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Thermoresponsive organometallic arene ruthenium complexes for tumour targeting

Chemical Science View Article Online Open Access Article. Published on 02 January 2014. Downloaded on 5/2/2026 2:00:06 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. EDGE ARTICLE Cite this: Chem. Sci., 2014, 5, 1097 View Journal | View Issue Thermoresponsive organometallic arene ruthenium complexes for tumour targeting† Catherine M. Clavel, Emilia Păunescu, Patrycja Nowak-Sliwinska and Paul J. Dyson* Received 19th November 2013 Accepted 23rd December 2013 Application of mild hyperthermia can increase the cytotoxicity of anticancer drugs in tumour cells. In this report, we describe low molecular weight thermoactive ruthenium-based drugs with fluorous chains that are selectively triggered by mild hyperthermia. The organometallic complexes were prepared, characterized, and evaluated for their in vitro cytotoxicity against a panel of human cancer cell lines and DOI: 10.1039/c3sc53185f non-cancerous immortalized cells. The compounds show considerable chemo-thermal selectivity towards www.rsc.org/chemicalscience cancer cells (ca. 5 mM versus >500 mM for healthy cells) for the compound with the longest fluorous chain. Introduction Platinum-based anticancer drugs including cisplatin, carboplatin and oxaliplatin lack selectivity towards cancerous cells and therefore their therapeutic application causes severe sideeffects such as nephrotoxicity,1–3 neurotoxicity,4,5 nausea and vomiting.6,7 In contrast, ruthenium-based chemotherapeutics present fewer side-effects compared to platinum-based drugs. Although ruthenium-based compounds are not currently employed in the clinic, two ruthenium(III) compounds, namely KP10198 and NAMI-A,9 completed phase I clinical trials and are currently in phase II trials. The different toxicity proles of platinum- and ruthenium-based compounds remain unclear, although several reasons have been proposed.10 Irrespective of the full mechanistic differences it is not unreasonable that DNA targeting by platinum compounds leads to the severe sideeffects due to the ubiquitous nature of this target. Interestingly, organoruthenium (piano-stool) complexes with the structural composition [RuII(h6-arene)X2(PTA)] (PTA ¼ 1,3,5-triaza-7phosphaadamantane), known as RAPTA compounds, exhibit anti-metastatic11 and anti-angiogenic12 properties coupled with a relatively low toxicity comparable to that observed for NAMI-A.13 In an effort to improve drug selectivity it is possible to enhance the activity of a compound at the tumour site by applying external techniques or inducers.14 One such strategy combines chemotherapy with tumour localised mild hyperthermia.15–17 A slight increase of the local temperature differentiates tissues, healthy ones adapting easily while cancerous cells, with a disorganized and compact vascular structure, have Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland. E-mail: paul.dyson@ep.ch † Electronic supplementary information (ESI) available: Detailed descriptions of the synthesis and characterization of all compounds, procedures for the cytotoxicity determination and cell uptake measurements by ICP-MS. See DOI: 10.1039/c3sc53185f This journal is © The Royal Society of Chemistry 2014 difficulties in dissipating the heat. Some chemotherapeutics exhibit increased activity under mild hyperthermia (40.5– 42  C),18 even though they are also cytotoxic under normal conditions, and were not intentionally designed for this application. The thermosensitivity of small molecule drugs can be enhanced by attaching them to thermoresponsive macromolecules, e.g. liposomal drug carriers19–24 or micelles that are insoluble at 37  C and become soluble under hyperthermia, enabling them to cross the cell membrane where they release their drug content.25,26 Replacing macromolecules with low molecular weight thermosensitive drugs remains an attractive alternative approach. As proof of concept, rationally designed thermoactive derivatives of the organic drug chlorambucil (CLB)27,28 have been recently designed and were found to be essentially inactive at 37  C and activated by mild hyperthermia (41  C) in vitro.29 Recently, the synthesis and biological evaluation (under normal conditions) of some short to medium length uorous chain bipyridine cisplatin derivatives have been reported.30,31 Similar types of compounds (amphiphilic uoroalkylated bipyridine platinum and palladium complexes) have also been tested in liposomal formulations.32–34 Liposomal formulations of platinum-based drugs, with the rational that liposomal delivery can increase drug bioavailability and also accumulation at the tumour site as a consequence of the enhanced permeability and retention (EPR) effect, are now in clinical trials.35–38 Herein, ruthenium(II)–arene derivatives (Fig. 1) modied with uorous chains in order to endow them with thermoresponsive properties39–41 are described. The general structure of these new ruthenium(II)–arene complexes is similar to that of RAPTA-C (Fig. 1) – the PTA ligand being replaced with the desired uorous or alkyl derivatized pyridine ligands. The two labile chloride ligands allow activation via hydrolysis following cellular internalization.11,42 Pyridine was selected as the coordinating moiety based on the widespread use of such ligands in the domain.43–51 The uorous and alkyl chains are connected to the pyridine ligand via an Chem. Sci., 2014, 5, 1097–1101 | 1097 View Article Online Open Access Article. Published on 02 January 2014. Downloaded on 5/2/2026 2:00:06 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Chemical Science Fig. 1 Edge Article Structure of RAPTA-C and the new ruthenium(II)–arene complexes derivatized with alkyl or fluoroalkyl ‘ponytails’. ester linker that may, in principle, be hydrolysed by intracellular enzymes such as esterases.41,52,53 Results and discussion The proposed approach implies a straightforward synthetic pathway and, consequently, the new derivatives, containing either an alkyl or uorous chain, were synthesized in two steps using modied pyridine ligands as shown in Scheme 1. The pyridine ligands were obtained in good yield (70–87%) using a standard procedure starting from commercially available 3-pyridine-propionic acid and the corresponding alkyl or uoroalkyl alcohols. In the second step the pyridine ligands were reacted with the dimer, [Ru(h6-p-cymene)Cl2]2, in anhydrous, degased dichloromethane in the dark under an inert atmosphere. The complexes were isolated by precipitation in good yield (71–87%). All the compounds have been fully characterized (1H, 13C and where appropriate 19F NMR spectroscopy, ESI mass spectrometry, IR spectroscopy and elemental analysis: see ESI for details†). The formation of the ester ligands (both alkyl and peruoroalkyl derivatives) is accompanied by a deshielding of around 0.4 ppm of the protons in the alpha position relative to the oxygen atom, and subsequent complexation to the ruthenium center via the pyridine N-atom is accompanied by a deshielding of ca. 0.4 ppm for the two pyridine protons in the alpha position to the nitrogen atom and of a deshielding of ca. 5 ppm for the respective carbon atoms. There is only little change in position of the proton signals of the p-cymene ring in comparison to those observed in the parent dimer [Ru(h6-pcymene)Cl2]2. The structures of the compounds were further corroborated by ESI-MS. The most abundant peaks observed in the spectra of the ligands are those assigned to [M + H]+ ions, whereas the spectra of the pyridine Ru(II)-p-cymene complexes are dominated by species assigned to [M  Cl]+ ions. Apart from Scheme 1 the 19F NMR spectra and the very specic 13C NMR prole, the presence of the uorous chain is also clearly evidenced from the IR spectra with the presence of a strong large peak between 1110 and 1250 cm1. A peak at ca. 1730 cm1 conrms the presence of the ester C]O group. In vitro anticancer activity The cytotoxicity of the modied pyridine ligands and their corresponding complexes has been assessed in various cancer cell lines (cisplatin-sensitive A2780 and resistant A2780cisR ovarian carcinoma, MCF-7 and MDA-MBA-231 breast carcinomas and A549 human lung carcinoma) and human embryonic kidney (HEK 293) cells (used as a model for normal cells). Cytotoxicity studies were carried out at 37  C for 72 hours and at 41  C for 2 hours followed by 70 hours at 37  C to simulate hyperthermia in the tested cell lines (Table 1). Distinct thermosensitive behaviour of the compounds is present, but needs to be evident against the majority of the tested cancerous cell lines in order to be considered as effective. In this respect, complex 2c exhibits considerable differences of up to at least two orders of magnitude (maximum concentrations tested were 500 mM) and hence exhibits ideal thermoresponsive behaviour. In all cases, complex 2c remains inactive at normal body temperature (IC50 values >500 mM) and becomes toxic towards tumour cells aer a 2 hour hyperthermia signal (IC50 values ranging from 5.0 to 42 mM in the various cancer cell lines). Strikingly, the ligand in 2c, i.e. L2c, shows no thermoactivity or cytotoxicity against the screened cell lines except on MCF-7 breast cancer with a negligible (non-thermoresponsive) toxicity of 237 mM at 37  C and 284 mM under hyperthermia. Moreover, 2c shows selectivity towards cancerous cells with a weak cytotoxicity under mild hyperthermia against HEK 293 cells. Synthesis of ligands L1a–L2c and the ruthenium-p-cymene complexes 1a–2c. 1098 | Chem. Sci., 2014, 5, 1097–1101 This journal is © The Royal Society of Chemistry 2014 View Article Online Edge Article Chemical Science IC50 values determined for the ligands L1a–L2c and complexes 1a–1d and 2a–2c in A2780, A2780cisR, A549, MCF-7, MDA-MB-231 and HEK 293 cell lines at 37  C and under hyperthermia (2 h at 41  C followed by 70 h at 37  C – labeled 41  C in the table) Table 1 Open Access Article. Published on 02 January 2014. Downloaded on 5/2/2026 2:00:06 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. A2780 (mM) A2780cisR (mM) MCF-7 (mM) MDA-MB-231 (mM) A549 (mM) HEK 293 (mM) Compound 37  C 41  C 37  C 41  C 37  C 41  C 37  C 41  C 37  C 41  C 37  C 41  C L1a L1b L1c L1d L2a L2b L2c 1a 1b 1c 1d 2a 2b 2c 263  12 98  7 69  4 40  4 181  12 243  35 >500 23  1 49  1 49  1 15  1 52  2 15  1 10  1 >500 224  23 69  4 88  5 141  9 192  7 >500 114  1 >500 84  1 >500 111  1 25  2 >500 389  27 >500 97  15 >500 >500 >500 >500 482  18 362  14 48  1 27  1 >500 21  1 42  2 >500 >500 315  48 301  75 209  5 >500 237  25 339  73 319  87 >500 >500 >500 38  2 >500 458  16 133  13 110  7 >500 136  1.8 >500 284  31 218  4 108  2 63  4 17  1 70  4 25  2 5.0  0.3 >500 >500 487  9 >500 362  24 >500 >500 328  22 >500 >500 70  8 275  19 36  2 >500 459  27 >500 >500 >500 >500 >500 >500 100  2 473  20 96  8 >500 67  9 31  2 36  5 303  17 >500 96  3 >500 364  8 >500 >500 >500 >500 391  14 >500 355  43 43  1 >500 358  20 323  18 100  9 >500 >500 >500 >500 >500 >500 123  8 42  12 >500 40  2 33  7 >500 >500 189  10 >500 >500 >500 >500 155  17 >500 86  10 >500 270  18 >500 >500 338  3 153  12 206  1 >500 >500 >500 >500 324  10 45  3 42  1 149  9 160  6 >500 132  5 >500 >500 >500 >500 >500 476  164 >500 >500 >500 113  2 >500 >500 44  1 >500 The incorporation of the uorous chain appears to give rise to the thermoactive effect. Indeed, complex 1c, the hydrocarbon analogue of 2c, exhibits a totally different prole to 2c. It is active in some cell lines at 37  C and also moderately toxic against non-tumourigenic HEK 293 cells with an IC50 value of 86 mM. Complexes with shorter alkyl chains, i.e. 1a and 1b, show generally poor activity against cancerous and non-cancerous cells. Complex 1d, with the longest alkyl chain, exhibits good thermoactivity except in the MDA-MB-231 cell line with an IC50 of 70 mM at 37  C and >500 mM under hyperthermia. Similarly, its ligand alone, L1d, is only thermoactive in A2780 cells. Indeed, against A2780cisR and MCF-7 cells, L1d is more active than the corresponding complex at normal body temperature, but loses activity under hyperthermia, a behaviour shared with ligands L1b, L2a, L2b and even complex 2a. Excluding the cisplatin-resistant cell line, only L2a is less cytotoxic under hyperthermia against MDA-MB-231 and A459 cells. Ligands L1d and L2b are inactive at both temperatures in the other cell lines. Ligands with the longest, bulky chains, i.e. L2b, L2c and L1d, are the least active ligands across the panel of cell lines. In A2780 cells the alkylated ligands show increasing cytotoxicity under hyperthermia as the chain length increases, possibly due to increased lipophilicity. Compounds containing the shorter uorinated chains do not exhibit a thermoactivity comparable to 2c. Consequently, the length of the uorous chain has a signicant impact on the potential thermoactive behaviour, which is consistent with the results from the study of chlorambucil modied with uorinated chains.29 Nevertheless, 2b is remarkably cytotoxic and selective towards cancerous cells compared to normal cells, whereas the activity of 2a is not affected by mild hyperthermia in a systematic manner, presumably due to the short uorous chain. was used to simulate the hyperthermia signal during a 24 hour incubation prior to measurement. At 37  C 2c is internalized three fold more in the A2780 ovarian cancer cell line compared to the normal HEK 293 cells (Fig. 2). Under mild hyperthermia, internalization of 2c in A2780 cells increases whereas heat has little impact on uptake into HEK 293 cells. These data are consistent with the tumour cell selectivity observed for 2c. It should be noted, however, that while uptake of 2c into cancer cells exceeds that in the HEK 293 cells, uptake alone does not explain the vast differences in cytotoxicity following heat treatment. In this context the difficulties cancer cells have dissipating heat54,55 must also make them more susceptible to cell death induced by the internalized compound. Cellular uptake Cellular uptake studies were conducted on the lead complex, i.e. 2c, to determine the dependency of uptake on temperature in cancerous and non-cancerous cells. A 2 hour heating at 41  C This journal is © The Royal Society of Chemistry 2014 Fig. 2 Cellular uptake of 2c in A2780 and HEK 293 cell lines with and without a 2 hour hyperthermia signal at 41  C. Error bars represent Standard Deviation. Chem. Sci., 2014, 5, 1097–1101 | 1099 View Article Online Chemical Science Open Access Article. Published on 02 January 2014. Downloaded on 5/2/2026 2:00:06 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Conclusions Organometallic ruthenium complexes with a long uorous appendage exert selective cytotoxicity toward tumour cells under mild hyperthermia. Long uorous chains are required to obtain relevant thermoresponsive behaviour. For the lead compound, i.e. 2c, it is noteworthy that the uorous ligand alone is not cytotoxic under any of the applied conditions whereas the ruthenium complex demonstrates considerable differences under normal and thermal conditions (ca. 5 mM versus >500 mM) and selectivity towards cancer cells over healthy HEK 293 cells. Discrimination between cancerous and normal cells may be attributed to more extensive internalization by cancer cells compared to normal cells combined with the fact that the tumoural cells are sensitized to the cytotoxic agents under mild hypothermia. This discovery opens the way towards the rational design of other thermoactive anticancer drugs. 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