Development of Ruthenium Antitumor Drugs that Overcome Multidrug Resistance Mechanisms Carsten A. Vock, ² Wee Han Ang, ² Claudine Scolaro, ² Andrew D. Phillips, ² Lucienne Lagopoulos, ‡ Lucienne Juillerat-Jeanneret,* ,‡ Gianni Sava, §,| Rosario Scopelliti, ² and Paul J. Dyson* ,² Institut des Sciences et Inge ´ nierie Chimiques, Ecole Polytechnique Fe ´ de ´ rale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland, UniVersity Institute of Pathology, Centre Hospitalier UniVersitaire Vaudois (CHUV), CH-1011 Lausanne, Switzerland, Callerio Foundation Onlus, Via A. Fleming 22-31, 34127, Trieste, Italy, and Dipartimento di Scienze Biomediche, UniVersita ` di Trieste, Via L. Giorgieri 7-9, 34127, Trieste, Italy ReceiVed January 11, 2007 Organometallic ruthenium(II) complexes of the general formula [Ru(η 6 -p-cymene)Cl 2 (L)] and [Ru(η 6 -p- cymene)Cl(L) 2 ][BPh 4 ] with modified phenoxazine- and anthracene-based multidrug resistance (MDR) modulator ligands (L) have been synthesized, spectroscopically characterized, and evaluated in vitro for their cytotoxic and MDR reverting properties in comparison with the free ligands. For an anthracene-based ligand, coordination to a ruthenium(II) arene fragment led to significant improvement of cytotoxicity as well as Pgp inhibition activity. A similar, but weaker effect was also observed when using a benzimidazole- phenoxazine derivative as Pgp inhibitor. The most active compound in terms of both Pgp inhibition and cytotoxicity is [Ru(η 6 -p-cymene)Cl 2 (L)], where L is an anthracene-based ligand. Studies show that it induces cell death via inhibition of DNA synthesis. Moreover, because the complex is fluorescent, its uptake in cells was studied, and relative to the free anthracene-based ligand, uptake of the complex is accelerated and accumulation of the complex in the cell nucleus is observed. Introduction Drug resistance, that is, the appearance of reduced or missing response of microorganisms as well as cancer cells to applied chemotherapeutic agents, is a serious problem for the treatment of different diseases. 1 The macroscopic phenomenon can be divided into intrinsic drug resistance, where the application of drugs has no effect at all, and acquired drug resistance, where a normal response is observed at the beginning of the therapy, which then diminishes quickly and often disappears completely after a certain period of time. 2,3 For the treatment of cancer, but other diseases also, multidrug resistance (MDR a ) plays a very important role. MDR corresponds to a particular form of drug resistance, characterized by the simultaneous appearance of resistance to the applied chemotherapeutic agent and cross-resistance to a number of functionally and structurally diverse hydrophobic drugs, with different mechanisms of action. 4,5 The cellular mechanisms leading to MDR are still not fully understood, 6 and several factors seem to be of importance. 7 Most frequently discussed are (a) lowering of the intracellular concentration of the drug either by blocking uptake or increasing efflux, 8 (b) increased rates of repair of the drug damage, 9 and (c) accelerated rates of drug inactivation by protein binding (e.g., metallothionine and glutathione-S-transferase) and conjugation to small molecules such as glutathione. 10 It has been shown that MDR cells overexpress certain efflux proteins, which leads to a significantly lower intracellular level of chemotherapeutical agents. 11 The most prominent examples of this superfamily of proteins, for which a similar mechanism of action is assumed, are P-glycoprotein (Pgp) and MDR protein (MRP1). 12 While Pgp mainly transports neutral and charged molecules in unmodified form, MRP1 is also able to accept metabolized substrates such as GSH, glucuronide, or sulfate conjugates. 13-15 Because the transport into the extracellular medium has to be carried out against a strong concentration gradient, the process requires energy, and all the known MDR proteins are ATP- dependent efflux pumps. 16 Experimental results indicate that ATP- and substrate binding to Pgp occur independently. 17,18 However, ATP-binding and hydrolysis are necessary to mediate the transport. 19 Due to the high importance of Pgp and MRPs for effective anticancer therapy, a lot of research has focused on developing MDR modulators, which function by blocking transporter- mediated drug efflux so that a concomitantly administered anticancer drug can cause tumor cell death. Interestingly, a huge structural variety is observed not only for the substrates, but also for the blockers of the MDR efflux proteins. One of the most promising MDR antagonists is verapamil 1 (Figure 1), which was the first compound found to reverse MDR in vitro 20 and to reach clinical trials. 21 It has also been coadministered with ruthenium compounds, resulting in a significant improvement of their toxicity to cancer cells. 22 Interestingly, the (R)-enantiomer of verapamil 1 exhibits the same MDR reversal activity as the (S)-enantiomer, but shows lower cardiovascular side effects. 23,24 A number of pharmaco- logically active compounds, for example, the potassium channel blocker amiodarone, 25 the CNS active agent fluphenazine, 26 and the important immunosuppressant cyclosporin A 27 (for struc- tures, see Supporting Information (SI)), have also been shown to be strong MDR reversal agents. However, due to their own strong pharmacological effects, these drugs are not suitable for coadministration with anticancer drugs. * To whom correspondence should be addressed. Dr. Lucienne Juillerat-Jeanneret, University Institute of Pathology, CHUV, Rue de Bugnon 25, CH-1011 Lausanne, Switzerland. Tel.: +41 21 314 7173. Fax: +41 21 314 7115. E-mail: lucienne.juillerat@chuv.ch. Prof. Paul J. Dyson, Institut des Sciences et Inge ´nierie Chimiques, Ecole Polytechnique Fe ´de ´rale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland. Tel.: +41 (0)21 693 98 54. Fax: +41 (0)21 693 98 85. E-mail: paul.dyson@epfl.ch. ² Ecole Polytechnique Fe ´de ´rale de Lausanne. ‡ Centre Hospitalier Universitaire Vaudois. § Callerio Foundation Onlus. | Universita ` di Trieste. a Abbreviations: anthraimid, N-(anthracen-9-yl)-imidazole; EtOAc, ethyl acetate; MDR, multidrug resistance/resistant; MTT, 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide; Pgp, P-glycoprotein; phenoximid, 2-(imidazol-1-yl)-1-(phenoxazin-10-yl)-ethanone; phenoxbenzimid, 2-(ben- zimidazol-1-yl)-1-(phenoxazin-10-yl)-ethanone; pta, 1,3,5-triaza-7-phos- phaadamantane. 2166 J. Med. Chem. 2007, 50, 2166-2175 10.1021/jm070039f CCC: $37.00 © 2007 American Chemical Society Published on Web 04/10/2007