The development of ruthenium anticancer compounds

Foreword

The development of new metal anticancer compounds is a challenge for inorganic chemists. We have to face the fact that four decades of research in this field have produced a dismayingly small number of clinically used compounds, most often developed through serendipity rather than through rational chemical design. Nevertheless, by virtue of the wealth of knowledge acquired in these years, medicinal inorganic chemistry is probably mature for making significant steps forward and there are great expectations for the future developments.

Those interested to the field of medicinal inorganic chemistry are referred to the recent book Bioinorganic Medicinal Chemistry, E. Alessio ed., Wiley-VCH, Weinheim, 2011.

Ruthenium anticancer compounds

The number of metal compounds in current clinical use for the treatment of cancer is extremely limited and concerns platinum compounds exclusively.
Only three Pt(II) compounds, i.e. cisplatin (cis-diamminedichloridoplatinum(II)), carboplatin (cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II)), and oxaliplatin (trans-R,R-cyclohexane-(1,2-diamine)oxalatoplatinum(II)) are used worldwide (Figure 1).

Figure 1. Platinum anticancer drugs used worldwide and year of FDA approval.

The use of Pt anticancer drugs is restricted by severe toxicity and by spontaneous or acquired resistance. With the aim of overcoming these limits, a huge number of other metal compounds was extensively investigated over the years.

Among them, those of ruthenium occupy a prevalent position. In the last 35 years, basically three main classes of active Ru compounds (i.e. compounds that have demonstrated effectiveness in vivo against animal models or transplanted human tumors) have been discovered: Ru-dmso compounds (developed by us), “Keppler-type” Ru(III) complexes such as KP1019, and organometallic Ru(II)-arene compounds such as RM175 and RAPTA-C (Figure 2). Typically these Ru-compounds are active against platinum-resistant tumors (at least in in vitro experiments).

Figure 2. Schematic structures of some of the most promising and thoroughly investigated anticancer Ru compounds: NAMI-A (top-left), KP1019 (top-right), RM175 (bottom-left), and RAPTA-C (bottom-right).

The Ru(III) complex developed by us in the 1990s, known as NAMI-A (Figures 2 and 3), was found to be particularly active against the development and growth of metastases of solid tumors. NAMI-A was the first ruthenium compound to be tested on humans (1999) in a phase 1 clinical trial. NAMI-A is currently being tested in a phase 1-2 combination study at the Netherland Cancer Institute of Amsterdam on patients affected by non-small cell lung carcinoma. This clinical investigation is expected to be completed within 2011.

Figure 3. Samples of NAMI-A

The discovery of NAMI-A and of the other Ru-anticancer compounds has strongly stimulated the worldwide research on Ru complexes in biological environment, as shown by the constantly increasing number of publications in this specific field.

Over the years the group has performed a thorough investigation of Ru-dmso complexes, that has afforded a deeper insight into the chemistry and anticancer activity of these compounds. The chemical and biological results are collected in several review articles, the most relevant of which are listed here:

• E. Alessio
  Synthesis and Reactivity of Ru-, Os-, Rh-, and Ir-halide-sulfoxide Complexes.
  Chem. Rev., 2004, 104, 4203 – 4242.
• E. Alessio, G. Mestroni, A. Bergamo, G. Sava
  Ruthenium antimetastatic agents.
  Curr. Topics Med. Chem., 2004, 4, 1525-1535.
• Bratsos, S. Jedner, T. Gianferrara, E. Alessio
  Ruthenium anticancer compounds: challenges and expectations
  Chimia, 2007, 61, 692-697.
• Bratsos, T. Gianferrara, E. Alessio, C. G. Hartinger, M. A. Jakupec, B. K. Keppler
  Ruthenium and Other Non-platinum Anticancer Compounds.
  in Bioinorganic Medicinal Chemistry, E. Alessio ed., Wiley-VCH, Weinheim, 2011, pp. 151-174.

The group is currently pursuing three main research lines in the field of ruthenium anticancer compounds:

1. Half-sandwich coordination complexes structurally similar to anticancer active organometallic compounds.


2. Photoactivated Ru compounds for chemotherapy


3. Ruthenium-porphyrin conjugates for improved targeting and phototoxicity

1.  Half-sandwich coordination complexes structurally similar to anticancer active
     organometallic compounds.

As said above, Ru(II)-arene compounds of the general formula [(η⁶-arene)Ru(YZ)X] (Figure 4, charge omitted), where YZ is typically a chelating bidentate ligand and X is a good leaving group (e.g. Cl^-), are widely investigated for their promising anticancer properties.

Figure 4. The generic structure of anticancer active Ru(II)-arene compounds.

The aim of this research line is to establish whether the η⁶-arene fragment of these organometallic half sandwich compounds might be effectively replaced by another neutral 6-electron donor face-capping ligand - or by three monodentate ligands (L) that form a stable fac-Ru(L)₃ fragment - while maintaining the other ligands unchanged. Thus, we are developing series of new half sandwich Ru(II) coordination compounds in which aromatic ligand is substituted by a neutral tridentate macrocycle, that occupies three facial coordination sites, such as 1,4,7-trithiacyclononane ([9]aneS₃) or 1,4,7-triazacyclononane ([9]aneN₃), or by three dmso κ-S (Figure 5). Two of the remaining coordination position are occupied either by a neutral N-N chelating ligand such as 1,2-diaminoethane (en), 2,2'-bipyridine (bipy) or by a chelating dicarboxylate ligand such as oxalate or malonate. The last coordination position is occupied by a leaving ligand that is either Cl or dmso.

Figure 5. Schematic representation of half sandwich organometallic and coordination compounds that differ for the nature of the face-capping fragment. From left to right:
η⁶-arene, [9]aneS3, [9]aneN3, and (dmso-S)₃.

So far, only complexes that are capable of hydrolyzing the monodentate ligand at a reasonable rate and of acting as hydrogen bond donors through the chelating ligand (e.g. chel = en or dach, where dach = trans-1,2-diaminocyclohexane) were found to have moderate antiproliferative activity in vitro.

The research is currently being extended also to the osmium analogues.

2.  Photoactivated Ru compounds for chemotherapy

The activity of most metal anticancer compounds is attributed to the direct coordination of the metal to the biological target. Usually these compounds show an unspecific toxicity, not limited to the tumor cells. A possible approach for reducing this disadvantage is selective activation: in this approach a prodrug, i.e. a non-toxic precursor of the active species, is administered, and is activated only in the tumor region. Provided that selective activation is possible, this strategy would have the clear advantage of limiting the undesired effects, thus increasing the therapeutic index of the compound. In ideal conditions, even if the prodrug distributes equally in the body, only the part activated at the tumor site would be highly cytotoxic. The use of light as activating agent of an anticancer compound is one of the strategies that are being investigated, that for this reason is called photoactivated chemotherapy (PACT).

We aim to use visible light for inducing ligand dissociation and thus activate the complexes. Coordinatively saturated and kinetically inert complexes are believed to be inactive because they are unable to bind to biological targets. This research line is aimed to prepare Ru compounds that are kinetically inert to hydrolysis and therefore inactive in the dark but that selectively dissociate the monodentate ligand upon irradiation with visible light, thus generating an active metal fragment. If these two requirements are achieved, the irradiation of the tumor site with laser light would lead to the release of active metal fragments directly into the cancer cells.

An example of photoactivation of a half-sandwich Ru(II) coordination complex with an inert monodentate ligand such as pyridine is reported in Figure 6.

Figure 6. Light-induced dissociation of pyridine in an inert half-sandwich coordination compound.

3.  Ruthenium-porphyrin conjugates for improved targeting and phototoxicity

The central role of natural and synthetic porphyrins and metalloporphyrins in the photodynamic therapy of cancer (PDT) is a well established issue. PDT is a binary therapy for cancer treatment that involves the activation of a tumor-localized sensitizer with visible light. In the absence of light, the photosensitizer should have negligible effect on either healthy or tumour cells. However, when the drug-localized tissue is irradiated, the drug becomes activated, typically generates singlet oxygen and other reactive oxygen species (ROS) that induce apoptosis and necrosis of targeted cells and tissues.

The conjugation of porphyrins to peripheral metal fragments is an intriguing strategy for making compounds that might combine the cytotoxicity of the metal moiety to the phototoxicity of the porphyrin chromophore for additive antitumor effects. In addition, they might have improved tumor selectivity, by virtue of the preferential uptake and retention of porphyrins in tumor tissues. Thus, porphyrins might behave as carrier ligands for the active transport of anticancer metal compounds into cancer cells. Finally, the fluorescence emission of the chromophore might be exploited for tracking the biodistribution of the metal in the extra- and intra-cellular environment of malignant cells through fluorescence microscopy.

We made and characterized several new Ru-porphyrin conjugates that bear either negatively charged NAMI-A-type Ru(III) fragments or positively charged half-sandwich Ru(II) coordination compounds. The connection between the tetrapyrrolic macrocycle and each peripheral metal center occurs either through a single N(pyridyl)-Ru bond or through a chelating bpy unit (Figure 7). The four peripheral bpy fragments are connected to the meso positions of the macrocycle through flexible linkers of different length and hydrophilicity.

Figure 7. Schematic structures of ruthenium-porphyrin conjugates.

The conjugates, tested on human breast cancer MDA-MB-231 and human non tumorigenic HBL-100 cells, have IC₅₀ values in the low micromolar range, that decrease of one order of magnitude upon irradiation of cell cultures with visible light (590-700 nm) and proved to have from moderate to good singlet oxygen quantum yields. According to fluorescence microscopy experiments, they accumulate in the cytoplasm of the breast cancer cells but do not penetrate significantly into the nucleus. One of the most active conjugates is depicted in Figure 8.

Figure 8. A water-soluble Ru-porphyrin conjugate that is phototoxic at low light and drug doses.