DOI: 10.1002/cmdc.201200341 Arene–Ru II Complexes of Curcumin Exert Antitumor Activity via Proteasome Inhibition and Apoptosis Induction Laura Bonfili,* [a] Riccardo Pettinari,* [b] Massimiliano Cuccioloni, [a] Valentina Cecarini, [a] Matteo Mozzicafreddo, [a] Mauro Angeletti, [a] Giulio Lupidi, [b] Fabio Marchetti, [c] Claudio Pettinari, [b] and Anna Maria Eleuteri [a] Introduction Curcumin, or diferuloylmethane, is an active compound isolat- ed from the rhizomes of Curcuma longa. It is a natural phenolic agent with multiple biological activities and cellular targets. In fact, it is well characterized for its anti-inflammatory, [1] antivi- ral, [2, 3] and neuroprotective actions, [4] and for its antioxidant ac- tivity. [5, 6] In addition, curcumin possesses antiproliferative ef- fects, targeting cell-cycle regulators and triggering apopto- sis. [6–8] As with other polyphenols, curcumin is able to inhibit proteasome function, the major non-lysosomal enzymatic com- plex, whose activity is fundamental for several cellular process- es including cell-cycle regulation, apoptosis, angiogenesis, and cell differentiation. [9, 10] The 20S proteasome is the proteolytic core of the ubiquitin–proteasome system and includes several peptidase activities: chymotrypsin-like activity (ChT-L, cleaving after hydrophobic residues), trypsin-like activity (T-L, cleaving after basic residues), peptidylglutamyl peptide-hydrolyzing ac- tivity (PGPH, cleaving after acidic residues), as well as branched-chain amino acid preferring (BrAAP) and small neu- tral amino acid preferring (SNAAP) components. [11] By virtue of all these properties, curcumin has recently become a very attractive compound for the treatment of a wide variety of disorders, including neurodegeneration and cancer. [12] In addition, numerous studies on human and animal models have revealed that curcumin is extremely safe and well tolerated even at high doses. [13] Nevertheless, the potential ap- plication of curcumin as a therapeutic agent is severely restrict- ed owing to its low water solubility, poor absorption and bio- availability, high rate of metabolism, and rapid clearance from the body. [13, 14] Several approaches have been taken to address these issues and to improve the efficacy of curcumin-based treatments, including the use of adjuvants, liposomal curcu- min, and the design of curcumin derivatives and analogues. [13] Metal complexes, including platinum, ruthenium, and galli- um offer a promising opportunity for the design of bioactive compounds with anticancer properties. [15, 16] Cisplatin deriva- tives are widely used in cancer treatment, but their inefficiency against platinum-resistant tumors and the associated severe side effects, such as nephrotoxicity, [17] have stimulated the quest for alternative strategies. Ruthenium complexes are con- sidered the most interesting alternatives. [18, 19] For example, imi- dazolium trans-[tetrachloro(dimethylsulfoxide)(1H-imidazole)- ruthenate(III)], known as NAMI-A, combines relatively low tox- icity with strong efficacy against solid tumor metastases and recently passed through a phase I clinical evaluation. [20–22] Inda- zolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], known Organometallic ruthenium(II) complexes of general formula [(h 6 -arene)Ru(curcuminato)Cl], with arene being p-iPrC 6 H 4 Me (1), C 6 H 6 (2), and C 6 Me 6 (3), were synthesized, characterized, and evaluated for their antitumor effects. Specifically, we ex- plored their ability to regulate the proteasome, a validated pharmacological target in cancer treatment. Ruthenium com- plexes inhibited isolated proteasomes to various extents, with the biological activity of these complexes depending on the nature of the bound arene; in particular, [(h 6 -arene)Ru(curcumi- nato)Cl] 2 suppressed proteasomal activities more potently than 1, 3, or free curcumin. Each complex also inhibited pro- teasomes in cultured colon cancer cells and consequently trig- gered apoptosis, with the [(h 6 -benzene)Ru(curcuminato)Cl] complex 2 being the most active. The influence on the oxida- tive status of HCT116 cells and the DNA binding ability of the [(h 6 -arene)Ru(curcuminato)Cl] complexes were studied. Com- plex 2 showed the highest antioxidant capacity; moreover, complexes 1 and 2 were shown to bind isolated DNA with higher affinity (up to threefold) than free curcumin. Collective- ly, our results demonstrate that the complexation of curcumin with ruthenium(II) is a promising starting point for the devel- opment of curcumin-based anticancer drugs. [a] Dr. L. Bonfili, Dr. M. Cuccioloni, Dr. V. Cecarini, Dr. M. Mozzicafreddo, Prof. M. Angeletti, Prof. A. M. Eleuteri School of Biosciences and Biotechnology, University of Camerino via Gentile III da Varano, 62032 Camerino (MC) (Italy) E-mail : laura.bonfili@unicam.it [b] Dr. R. Pettinari, Prof. G. Lupidi, Prof. C. Pettinari School of Pharmacy, University of Camerino via S. Agostino 1, 62032 Camerino (MC) (Italy) E-mail : riccardo.pettinari@unicam.it [c] Prof. F. Marchetti School of Science and Technology, University of Camerino via S. Agostino 1, 62032 Camerino (MC) (Italy) 2010  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemMedChem 2012, 7, 2010 – 2020 MED