Chemistry and catalysis advances in organometallic chemistry and catalysis
ON THE TRACK TO CANCER THERAPY: PAVING
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43 ON THE TRACK TO CANCER THERAPY: PAVING NEW WAYS WITH RUTHENIUM ORGANOMETALLICS T ˆania S. Morais and M. Helena Garcia *
In the field of search for metallodrugs as anticancer agents, ruthenium chemistry has been an attracting field because of the promising results obtained for some families of the inorganic octahedral Ru(III) compounds. In this frame, NAMI-A [1] and KP1019 [2] appeared as the most representative examples being already in clinical evaluation. Although the original appeal for ruthenium drugs have circumvented the noxious effects of the highly toxic chemotherapy based on cisplatin drugs, the success in the treatment of metastases by some ruthenium compounds pointed out the important potential of these drugs in the field of innovative chemotherapies. At present, there is no drug capable of controlling metastases growth, thus making the fight against metastases one of the main problems in the battle against cancer that urge to be solved. The strategy of synthesis for metallodrugs has been following the platinum models envisaging the cell death by interaction with DNA, preferentially by a different mechanism from that of cisplatin, to overcome tumor cell resistance. In this scenario, a variety of ruthenium(II) organometallic complexes have been designed viewing the classical target, the DNA. Two main families of organometallic ruthenium(II)-arene complexes (see Fig. 43.1) were developed and studied by Sadler [3, 4] (RM complexes) and Dyson [5, 6] (RAPTA complexes), in which the basic structures present the piano-stool geometry and their reactivity toward biological targets is based on the leaving chloride ligands, as happens with cisplatin. Nevertheless, these two families of compounds besides the similarity of their structure present different characteristics. While RM complexes reveal quite good cytotoxicity in vitro, RAPTA complexes show low cytotoxicity but are very efficient against invasion and metastasis effects. A third family of organometallic compounds was studied by Meggers [7, 8] (DW-complexes) and involves pyridocarbazole ligands (to mimic stausporine) as organic moiety coordinated to the ruthenium(II)-cyclopentadienyl ligand fragment (see Fig. 43.1), which revealed strong and selective inhibitors of protein kinases GSK-3 and Pim-1, thus showing their potentiality as anticancer agents. In spite of the development of ruthenium drugs and all the studies in some large classes of compounds, essentially typified in Fig. 43.1, there is not yet any direction concerning structure–activity relationship to serve as a guide line for future research in the field. However, all results gathered up now certainly point out for an added value of the ruthenium ion on the overall interaction in spite of the nonexistence of any plausible direct interaction with the target. Advances in Organometallic Chemistry and Catalysis: The Silver/Gold Jubilee International Conference on Organometallic Chemistry Celebratory Book, First Edition. Edited by Armando J. L. Pombeiro. © 2014 John Wiley & Sons, Inc. Published 2014 by John Wiley & Sons, Inc.
582 ON THE TRACK TO CANCER THERAPY: PAVING NEW WAYS WITH RUTHENIUM ORGANOMETALLICS Figure 43.1 Chemical structures of some ruthenium compounds. 43.2 OUR STRATEGY IN BIOORGANOMETALLIC CHEMISTRY In the frame of organometallic ruthenium drugs, our research group started to develop a new family of compounds based on the so-called piano-stool geometry, in which the reactivity mechanism is expected to be different from the complexes presented earlier because of the absence of any chloride leaving groups. The ruthenium–carbon bond of the “piano” is built with a cyclopentadienyl group (Cp), in which the negative charge gives additional electrostatic stability to the “Ru(II)Cp” fragment, compared to the equivalent “Ru(II)-arene” “piano” structure (see Fig. 43.2). Stability of the fragment “RuCp” was corroborated by our studies in gas phase by electrospray ionization mass spectrometry (ESI-MS), carried out with several complexes of the family [RuCp(PP) n L][CF
3 SO 3 ] (where PP = phosphane, L = heteroaromatic ligand, and n = 1 or 2), which in all the cases originate “RuCp” as final species, impossible to dissociate even at very high values of energy [9]. This general piano-stool structure allows us to play with the nonleaving groups on the legs of the piano, which were chosen to be phosphane ligands (mono or bidentate) and heteroaromatic ligands coordinated by N, O, S, etc. where the coordination can be by one or two atoms. The design of these compounds can lead to a significant structural diversity as we can play with each part of the molecule, such as functionalization on the Cp ring, coordination number, and nature of the coligands, and also with the counter-ion itself (Fig. 43.3). Several of these features can constitute an important advantage to control the solubility of the compounds, this being the most important to optimize the biological activity.
Building of piano-stool structures based on cyclopentadienyl and arene groups. DISTRIBUTION IN THE BLOOD 583 Figure 43.3 Examples of several structures of “RuCp” compounds showing different coordination for the ligands. TABLE 43.1 IC 50 Values for Some “RuCp” Based Compounds for Several Human Cancer Cell Lines (72 h, 37 ◦
Tumor Cell Lines IC 50 , μM Compound
A2780 A2780CisR MCF7 MDAMDB231 HT29 PC3
4 0.19
± 0.03 0.21
± 0.04 0.05
± 0.01 0.03
± 0.01 0.08
± 0.01 0.41
± 0.08 5 0.46
± 0.11 0.47
± 0.16 0.41
± 0.09 0.23
± 0.07 0.53
± 0.14 1.9
± 0.3 TM34 0.14
± 0.01 0.07
± 0.02 0.29
± 0.01 0.72
± 025 0.41
± 0.07 0.54
± 0.10 Cisplatin 2.00 ± 0.10
17 ± 3.0
28 ± 6.0
39 ± 5.0
7.0 ± 2.0
51 ± 7.0
43.3 BIOLOGICAL ACTIVITY Cytotoxicity studies for these “RuCp” compounds, carried out in vitro with several human cancer cell lines, namely, A2780 (ovarian carcinoma), A2780CisR (ovarian carcinoma, cisplatin resistant), HT29 (colon adenocarcinoma), MCF7 (breast adenocarcinoma— hormone dependent, ER α+), MDAMB231 (breast adenocarcinoma—hormone independent), and PC3 (prostate cancer) revealed excellent antitumor activities with IC 50 values much lower than those found for cisplatin [10, 11] (Table 43.1). Noteworthy, the excellent activity found for cisplatin-resistant cancer cells suggests that a mechanism of action different from cisplatin might be involved in the present case. It is important to mention the excellent values obtained with MDAMB231 cancer cells that have highly metastatic properties.
Protein-drug binding greatly influences the distribution and pharmacological properties of a drug [12]. Human serum albumin (HSA) is the principal and most abundant protein of the circulatory system [12, 13]. The main function of albumin is to transport fatty acids and a broad range of drug molecules to its targets [14, 15]. HSA often increases the solubility of hydrophobic drugs in plasma [16]. As HSA serves as a drug transport carrier, the knowledge of the kind of interaction between drugs and plasma proteins is of major importance to understand the drug pharmacokinetics and pharmacodynamics. 584 ON THE TRACK TO CANCER THERAPY: PAVING NEW WAYS WITH RUTHENIUM ORGANOMETALLICS (a) (b)
(c) Figure 43.4 Effect of HSA on the cytotoxicity of TM34 on A2780 (a) and A2780CisR cells (c) after a 24-h challenge. A27890 cells were treated with TM34 at 1 μM (a and c) and 5 μM (b) preincubated with HSA at 1 : 1, 1 : 5, and 1 : 10 complex-to-protein molar ratios. Data shown are the mean values ( ±SD) of two independent experiments, each performed with at least six replicates. Control indicates cells with no treatment (negative control), HSA (5, 25, and 50 μM) indicates cells treated with HSA alone in the concentrations indicated, TM34 (1 and 5 μM) are positive controls (cell treated with the complex in the absence of albumin). Adapted from Reference 11. The binding of several “RuCp” compounds to HSA was studied as a first approach to outline its pharmacokinetics and had been investigated by spectroscopic methods (absorption and fluorescence) and ultrafiltration-UV–vis. as well. It was found that TM34 binds to HSA forming a 1 : 1 adduct; the stability constant calculated for this {HSA–TM34} adduct was log Kb = 4. This value is similar to that found for KP1019 and shows that the complex can be transported in the blood by HSA [11]. As the binding to HSA can affect the drug biological activity and toxicity, it was important to check the cytotoxicity in the presence of albumin. Our experiments carried out with the {HSA–TM34} adduct did not significantly affect the activity of TM34 in the A2780 ovarian adenocarcinoma cells even in the sensitivity or resistance to cisplatin [11] (see Fig. 43.4).
Information concerning drug distribution and concentration in the tumor cells is of primordial importance for understanding the drug mechanism of action. Drugs must not only enter into the cell but also concentrate in the cell compartments where the target is localized. For many traditional and new anticancer agents, these targets are localized either in the cell membrane and cytosol or in the nucleus. Studies carried out for some of our “RuCp” complexes by inductively coupled plasma mass spectrometry (ICP-MS) to quantify the amount of ruthenium ion in the several cell fragments, after incubation with different cancer cell lines, showed that ruthenium preferentially accumulates in the cell membrane. Nevertheless, a significant amount of this metal is also found in cytosol and nucleus. Comparison with results obtained for cisplatin reference, used in our studies in the same experimental conditions, reveals that Pt barely reaches these three compartments. 43.6 MECHANISMS OF ACTION The study of metallodrugs has been inspired in the “cisplatin model” that is until now the only metallodrugs available for chemotherapy treatments. The ruthenium(III) complexes (NAMI-A [1] and KP1019 [2]) that are currently in clinical trials were synthesized having in view DNA as the main target. However, some evidences have been found for the involvement of other biomolecule targets such as proteins and enzymes besides DNA. In this frame, our studies were thought to envisage both classical and nonclassical targets thus being focused on the interaction studies with DNA, PARP-1 enzyme, and some cell proteins.
Our first approach in the interaction studies with DNA involved comparison of atomic force microscopy (AFM) images of the plasmid pBR322 DNA incubated with our “RuCp” compounds [17–20], which revealed several types of strong
MECHANISMS OF ACTION 585 OC CCC (a) (b)
2.00 1.00
0 2.00
1.00 0 0 1.00 2.00
0 1.00
2.00 μm μm Figure 43.5 AFM image of the (a) free plasmid pBR322 DNA and (b) plasmid pBR322 DNA incubated with the complex TM34 ([RuCp(PPh 3 )(2,2 -bipy)][CF 3 SO 3 ]) after 1 h, showing supercoiled forms (b). Adapted from Reference 20. interactions when compared with the free plasmid. In fact, the images of TM34 displayed several supercoiled forms of plasmid DNA [20] (see Fig. 43.5) and were similar to the images previously observed for typical intercalating molecules such as 9-aminoacridine [21, 22] showing that TM34 was able to modify the DNA structure. Moreover, also a strong interaction was observed when compounds of this family were incubated with calf thymus DNA, which solutions presented visible aggregates slightly colored by the compound. This feature did not allow us to pursue further studies by other techniques, such as viscosity, fluorescence, and UV–vis spectroscopies, to collect experimental data concerning the type of interaction of “RuCp” with DNA. The observed formation of colored DNA aggregates is certainly a clear evidence of a strong interaction. Therefore, it might be concluded that DNA is a possible target of this family of compounds.
The increasing evidence in the literature concerning the importance of enzymes and proteins as relevant targets for the mode of action of nonplatinum anticancer metallodrugs (for which multiple biological pathways have been proposed) [23, 24] led us to explore the role of proteins and enzymes in the antitumor activity of “RuCp” compounds [11]. PARP (poly-(adenosinediphosphate(ADP)-ribose)polymerases) enzymes play a key role in DNA repair mechanisms by detecting and initiating repair after DNA strand breaks [25]. PARP inhibitors have been evaluated as drugs for use in combinatorial therapies with DNA-damaging agents [26] and to sensitize cancer cells to subsequent treatment with cisplatin and carboplatin [27]. Phases I and II clinic trials evaluating the use of PARP inhibitors in combination therapies with platinum drug are currently underway [28]. It is known that PARP-1, the most studied member of the PARP family, is activated by mild DNA damage and is involved in DNA repair process, so that the cell survives. Moreover, PARP-1 may cause cellular death via necrosis or apoptosis, depending on the type, strength, and duration of genotoxic stimuli and the cell type. The ruthenium complex TM34 presents an IC 50 value for PARP-1 inhibition of 1 μM [11] (see Fig. 43.6), considerably lower than 33
μM found for the classical inhibitor 3-aminobenzamide [29], for the ruthenium complexes RAPTA and NAMI-A (28 and 18,9
μM, respectively) and cisplatin (12.3 μM) [30]. Comparing these metallodrugs, TM34 is the strongest ruthenium inhibitor of PARP-1, its effect clearly surpassing those of RAPTA-T and NAMI-A and that of cisplatin as well (these being circa 30–10 times less effective) [11]. Apoptosis (programmed cell death) is involved in the development and elimination of the damaged cells. Deregulation of apoptosis can cause diseases such as cancers. Ubiquitin and cytochrome c are important in the mechanism of cell death being involved in the first steps of apoptosis. Apoptosis is executed by a subfamily of cysteine proteases known as caspases. In mammalian cells, a major caspase activation is the cytochrome c initiated pathway [31]. On the other hand, ubiquitin is used by cells as a covalent modifier of other proteins both to activate their function and to target them for degradation [32]. Our most recent studies by ESI-MS of “RuCp” complexes with these proteins show significant interaction and importantly reveal that ruthenium compounds preserve their initial structure. 586 ON THE TRACK TO CANCER THERAPY: PAVING NEW WAYS WITH RUTHENIUM ORGANOMETALLICS Figure 43.6 IC 50 value for TM34 inhibition compared to the reference benchmark PARP-1 inhibitor 3-aminobenzamide (3-AB) [29], cisplatin, and ruthenium complexes that exhibit antimetastatic activity in vivo NAMI-A and RAPTA-T (data for comparison taken from [30]). Adapted from Reference 11.
Organometallic compounds offer much potential for the search of anticancer drugs in particular with relevance for the treatment of metastases and thus for an innovative chemotherapy. Results published in the literature involving ruthenium complexes show that inorganic (e.g., NAMI-A and KP1019) and organometallic compounds (e.g., RM and RAPTA complexes) can be considered promising metallodrugs to circumvent the noxious effects of platin drugs and therefore present some potentialities to be used in chemotherapy. In this context, our “RuCp” compounds appear exhibiting excellent anticancer activity in vitro and their kinetic stability gives them a vital advantage for a possible therapeutic formulation. The framework of this family of compounds provides considerable scope for optimizing the design of new molecules, by the introduction of substituent groups in Cp ring, using different hapticity of the coligands and varying the counter-ion. Albumin (HSA) revealed to be a good carrier without influencing significantly the cytotoxicity of the compounds. Our most recent studies showed that “RuCp” compounds can reach the nucleus and cytosol besides their preferential accumulation in the cell membrane. The excellent values obtained for some of the studied compounds with MDAMB231 cancer cells, which have highly metastatic properties, can pave the way for the potentiality of these compounds in metastasis treatments. Notably, TM34 was found to be the most efficient PARP-1 inhibitor compared with the published ruthenium and cisplatin compounds. All the results gathered so far may possibly foresee a promising role of “RuCp” compounds as metallodrugs in innovative chemotherapies.
We thank Fundac¸˜ao para a Ciˆencia e Tecnologia for financial support (PTDC/QUI-QUI/101187/2008, PTDC/QUI- QUI/118077/2010, PEst-OE/QUI/UI0536/2011). Tˆania S. Morais thanks FCT for her Ph.D. Grant (SFRH/BD/45871/2008). The ruthenium(II) cyclopentadienyl compounds from our laboratory described in this review are the subject of a patent application PCT/IB2012/054914.
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