3c02606\n Inorg. Chem. 2023, 62, 18510\u221218523\n\f", "pages_extracted": 14, "text_length": 108975}