Cyclopentadienyl Half‐Sandwich Rhodium(III) Azopyridine Anticancer Complexes with Activity Tuned by Ligand Substituents

{"full_text": " Research Article\nChemCatChem doi.org/10.1002/cctc.202401863\n\n\n www.chemcatchem.org\n\n\n Cyclopentadienyl Half-Sandwich Rhodium(III) Azopyridine\n Anticancer Complexes with Activity Tuned by Ligand\n Substituents\n Edward C. Lant,[a] Russell J. Needham,[a] Zijin Zhang,[a, b] James P. C. Coverdale,[a, d]\n Guy J. Clarkson,[a] Ian Bagley,[c] Robert Dallmann,[b] and Peter J. Sadler*[a]\n\n We report the synthesis and characterization of ten novel half- selectivity (>3x) for A549 cancer cells versus normal cells. The\n sandwich Rh(III) azopyridine complexes as potential anticancer highly active, lipophilic complex 2 was strongly accumulated\n agents, with the general formula [(\u03b75 -Cpx )Rh(4-R2 -phenylazopy- by cells and catalyzed the oxidation of NADH (reduced nicoti-\n 5-R1 )Cl]PF6 , where Cpx = Cp*, CpxPh or CpxPhPh , R1 = H, Br, or namide adenine dinucleotide) to NAD+ , and GSH (glutathione)\n CF3 , and R2 = H, OH or NMe2 . X-ray crystallographic data for to GSSG. Notably, complex 2 is almost an order of magni-\n complex 2 (R1 = Br, R2 = OH, Cpx = CpxPh ) and complex 3 tude less toxic toward zebrafish in vivo than cisplatin, despite\n (R1 = CF3 , R2 = OH, Cpx = CpxPh ) confirm their typical half- being 10-fold more active in A549 cells. These studies demon-\n sandwich \u201cpiano-stool\u201d geometry. The substituents have a major strate how the chemical and biological activities of this series\n influence on the cytotoxicity of these complexes toward human of half-sandwich organorhodium(III) complexes can be finely\n ovarian (A2780 and cisplatin-resistant A2780cis), lung (A549) and tuned by the choice of substituents on the cyclopentadienyl and\n prostate (PC-3) cancer cells, and non-cancerous human lung azopyridine ligands. The complexes appear to have an unusual\n fibroblasts (MRC-5). Potencies range from sub-micromolar to mechanism of anticancer activity, associated not only with Rh(III)\n inactive (>50 \u03bcM). They were non-cross-resistant with cisplatin, but also with the phenylazopyridine, cyclopentadienyl, and the\n and complex 9 (R1 = H, R2 = NMe2 , Cpx = Cp*) showed some chlorido ligands.\n\n\n\n 1. Introduction in biological systems, and offer new avenues for targeted\n therapy.[5,6] Examples include cyclometalated phenylpyridine\n The success of platinum drugs, cisplatin and its analogues Os(II) half-sandwich anticancer complexes, which promote\n (carboplatin and oxaliplatin), has led to a search for the next rapid hydrolysis and activation,[7] and metal carbene complexes\n generation of transition metal-based anticancer agents, with for in-cell catalysis and synthesis.[8,9] Transfer hydrogenation\n the goal of reducing doses and side effects, and combat- catalysis in cells is an emerging approach, involving the mod-\n ting resistance by introducing new mechanisms of action.[1\u20134] ulation of cellular redox potentials via the NAD+ /NADH couple\n Recent studies have highlighted the design and application by Ru(II) catalysts,[10] or the stereospecific reduction of pyru-\n of organometallic metal complexes that can catalyze reactions vate by Os(II) catalysts.[11] These studies collectively underline\n the dual role of organometallic complexes in catalysis and\n therapeutic intervention, demonstrating their growing impor-\n [a] E. C. Lant, R. J. Needham, Z. Zhang, J. P. C. Coverdale, G. J. Clarkson, tance in innovative cancer treatments and other biomedical\n P. J. Sadler applications. Organorhodium compounds have shown promis-\n Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK\n ing cytotoxic and potential anticancer properties.[12\u201324] It is\n E-mail: p.j.sadler@warwick.ac.uk\n important to investigate the dependence of their chemical\n [b] Z. Zhang, R. Dallmann\n and biological reactivity on the oxidation state of the metal,\n Biomedical Sciences, Warwick Medical School, University of Warwick,\n Coventry CV4 7AL, UK its coordination geometry, and the types and number of lig-\n [c] I. Bagley ands, including ligand substituents. Fine-tuning the electronic\n Biomedical Service Unit Research Technology Platform (BSU RTP), Biomedical and steric properties of the bound ligands, and/or varying\n Sciences, University of Warwick, Coventry CV4 7AL, UK the metal and its oxidation state, are important for drug\n [d] J. P. C. Coverdale design and the construction of structure-activity relation-\n School of Pharmacy, School of Health Sciences, College of Medicine and ships. For example, changing Ru(II) to Rh(III) in the candidate\n Health, University of Birmingham, Edgbaston B15 2TT, UK anticancer drug RAPTA-C [Ru(\u03b76 -p-cymene)(pta)Cl2 ]), (where\n Supporting information for this article is available on the WWW under pta = 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane), results in\n https://doi.org/10.1002/cctc.202401863\n similar cytotoxicity toward A549 lung cancer cells.[23] When\n \u00a9 2025 The Author(s). ChemCatChem published by Wiley-VCH GmbH. This is co-administrated with a non-toxic dose of sodium formate\n an open access article under the terms of the Creative Commons Attribution\n as a source of reducing hydride, Rh(III) complexes of the\n License, which permits use, distribution and reproduction in any medium,\n provided the original work is properly cited. general structure [(Cpx )Rh(N\u02c6N)Cl]+ (Cpx = Cp* or CpxPh ,\n\n\n ChemCatChem 2025, 17, e202401863 (1 of 10) \u00a9 2025 The Author(s). ChemCatChem published by Wiley-VCH GmbH\n\f 18673899, 2025, 11, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cctc.202401863 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\nChemCatChem doi.org/10.1002/cctc.202401863\n\n\n\n\n Scheme 1. Rh(III) azopyridine complexes [(\u03b75 -Cpx )Rh(4-R2 -phenylazopy-5-R1 )Cl]PF6 (1\u201310) studied in this work and their synthetic route.\n\n N\u02c6N = ethylenediamine, 2,2\u0002 -bipyridine, 2,2\u0002 -dimethylbipyridine, related N\u02c6N chelated Rh-CpxPh distances are close to those in\n or 1,10-phenanthroline) can catalyze the reduction of intracel- similar diamine complexes.[29,32] The N\u2550N azo bonds are slightly\n lular biomolecules such as NAD+ via a transfer hydrogenation lengthened (1.275\u20131.277 \u00c5) compared to the uncoordinated ligand\n mechanism.[25\u201328] (1.25 \u00c5).[29,32]\n The methyl substituents of the Cpx ring in [(Cpx )Rh(N\u02c6N)Cl]+\n complexes (N\u02c6N = e.g. bipyridine) have unusual properties.[29]\n They can be activated by intramolecular proton abstraction 2.2. Hydrolysis\n by a Rh(III)\u2500OH group following Rh(III)\u2500Cl hydrolysis and\n undergo rapid H/D exchange, involving Rh(I) fulvene inter- Hydrolysis is a common activation mechanism for chlorido\n mediates. These intermediates can be trapped by the forma- transition metal anticancer complexes, for example, for cis-\n tion of Diels\u2013Alder adducts with conjugated dienes, includ- platin Pt(II)\u2500OH2 bonds are more reactive than Pt(II)\u2500Cl\n ing biologically important dienes, so providing novel reaction bonds.[17] The differences in ligand lability across various\n pathways for half-sandwich Rh(III) complexes.[30,31] Here, ten metal ions are reflected in their aqua ligand exchange rates.\n novel half-sandwich organorhodium(III) chlorido complexes 1\u2013 Appropriate choices of ligands can modulate hydrolysis\n 10 (Scheme 1) bearing various substituted bidentate azopyri- activity.[17]\n dine ligands have been synthesized, and characterized by 1 H Aquation of chlorido complexes 1\u201310 in 20% MeOD-d4 / 80%\n and 13 C NMR spectroscopy, high-resolution mass spectrome- D2 O (1 mM, 310 K) was studied by 1 H NMR with peak assignments\n try (HRMS), and HPLC. Their chemical and anticancer proper- assisted by comparison of spectra before and after removal of\n ties have been investigated, including the catalytic oxidation the chlorido ligand by reaction with 1 mol equiv of AgNO3 .\n of the intracellular coenzyme NADH and abundant tripep- After 24 h there was ca. 63% hydrolysis for 1, which was readily\n tide GSH (\u03b3 -L-Glu-L-Cys-Gly). The substituents on the Cp and reversed by the addition of 2 mol equiv of NaCl (Figures S3\u2013S5).\n azpy ligands are shown to have dramatic effects on their After 24 h, complexes 1\u20136 and 9\u201310 all reached equilibrium for\n activities. hydrolysis (Table 1). Complexes 7 and 8 were not soluble to 1 mM\n in MeOD-d4 for this 1 H NMR study. 1 H NMR spectra acquired\n after 24 h incubation at 310 K showed the presence of both the\n 2. Results and Discussion starting complex containing the chelated ligand (4-R2 -phenyl-\n azopyridine-5-R1 ) and the corresponding aqua complex.\n 2.1. Synthesis and Characterization of Complexes The extent of hydrolysis over 24 h for complexes varied\n between 0%\u2013100% at equilibrium, based on 1 H NMR integrations,\n Complexes 1\u201310 were synthesized by reactions of the azopyridine as shown for 1 in Figures S3\u2013S5. No apparent hydrolysis was\n ligands with the appropriate chlorido-bridged dimer (Scheme 1) observed for a 1 mM solution of complex 10, however, hydroly-\n in good yields of >78%, with an HPLC purity of >97% (see sis was observed at the lower concentrations (20 \u03bcM) by UV\u2013vis\n S1.2). NMR and HRMS data were consistent with the proposed spectroscopy at 310 K (Figure S7). The pKa values of the coordi-\n structures (see S1.2, Figures S1 and S2). Single crystals suitable nated water in the aqua adduct or phenolic groups on the azpy\n for X-ray diffraction of complexes 2\u00b7Et2 O and 3\u00b7Et2 O, as PF6 \u2212 ligands in these complexes were not investigated in this study.\n salts, were obtained at ambient temperature by slow diffusion If the pKa values for the aqua species are <7, then this is likely\n of Et2 O into saturated dichloromethane solutions (Figure 1 and to result in the formation of hydroxido species at physiologi-\n X-ray crystallographic data listed in Table S8, and selected bond cal pH (7.4), which might be prone to the formation of bridged\n lengths and bond angles in Table S9). The complexes adopt a species at higher concentrations. The pKa values for the Ph\u2500OH\n typical pseudo-octahedral \u201cpiano-stool\u201d geometry. Rh-Cl bond fragment are likely to lie close to the range 3.90\u20136.49, based on\n lengths (2.3745(14) and 2.3763(10) \u00c5 for 2\u00b7Et2 O and 3\u00b7Et2 O, respec- published data for related Ir(III) Cpx azpy complexes.[32] These\n tively) and Rh-Cpx centroid distances (1.495 and 1.500 \u00c5) and deprotonations might have an effect on the hydrolysis equilib-\n\n\n ChemCatChem 2025, 17, e202401863 (2 of 10) \u00a9 2025 The Author(s). ChemCatChem published by Wiley-VCH GmbH\n\f 18673899, 2025, 11, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cctc.202401863 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\nChemCatChem doi.org/10.1002/cctc.202401863\n\n\n\n\n Figure 1. X-ray crystal structures of complexes (a) [(\u03b75 -Cp*)Rh(HO-azpy-Br)Cl]PF6 (2\u00b7Et2 O) and (b) [(\u03b75 -Cp*)Rh(HO-azpy-CF3 )Cl]PF6 (3\u00b7Et2 O), with thermal\n ellipsoids drawn at 50% probability. The hydrogen atoms, counter anions, and solvent molecules are omitted for clarity.\n\n\n Table 1. Hydrolysis of complexes 1\u201310 studied by 1 H NMR in 20% MeOD-d4 /80% D2 O (1 mM, 310 K) and UV\u2013vis spectroscopy in 10% MeOH/90% H2 O (310 K).\n\n Complex Ligand Cpx Extent (%)a) kb) (min\u22121 ) t1/2 (min)\n\n 1 HO-azpy Cp xPh\n 63 0.0038 \u00b1 0.0002 182\n 2 HO-azpy-Br CpxPh 50 0.0030 \u00b1 0.0001 224\n 3 HO-azpy-CF3 CpxPh 73 0.0088 \u00b1 0.0001 78.6\n 4 azpy-F Cp xPh\n 100 0.0062 \u00b1 0.0002 111\n 5 HO-azpy Cp* 100 0.0034 \u00b1 0.0001 203\n 6 HO-azpy CpxPhPh 100 0.015 \u00b1 0.003 46,2\n 7 azpy-Br Cp* \u2013 0.015 \u00b1 0.001 46.2\n 8 azpy-Br Cp xPh\n \u2013 0.012 \u00b1 0.002 57.7\n 9 Me2 N-azpy Cp* 100 0.051 \u00b1 0.001 13.6\n 10 Me2 N-azpy CpxPh 0 0.032 \u00b1 0.001 21.7\n\n a)\n Determined by 1 H NMR peak integrals of the aqua adduct at equilibrium for 1 mM solutions in 20% MeOD-d4 /80% D2 O after 24 h at 310 K;\n b)\n Apparent pseudo-first-order rate constant determined by fitting the UV\u2013vis absorption changes versus time for 20 \u03bcM solutions of the complex in 10%\n MeOH/90% H2 O (310 K). UV\u2013vis data are shown in Figure S6 for 2 and 9.\n\n\n\n ria, and the species present at pH 7 under biological screening (\u2500N\u2550N\u2500 + e\u2212 \u2192 {\u2500N\u2500N\u2500}\u2212 ).[33] The relative order therefore\n conditions. reflects the decreasing \u03c0 -acceptor capability of the substituted\n Changes in the UV\u2013vis spectra were monitored for 20 \u03bcM azpy ligands with the addition of electron-donating groups onto\n solutions of the complexes in MeOH/H2 O v/v. Half-lives varied the phenyl ring.[33,34] The irreversible second reduction step is\n between ca. 14 and 224 min for complexes 9 and 2, respectively. assigned to conversion to the dianionic species ({\u2500N\u2500N\u2500}2 \u2212 ).\n Complex 9 has the electron-donating NMe2 substituent on the Azo groups usually give rise to two separate electrochemical\n azopyridine and a Cp*, whilst 2 has an electron-withdrawing reductions in polar aprotic solvents.[33,34] In aqueous media, the\n Br group on the pyridine and a phenyl group on the Cpx two-electron reduction of azo groups is accompanied by proton\n ring. transfer to give hydrazo groups (NH\u2500NH) in a single step.[33,34]\n In comparison with reported electrochemical data on related\n 2.3. Electrochemical Reduction half-sandwich azpy complexes (Table S2), the first reduction\n potentials for the Ru(II) complex [(\u03b76 -pcym)Ru(Me2 N-azpy)I]+\n Electrochemical reduction of complexes 2 and 9 in methanol was (Ru-a)[35] and Os(II) complex [(\u03b76 -pcym)Os(Me2 N-azpy)l]+ (Os-\n studied by cyclic voltammetry (CV) under N2 versus a Ag+ /Ag b)[36] are higher than for the Rh(III) complex [(\u03b75 -Cp*)Rh(Me2 N-\n reference electrode. For both complexes, the first reduction azpy)Cl]+ (9): 9 (MeOH) < Os-b (CH3 CN) < Ru-a (DMF). Although\n step was quasi-reversible (\u22120.586 and \u22120.795 V for 2 and 9, the solvents used are different, they have similar dielectric con-\n respectively), but the second was weak and irreversible Table stants (33\u201337).[37] Since the complexes all have the same azpy\n S1 and Figure S8. The first reduction potential becomes more ligand, the data illustrate that the metal ion, arene/Cpx , and\n positive as the Cpx ring is extended from Cp* to CpxPh and monodentate (Cl/I) ligands can all modulate the overall redox\n as the chelating azo ligand is changed from Me2 N-azpy, to properties of the complexes. The first reduction potentials are\n HO-azpy-Br. This reduction potential can be assigned to the addi- lower than the biologically relevant range, for example, for\n tion of an electron into the \u03c0 * orbital centered on the azo GSH/GSSG \u2212240 mV at pH 7,[38] noting that the latter value is\n group of the azopyridine ligands to form the azo anion radical for an aqueous medium in comparison to MeOH used here.\n\n\n\n\n ChemCatChem 2025, 17, e202401863 (3 of 10) \u00a9 2025 The Author(s). ChemCatChem published by Wiley-VCH GmbH\n\f 18673899, 2025, 11, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cctc.202401863 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\nChemCatChem doi.org/10.1002/cctc.202401863\n\n\n\n Table 2. IC50 values for complexes 1\u201310 and cisplatin (CDDP) in human A549 lung, PC-3 prostate, A2780 ovarian, and A2780cis cisplatin-resistant ovarian\n cancer cells, and normal MRC-5 fetal lung fibroblasts. Growth inhibition curves are shown in Figures S14\u2013S21.\n\n Complex A549 PC-3 A2780 A2780cis MRC-5\n IC50 /(\u03bcM)a) (RF)b)\n\n 1 3.7 \u00b1 0.1 6\u00b11 \u2013 \u2013 \u2013\n 2 0.8 \u00b1 0.1 1.4 \u00b1 0.1 1.4\u00b1 0.1 0.35 \u00b1 0.03 (0.2) 1.4 \u00b1 0.4\n 3 1.5 \u00b1 0.2 2.9 \u00b1 0.4 4.5 \u00b1 0.1 0.8 \u00b1 0.2 (0.2) 1.6 \u00b1 0.1\n 4c) >50 >50 \u2013 \u2013 \u2013\n 5c) >50 >50 \u2013 \u2013 \u2013\n 6 5.5 \u00b1 1.3 4.0 \u00b1 0.9 \u2013 \u2013 \u2013\n 7 33 \u00b1 12 19.6 \u00b1 0.8 \u2013 \u2013 \u2013\n 8 13 \u00b1 1 11.9 \u00b1 0.2 \u2013 \u2013 \u2013\n 9 29.9 \u00b1 0.5 18 \u00b1 4 8 \u00b1 0.5 7 \u00b1 1 (0.9) >100\n 10 6\u00b12 12 \u00b1 2 \u2013 \u2013 \u2013\n CDDP 6.6 \u00b1 0.1 1.7 \u00b1 0.4 1.4 \u00b1 0.1 12.2 \u00b1 0.2 (8.8) 7.6 \u00b1 0.6\n\n a)\n IC50 values are the mean \u00b1 standard deviations for two independent experiments, each carried out in triplicate. Cancer cells were exposed to the test\n complex for 24 h followed by 72 h recovery time in fresh medium.\n b)\n Resistance factor RF = IC50 (A2780cis)/IC50 (A2780).\n c)\n 100 \u03bcM highest test dose, see Figures S14d and S15e.\n\n\n tain an extended CpxPh ring and a hydroxyl substituent on the\n phenyl group of the azpy ligand. Under physiological conditions,\n it is likely that the OH group is deprotonated (pKa <7), as is the\n case for the analogous Ir(III) complexes,[32] hence the chlorido\n complexes would be neutral, enhancing cellular uptake. They\n would also be zwitterions. Guo et al. have reported potent activ-\n ity for zwitterionic Rh(III) half-sandwich sulfonated iminopyridine\n complexes, which initially target lysosomes.[40]\n A similar order of potency was observed for PC-3 prostate\n cancer cells (Table 2): 2 (most potent), CDDP, 3, 6, 1 > 8, 10 >\n 9, 7 > 4, and 5 (least potent).\n The relative potency toward the normal lung cell line MRC-5\n Figure 2. Accumulation of Rh (ng/106 cells) in A549 human lung cancer for complexes 2 and 3 versus the cancer cell line A549, indicates\n cells after 24 h treatment with complex 2 or 9 at concentrations of 0.5x a poor selectivity, perhaps influenced by their lipophilicities and\n IC50 , 1x IC50 , 2x IC50 , and 4x IC50 . The values represent mean \u00b1 standard\n cell uptake. Complex 9 showed promising selectivity of >3 for\n deviations for three independent biological replicates (N = 3).\n cancer cells over non-cancerous cells: IC50 29.9 \u00b1 0.5 \u03bcM in A549\n 2.4. Antiproliferative Activity (IC50 ) lung cancer cells, compared to >100 \u03bcM in MRC-5 lung fibroblast\n cells.\n Since for biological studies the rhodium complexes were pre- The accuracy by which some of the IC50 values could be\n pared in 5% DMSO, 95% DMEM, v/v, to aid solubility, the determined was limited by the shapes of some of the dose-\n stability of complexes 1\u201310 in DMSO for 24 h was studied by 1 H response sigmoidal plots, Figures S14\u2013S21. Notably, the highly\n NMR (400 MHz, DMSO-d6 ). No spectral changes were observed active complexes 1, 2, and 6, exhibited an abrupt decrease in\n indicative of their high stability in this solvent. cell viability as the administered dose was increased (Figures S15\n The antiproliferative activities of complexes 1\u201310 and cis- A549, S17, S18 PC-3, S20 A2780 and S21 MRC-5). This sharp, rapid\n platin were determined toward human lung A549 (Table 2), and decrease in cell percentage viability relative to the untreated\n prostate PC-3 cancer cell lines using the SRB assay[39] (Table 2). control may be correlated with the mechanism of cell death and\n Some of these complexes display potent cytotoxicity, with IC50 suggests possible necrosis, involving a sudden disintegration\n values in the range of 0.2 to 29.9 \u03bcM (Table 2). The order of of the plasma membrane.[41,42] Further investigation of plasma\n potency in A549 cells was: 2 (most potent) > 3 > 1, CDDP, 6, membrane targeting is underway, but beyond the scope of the\n 10 > 8 > 9, 7 > 4, and 5 (least potent). present study.\n Complex 2 [(CpxPh )Rh(HO-azpy-Br)Cl]PF6 was 10-fold more Based on their high in vitro activity, complexes 2 and 3, along\n active than cisplatin. In contrast, complexes 7 [(CpxPh )Rh(azpy- with complex 9, were selected for further screening against\n Br)Cl]PF6 and 9 [(Cp*)Rh(Me2 N-azpy)Cl]PF6 were less active. human ovarian cancer cells (A2780) and cisplatin-resistant A2780\n Interestingly, complexes 4 and 5 both showed poor activity ovarian cancer cells (A2780cis) to examine cross-resistance with\n (IC50 > 50 \u03bcM). The most active complexes (2, 3, and 1) all con- cisplatin (Table 2). Complexes 2 and 3 were ca. 60\u00d7 and\n\n\n ChemCatChem 2025, 17, e202401863 (4 of 10) \u00a9 2025 The Author(s). ChemCatChem published by Wiley-VCH GmbH\n\f 18673899, 2025, 11, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cctc.202401863 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\nChemCatChem doi.org/10.1002/cctc.202401863\n\n\n\n 15\u00d7 more potent than cisplatin toward the drug-resistant cell complex 2 or 9 using flow cytometry (Figure 4). Superoxide pro-\n line, while complexes 2,3, and 9 were not cross-resistant with duction was monitored in the orange channel FL1, and total ROS\n cisplatin, with resistance factors IC50 (A2780cis)/IC50 (A2780) of 0.3, species (including H2 O2 , peroxy and hydroxyl radicals, and per-\n 0.7, and 0.9, respectively. oxynitrite and NO) were monitored by the green channel FL2.\n It is notable that within a family of reported half-sandwich After 24 h exposure, an increase in ROS levels was observed in\n azpy Ru(II), Os(II), Rh(III), and Ir(III) complexes (Ru-a, Os-a, Os-b, cells treated with 2 or 9, relative to the untreated (stained) nega-\n and Ir-c, Table S3), the anticancer activity in, for example, A549 tive control. Elevated levels of superoxide were also observed in\n lung cancer cells can vary from highly potent nanomolar activity cells treated with complex 2 but not 9. Similarly, superoxide (SO)\n for Ir-c,[32] and Rh 2, which differ only in the I/Cl monodentate lig- generation has been reported for related cytotoxic azpy com-\n and), to inactivity (Rh 4, Ru-a).[35] This illustrates the fine-tuning plexes of Os(II), Ru(II), and Ir(III).[32,35,43] The observation of the\n available within this family of half-sandwich complexes as a plat- burst of SO generation observed in the Ir(III) azpy analogues[32]\n form for drug design for consideration of both activity, targeting, strongly points toward the importance of the azpy ligands in the\n and minimizing side effects. production of these species.\n\n\n 2.5. Cellular Rh Accumulation\n\n To elucidate factors that influence the observed anticancer activ- 2.8. In Vivo Activity\n ities, cellular Rh accumulation was quantified in A549 cells by\n ICP-MS after 24 h treatment with complexes 2 and 9 at equipo- Since the compounds showed promise as anticancer therapeu-\n tent concentrations of 0.5\u00d7,1\u00d7, 2\u00d7, and 4\u00d7 IC50 at 310 K (Table tics in vitro, in vivo toxicities were investigated using zebra\n S4). Rh accumulation increased in a concentration-dependent fish embryos. The zebra fish model has been shown to closely\n manner. After 24 h, cellular Rh accumulation in cells treated with model toxicity in humans.[44,45] LD50 concentrations were deter-\n equipotent concentrations of more potent complex 2 was ca. mined following the well-established zebra fish embryo tox-\n eightfold higher than in cells treated with less potent complex icity test (Figure 5). Strikingly, rhodium complexes 2 and 9\n 9 (Figure 2). Hence, the extent of drug accumulation may play a (LD50 = 4.8 \u00b1 0.2 \u03bcM and 1.1 \u00b1 0.1 \u03bcM, respectively) were up\n major role in the activity. to eightfold less toxic toward zebra fish embryos than cisplatin\n (LD50 = 0.6 \u00b1 0.2 \u03bcM in SG-WT). The second-generation plat-\n inum anticancer drug, carboplatin, was an order of magnitude\n 2.6. Lipophilicity and Anticancer Activity less toxic than cisplatin (LD50 = 5.7 \u00b1 0.9 \u03bcM in SG-WT) toward\n zebra fish.[43]\n Capacity factors (K) measure the affinity of a compound toward Whole-mount zebra fish embryos were exposed to equipo-\n the HPLC stationary phase, and hence provide a measure of the tent solutions of Rh(III) complexes 2 and 9 (1.0 \u00d7 IC50 ) for 96 h\n relative lipophilicity of complexes. Capacity factors were deter- and anesthetized. Levels of reactive oxygen species were deter-\n mined for complexes 2, 3, 9, and 10 (as examples of complexes mined using a green fluorescent probe to detect ROS (including\n with either high or low antiproliferative activity) using an iso- H2 O2 , peroxynitrite, and hydroxyl radicals) and analyzed using\n cratic HPLC method at 298 K, relative to the retention time of confocal microscopy. Embryos were stained using the reagent\n uracil, Table S6. The mobile phase was H2 O: MeOH (1:1, v/v) of the ROS/Superoxide Detection Assay (Enzo Life Sciences) for\n containing 50 mM NaCl, and the stationary phase was a reverse- ROS detection with excitation at 458, 488, and 561 nm and green\n phase C18 column (250 \u00d7 4.6 mm column with a pore size emission for ROS at 493\u2013550 nm.[46] Pyocyanin was used as a\n of 5 \u03bcm). Correlations between antiproliferative activity toward positive control. Dye localization was observed in the positive\n A549 cells and capacity factor are shown in Figure 3. control, (Figure 5c, zebra fish embryos exposed to 50 \u03bcM of\n A strong inverse correlation is observed (\u22120.92, Figure 3); pyocyanin for 5 min).\n that is, when the relative lipophilicity increases, the IC50 value In general, high intracellular ROS generation causes cell\n decreases (increased activity), which is overall likely due to the death by activating cell death pathways (both mitochondrial-\n improved cellular accumulation of complexes exhibiting higher dependent and independent).[47\u201349] However, low levels of ROS\n lipophilicity. Complex 2 showed the highest capacity factor and act as signaling molecules that facilitate cell survival.[47\u201349] There\n hence was the most lipophilic complex. The trend for increasing are distinct differences in the extent and localization of the sig-\n lipophilicity is 9 <10 <3 <2, that is, increasing with extension to nal attributed to the production of ROS in embryos treated\n the substituent on the Cpx ring (i.e., CpxPh >Cp*). This is clearly with 2 and 9. Embryos incubated with 2 exhibited fluorescence\n demonstrated by complexes 9 and 10, both of which contain the localized in a region of the rhombencephalon or hindbrain (hb)\n same azpy ligand substituents. (Figure 5b), whereas embryos treated with 9 displayed a much\n greater concentration of ROS fluorescence extending from the\n yolk sac (ys) to the caudal vein (cv) (Figure 5d). The differences\n 2.7. ROS Studies in ROS signals between 2 and 9 correlate with the in vitro ROS\n induction study (Table 2 and Figure 4) whereby 9 induces greater\n Levels of reactive oxygen species (ROS) and superoxide (SO) ROS levels in comparison to 2, which may be related to the\n were quantified in A549 human lung cancer cells treated with efficiency of radical generation.\n\n\n ChemCatChem 2025, 17, e202401863 (5 of 10) \u00a9 2025 The Author(s). ChemCatChem published by Wiley-VCH GmbH\n\f 18673899, 2025, 11, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cctc.202401863 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\nChemCatChem doi.org/10.1002/cctc.202401863\n\n\n\n\n Figure 3. Plot of antiproliferative activity (IC50 toward A549 cells, \u03bcM) versus capacity factor (K), for complexes 2, 3, 9, and 10 in which substituents on the\n azpy and Cpx ligands are varied. In general, the most lipophilic complexes are the most active. Highly active complex 2 is likely to have a deprotonated\n phenoxide group at pH 7.4. It has a higher lipophilicity (capacity factor, Table S6) and cell uptake (Figure 3) than complex 9.\n\n\n\n\n Figure 4. Cell population for total ROS and superoxide production in A549\n cancer cells exposed to complex 2 or 9 at IC50 concentrations of 2 (0.5 \u03bcM)\n and 9 (29.9 \u03bcM) for 24 h with untreated cells as the negative control. FL1\n channel detects superoxide production, and FL2 channel detects total\n oxidative stress. Normalized population data are presented as the\n mean \u00b1 SD of triplicate samples for one experiment. p-Values were\n calculated after a t-test against the negative control data, *p < 0.05, **p <\n Figure 5. Fluorescence imaging of ROS (green) for whole-mount SG-WT\n 0.01. See Table S5 for full numerical data.\n zebra fish (Danio rerio) treated with Rh(III) complexes (1.0 \u00d7 IC50 ) (b) 2 and\n (d) 9 for 96 h. (b) Embryos incubated with 2 exhibited fluorescence\n 2.9. Catalytic Oxidation of NADH localized in the hindbrain (hb). (d) Whereas embryos treated with 9\n displayed fluorescence extending from the yolk sac (ys) to the caudal vein\n (cv). Confocal images were acquired using a Zeiss LSM880 confocal\n The cofactor 1,4-NADH is an important hydride source in microscope. Embryos were stained using the reagent of the\n cells.[10,50,51] Its oxidation to NAD+ by Rh(III) complexes 2 and 9 ROS/Superoxide Detection Assay (Enzo Life Sciences) for ROS detection.\n was monitored using UV\u2013visible spectroscopy for 24 h at 298 K, Excitation at 458 and 488 nm; green emission for ROS at 493\u2013550 nm.[46]\n Pyocyanin was used as a positive control; the negative control was stained\n with 25, 50, and 100 \u03bcM 1,4-NADH (Figure 6). Conversion was using 2 \u03bcM of ROS detection reagent.\n determined by measuring the absorbance at 339 nm, corre-\n sponding to 1,4-NADH (Figure 6). Turnover numbers (moles of equiv.) compared to catalyst 2 (TOF = 1.43 h\u22121 ). This may be\n NADH converted per mol of catalyst in 24 h) and maximum related to the faster rate of hydrolysis of complex 9 compared\n turnover frequencies (TOFmax /h\u22121 ) were then calculated (Figure 7 to complex 2 and the higher reactivity of the aqua species.\n and Table 3). Both complexes catalyze the oxidation of 1,4-NADH In the presence of 25 or 50 \u03bcM NADH, complex 9 (bearing a\n to NAD+ . The initial rate was greater for catalyst 9 for all three Cp* unit) showed a respective ca. twofold or ca. fourfold higher\n concentrations of 1,4-NADH (TOFmax = 4.93 h\u22121 for 100 mol TOFmax than CpxPh complex 2. This is similar to previous stud-\n\n\n ChemCatChem 2025, 17, e202401863 (6 of 10) \u00a9 2025 The Author(s). ChemCatChem published by Wiley-VCH GmbH\n\f 18673899, 2025, 11, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cctc.202401863 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\nChemCatChem doi.org/10.1002/cctc.202401863\n\n\n\n\n Figure 6. UV\u2013vis absorption spectra recorded every hour for 25, 50, or 100 mol equiv of NADH in the presence of 2 \u03bcM complex (a) control (ca. 200 \u03bcM\n NADH) (b) 9 with 25 mol equiv NADH (c) 9 with 50 mol equiv, and (d) 9 with 100 mol equiv after 24 h in 1.6% MeOH/98.4% 5 mM phosphate buffer (pH\n 7.4) at 310 K. The turnover number (TON) and maximal turnover frequency (TOFmax ) were determined based on the decrease in absorption of NADH at\n 339 nm due to the conversion of NADH to NAD+ over 24 h. In (a) and (d), the intensity of the peak at 252 nm is affected by the limit of detection.\n\n\n\n\n Figure 7. Plots of turnover number versus time for the catalytic oxidation of NADH at three different molar equivalents of NADH (25, 50 or 100 \u03bcM) by\n complexes (a) 9 (2 \u03bcM) and (b) 2 (2 \u03bcM) in 1.6% MeOH/98.4% v/v, 5 mM phosphate buffer (pH 7.4) at 293 K.\n\n Table 3. TONs and TOFs for oxidation of NADH to NAD+ by 2 and 9 in ligand. In a related study involving the generation of NADH from\n 1.6% MeOH/98.4% 5 mM Na2 HPO4 -NaH2 PO4 buffer (pH 7.4). NAD+ using formate as the hydride source and Rh(III) complexes\n Catalyst NADH (mol equiv) TON TOFmax (h\u22121 )\n with N\u02c6N-substituted 2,2\u0002 -bipyridine ligands, the substituents\n strongly influenced catalytic activity.[51] Hydrogen peroxide was\n 2 25 2 0.08 \u00b1 0.01 also detected semi-quantitatively (appearance of blue color on\n 2 50 30 1.23 \u00b1 0.05 Quantofix test sticks) in a reaction mixture of complex 9 (ca.\n 2 100 34 1.43 \u00b1 0.06 1 mM) with 3.0 mol equiv. NADH in MeOH/H2 O (1/1 v/v) after\n 9 25 8 0.31 \u00b1 0.01 24 h at 310 K (Figure S9) showing ca. 5 mg/L H2 O2 , implicating a\n 9 50 74 3.06 \u00b1 0.01 role for O2 in the catalytic cycle.\n 9 100 118 4.93 \u00b1 0.02\n\n 2.10. Reactions with Glutathione\n\n ies of [(Cpx )Rh(diamine)Cl)]+ complexes, for which the catalytic In cells, a major reducing agent is the intracellular tripeptide\n activity (TOF) for the reduction of NADH increased in the order glutathione (GSH, \u03b3 -L-Glu-L-Cys-Gly), a thiol present at millimo-\n en <phen <bpy and the TOF was also lower for extended CpxPh lar concentrations which acts as a detoxification agent and\n or CpxPhPh rings.[51] ROS scavenger.[52] Reactions of 9 [(Cp*)Rh(Me2 N-azpy)Cl]+ and\n These reactions may also be influenced by steric factors 2 [(CpxPh )Rh(HO-azpy-Br)Cl]+ (100 \u03bcM) with GSH (10 mM) in\n caused by the size of the extended Cpx ring and the chelated phosphate buffer (20 mM, pH 7.4) were monitored over 24 h\n\n\n ChemCatChem 2025, 17, e202401863 (7 of 10) \u00a9 2025 The Author(s). ChemCatChem published by Wiley-VCH GmbH\n\f 18673899, 2025, 11, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cctc.202401863 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\nChemCatChem doi.org/10.1002/cctc.202401863\n\n\n\n by HPLC/LC-MS (Figure S10). From the ESI-MS of the HPLC by the X-ray crystal structures of highly cytotoxic complexes 2\n peaks (Figure S11 and Table S7) ca. 95% of 9 was converted to and 3.\n the glutathione adduct [(Cp*)Rh(Me2 N-azpy)(SG)]+ (m/z 768.21 This study reveals how significant variations in the cytotox-\n [M + SG]+ ), (9-SG) within the first 10 min, and was the major icity of Rh(III) azopyridine complexes depend on both ligand\n species in solution over 24 h. In contrast, no adduct formation substituents and cellular interactions. For example, complexes 1,\n was observed for 2 after 24 h, correlating with its slower rate of 5, and 6 containing the HO-azpy ligand differ only in the exten-\n hydrolysis (Table 1). sion of the Cpx ligand, with 1 containing Ph, 5 CH3 , and 6 PhPh\n as substituents. However, this results in 30x and 1.5x increase in\n cytotoxicity for 1 and 6, respectively, compared to 5 in A549 cells.\n 2.11. Catalytic Oxidation of Glutathione Similarly, for complexes 9 and 10, which both contain the Me2 N-\n azpy ligand and again differ only in the extension of the Cpx\n Reactions of complex 2 and 9 (100 \u03bcM) with glutathione (10 mM) ligand, with 10 containing an extended Ph substituent. A similar\n in phosphate-buffered saline (20 mM, pH* 7.4) at 310 K were increase in cytotoxicity is observed, with 10 being slightly more\n also monitored by 1 H NMR for 24 h. The formation of oxidized active than 9 in A549 and PC-3 cells (Table 2). Another example\n glutathione (GSSG) was evident from the appearance of new res- shows that in a comparison of the cytotoxicity of 2 with 1, which\n onances at \u03b4 = 3.30 ppm, corresponding to the \u03b2 -CH2 of GSSG differ only in the addition of a Br group on the azpy ligand in 2\n from the reaction of complex 9 (TON = 27 \u00b1 1, Figure S12). In and there is a 5x increase in activity in A549 and PC-3 cells.\n contrast, no oxidation was observed in the presence of complex Importantly, neither complex 2 nor 9 was cross-resistant with\n 2. There are reports of azo compounds facilitating the oxidation cisplatin, suggesting a distinct mechanism of action which may\n of GSH, including the Hodgkin\u2019s and non-Hodgkin\u2019s lymphoma not involve DNA targeting, the classical mechanism for current\n drug procarbazine, a hydrazo compound that is readily oxidized clinical platinum drugs cisplatin and carboplatin. The sudden\n to the corresponding azo compound and re-reduced via GSH onset of cell death apparent in some cell growth inhibition plots\n oxidation to GSSG.[53] Interestingly, analogous GSH adducts of (Figures S14\u2013S21) suggests that necrosis may play a role in the\n Ru(II) and Os(II) arene complexes can undergo oxidation at the mechanism of cell death for some of the complexes studied\n coordinated sulfur.[54,55] The re-oxidation of this hydrazo group here, and correlate with the ability of these complexes to target\n is facilitated by O2 inside cells, which is hydrogenated to form cell membranes.\n H2 O2 .[54,55] Future investigations into the role of the azo bond in Despite the ability of 9 to catalyze the oxidation of GSH and\n the catalytic cycle are therefore warranted. NADH, its IC50 (29.9 \u03bcM in A549) was significantly higher than\n Studies have also shown that the oxidation of GSH to GSSG is that of 2. However, 9 exhibited promising selectivity for cancer\n not induced by the free azopyridine ligands.[54,55] This is perhaps cells over non-cancerous cells. In vivo zebra fish toxicity studies\n not surprising because electron-donating groups attached to the revealed that both 2 and 9 were significantly less toxic than cis-\n azo ligands (in this instance OH or NMe2 ) decrease their ability platin. ROS generated by complex 2 were localized primarily in\n to oxidize GSH.[39] The first step in the electrochemical reduc- the zebra fish hindbrain, whereas ROS generated by 9 were more\n tion of azo ligands can be assigned to the one-electron addition widespread, extending from the yolk sac to the caudal vein.\n into the \u03c0 * orbital centered on the azo group to give the azo These results are consistent with ROS induction studies in A549\n anion radical.[34,56,57] The second one-electron reduction gives lung cancer cells. Complex 2 demonstrated eightfold greater\n rise to the dianionic species ([\u2500N\u2500N\u2500]\u22122 ). In aqueous media, rhodium accumulation in A549 cells compared to 9 (Table 2),\n two-electron reduction is also accompanied by proton transfer likely a consequence of its higher relative lipophilicity. Nonethe-\n to give hydrazo groups [\u2500NH\u2500NH\u2500].[34] Since the redox poten- less, complex 9 exhibited the highest catalytic activity for NADH\n tial for GSH/GSSG (\u2212240 mV at pH 7)[34,58,59] is less negative than oxidation. Complex 9 also reacted more rapidly with GSH, which\n those of the azpy Rh(III) complexes 2 and 9, (Table S1) and out- might result in greater cellular deactivation, and thus contribute\n side the biologically relevant region of \u2212430 and \u2212273 mV,[38] to the higher potency of 2 compared to 9 in lung cancer cells.\n these complexes are less easily reduced. This suggests that these However, for the analogous Ir(III) azpy complexes, DFT calcu-\n complexes are less likely to be reduced by GSH than in their Ir(III) lations have suggested that a plausible pathway for the GSSG\n analogues.[32] Overall, the catalytic oxidation of GSH to GSSG by formation involves the initial formation of the GSH adduct.[32]\n Rh(III) complexes is not as efficient as for their Ir(III) analogues, The findings in this study illustrate that the rational design\n and may point toward a different mechanism of action for this of these half-sandwich anticancer complexes as complexes with\n class of Rh(III) Cpx azopyridine compounds. unusual mechanisms of action can be fine-tuned by the choice\n of substituents on the azpy and cyclopentadienyl ligands.\n\n 3. Conclusions 4. Experimental Section\n The synthesis and characterization of novel azopyridine Rh(III) 4.1. General Procedures\n anticancer complexes 1\u201310 has allowed the dependence of their Rhodium chlorido complexes 1\u201310 (Scheme 1) were synthesized\n chemical and biological activities on substituents on the Cp ring by the same general procedure as follows: the ligand (2.1 mol\n and chelated azpy ligands to be studied. The complexes have equiv) was added to a 10.0 mL ethanol solution of [(\u03b75 -Cpx )Rh(\u03bc-\n typical \u201cpiano-stool\u201d half-sandwich configurations, as exemplified Cl)Cl]2 (Cpx = Cp* or CpxPh , 1.0 mol equiv). The mixture immediately\n\n\n ChemCatChem 2025, 17, e202401863 (8 of 10) \u00a9 2025 The Author(s). ChemCatChem published by Wiley-VCH GmbH\n\f 18673899, 2025, 11, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cctc.202401863 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\nChemCatChem doi.org/10.1002/cctc.202401863\n\n\n\n turned to dark red and was stirred at ambient temperature for Keywords: Azopyridine ligands \u2022 Cancer cell cytotoxicity \u2022 Half-\n 24 h. Substituted azopyridine ligands HO-azpy-Br, HO-azpy-CF3 and\n sandwich complexes \u2022 Reactions with biomolecules \u2022 Rh(III)\n HO-azpy, azpy-Br and azpy-F were synthesized and characterized\n cyclopentadienyl catalysts\n according to reported procedures as reported in the Supporting\n Information.[2,3]\n [1] K. Peng, Y. Zheng, W. Xia, Z. W. Mao, Chem. Soc. Rev. 2023, 52, 2790\u20132832.\n [2] A. M. Florea, D. B\u00fcsselberg, Cancers 2011, 3, 1351\u20131371.\n 4.2. X-Ray Crystallography [3] S. Dasari, P. B. Tchounwou, Eur. J. Pharmacol. 2014, 740, 364\u2013378.\n [4] M. Hanif, C. G. Hartinger, Future Med. Chem. 2018, 10, 615\u2013617.\n X-ray crystallographic data for complexes 2 and 3 have been [5] S. Alonso-de Castro, A. Terenzi, J. Gurruchaga-Pereda, L. Salassa, Chem.\n deposited in the Cambridge Crystallographic Data Centre under - Eur. J. 2019, 25, 6651\u20136660.\n the accession numbers CCDC 2390246\u20132390247, respectively. X- [6] A. L. Noffke, A. Habtemariam, A. M. Pizarro, P. J. Sadler, Chem. Commun.\n 2019, 48, 5219\u20135246.\n ray crystallographic data in CIF format are available from the\n [7] T. C. Chang, K. Tanaka, Bioorg. Med. Chem. 2021, 46, 116353.\n Cambridge Crystallographic Data Centre (http://www.ccdc.cam.ac\n [8] S. Infante-Tadeo, V. Rodr\u00edguez-Fanjul, C. C. Vequi-Suplicy, A. M. Pizarro,\n .uk/). Inorg. Chem. 2022, 61, 18970\u201318978.\n More detailed experimental procedures can be found in the [9] S. Guti\u00e9rrez, M. Tom\u00e1s-Gamasa, J. L. Mascare\u00f1as, Chem. Sci. 2022, 13,\n Supporting Information where the authors have cited additional 6478\u20136495.\n references. [10] S. Banerjee, P. J. Sadler, RSC Chem. Biol. 2021, 2, 12\u201329.\n [11] J. P. Coverdale, I. Romero-Canel\u00f3n, C. Sanchez-Cano, G. J. Clarkson,\n A. Habtemariam, M. Wills, P. J. Sadler, Nat. Chem. 2018, 10, 347\u2013\n 354.\n Author Contributions [12] Y. B. Peng, W. He, Q. Niu, C. Tao, X. L. Zhong, C. P. Tan, P. Zhao, Dalton\n Trans. 2021, 50, 9068\u20139075.\n Edward C. Lant and Peter J. Sadler conceived the project, [13] M. C. Risi, J. Stj\u00e4rnhage, W. Henderson, J. R. Lane, C. G. Hartinger, G. C.\n Saunders, Dalton Trans. 2025, 54, 539\u2013549.\n planned the experiments, and interpreted data. Edward C. Lant [14] C. H. Leung, H. J. Zhong, D. S. H. Chan, D. L. Ma, Coord. Chem. Rev. 2013,\n and Russell J. Needham designed and synthesized the ligands 257, 1764\u20131776.\n and complexes, characterized by Edward C. Lant. Guy J. Clarkson [15] Y. Geldmacher, M. Oleszak, W. S. Sheldrick, Inorganica Chim. Acta 2012,\n 393, 84\u2013102.\n carried out the X-ray crystallography. Hydrolysis and redox reac-\n [16] G. Gasser, I. Ott, N. Metzler-Nolte, J. Med. Chem. 2011, 54, 3\u201325.\n tions were carried out by Edward C. Lant. Cytotoxicity screening [17] E. J. Anthony, E. M. Bolitho, H. E. Bridgewater, O. W. Carter, J. M.\n was carried out by Zijin Zhang, Edward C. Lant and Robert Dall- Donnelly, C. Imberti, E. C. Lant, F. Lermyte, R. J. Needham, M. Palau, P.\n mann, Rh accumulation assays by Zijin Zhang and Edward C. J. Sadler, Chem. Sci. 2020, 11, 12888\u201312917.\n [18] C. M. Bernier, C. M. DuChane, J. S. Martinez, J. O. I. I. Falkinham, J. S.\n Lant, and zebrafish screening by James P. C. Coverdale, Edward\n Merola, Organometallics 2021, 40, 1670\u20131681.\n C. Lant, and Ian Bagley. The paper was drafted by Edward C. [19] V. S. da Silva, R. M. Dantas, M. Jos\u00e9, S. Mesquita, B. A. Rodrigues, S. C.\n Lant and Peter J. Sadler and all authors contributed to the final Correia, D. O. da Silva Mendes, A. C. de Melo Cotrim, J. L. da Silva Gon\u00e7,\n version. W. B. dos Santos, Adv. Biol. Chem. 2025, 15, 1\u20137.\n [20] P. \u0160tarha, Z. Dvor\u030c\u00e1k, Z. Tr\u00e1vn\u00edc\u030cek, J. Organomet Chem. 2018, 872, 114\u2013122.\n [21] J. Liang, A. Levina, J. Jia, P. Kappen, C. Glover, B. Johannessen, P. A. Lay,\n Inorg. Chem. 2019, 58, 4880\u20134893.\n [22] G. J. Yang, W. Wang, S. W. F. Mok, C. Wu, B. Y. K. Law, X. M. Miao, K. J.\n Acknowledgements Wu, H. J. Zhong, C. Y. Wong, V. K. W. Wong, D. L. Ma, Angew. Chem., Int.\n Ed. 2018, 57, 13349.\n We thank the Engineering and Physical Sciences Research Coun- [23] A. Dorcier, W. H. Ang, S. Bolano, L. Gonsalvi, L. Juillerat-Jeannerat,\n cil (EPSRC, grant no. EP/F034210/1 and EP/P030572/1), Anglo G. Laurenczy, M. Peruzzini, A. D. Phillips, F. Zanobini, P. J. Dyson,\n Organometallics 2006, 25, 4090\u20134096.\n American Platinum, China Scholarship Council (Studentship for [24] J. J. Soldevila-Barreda, P. J. Sadler, Curr. Opin. Chem. Biol. 2015, 25, 172\u2013\n ZZ), the Royal Society of Chemistry (grant no. E22-1637945680 for 183.\n JPCC) and Warwick Analytical Science CDT/Bruker (studentship [25] A. Marrone, R. H. Fish, Organometallics 2023, 42, 288\u2013306.\n for ECL) for funding. We thank Dr. Lijiang Song for assistance [26] W. Y. Zhang, H. E. Bridgewater, S. Banerjee, J. J. Soldevila-Barreda, G. J.\n Clarkson, H. Shi, C. Imberti, P. J. Sadler, Eur. J. Inorg. Chem. 2020, 1052\u2013\n with mass spectrometry, Dr. Ivan Prokes with NMR spectroscopy, 1060.\n and Drs. Oliver Carter and Cinzia Imberti with cell culture and [27] M. M. Milutinovic\u0301, J. V. Bogojeski, O. Klisuric\u0301, A. Scheurer, S. K. Elmroth,\n cytotoxicity assays. \u017d. D. Bugarc\u030cic\u0301, Dalton Trans. 2016, 45, 15481\u201315491.\n [28] J. J. 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