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In-cell Catalysis by Tethered Organo-Osmium Complexes Generates Selectivity for Breast Cancer Cells.
{"full_text": " Research Article\nChemBioChem doi.org/10.1002/cbic.202400374\n\n www.chembiochem.org\n\n\n In-cell Catalysis by Tethered Organo Osmium Complexes\n Generates Selectivity for Breast Cancer Cells\n J. P. C. Coverdale,*[a, b] R. A. Bedford,[a] O. W. L. Carter,[b] S. Cao,[a] M. Wills,[b] and P. J. Sadler[b]\n\n Anticancer agents that exhibit catalytic mechanisms of action bound in the extracellular medium, while cellular accumulation\n offer a unique multi-targeting strategy to overcome drug studies identified an energy-dependent, protein-mediated Os\n resistance. Nonetheless, many in-cell catalysts in development accumulation pathway, consistent with albumin-mediated up-\n are hindered by deactivation by endogenous nucleophiles. We take. Importantly, the tethered Os complex was active for in-cell\n have synthesised a highly potent, stable Os-based 16-electron transfer hydrogenation catalysis, initiated by co-administration\n half-sandwich (\u2018piano stool\u2019) catalyst by introducing a perma- of a non-toxic dose of sodium formate as a source of hydride,\n nent covalent tether between the arene and chelated diamine indicating that the Os catalyst is delivered to the cytosol of\n ligand. This catalyst exhibits antiproliferative activity compara- cancer cells intact. The mechanism of action involves the\n ble to the clinical drug cisplatin towards triple-negative breast generation of reactive oxygen species (ROS), thus exploiting the\n cancer cells and can overcome tamoxifen resistance. Speciation inherent redox vulnerability of cancer cells, accompanied by\n experiments revealed Os to be almost exclusively albumin- selectivity for cancerous cells over non-tumorigenic cells.\n\n\n Introduction catalytic oxidation of biomolecules; for which various half-\n sandwich organometallic catalysts of Rh, Ru, Ir, and Os have\n Catalysts lower activation energies and increase rates of been described.[5] Those bearing iminopyridine or azopyridine\n reactions that would otherwise take place very slowly, if at all. bidentate ligands can achieve high potencies towards cancer\n In nature, enzymes (mainly proteins but also some ribonucleic cells, involving the catalytic generation of reactive oxygen\n acids) carry out biological transformations, and around a third species, and the catalytic oxidation of NADH to NAD +.[6]\n are metalloenzymes.[1] These biological catalysts contain metal Transition metal complexes can also catalyse transfer hydro-\n centres which play important roles in the overall mechanism of genation (reduction) reactions in the presence of a suitable\n action. This has inspired the design of artificial enzymes and hydride donor (commonly sodium formate).[2d,7] We have\n small molecule catalysts, and their possible use as anticancer previously reported a new class of 16-electron Os arene\n agents has recently gained significant attention.[2] For example, complexes with the general formula [Os(arene)(diamine)], which\n Pd-mediated bioorthogonal activation of biochemically stable can act as in-cell transfer hydrogenation (TH) catalysts,[8] and\n prodrugs has been described for the in vitro activation of can achieve the enantioselective reduction of pyruvate to\n precursors of 5-fluorouracil and gemcitabine to their active unnatural D-lactate in cancer cells (Figure 1, Os catalyst 4).[9] To\n forms.[3] A similar strategy was employed for the in-cell catalytic improve the efficiency of the catalytic activity in cells, a more\n activation of allyl carbamate protected amines to liberate comprehensive understanding of their distribution and their\n deprotected doxorubicin using a Ru catalyst, achieving excel- stability is required. Inductively coupled plasma mass spectrom-\n lent bioorthogonal specificity and high substrate turnover, even\n in the presence of millimolar concentrations of thiols.[4] Another\n approach to achieve catalytic anticancer activity involves the\n\n\n [a] Dr. J. P. C. Coverdale, R. A. Bedford, S. Cao\n School of Pharmacy, Institute of Clinical Sciences, College of Medical and\n Dental Sciences\n University of Birmingham\n Edgbaston, B15 2TT, UK\n E-mail: j.p.coverdale@bham.ac.uk\n [b] Dr. J. P. C. Coverdale, Dr. O. W. L. Carter, Prof. M. Wills, Prof. P. J. Sadler\n Department of Chemistry\n University of Warwick\n Coventry, CV4 7AL, UK\n Supporting information for this article is available on the WWW under\n Figure 1. The structure of tethered Os(II) catalyst 5 studied in this work.\n https://doi.org/10.1002/cbic.202400374\n Catalyst 4 and an 18-electron reversible-tethered Os(II) complex (left) have\n \u00a9 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH. This is been previously reported to carry out the in-cell catalytic reduction of\n an open access article under the terms of the Creative Commons Attribution pyruvate to lactate.[9] Structurally similar Ru(II) N-\n License, which permits use, distribution and reproduction in any medium, tosyl diphenylethylenediamine (TsDPEN) tethered and non-tethered com-\n provided the original work is properly cited. plexes (right) are known to be active for transfer hydrogenation catalysis.[7]\n\n\n ChemBioChem 2024, 25, e202400374 (1 of 11) \u00a9 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH\n\f 14397633, 2024, 15, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cbic.202400374 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nChemBioChem doi.org/10.1002/cbic.202400374\n\n\n etry (ICP-MS) and synchrotron X-ray fluorescence (XRF) elemen- for the synthesis of untethered Os complex 4 from TsDPEN and\n tal mapping experiments have identified an intracellular catalyst [OsCl2(p-cymene)]2 dimer (Scheme 1).[8a]\n decomposition pathway involving loss of the diamine.[10] To understand how the inclusion of a 3-carbon tether would\n Deactivation by loss of the arene has also been reported for affect the geometry of the pseudo-planar 16-electron species,\n Ru(II) analogues.[11] Such a loss of a chelated ligand would be density functional theory calculations of complexes 4 (non-\n expected to lead to loss of catalytic activity. Hence, structural tethered) and 5 (tethered) were performed using Gaussian 16\n modifications are being explored to design organo metallic (Figure 2).[20]\n catalysts with improved stability in intracellular media. One Molecular structures were generated using GaussView 6.0\n strategy involves the inclusion of the active metal complex based on previously reported crystallographic data for complex\n inside a protective protein scaffold, analogous to that of a 4 and a structurally similar tethered ruthenium(II) TsDPEN\n natural metalloenzyme.[12] Nanoscale formulations have also complex (CCDC 273937).[8a,21] Geometries were optimized using\n been explored to improve tumor cell delivery and selectivity.[13] the hybrid Perdew-Burke-Ernzerhof functional (PBE0) with the\n Alternatively, the metal complex could be stabilized by Lanl2DZ (including effective core potential for Os) and 6-31 +\n introduction of a covalent tether between the coordinated G** (all other atoms) basis sets. Hessian analysis of the\n arene and the bidentate ligand, exploiting the chelate effect to optimized structures showed no imaginary frequencies, con-\n improve complex stability. Complexes bearing a pH-sensitive firming that the structures represented true energy minima.\n reversible tether between the \u03b75 (Ir) or \u03b76 (Ru or Os) ligand and Os N bond lengths were highly comparable between both\n the monodentate coordination site have been shown to structures (Os N(Ts): 2.01 \u00c5 for both complexes; Os N: 1.91 \u00c5\n improve aqueous solution stability and can catalyse the in-cell\n reduction of pyruvate to lactate (Figure 1),[14] however, this\n reversible strategy does not address loss of the bidentate\n ligand. Ru(II) complexes bearing an irreversible (permanent)\n tether between the \u03b76-arene and the bidentate diamine ligand\n are well known (Figure 1, Ru TsDPEN catalysts).[15] Some\n examples of tethered catalysts have been explored for in-cell\n catalysis and were shown to modulate catalytically the intra-\n cellular NADH/NAD + ratio.[16] Non-tethered Os(II) TsDPEN cata-\n lysts are known,[8a,9] but arene diamine-tethered Os(II) ana-\n logues of the aforementioned Ru(II) TsDPEN catalysts have not\n been synthesised previously (Figure 1).\n Here, the first example of a covalently-tethered Os(II)\n complex is described, which can catalyse in-cell transfer hydro-\n genation reactions using formate as a H source. Its extracellular Scheme 1. Synthetic routes to either Os(II) arene complex 4 (non-tethered)\n or 5 (tethered) from chirally pure N-tosyl-1,2-diphenylethylenediamine\n speciation was studied by LC-ICP-MS to investigate the stability (TsDPEN). Only (R,R)-configured enantiomers were prepared. Complex 4 was\n of the complex, as well as interactions with serum proteins obtained via a one-pot biphasic reaction similar to a previous report.[8a]\n which might play a role in its cellular accumulation. Complex 5 first requires conversion of TsDPEN to tethered ligand 1, which is\n subsequently reacted with OsCl3 hydrate under microwave conditions to\n afford tethered dimer complex 3. Conversion of 3 to afford 5 proceeds via a\n similar one-pot biphasic reaction.\n Results and Discussion\n\n Initial efforts to synthesize a tethered Os(II) complex via an\n arene-exchange mechanism, as is routinely used for the syn-\n thesis of analogous Ru(II) complexes, proved ineffective.[17]\n Instead, tethered Os complexes were prepared from 3-\n (cyclohexadienyl)propan-1-ol, which was first converted to the\n corresponding triflate and then reacted with TsDPEN to afford\n tethered ligand 1 via a previously reported synthesis.[18]\n However, formation of the Os dimer, complex 3, was unsuccess-\n ful using conventional reflux conditions. Nonetheless, dimer 3\n was successfully obtained using microwave reaction conditions\n using OsCl3 hydrate, as reported for similar metal arene dimer\n complexes.[14c,19] Crucially, Os dimer 3 was prepared under acidic\n conditions to afford its amine salt and prevent premature Figure 2. Density functional theory (DFT) calculations of the structure,\n coordination of the diamine ligand to the metal centre. electrostatic potential (mapped onto total electron density, 0.10 to + 0.30),\n Conversion of dimer 3 proceeded via a one-pot biphasic and HOMO/LUMO molecular orbitals for Os complexes 4 (non-tethered) and\n 5 (tethered) using the hybrid Perdew-Burke-Ernzerhof functional (PBE0) with\n reaction in dichloromethane/water with potassium hydroxide the Lanl2DZ (including effective core potential for Os) and 6-31 + G** (all\n gave complex 5 as a red crystalline solid, as similarly reported other atoms) basis sets, using GaussView 6.0 and Gaussian 16.[20]\n\n\n ChemBioChem 2024, 25, e202400374 (2 of 11) \u00a9 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH\n\f 14397633, 2024, 15, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cbic.202400374 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nChemBioChem doi.org/10.1002/cbic.202400374\n\n\n for complex 4, 1.92 \u00c5 for complex 5), and bond angles were investigated to assess its suitability for in-cell catalysis.\n calculated in the carbon tether (ranging from 113.1\u2013115.5\u00b0) are Aqueous solutions of 5 were prepared using 5 % v/v DMSO to\n comparable to those determined experimentally in a structur- aid solubility. Data obtained using UV-visible spectroscopy\n ally similar Ru(II) 3-carbon tethered complex (112.5\u2013115.2\u00b0, showed that complex 5 remained highly stable in cell culture\n CCDC 273937).[21] Electrostatic potential surfaces (EPS) showed medium (supplemented with 10 % foetal calf serum) and\n similar charge distributions between complexes 4 and 5, human serum (10 % v/v prepared in DPBS) in the presence of\n however, the calculated geometries revealed that the presence 5 % v/v DMSO over a 24 h period (Supporting Information,\n of an arene diamine tether may hinder accessibility of the Figure S2). Speciation was subsequently investigated by devel-\n nitrogen atom for transfer hydrogenation. The critical impor- oping an offline LC-ICP-MS methodology, employing a TSKgel\n tance of the N H bond in TsDPEN-bearing catalysts for Q-STAT anion exchange column with an ammonium acetate/\n asymmetric transfer hydrogenation (ATH) is well known; similar Tris binary mobile phase at physiological pH to quantify\n Ru(II) tethered diamine arene catalysts have been shown to interactions between the Os catalyst and serum proteins\n maintain catalytic activity for ATH reactions while Ru(II) (Figure 3). Extracellular speciation of 5 was determined in the\n complexes containing two alkyl groups on the non-tosylated presence of bovine serum albumin (BSA; 0.6 mM in DPBS) or\n nitrogen atom are particularly poor catalysts.[22] Frontier molec- foetal bovine serum to replicate cell culture experimental\n ular orbitals calculated for complexes 4 and 5 were also conditions, and additionally for comparison using human\n comparable (calculated HOMO-LUMO energy gaps of serum. Analysis of BSA incubated with complex 5 (Figure 3a)\n 4.13885 eV and 4.0289 eV respectively) emphasizing the mar- demonstrated Os to be almost exclusively BSA-bound (tR = 10\u2013\n ginal impact of tether introduction on the overall complex. 13 min), while speciation measurements using FBS (Figure 3b)\n The catalytic activity of tethered Os(II) complex 5 was and human serum (Figure 3c) revealed the major Os-containing\n determined for transfer hydrogenation from formic acid to fractions (ca. 94\u201395 % of total Os) to also coincide with the\n acetophenone, a common test substrate for transfer hydro- retention time of albumin, with the remaining Os (ca. 5\u20136 %)\n genation catalyst development (Table 1). Using a 0.5 mol% recovered in the flow-through fractions (tR = 0\u20133 min). Good Os\n catalyst loading at 310 K, maximal turnover frequencies (TOFmax) recovery was achieved under all conditions (101\u2013105 %).\n were determined using time-dependent 1H NMR spectroscopy Retention times for two abundant metalloproteins were also\n by evaluating the formation of a new quartet resonance for 2- determined using single protein standards (human transferrin:\n phenylethanol and a decrease in intensity of the singlet methyl tR = 6\u20138 min, human albumin: tR = 10\u201312 min, Figure 3d). Impor-\n resonance of acetophenone. For comparison, TOFmax values tantly, the high abundance of albumin in serum (35\u201350 g \u00b7 L 1\n were also determined for non-tethered Os complex 4. The physiological concentration) also means albumin is readily\n measured TOFmax for tethered Os complex 5 (20 \ufffd 1 h 1) was available to bind Os.[25] Although LC-ICP-MS studies detect only\n significantly lower than that of non-tethered Os complex 4 Os protein binding (and not that of complex 5 specifically),\n (63 \ufffd 2 h 1, > 99 % conversion achieved within 24 h), which albumin has been shown to bind other metallodrugs, including\n correlates with the steric hindrance predicted from DFT two clinically trialed Ru(III) anticancer complexes, NAMI A and\n calculations. In contrast, the introduction of a covalent tether KP1019/NKP1339, which retain biological activity after albumin\n between the arene and diamine ligands in structurally similar binding.[26]\n Ru(II) arene complexes has been found to significantly enhance Antiproliferative activities (IC50/\u03bcM) of Os complexes 4 and 5,\n the rate of catalytic reduction of acetophenone.[15a] The origin of and the clinically used Pt drug, cisplatin were next determined\n this difference between analogous Ru and Os tethered/non- towards seven human cell lines: A2780 ovarian cancer cells,\n tethered complexes remains unclear, but nonetheless highlights A549 lung cancer cells, HCT-116 colorectal cancer cells, MCF7\n how the metal centre can have a significant impact on breast cancer cells, MCF7 TAMR-1 tamoxifen-resistant breast\n physicochemical properties. cancer cells, MCF10-A non-tumorigenic breast cells, and\n With the knowledge that tethered Os catalyst 5 is active in MDA MB-231 triple negative breast cancer (TNBC) cells (Ta-\n an organic phase system (5 : 2 formic acid triethylamine ble 2). Tethered Os complex 5 was more potent than non-\n azeotrope), the stability and speciation of 5 in aqueous media tethered complex 4 and exhibited activity comparable to\n cisplatin towards breast cancer cell lines. The greater potency of\n the tethered complex compared to its non-tethered counterpart\n Table 1. Reduction of acetophenone, a model substrate for transfer may be related to the greater structural stability offered by\n hydrogenation catalysis, using either non-tethered Os 16-electron sulfona- covalently linking the tosyl diamine and \u03b76-arene, afforded by\n mide catalyst 4 or tethered Os 16-electron sulfonamide analogue 5\n (0.5 mol%) in the presence of formic acid triethylamine azeotrope (source\n the chelate effect. De-coordination of the 1,3-5-trimethylphenyl\n of hydride). Maximum turnover frequencies (TOFmax) were determined arene in structurally similar TH complexes has been described\n using time-dependent 1H NMR spectroscopy (400 MHz, C6D6, 310 K). as being consistent with increased stability observed for of Ru\n Catalyst S/C[a] Temp/K Tether TOFmax/h 1 tethered-arene catalysts.[11] Furthermore, we have previously\n 4 200 310 No 63 \ufffd 2\n shown that the release of a non-tethered diamine from Os\n coordination in the presence of endogenous thiols occurs inside\n 5 200 310 Yes 20 \ufffd 1\n cell lysosomes.[10] However, other factors such as uptake and\n [a] S/C substrate to catalyst ratio (S/C = 200 equivalent to 0.5 mol% distribution within the cell may also be important and warrant\n catalyst loading).\n further investigation.\n\n\n ChemBioChem 2024, 25, e202400374 (3 of 11) \u00a9 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH\n\f 14397633, 2024, 15, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cbic.202400374 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nChemBioChem doi.org/10.1002/cbic.202400374\n\n\n\n\n Figure 3. Extracellular speciation of tethered osmium catalyst 5 (15 \u03bcM, 310 K, 24 h), determined using offline LC-ICP-MS fitted with an anion exchange\n column (TSKgel Q-STAT). (a) Bovine serum albumin (BSA) incubated with 5 for 1 h. (b) Foetal bovine serum (FBS) incubated with 5 for 1 h. (c) Human serum\n incubated with 5 for 1 h. Recovery of Os by offline LC-ICP-MS speciation: BSA = 104.2 %, FBS = 101.6 %, human serum = 103.1 %. (d) UV-visible chromatograms\n (absorbance at 280 nm, referenced to 360 nm) of single protein standards for human holo-transferrin (tR = 6\u20138 min) and human serum albumin (tR = 10\u2013\n 13 min) alongside human serum.\n\n\n\n Table 2. Antiproliferative activities (IC50/\u03bcM) determined for Os complexes 4 and 5, cisplatin, and tamoxifen towards seven human cell lines: A2780 (ovarian\n carcinoma), A549 (lung carcinoma), HCT-116 (colorectal carcinoma), MCF7 (breast carcinoma; ER + , PR + HER2 ),[23] MCF7 TAMR-1 (tamoxifen-resistant\n breast carcinoma), MCF10-A (non-tumorigenic breast cells) and MDA MB-231 (breast carcinoma; ER-, PR- HER2-).[23] Cells were treated for 24 h and allowed\n 72 h recovery time in drug-free medium. Cell viability was determined using the SRB assay.[24] N.D. = not determined.\n Antiproliferative activity/\u03bcM\n Cell line Description Complex 4 Complex 5 Cisplatin Tamoxifen\n\n A2780 Ovarian 15.5 \ufffd 0.5 10.5 \ufffd 0.1 1.2 \ufffd 0.3 12.4 \ufffd 0.1\n A549 Lung 21.1 \ufffd 0.3 14.1 \ufffd 0.3 3.2 \ufffd 0.1 13.5 \ufffd 0.1\n HCT-116 Colorectal 37 \ufffd 1 36.4 \ufffd 0.2 5.2 \ufffd 0.3 19 \ufffd 3\n MCF7 Breast (ER + , PR + HER2 ) 11.0 \ufffd 0.3 8.1 \ufffd 0.2 6.6 \ufffd 0.2 5.9 \ufffd 0.4\n MCF7 TAMR-1 Breast (tamoxifen-resistant) N.D. 9\ufffd1 6.0 \ufffd 0.7 14.5 \ufffd 0.1\n MCF10-A Breast (non-tumorigenic) N.D. 30.9 \ufffd 0.4 6\ufffd1 25.0 \ufffd 0.2\n MDA MB-231 Breast (ER-, PR- HER2-) 15 \ufffd 1 9.1 \ufffd 0.9 9.6 \ufffd 0.4 12.9 \ufffd 0.2\n\n\n\n\n The activity of 5 was similar towards tamoxifen-resistant recurrence, metastases, and poor clinical prognosis,[28] and are\n MCF7 TAMR-1 breast cancer cells compared to tamoxifen- considered the most aggressive subtype of breast cancers.[27]\n sensitive MCF7 cells (P = 0.52), while a 2.4-fold activity decrease Though platinum therapies are effective in neoadjuvant chemo-\n was observed for cells treated with tamoxifen (Table 2) therapy regimens toward TNBCs,[29] they are associated with\n confirming the MCF7 TAMR-1 resistance phenotype. Interest- increased hematological toxicity.[30] As such, the ability of 5 to\n ingly, 5 also displayed excellent activity towards MDA MB-231 overcome drug resistance and maintain activity towards\n TNBC cells, with potency comparable to that of cisplatin (IC50 = MCF7 TAMR-1 tamoxifen-resistant cells and MDA MB-231\n 9.1 \ufffd 0.9 \u03bcM for 5, compared to 9.6 \ufffd 0.4 \u03bcM for cisplatin, TNBC cells is highly promising. Encouragingly, 5 was signifi-\n Table 2). TNBCs lack expression of oestrogen (ER), progesterone cantly also less toxic towards non-tumorigenic breast cells\n (PR), and human epidermal growth factor receptor 2 (HER2) (MCF10-A, IC50 = 30.9 \ufffd 0.4 \u03bcM) compared to breast cancer cells\n receptors,[27] and are associated with a high incidence of (MCF7, MCF7 TAMR-1, MDA MB-231, IC50 8.1\u20139.1 \u03bcM). This was\n\n\n ChemBioChem 2024, 25, e202400374 (4 of 11) \u00a9 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH\n\f 14397633, 2024, 15, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cbic.202400374 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nChemBioChem doi.org/10.1002/cbic.202400374\n\n\n not the case for cisplatin, which exhibited similar potency tent treatment was found to be ca. 2.5-fold greater for cells\n towards cancerous and non-cancerous breast cells (6.6 \ufffd 0.2 \u03bcM treated with 4 (complex 4: 33 \ufffd 2 fg \u00b7 cell 1, complex 5: 13.1 \ufffd\n and 6 \ufffd 1 \u03bcM toward MCF7 and MCF10-A cells, respectively). 0.5 fg \u00b7 cell 1). In addition, to replicate the experimental con-\n The zebrafish embryo model has been shown to successfully ditions used during antiproliferative activity studies, cells were\n and reliably predict drug toxicity in humans,[31] supported by also exposed to complex 5 for 24 h and then further incubated\n correlations in cardiotoxicity, hepatotoxicity and nephrotoxicity for 72 h in Os-free medium (\u2018recovery time\u2019). After 72 h, over ca.\n between zebrafish and humans.[32] The genomes of zebrafish 95 % of the maximal Os accumulated (t = 6 h) was no longer\n and humans are highly comparable,[33] with around 75 % of present.\n human genes having at least one zebrafish orthologue.[34] Acute To explore the mechanism of cellular efflux, experiments\n in vivo toxicities of complex 5 and cisplatin were determined were repeated in the presence of 20 \u03bcM verapamil (a selective\n according to OECD Test 236, where 5 was found to exhibit 9\u00d7 inhibitor of the P-gp efflux transporter) in the recovery medium\n less acute toxicity towards zebrafish embryos compared to (Figure 4a, dashed line).[37] The presence of verapamil did\n cisplatin (Complex 5 LC50 = 5.2 \ufffd 0.3 \u03bcM; cisplatin LC50 = 0.6 \ufffd increase the intracellular Os concentration (P < 0.001) relative to\n 0.2 \u03bcM), supporting its suitability for future therapeutic develop- the verapamil-free control, yet the majority of Os was still\n ment. exported after 72 h recovery. As such, the contribution of P-gp\n Focusing on the specific application of tethered complex 5 to the overall efflux of 5 is likely minor, which was also\n in breast cancer cells, metal accumulation studies were carried observed previously in A2780 cells using complex 4.[9]\n out using MCF7 cells treated with 5 (fixed concentration equal Cellular accumulation studies were repeated in MDA\u2013MB-\n to 1\u00d7IC50 = 8.1 \u03bcM) and Os quantification was achieved using 231 TNBC cells to investigate correlations between potency and\n ICP-MS analysis of acid-digested cell pellets. Time-dependent accumulation in breast cancer cells. In agreement with the\n measurements (Figure 4a) revealed the maximal Os accumu- similar IC50 values determined for complex 5 in MCF7 (8.1 \ufffd\n lation occurs 6 h after commencing exposure to complex 5, 0.2 \u03bcM) and MDA\u2013MB-231 (9.1 \ufffd 0.9 \u03bcM), cellular Os accumula-\n with only ca. 66 % of the maximum Os accumulated being tion in TNBC cells (14.9 \ufffd 0.8 fg \u00b7 cell 1) was remarkably compara-\n present after 24 h exposure. Similar time-dependent accumu- ble to that of MCF7 cells (13.1 \ufffd 0.5 fg \u00b7 cell 1). This may indicate\n lation profiles have been reported for non-tethered complex 4 that there is a common mechanism of cellular accumulation,\n and structurally similar Os azopyridine complexes in A2780 and perhaps also a similar in-cell mechanism of action.\n ovarian cancer cells, which also achieved maximal Os accumu- To probe the mechanism of cellular uptake, Os accumu-\n lation after 6 h.[9,36] Non-tethered complex 4 is less potent than lation was determined in MCF7 breast cancer cells treated with\n tethered complex 5 (15.5 \ufffd 0.5 \u03bcM vs. 10.5 \u03bcM \ufffd 0.1 in A2780), tethered complex 5 in the presence of various known uptake\n and accordingly, Os accumulation in MCF7 cells after equipo- (influx) pathway inhibitors (Figure 4b).[35] Co-administration with\n\n\n\n\n Figure 4. Cellular Os accumulation in MCF7 breast cancer cells treated with tethered complex 5 (1\u00d7IC50). (a) Time-dependent Os accumulation: 0\u201324 h\n exposure to complex 5 followed by 72 h recovery time in Os-free medium. Solid line indicates complex 5 only, dashed line indicates recovery time (72 h) in\n the presence of 20 \u03bcM verapamil, a potent inhibitor of the P-gp efflux transporter. Os accumulation reaches a maximum concentration after 6 h exposure,\n after which time intracellular Os decreases. After 72 h recovery, > 95 % of Os has been exported from cells. (b) Os accumulation (24 h) in cells treated with\n complex 5 in combination with: 20 \u03bcM amphotericin B (causes membrane disruption as a model for protein-mediated uptake), 0.2 mM Cu(II) chloride\n (competitive uptake via Ctr1), 0.2 mM ouabain (inhibitor of Na + /K + pump to investigate the role of membrane potential), and 1 mM \u03b2-methyl cyclodextrin\n (inhibitor of caveolin-mediated endocytosis).[35] The accumulation of Os in cells treated with complex 5 is significantly increased by the presence of\n amphotericin B. (c) Temperature-dependent Os accumulation (3\u20136 h) experiments in cells treated with complex 5 reveal significantly lower accumulation at\n lower temperature, suggesting the contribution of energy-dependent (active-transport) cellular uptake mechanisms. Data shown are mean values with error\n bars representing \ufffd 1 standard deviation. Full numerical and statistical data can be found in the Supporting Information. Statistical significances were\n determined using a two-tailed t-test assuming unequal sample variances (*p < 0.05).\n\n\n ChemBioChem 2024, 25, e202400374 (5 of 11) \u00a9 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH\n\f 14397633, 2024, 15, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cbic.202400374 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nChemBioChem doi.org/10.1002/cbic.202400374\n\n\n non-toxic concentrations of Cu(II) chloride (0.2 mM, a compet- the structural similarity between complexes 4 and 5, this\n itive substrate for Ctr1 which is known to contribute to the mechanistic commonality is perhaps unsurprising, but nonethe-\n uptake of Pt drugs), ouabain (0.2 mM, Na + /K + pump inhibitor, less encouraged the further exploration of complex 5 as a\n to investigate the role of membrane potential in metallodrug redox- modulating agent. This qualitative analysis of intra-\n uptake) and \u03b2-methyl cyclodextrin (1 mM, inhibitor of caveolin- cellular ROS illustrates that the generation of intracellular ROS is\n mediated endocytosis) had no significant effect on the accumu- not hindered by the introduction of the covalent tether. Future\n lation of Os after 24 h. However, co-administration of complex 5 studies should explore ROS generation in triple-negative\n with amphotericin B (20 \u03bcM), which causes membrane disrup- MDA MB-231 cells, though such a detailed mechanistic study\n tion as a model for protein-mediated uptake, was found to goes beyond the scope of this work. Chemical catalysis using\n significantly increase the accumulation of Os, relative to cells formic acid as a source of hydride (Table 1) demonstrated that\n administered with complex 5 alone (P = 0.0277). Os accumu- complex 5 can catalyse TH reactions. Using the sodium salt of\n lation determined at 277 K (Figure 4c) further highlighted the formic acid as a hydride donor, in-cell catalysis was studied by\n significant contribution of energy-dependent transport mecha- co-administration of a sub-lethal dose of 5 (0.5\u00d7IC50) alongside\n nisms to the accumulation of Os. In summary, extracellular a non-toxic concentration of formate (0\u20132 mM) in MCF7 cells. In\n speciation studies and intracellular accumulation measurements the absence of Os complex (Figure 6a), formate had no\n demonstrate that Os from complex 5 is almost exclusively significant effect on cell survival, confirming that the co-factor is\n albumin-bound in the extracellular environment and appears to non-toxic in this concentration range. However, in the presence\n be accumulated by cells via a protein-mediated, energy- of tethered complex 5 (Figure 6c), cell survival significantly\n dependent transport mechanism. decreased with increasing formate concentration. The specific\n Many metal arene complexes are known to perturb the role of formate as a hydride donor in a TH mechanism was\n cellular redox balance as part of a multi-targeting mechanism of confirmed by substituting formate by acetate, which cannot\n action.[38] Since complex 5 was shown to be an active catalyst donate hydride: in this case, there was no significant impact on\n for transfer hydrogenation reactions in a chemical system, the cell survival irrespective of the presence or absence of complex\n qualitative level of reactive oxygen species (ROS) generated by 5 (Figures 6b and 6d). Importantly, accumulation of Os in cells is\n complex 5 in cells was explored using fluorescence microscopy. not affected by the presence of formate, demonstrating that\n After 24 h exposure to 5, MCF7 cells were stained using 2\u2019,7\u2019- the co-factor does not modulate cellular accumulation of\n dichlorodihydrofluorescein diacetate (H2 DCFDA), a membrane complex 5, thus the observed reduction in cell survival is likely\n permeable non-fluorescent precursor which undergoes intra- attributable to an in-cell catalytic mechanism of action (Support-\n cellular decarboxylation and is then oxidised by various intra- ing Information Table S2).\n cellular reactive oxygen-based or reactive nitrogen-based Activity modulation experiments using sodium formate and\n species (ROS and RNS, respectively) to fluorescent DCF (\u03bbEx/Em = sodium acetate were repeated in MDA MB-231 (TNBC) cells\n 485/530 nm).[39] The assay was validated using a known inducer (Figures 6e\u2013h and Supporting Information), where the in-cell\n of cellular ROS (50 \u03bcM Antimycin A, 30 min, Figure 5).[39b,40] DCF catalytic mechanism was conserved. Decreased cell viability was\n fluorescence was measured in cells treated with complex 5, observed by increasing formate co-administration (Figure 6g),\n where increased fluorescence indicated significantly elevated while no effect was observed in the presence of acetate\n ROS relative to the untreated control. This was also previously (Figure 6h). In achieving in-cell catalysis in TNBC cells, which has\n observed for non-tethered complex 4 in ovarian and breast also been observed with structurally related Os arene\n cancer cells,[41] consequently identifying a commonality in the catalysts,[14c] we demonstrate how this in-cell catalytic mecha-\n mechanism of action of Os(II) tosyl diamine complexes. Given nism of action could be applied to treat cancers which currently\n have limited treatment options. Finally, activity modulation\n experiments using formate and acetate were repeated using\n MCF10-A non-tumorigenic cells (Figures 6i\u2013l and Supporting\n Information) as a model of healthy cells. Similarly to previous\n observations using untethered Os catalyst 4 in a healthy ovarian\n cell model,[9] formate co-administration did not enhance the\n potency of complex 5 (Figure 6k). This critical finding highlights\n the potential of in-cell transfer hydrogenation to offer a unique\n approach to enhance selectivity for cancer cells over non-\n cancerous cells.\n The successful delivery of an intact metallodrug is essential\n Figure 5. Detection of ROS in MCF7 breast cancer cells by fluorescence\n to achieve in-cell catalysis. The ability of complex 5 to facilitate\n microscopy (10\u00d7 magnification): (a) negative control (no test compound), (b) in-cell catalysis in breast cancer cells (MCF7 and MDA- MB-231)\n treated with Antimycin A (positive control, 50 \u03bcM, 30 min), (c) treated with suggests that the extracellular albumin-bound Os species,\n tethered Os complex 5 (1\u00d7IC50, 8 \u03bcM, 24 h). Cells were stained using 2\u2019,7\u2019-\n dichlorodihydrofluorescein diacetate (H2 DCFDA, 20 min in the dark), a\n which is accumulated in cells via a protein-mediated uptake\n membrane permeable indicator of intracellular ROS and were washed twice mechanism, must result in the successful delivery of the intact\n with DPBS prior to imaging to remove excess H2 DCFDA. Cells were imaged active transfer hydrogenation catalyst 5 to cells. This is\n using an EVOS FL fluorescence microscope fitted with a GFP filter cube (\u03bb\n consistent with our previous work using a Br-containing\n Ex/Em = 470/510 nm, exposure time 0.5 sec, intensity 50 %).\n\n\n\n ChemBioChem 2024, 25, e202400374 (6 of 11) \u00a9 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH\n\f 14397633, 2024, 15, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cbic.202400374 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nChemBioChem doi.org/10.1002/cbic.202400374\n\n\n\n\n Figure 6. Activity modulation of complex 5 in MCF7 (breast cancer, a\u2013d), MDA MB-231 (TNBC breast cancer, e\u2013h) and MCF10-A (non-tumorigenic breast, i\u2013l)\n cells using non-toxic 0\u20132 mM concentrations of sodium formate (a, c, e, g, i, k) or sodium acetate (b, d, f, h, j, l). Cells were either treated with formate or\n acetate in the absence of complex 5 (a, b, e, f, i, j) or a sub-lethal concentration of complex 5 equal to 0.5\u00d7IC50 (c, d, g, h, k, l). Cell survival (%) was determined\n using the sulforhodamine B assay after 24 h exposure + 72 h recovery time in fresh culture medium. Data shown are mean values of three biological\n replicates with error bars representing \ufffd 1 standard deviation. Full numerical and statistical data are found in the Supporting Information. Statistical\n significances were determined using a two-tailed t-test assuming unequal sample variances (*p < 0.05).\n\n\n\n\n diamine analogue of non-tethered complex 4, where X-ray exclusively albumin-bound in human serum, in agreement with\n fluorescence elemental mapping revealed the Br-containing the protein-mediated, energy-dependent uptake pathway iden-\n diamine and Os to be co-localized in cells.[10] tified in cellular Os accumulation studies. The mechanism of\n action of complex 5 is likely to be multi-targeted, involving\n modulation of cellular redox balance (as evidenced by the\n Conclusions generation of ROS inside cells) and an in-cell catalytic mecha-\n nism which was conserved in both triple-positive and triple-\n Novel covalently-tethered organo osmium TsDPEN catalyst 5 negative breast cancer cells. Importantly, we highlight that the\n was synthesized using microwave radiation of a chlorido catalytic mechanism generates selectivity for cancer cells over\n bridged Os dimer intermediate. Catalytic rates (TOFmax) meas- non-tumorigenic cells. Future work will involve characterizing\n ured for the transfer hydrogenation of acetophenone by the nature of the extracellular albumin Os binding, further\n tethered complex 5 were slower than for non-tethered Os characterization of the specific albumin-mediated uptake path-\n complex 4. However, tethered Os complex 5 was more potent way, and exploration of the intracellular Os speciation in\n towards cancer cells than its non-tethered analogue, in some relation to catalyst deactivation and detoxification.\n instances achieving potency comparable to cisplatin. Extracel-\n lular speciation measurements revealed Os to be almost\n\n\n ChemBioChem 2024, 25, e202400374 (7 of 11) \u00a9 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH\n\f 14397633, 2024, 15, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cbic.202400374 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nChemBioChem doi.org/10.1002/cbic.202400374\n\n\n Experimental Section to afford a dark red oil. The oil was recrystallized in dichloro-\n methane/hexane to afford a red crystalline solid (68 mg, 0.10 mol,\n Synthesis of Tethered Ligand 1 75 %). 1H NMR (400 MHz, CDCl3, 25 \u00b0C, TMS): \u03b4 = 7.41 (d, ArH,\n 3\n J(H,H) = 7.6 Hz, 2H), 7.05\u20137.20 (m, ArH, 10H), 6.82 (d, ArH, 3J(H,H) =\n Ligand 1 was prepared as previously reported.[21] To a cooled (0 \u00b0C) 8.0 Hz, 2H), 6.80 (br s, NH, 1H), 5.79 (d, Os ArH, 3J(H,H) = 5.6 Hz, 1H),\n solution of 3-(1,4-cyclohexadien-1-yl)-1-propanol (553 mg, 5.62 (d, Os ArH, 3J(H,H) = 5.6 Hz, 1H), 5.52 (d, Os ArH, 3J(H,H) =\n 4.0 mmol, 1.6 equiv.) and 2,6-lutidine (613 \u03bcL, 5.25 mmol, 5.6 Hz, 1H), 5.42 (d, Os ArH, 3J(H,H) = 5.6 Hz, 1H), 4.42 (s,\n 2.10 equiv.) in anhydrous dichloromethane (20 mL) was added CHCHNH2,1H), 3.94 (d, TsNCH, 3J(H,H) = 4.3 Hz, 1H), 2.45 (sept,\n slowly a solution of trifluoromethane sulfonic anhydride (728 \u03bcL, CH(CH3)2, 3J(H,H) = 6.9 Hz, 1H), 2.23 (s, CH3, 3H), 2.22 (s, CH3, 3H),\n 4.3 mmol, 1.7 equiv.) in anhydrous dichloromethane (10 mL). The 1.23 (d, CH(CH3)2), 3J(H,H) = 6.9 Hz, 3H) 1.17 (d, CH(CH3)2, 3J(H,H) =\n reaction temperature was maintained at 0 \u00b0C for 30 min, followed 6.9 Hz, 3H); 13C NMR (100 MHz, CDCl3, 25 \u00b0C, TMS) \u03b4 = 127.4, 127.0,\n by warming to ambient temperature for 1 h. After re-cooling to 126.8, 126.0, 125.9, 125.9, 125.4, 81.7, 76.2, 72.4, 70.7, 70.0, 66.2,\n 0 \u00b0C, a solution of (R,R)-TsDPEN (915 mg, 2.5 mmol, 1.0 equiv.) and 22.5, 22.4, 20.2; UV/Vis: \u03bbmax 260, 410 and 478 nm; HR-MS (ESI): m/z\n triethylamine (838 \u03bcL, 6.0 mmol, 2.4 equiv.) in anhydrous dichloro- calculated for C31H35N2O2OsS [M + H] + : 691.2028. Found: 691.2031.\n methane (20 mL) was added dropwise and stirred for 3 h. The Elemental analysis (calculated, found for C31H34N2O2OsS): C (54.05,\n resultant solution was diluted with additional dichloromethane 53.66), H (4.97, 4.88), N (4.07, 3.95).\n (50 mL) and washed with saturated aqueous sodium hydrogen\n carbonate (3\u00d7100 mL), water (2\u00d7100 mL) and brine (1\u00d7100 mL). The\n organic phase was dried over magnesium sulphate and concen- Synthesis of Tethered Osmium Complex 5\n trated to afford an amber oil. The oil was recrystallized in hot\n To a solution of Os pre-tether dimer 3 (149 mg, 0.10 mmol,\n ethanol and filtered to obtain 1 as a white amorphous solid, which\n 0.5 equiv.) in dichloromethane (25 mL) was added water (25 mL)\n was washed with cold ethanol and diethyl ether (968.4 mg,\n and potassium hydroxide pellets (112 mg, 1 mmol, 10 equiv.) with\n 2.00 mmol, 80 %).1H NMR (400 MHz, (CD3)2SO, 25 \u00b0C, TMS): \u03b4 = 7.37\n stirring for 10 min. The organic phase was washed with water\n (d, ArH, 3J(H,H) = 8.1 Hz, 2H), 7.13 (m, ArH, 3H), 7.04 (m, ArH, 5H),\n (2\u00d750 mL), then dried over magnesium sulphate and concentrated\n 6.95 (m, ArH, 2H), 6.91 (m, ArH, 2H), 6.28 (br s, NH, 1H), 5.69 (m, CH,\n to afford a dark red oil. The oil was recrystallised in dichloro-\n 2H), 5.31 (s, CH, 1H), 4.24 (d, CH, 3J(H,H) = 7.8 Hz, 1H), 3.60 (d, CH,\n 3 methane/hexane to afford a red semi-crystalline solid (106 mg,\n J(H,H) = 7.8 Hz, 1H), 2.64 (m, CH2, 2H), 2.52 (m, CH2, 2H), 2.34 (s,\n 0.16 mol, 79 %). 1H NMR (400 MHz, CDCl3, 25 \u00b0C, TMS): \u03b4 = 6.85\u20137.51\n CH3, 3H), 1.88 (m, CH2, 2H), 1.51 (m, CH2, 4H). ESI-MS: m/z = 487.2.\n (m, 14H, ArH), 6.01\u20136.09 (m, 1H, Os ArH), 5.92\u20136.01 (m, 2H,\n Os ArH), 5.81\u20135.89 (m, 1H, Os ArH), 5.33\u20135.42 (m, 1H, Os ArH),\n Synthesis of Non-Tethered [OsCl2(p-Cymene)]2 Dimer 2 4.43 (s, 1H, CH), 3.73 (s, 1H, CH), 2.41\u20132.59 (m, 2H, CH2), 2.32 (s, 3H,\n CH3), 2.19\u20132.29 (m, 2H, CH2), 1.81\u20131.89 (m, 2H, CH2); 13C NMR\n Os dimer 2 was prepared as previously reported.[19] To a solution of (100 MHz, CDCl3, 25 \u00b0C, TMS) \u03b4 = 146.4, 146.3, 141.5, 141.4, 140.5,\n Os(III) chloride trihydrate (1.00 g, 2.8 mmol, 2 equiv.) in methanol 129.1, 128.4, 128.4, 128.3, 128.3, 128.3, 128.3, 127.9, 127.6, 127.4,\n (10 mL) was added \u03b1-phellandrene (3.80 g, 28 mmol, 20 equiv.). The 127.3, 127.1, 126.8, 126.5, 92.1, 88.3, 73.3, 70.2, 69.1, 65.4, 64.9, 59.6,\n solution was subject to microwave-assisted reaction (CEM Discov- 34.2, 30.2, 21.4; HR-MS (ESI): m/z calculated for C30H30KN2O2OsS [M +\n ery-SP microwave, 150 W, 250 psi, 413 K, 10 min) and then cooled K] + : 713.1280. Found: 713.1278. HPLC Purity (254 nm): 98.3 %.\n to ambient temperature. An orange crystalline precipitate was\n obtained by addition of n-pentane (3\u00d710 mL) and agitation, which\n was collected by filtration and washed with n-pentane and diethyl Density Functional Theory Calculations\n ether (863 mg, 1.11 mmol, 79 %). Characterisation data matched\n Initial input structures for tethered complex 5 were achieved by\n those previously reported for the [OsCl2(p-cymene)]2 dimer.[8a]\n structural modification of crystallographic data for a similar 18e Ru\n tethered complex (CCDC 913682) using GaussView 6.0.[20] Geometry\n Synthesis of Pre-Tether Osmium Dimer 3 optimization calculations were carried out in the gas phase using\n Gaussian 16, using the hybrid Perdew-Burke-Ernzerhof functional\n To a solution of Os(III) chloride trihydrate (175 mg, 0.50 mmol, (PBE0) with the Lanl2DZ (including effective core potential for Os)\n 1 equiv.) in methanol (5 mL) was added tethered ligand 1 (292 mg, and 6-31 + G** (all other atoms) basis sets. Energy minima were\n 0.60 mmol, 1.2 equiv.) and hydrochloric acid (75 \u03bcL 37 % HCl, confirmed by the lack of imaginary vibrational modes.\n 0.90 mmol, 1.8 equiv.). The solution was subjected to a microwave-\n assisted reaction (CEM Discovery-SP microwave, 150 W, 250 psi,\n 413 K, 4\u00d710 min reaction cycles) and then cooled to ambient Transfer Hydrogenation of Acetophenone\n temperature. The solution was filtered to remove insoluble particles\n Os complex 5 (1 mol. equiv.) was weighed into a round bottomed\n and the solvent removed under reduced pressure to afford a pale\n flask and placed under an inert atmosphere of nitrogen, to which\n brown amorphous solid (536 mg, 0.36 mmol, 72 %). This intermedi-\n was added 5 : 2 v/v formic acid/triethylamine azeotrope (0.5 mL)\n ate was used directly without further purification for the prepara-\n and d6-benzene (100 \u03bcL) by syringe and the catalyst stirred at\n tion of catalyst 5.\n 310 K. After 5 min, 120 \u03bcL of the catalytic substrate, acetophenone\n (200 mol. equiv.) was added by syringe and the mixture transferred\n Synthesis of [Os(p-Cymene)(TsDPEN)] Complex 4 to a 5 mm NMR tube (t = 0 h). 1H NMR spectra were recorded at\n 73 s intervals for 3 h at 310 \ufffd 0.5 K using a Bruker AV-400\n Complex 4 was prepared as previously reported.[8a] To a solution of spectrometer. 1H NMR data were processed using TopSpin 3.2 for\n non-tethered Os dimer 2 (51.4 mg, 0.065 mmol, 1 equiv.) and (R,R)- Windows. Substrate conversion was determined by measuring the\n TsDPEN (51.3 mg, 0.14 mmol, 2.1 equiv.) in dichloromethane ratio of integrals of peaks from 2.25\u20132.65 ppm (substrate CH3\n (10 mL) was added water (10 mL) and potassium hydroxide pellets singlet) and 4.55\u20135.00 ppm (product CH(OH) quartet), which in turn\n (56.1 mg, 1 mol, 15 equiv.) with stirring for 10 min. The organic was converted to turnover number and turnover frequency, given\n phase was diluted with dichloromethane (40 mL) and washed with the substrate : catalyst ratio (200 : 1). The experiment was carried\n water (2\u00d750 mL), dried over magnesium sulphate, and concentrated\n\n\n ChemBioChem 2024, 25, e202400374 (8 of 11) \u00a9 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH\n\f 14397633, 2024, 15, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cbic.202400374 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nChemBioChem doi.org/10.1002/cbic.202400374\n\n\n out in triplicate with mean and the associated standard deviations Modulation of Antiproliferative Activity\n reported.\n In a modification of the antiproliferative activity determination\n protocol, MCF7, MCF7 TAMR-1, MCF10-A or MDA MB-231 cells\n Extracellular Speciation Analysis were treated with a fixed sub-lethal concentration (0.5\u00d7IC50) of Os\n catalyst 5 in the presence of sodium formate or sodium acetate (0,\n Briefly, 10 \u03bcM solutions of Os complex 5 were prepared in three 0.5, 1 or 2 mM) for 24 h. Sodium formate and sodium acetate were\n biological matrixes (600 \u03bcM bovine albumin, foetal calf serum, or added independently of the Os catalyst but within 5 min. Cell\n human serum; all containing 5 % DMSO to aid solubility) and were viability was determined using the SRB assay as previously\n incubated at 310 K for 24 h. After this time, samples were analysed described. Cell survival was calculated relative to the formate-free\n using an optimised offline LC-ICP-MS methodology. Samples were (or acetate-free) cell population and standard deviations were\n diluted 10-fold with 50 mM Tris solution (pH 7.4) prior to analysis. calculated. Experiments were carried out in triplicate as part of two\n The liquid chromatographic separation (LC) was achieved using an independent experiments (duplicate of triplicate analysis). Statistics\n Agilent 1200 series HPLC fitted with a TSKgel Q-STAT strong anion were calculated using a two-tailed t-test assuming unequal sample\n exchange column (7 \u03bcm, 10 cm\u00d74.6 mm i.d.) with a flow rate of variances (Welch\u2019s t-test).\n 0.7 mL \u00b7 min 1 and an injection volume of 100 \u03bcL. Buffer A: 50 mM\n Tris base, pH 7.4. Buffer B: 50 mM Tris base + 1 M ammonium\n acetate, pH 7.4. Gradient (linear): 0\u20133 min, 0 % B; 3\u20139 min, 20 % B; Os Accumulation in Cancer Cells\n 9\u201313.5 min, 50 % B; 13.5\u201316.5 min, 100 % B; 16.5\u201322.5 min, 100 % B;\n 22.5\u201327 min, 0 % B; 27\u201330 min, 0 % B (re-equilibration). Elemental Briefly, 4\u00d7106 MCF7 or MDA MB-231 breast cancer cells were\n data for 56Fe (He-gas mode), 63Cu (He-gas mode), 66Zn (He-gas seeded in P100 plates using 10 mL of culture medium per plate and\n mode) and 189Os (no-gas mode) were obtained offline using an incubated for 24 h, after which time the supernatant media was\n Agilent 7900 series ICP-MS, with calibration standards prepared removed and cells were treated with a fixed concentration (1\u00d7IC50)\n from 1000 mg \u00b7 L 1 certified reference materials using 50 mM Tris of Os catalyst 5 for 24 h (310 K) without recovery time. After\n solution (pH 7.4) as a diluent. Data were acquired and processed exposure, the Os-containing medium was removed by aspiration,\n using MassHunter 4.4 (version C.01.04, build 544.8, Agilent Tech- cells were washed with DPBS and harvested using trypsin/EDTA. A\n nologies, Inc.). cell count was performed for sample normalization. Cell pellets\n were obtained by centrifugation (1000 rpm, 5 min) which were re-\n suspended in DPBS (1 mL) prior to final centrifugation (1000 rpm,\n Antiproliferative Activity Determination 5 min) to obtain pellets for chemical analysis. Cell pellets were\n obtained in triplicate (three biological experimental replicates). This\n Initially, 5\u00d7103 cells per well were seeded in 96 well plates and experiment was repeated with the following modifications based\n incubated for 48 h (310 K). Solutions of test compounds were on a previously reported methodology.[35a] Untreated (negative\n prepared in culture medium containing < 5% DMSO (v/v) to aid control): Cells were not treated with Os catalyst 5. Cells were\n solubility (\u201cAqueous stability studies\u201d in the Supporting Information). incubated for 24 h (310 K) prior to harvesting cell pellets. Time-\n Solution concentrations were determined as described below dependent Os accumulation: Cells were treated with a fixed\n (\u201cDetermination of Os concentrations\u201d). The supernatant was concentration (1\u00d7IC50) of Os catalyst 5 for 3 h or 6 h (310 K) without\n removed and replaced with medium containing six known recovery time. Cells were also treated with Os catalyst 5 for 24 h,\n concentrations of test compound (typically 0.1\u2013100 \u03bcM, 200 \u03bcL per after which time the cells were washed with DPBS and allowed a\n well) and cells were exposed for 24 h. After this time, the super- further 72 h recovery time in Os-free medium. Temperature-depend-\n natant was removed by aspiration, cells were washed with DPBS ent Os accumulation: Cells were treated with a fixed concentration\n and allowed 72 h recovery time in fresh medium (in the absence of (1\u00d7IC50) of Os catalyst 5 for 3 h or 6 h (277 K) without recovery time.\n test compound). Cells were fixed by addition of trichloroacetic acid Involvement of Na + /K + pump in Os accumulation: Cells were treated\n (50 mM, 50 \u03bcL per well, 1 h, 277 K) and cell viability was determined with a fixed concentration (1\u00d7IC50) of Os catalyst 5 for 24 h (310 K)\n using the SRB assay as previously described.[24] Experiments were without recovery time in the presence of a non-toxic concentration\n carried out in triplicate as part of two independent experiments of ouabain octahydrate (0.2 mM). Involvement of protein-mediated\n (duplicate of triplicate). Half-maximal inhibitory concentrations transport in Os accumulation: Cells were treated with a fixed\n (IC50) were determined relative to the untreated negative control, concentration (1\u00d7IC50) of Os catalyst 5 for 24 h (310 K) without\n and standard deviations were calculated. Exemplar dose-response recovery time in the presence of a non-toxic concentration of\n sigmoidal curve fits are found in the Supporting Information. amphotericin B (20 \u03bcM). Involvement of caveolin-mediated endocy-\n tosis in Os accumulation: Cells were treated with a fixed concen-\n tration (1\u00d7IC50) of Os catalyst 5 for 24 h (310 K) without recovery\n Determination of Os Concentrations time in the presence of a non-toxic concentration of \u03b2-methyl\n Stock solutions from antiproliferative activity determinations were cyclodextrin (1 mM). Involvement of the Ctr1 transporter in Os\n analysed using a Perkin Elmer Optima 5300 DV Series Inductively accumulation: Cells were treated with a fixed concentration (1\u00d7IC50)\n Coupled Plasma Optical Emission Spectrophotometer (ICP-OES). of Os catalyst 5 for 24 h (310 K) without recovery time in the\n Calibration standards for Os and Pt were freshly prepared in 3.6 % v/ presence of a non-toxic concentration of copper(II) chloride\n v nitric acid supplemented with thiourea (10 mM) and ascorbic acid (0.2 mM). Involvement of formate or acetate in Os accumulation: Cells\n (50 mg \u00b7 L 1). Samples were diluted to within the calibration range were treated with a fixed concentration (1\u00d7IC50) of Os catalyst 5 for\n (50\u2013700 ppb), with the matrix of the calibration standards and 24 h (310 K) without recovery time in the presence of a non-toxic\n calibration blanks adjusted by standard addition of sodium chloride concentration of sodium formate or sodium acetate (2 mM).\n (99.999 % trace metal basis) to match that of the samples. Data Involvement of P-gp in Os efflux: Cells were treated with a fixed\n were acquired and processed using WinLab32V3.4.1 for Windows. concentration (1\u00d7IC50) of Os catalyst 5 for 24 h, after which time the\n cells were washed with DPBS and allowed a further 72 h recovery\n time in Os-free medium containing 20 \u03bcM verapamil hydrochloride,\n a selective inhibitor of the p-glycoprotein (P-gp) efflux transporter.\n Cell pellets were subjected to acidic digestion (200 \u03bcL of 72 % ultra-\n\n\n\n ChemBioChem 2024, 25, e202400374 (9 of 11) \u00a9 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH\n\f 14397633, 2024, 15, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cbic.202400374 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nChemBioChem doi.org/10.1002/cbic.202400374\n\n\n pure nitric acid, 353 K, overnight) prior to dilution to a working acid Supporting Information\n concentration of 3.6 % v/v using Type I Milli-Q water supplemented\n with thiourea (10 mM) and ascorbic acid (50 mg \u00b7 L 1) to stabilize Os The Supporting Information is available free of charge online.\n in nitric acid solution. Samples were analysed using an Agilent\n Technologies 7900 series ICP-MS, operated in He-gas mode (189Os,\n Computational data (density functional theory calculations) are\n 66\n Zn, 63Cu) and H2-gas mode (56Fe) with an internal standard inline available upon reasonable request to the authors. The authors\n infusion of 50 ppb 166Er. External calibrants for Os, Zn, Cu and Fe have cited additional references within the Supporting\n were prepared from certified reference materials (1000 mg \u00b7 L 1) Information.[8a,19,21,24,35a,39b,41]\n ranging from 0.1\u20131000 \u03bcg \u00b7 L 1 in 3.6 % v/v HNO3 supplemented with\n thiourea (10 mM) and ascorbic acid (50 mg \u00b7 L 1). ICP-MS data were\n acquired as instrumental (technical) triplicates for each experimen-\n tal replicate. Data were normalised by cell count and reported as Acknowledgements\n the mean value (femtograms of osmium per cell) with the\n associated standard deviation (N = 3). We thank the Royal Society of Chemistry (grant no. E22-\n 1637945680) and the University of Birmingham for support. PJS\n research is supported by Anglo American Platinum and the\n Fluorescence Microscopy\n EPSRC (grant no. EP/P030572/1). We thank the University of\n Briefly, 1\u00d7104 MCF7 breast cancer cells were seeded in a 24 well Warwick Analytical Science CDT and GoldenKeys High-Tech\n plate using 0.5 mL per well, and incubated for 48 h at 310 K. After Materials Co. Ltd for a PhD CASE studentship for OWLC.\n this time, cells were treated with test compounds (1\u00d7IC50, 0.5 mL\n per well) for 24 h (or cell culture medium only for the negative\n untreated control). Positive control wells were treated with 50 \u03bcM\n Antimycin A for 30 min in the dark prior to staining.[39b] All cells Conflict of Interests\n were washed with DPBS (1\u00d70.5 mL) and stained using 2\u2019,7\u2019-\n dichlorodihydrofluorescein diacetate (H2 DCFDA, 10 \u03bcg \u00b7 mL 1) for The authors declare no conflict of interest.\n 20 min in the dark at 310 K. Cells were washed using DPBS\n (2\u00d70.5 mL) to remove excess dye and imaged using an EVOS FL\n fluorescence microscope fitted with a GFP filter cube (\u03bbEx/Em = 470/\n 510 nm, exposure time 0.5 sec, intensity 50 %). Data Availability Statement\n\n The data that support the findings of this study are available in\n Acute in Vivo Zebrafish Embryo Toxicity the supplementary material of this article.\n In vivo experiments were carried out using Singapore wild-type\n zebrafish embryos under project AWERB.85/21-22. The University of\n Keywords: osmium \u00b7 in-cell catalysis \u00b7 cancer \u00b7 transfer\n Warwick is a member of the Institute of Animal Technology and the\n Laboratory Animal Science Association. Zebrafish were maintained hydrogenation \u00b7 redox\n in accordance with ASPA 1986 by Mr. Ian Bagley, using 3.5 L tanks\n (checked daily for water quality) in a 14 h light cycle, and provided\n with food (live and powder) four times a day during the week and [1] M. Hoppert, in Encyclopedia of Geobiology (Eds.: J. Reitner, V. Thiel),\n twice a day during weekends. Fish were mated in breeding tanks Springer Netherlands, Dordrecht, 2011, pp. 558\u2013563.\n fitted with a divider. Two mating pairs were used per breeding [2] a) J. J. Soldevila-Barreda, P. J. Sadler, Curr. Opin. Chem. Biol. 2015, 25,\n tank, and the divider was removed at dawn. Acute in vivo toxicities 172\u2013183; b) Z. Yu, J. A. Cowan, Chem. Eur. J. 2017, 23, 14113\u201314127;\n were determined according to OECD Test 236: \u201cFish Embryo Acute c) E. J. Anthony, E. M. Bolitho, H. E. Bridgewater, O. W. L. Carter, J. M.\n Toxicity (FET)\u201d and as previously reported.[41] Freshly harvested Donnelly, C. Imberti, E. C. Lant, F. Lermyte, R. J. Needham, M. Palau, P. J.\n embryos were collected at dawn and fertilized embryos were Sadler, H. Shi, F. X. Wang, W. Y. Zhang, Z. Zhang, Chem. Sci. 2020, 11,\n 12888\u201312917; d) J. J. Soldevila-Barreda, N. Metzler-Nolte, Chem. Rev.\n seeded at a density of 2\u20133 embryos per well in 24 well plates. Serial\n 2019, 119, 829\u2013869; e) S. Alonso-de Castro, A. Terenzi, J. Gurruchaga-\n dilutions of complex 5 and cisplatin were prepared in egg water Pereda, L. Salassa, Chem. Eur. J. 2019, 25, 6651\u20136660.\n (typically 0.01\u2013100 \u03bcM, 1 mL per well, 20 wells per test concen- [3] a) J. T. Weiss, J. C. Dawson, K. G. Macleod, W. Rybski, C. Fraser, C. Torres-\n tration) and embryos were exposed for 96 h at 301 K. The experi- S\u00e1nchez, E. E. Patton, M. Bradley, N. O. Carragher, A. Unciti-Broceta, Nat.\n ment included two positive (2,3-dichloroaniline) and two negative Commun. 2014, 5, 3277; b) J. T. Weiss, J. C. Dawson, C. Fraser, W. Rybski,\n control wells (untreated embryos, egg water only) on each 24-well C. Torres-S\u00e1nchez, M. Bradley, E. E. Patton, N. O. Carragher, A. Unciti-\n plate. Embryo viability was determined according to four criteria \u2013 Broceta, J. Med. Chem. 2014, 57, 5395\u20135404.\n [4] T. V\u00f6lker, F. Dempwolff, P. L. Graumann, E. Meggers, Angew. Chem. Int.\n if any (or multiple) attributes were observed, the embryo was\n Ed. 2014, 53, 10536\u201310540.\n considered non-viable \u2013 (1) lack of heartbeat, (2) coagulation of [5] a) J. J. Soldevila-Barreda, K. B. Fawibe, M. Azmanova, L. Rafols, A. Pitto-\n fertilized egg, (3) lack of somite formation, (4) lack of tail-bud Barry, U. B. Eke, N. P. E. Barry, Molecules 2020, 25(19), 4540, DOI:\n detachment from the yolk sac. A sigmoidal dose-response curve 10.3390/molecules25194540; b) W.-Y. Zhang, H. E. Bridgewater, S. Bane-\n was obtained (embryo survival as a function of concentration) and rjee, J. J. Soldevila-Barreda, G. J. Clarkson, H. Shi, C. Imberti, P. J. Sadler,\n fitted using GraphPad Prism 10. This experiment was carried out as Eur. J. Inorg. Chem. 2020, 2020, 1052\u20131060; c) R. A. Sarfraz, T. G. Kazi, S.\n part of two independent biological experiments, each with 20 Iqbal, H. I. Afridi, M. K. Jamali, N. Jalbani, M. B. Arain, Appl. Organomet.\n replicates per test concentration. Data were reported as the Chem. 2008, 22, 187\u2013192.\n [6] a) D. Kong, M. Tian, L. Guo, X. Liu, S. Zhang, Y. Song, X. Meng, S. Wu, L.\n average of the two independent experiments, with an associated\n Zhang, Z. Liu, J. Biol. Inorg. Chem. 2018, 23, 819\u2013832; b) S. J. Dougan, A.\n standard deviation. Dose-response sigmoidal curve fits can be Habtemariam, S. E. McHale, S. Parsons, P. J. Sadler, Proc. Natl. Acad. Sci.\n found in the Supporting Information. USA 2008, 105, 11628\u201311633; c) W. Y. Zhang, S. Banerjee, G. M. Hughes,\n H. E. Bridgewater, J. I. Song, B. G. Breeze, G. J. Clarkson, J. P. C. Cover-\n dale, C. Sanchez-Cano, F. Ponte, E. Sicilia, P. J. Sadler, Chem. Sci. 2020,\n 11, 5466\u20135480.\n\n\n ChemBioChem 2024, 25, e202400374 (10 of 11) \u00a9 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH\n\f 14397633, 2024, 15, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cbic.202400374 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nChemBioChem doi.org/10.1002/cbic.202400374\n\n\n [7] a) J. J. Soldevila-Barreda, I. Romero-Canel\u00f3n, A. Habtemariam, P. J. [28] W. Feng, Y. He, J. Xu, H. Zhang, Y. Si, J. Xu, S. Li, Anticancer Drugs 2022,\n Sadler, Nat. Commun. 2015, 6, 6582; b) R. Kushwaha, A. Kumar, S. Saha, 33(1), e52\u2013e60.\n S. Bajpai, A. K. Yadav, S. Banerjee, Chem. Commun. 2022, 58, 4825\u20134836. [29] L. Bian, P. Yu, J. Wen, N. Li, W. Huang, X. Xie, F. Ye, NPJ Breast Cancer\n [8] a) J. P. C. Coverdale, C. Sanchez-Cano, G. J. Clarkson, R. Soni, M. Wills, 2021, 7, 157.\n P. J. Sadler, Chem. Eur. J. 2015, 21, 8043\u20138046; b) E. M. Bolitho, N. G. [30] F. Poggio, M. Bruzzone, M. Ceppi, N. F. Pond\u00e9, G. La Valle, L. Del Mastro,\n Worby, J. P. C. Coverdale, J. A. Wolny, V. Sch\u00fcnemann, P. J. Sadler, E. de Azambuja, M. Lambertini, Ann. Oncol. 2018, 29, 1497\u20131508.\n Organometallics 2021, 40, 3012\u20133023. [31] a) S. Zhao, J. Huang, J. Ye, J. Exp. Clin. Cancer Res. 2015, 34, 80; b) P. M.\n [9] J. P. C. Coverdale, I. Romero-Canel\u00f3n, C. Sanchez-Cano, G. J. Clarkson, A. Eimon, A. L. Rubinstein, Expert Opin. Drug Metab. Toxicol. 2009, 5, 393\u2013\n Habtemariam, M. Wills, P. J. Sadler, Nat. Chem. 2018, 10, 347\u2013354. 401.\n [10] E. M. Bolitho, J. P. C. Coverdale, H. E. Bridgewater, G. J. Clarkson, P. D. [32] C. A. MacRae, R. T. Peterson, Nat. Rev. Drug Discovery 2015, 14, 721\u2013731.\n Quinn, C. Sanchez-Cano, P. J. Sadler, Angew. Chem. Int. Ed. 2021, 60, [33] a) G. J. Lieschke, P. D. Currie, Nat. Rev. Genet. 2007, 8, 353\u2013367; b) W. B.\n 6462\u20136472. Barbazuk, I. Korf, C. Kadavi, J. Heyen, S. Tate, E. Wun, J. A. Bedell, J. D.\n [11] A. M. R. Hall, P. Dong, A. Codina, J. P. Lowe, U. Hintermair, ACS Catal. McPherson, S. L. Johnson, Genome Res. 2000, 10, 1351\u20131358.\n 2019, 9, 2079\u20132090. [34] K. Howe, M. D. Clark, C. F. Torroja, J. Torrance, C. Berthelot, M. Muffato,\n [12] a) H. J. Davis, T. R. Ward, ACS Cent. Sci. 2019, 5, 1120\u20131136; b) F. J. E. Collins, S. Humphray, K. McLaren, L. Matthews, S. McLaren, I. Sealy,\n Schwizer, Y. Okamoto, T. Heinisch, Y. Gu, M. M. Pellizzoni, V. Lebrun, R. M. Caccamo, C. Churcher, C. Scott, J. C. Barrett, R. Koch, G.-J. Rauch, S.\n Reuter, V. K\u00f6hler, J. C. Lewis, T. R. Ward, Chem. Rev. 2018, 118, 142\u2013231. White, W. Chow, B. Kilian, L. T. Quintais, J. A. Guerra-Assun\u00e7\u00e3o, Y. Zhou,\n [13] a) N. Soliman, G. Gasser, C. M. Thomas, Adv. Mater. 2020, 32, 2003294; Y. Gu, J. Yen, J.-H. Vogel, T. Eyre, S. Redmond, R. Banerjee, J. Chi, B. Fu, E.\n b) S. R. Alves, A. Colquhoun, X. Y. Wu, D. de Oliveira Silva, J. Inorg. Langley, S. F. Maguire, G. K. Laird, D. Lloyd, E. Kenyon, S. Donaldson, H.\n Biochem. 2020, 205, 110984; c) J. Karges, D. D\u00edaz-Garc\u00eda, S. Prashar, S. Sehra, J. Almeida-King, J. Loveland, S. Trevanion, M. Jones, M. Quail, D.\n G\u00f3mez-Ruiz, G. Gasser, ACS Appl. Bio Mater. 2021, 4, 4394\u20134405. Willey, A. Hunt, J. Burton, S. Sims, K. McLay, B. Plumb, J. Davis, C. Clee, K.\n [14] a) F. Mart\u00ednez-Pe\u00f1a, S. Infante-Tadeo, A. Habtemariam, A. M. Pizarro, Oliver, R. Clark, C. Riddle, D. Elliott, G. Threadgold, G. Harden, D. Ware, S.\n Inorg. Chem. 2018, 57, 5657\u20135668; b) A. C. Carrasco, V. Rodr\u00edguez-Fanjul, Begum, B. Mortimore, G. Kerry, P. Heath, B. Phillimore, A. Tracey, N.\n A. M. Pizarro, Inorg. Chem. 2020, 59, 16454\u201316466; c) S. Infante-Tadeo, V. Corby, M. Dunn, C. Johnson, J. Wood, S. Clark, S. Pelan, G. Griffiths, M.\n Rodriguez, A. Habtemariam, A. M. Pizarro, Chem. Sci. 2021, 12, 9287\u2013 Smith, R. Glithero, P. Howden, N. Barker, C. Lloyd, C. Stevens, J. Harley,\n 9297. K. Holt, G. Panagiotidis, J. Lovell, H. Beasley, C. Henderson, D. Gordon, K.\n [15] a) D. J. Morris, A. M. Hayes, M. Wills, J. Org. Chem. 2006, 71, 7035\u20137044; Auger, D. Wright, J. Collins, C. Raisen, L. Dyer, K. Leung, L. Robertson, K.\n b) J. Hannedouche, G. J. Clarkson, M. Wills, J. Am. Chem. Soc. 2004, 126, Ambridge, D. Leongamornlert, S. McGuire, R. Gilderthorp, C. Griffiths, D.\n 986\u2013987. Manthravadi, S. Nichol, G. Barker, S. Whitehead, M. Kay, J. Brown, C.\n [16] a) R. Soni, T. H. Hall, B. P. Mitchell, M. R. Owen, M. Wills, J. Org. Chem. Murnane, E. Gray, M. Humphries, N. Sycamore, D. Barker, D. Saunders, J.\n 2015, 80, 6784\u20136793; b) F. Chen, I. Romero-Canel\u00f3n, J. J. Soldevila- Wallis, A. Babbage, S. Hammond, M. Mashreghi-Mohammadi, L. Barr, S.\n Barreda, J. I. Song, J. P. C. Coverdale, G. J. Clarkson, J. Kasparkova, A. Martin, P. Wray, A. Ellington, N. Matthews, M. Ellwood, R. Woodmansey,\n Habtemariam, M. Wills, V. Brabec, P. J. Sadler, Organometallics 2018, 37, G. Clark, J. D. Cooper, A. Tromans, D. Grafham, C. Skuce, R. Pandian, R.\n 1555\u20131566. Andrews, E. Harrison, A. Kimberley, J. Garnett, N. Fosker, R. Hall, P.\n [17] R. Soni, K. E. Jolley, G. J. Clarkson, M. Wills, Org. Lett. 2013, 15, 5110\u2013 Garner, D. Kelly, C. Bird, S. Palmer, I. Gehring, A. Berger, C. M. Dooley, Z.\n 5113. Ersan-\u00dcr\u00fcn, C. Eser, H. Geiger, M. Geisler, L. Karotki, A. Kirn, J. Konantz,\n [18] K. E. Jolley, A. Zanotti-Gerosa, F. Hancock, A. Dyke, D. M. Grainger, J. A. M. Konantz, M. Oberl\u00e4nder, S. Rudolph-Geiger, M. Teucke, C. Lanz, G.\n Medlock, H. G. Nedden, J. J. M. Le Paih, S. J. Roseblade, A. Seger, V. Raddatz, K. Osoegawa, B. Zhu, A. Rapp, S. Widaa, C. Langford, F. Yang,\n Sivakumar, I. Prokes, D. J. Morris, M. Wills, Adv. Synth. Catal. 2012, 354, S. C. Schuster, N. P. Carter, J. Harrow, Z. Ning, J. Herrero, S. M. J. Searle,\n 2545\u20132555. A. Enright, R. Geisler, R. H. A. Plasterk, C. Lee, M. Westerfield, P. J. de\n [19] J. T\u00f6nnemann, J. Risse, Z. Grote, R. Scopelliti, K. Severin, Eur. J. Inorg. Jong, L. I. Zon, J. H. Postlethwait, C. N\u00fcsslein-Volhard, T. J. P. Hubbard,\n Chem. 2013, 2013, 4558\u20134562. H. R. Crollius, J. Rogers, D. L. Stemple, Nature 2013, 496, 498\u2013503.\n [20] a) G. W. T. M. J. Frisch, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. [35] a) I. Romero-Canel\u00f3n, A. M. Pizarro, A. Habtemariam, P. J. Sadler, Metal-\n Cheeseman, G. Scalmani, V. Barone, G. A. Petersson, H. Nakatsuji, X. Li, lomics 2012, 4, 1271\u20131279; b) L. Chen, H. Wang, X. Li, C. Nie, T. Liang, F.\n M. Caricato, A. V. Marenich, J. Bloino, B. G. Janesko, R. Gomperts, B. Xie, K. Liu, X. Peng, J. Xie, RSC Adv. 2018, 8, 35246\u201335256.\n Mennucci, H. P. Hratchian, J. V. Ortiz, A. F. Izmaylov, J. L. Sonnenberg, D. [36] A. Ballesta, F. Billy, J. P. C. Coverdale, J. I. Song, C. Sanchez-Cano, I.\n Williams-Young, F. Ding, F. Lipparini, F. Egidi, J. Goings, B. Peng, A. Romero-Canel\u00f3n, P. J. Sadler, Metallomics 2019, 11, 1648\u20131656.\n Petrone, T. Henderson, D. Ranasinghe, V. G. Zakrzewski, J. Gao, N. Rega, [37] M. A. Summers, J. L. Moore, J. W. McAuley, Ann. Pharmacother. 2004, 38,\n G. Zheng, W. Liang, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. 1631\u20131634.\n Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, [38] a) M. M. Gonz\u00e1lez-Ballesteros, C. Mej\u00eda, L. Ruiz-Azuara, FEBS Open Bio\n K. Throssell, J. A. Montgomery, Jr., J. E. Peralta, F. Ogliaro, M. J. Bearpark, 2022, 12, 880\u2013899; b) U. Jungwirth, C. R. Kowol, B. K. Keppler, C. G.\n J. J. Heyd, E. N. Brothers, K. N. Kudin, V. N. Staroverov, T. A. Keith, R. Hartinger, W. Berger, P. Heffeter, Antioxid. Redox Signaling 2011, 15,\n Kobayashi, J. Normand, K. Raghavachari, A. P. Rendell, J. C. Burant, S. S. 1085\u20131127; c) X. Li, Y. Wang, M. Li, H. Wang, X. Dong, Molecules 2021,\n Iyengar, J. Tomasi, M. Cossi, J. M. Millam, M. Klene, C. Adamo, R. Cammi, 27(1), 148, https://doi.org/10.3390/molecules27010148.\n J. W. Ochterski, R. L. Martin, K. Morokuma, O. Farkas, J. B. Foresman, D. J. [39] a) E. Eruslanov, S. Kusmartsev, in Advanced Protocols in Oxidative Stress II\n Fox, Gaussian 16, Revision C.01, Gaussian, Inc., Wallingford CT, 2016; (Ed.: D. Armstrong), Humana Press, Totowa, NJ, 2010, pp. 57\u201372; b) A.\n b) T. A. K. R. Dennington, J. M. Millam, GaussView, Version 6, Semichem Wojtala, M. Bonora, D. Malinska, P. Pinton, J. Duszynski, M. R.\n Inc, Shawnee Mission, KS, 2016. Wieckowski, Methods Enzymol. 2014, 542, 243\u2013262.\n [21] A. M. Hayes, D. J. Morris, G. J. Clarkson, M. Wills, J. Am. Chem. Soc. 2005, [40] W. H. Park, Y. W. Han, S. H. Kim, S. Z. Kim, J. Cell. Biochem. 2007, 102, 98\u2013\n 127, 7318\u20137319. 109.\n [22] R. Soni, F. K. Cheung, G. C. Clarkson, J. E. D. Martins, M. A. Graham, M. [41] J. P. C. Coverdale, H. E. Bridgewater, J. I. Song, N. A. Smith, N. P. E. Barry,\n Wills, Org. Biomol. Chem. 2011, 9, 3290\u20133294. I. Bagley, P. J. Sadler, I. Romero-Canel\u00f3n, J. Med. Chem. 2018, 61, 9246\u2013\n [23] X. Dai, H. Cheng, Z. Bai, J. Li, J. Cancer 2017, 8, 3131\u20133141. 9255.\n [24] V. Vichai, K. Kirtikara, Nat. Protoc. 2006, 1, 1112\u20131116.\n [25] S.-C. Chien, C.-Y. Chen, C.-F. Lin, H.-I. Yeh, Biomark. Res. 2017, 5, 31.\n [26] a) V. Novohradsk\u00fd, A. Bergamo, M. Cocchietto, J. Zajac, V. Brabec, G.\n Mestroni, G. Sava, Dalton Trans. 2015, 44, 1905\u20131913; b) O. D\u00f6m\u00f6t\u00f6r,\n C. G. Hartinger, A. K. Bytzek, T. Kiss, B. K. Keppler, E. A. Enyedy, J. Biol.\n Inorg. Chem. 2013, 18, 9\u201317; c) J. Lu, A. J. Stewart, P. J. Sadler, T. J. Manuscript received: April 23, 2024\n Pinheiro, C. A. Blindauer, Biochem. Soc. Trans. 2008, 36, 1317\u20131321. Revised manuscript received: May 23, 2024\n [27] J.-J. Qin, L. Yan, J. Zhang, W.-D. Zhang, J. Exp. Clin. Cancer Res. 2019, 38, Accepted manuscript online: May 24, 2024\n 195. Version of record online: July 9, 2024\n\n\n\n\n ChemBioChem 2024, 25, e202400374 (11 of 11) \u00a9 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH\n\f", "pages_extracted": 11, "text_length": 89155}