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Review Article |
Iron Metabolism and Chelation Program, Children's Cancer Institute Australia for Medical Research, Sydney, New South Wales, Australia
Abstract
Abstract I. Introduction II. Redox Activity of Iron III. Iron Metabolism in Normal Cells A. Iron Absorption from the Gut B. Iron Uptake C. The Labile Iron Pool and Iron Storage D. Iron Homeostasis 1. Iron-Regulatory Proteins 1 and 2. 2. Iron-Regulatory Protein Binding: Control of Iron Homeostasis. IV. Iron Overload Disease A. {beta}-Thalassemia B. Friedreich's Ataxia V. Targeting Iron in Cancer Therapy: Iron Metabolism in Neoplastic Cells A. Antiproliferative Activity and Lipophilicity B. Antiproliferative Activity and Redox Activity VI. The Evolution of Iron Chelators A. Siderophores 1. Desferrioxamine. 2. Desferrithiocin. a. A structure-activity relationship examination of the desferrithiocin scaffold. b. A structure-activity relationship examination of chemical parameters. c. Hexadentate desferrithiocin analog. d. Desferrithiocin as a selective antiproliferative agent. 3. Desferri-exochelin. B. Synthetic Iron Chelators 1. ICL670A. 2. Deferiprone and Hydroxypyridinone Analogs. a. Combination therapy: deferiprone and desferrioxamine. b. Deferiprone metabolism. c. CP94. d. Hydroxypyridinone ester prodrugs. e. Increasing pFe(III) values of hydroxypyridinones. f. Hexadentate hydroxypyridinone analogs. 3. Tachpyridine. a. Novel tachpyridine analogs. 4. Aroylhydrazones: Pyridoxal Isonicotinoyl Hydrazone and Analogs. a. 100 series analogs. b. 200 series analogs. c. 300 series analogs. d. 2-Pyridylcarboxaldehyde isonicotinoyl hydrazone analogs. e. Di-2-pyridylketone isonicotinoyl hydrazone analogs. 5. Thiosemicarbazones. a. Triapine. b. Development of hybrid chelators derived from thiosemicarbazones and aroylhydrazones: the 2-hydroxy-1-naphthylaldehyde-3-thiosemicarbazone series. c. Development of hybrid chelators derived from thiosemicarbazones and aroylhydrazones: the di-2-pyridylketone thiosemicarbazone analogs. VII. Conclusions
The evolution of iron chelators from a range of primordial siderophores and aromatic heterocyclic ligands has lead to the formation of a new generation of potent and efficient iron chelators. For example, various siderophore analogs and synthetic ligands, including ICL670A [4-[3,5-bis-(hydroxyphenyl)-1,2,4-triazol-1-yl]-benzoic acid], 4'-hydroxydesazadesferrithiocin, and Triapine, have been developed from predecessors and illustrate potent iron-mobilizing or antineoplastic activities. This review focuses on the evolution of iron chelators from initial lead compounds through to the development of novel chelating agents, many of which show great potential to be clinically applied in the treatment of iron overload disease and cancer.
Iron chelation therapy involves the use of ligating drugs that avidly bind iron for the treatment of potentially fatal conditions, namely iron overload disease and cancer (for reviews, see Buss et al., 2003c
, 2004a; Tam et al., 2003). By coordinating with intracellular and extracellular iron, these ligands promote the excretion and subsequent depletion of this transition metal in biological systems. These iron-chelating agents consist of a range of bidentate, tridentate, and hexadentate ligands in which two, three, or six atoms, respectively, are able to coordinate with iron, forming octahedral complexes (Liu and Hider, 2002a; Tam et al., 2003). Oxygen and nitrogen donor atoms within ligands are able to bind tightly with iron, and this is illustrated within the porphyrin-ring of heme-containing proteins, where Fe-N bonds anchor the iron center in place.
As iron exists in the environment in an insoluble form, microbes have overcome this accessibility problem by excreting low molecular weight ligands, known as siderophores, to specifically sequester iron in a useable form (Reid et al., 1993; Bergeron et al., 2003d; for review, see Neilands, 1995). Many of these siderophores have become lead compounds in the search for more efficient and orally active iron chelators, as these molecules have evolved through natural selection to specifically chelate iron. Currently, the siderophore, desferrioxamine (DFO1), is clinically used for the treatment of iron overload disease, such as 
-thalassemia major (for reviews, see Olivieri and Brittenham, 1997; Wong and Richardson, 2003). In addition, DFO has also shown antiproliferative activity against aggressive tumors in clinical trials, including neuroblastoma (NB) and leukemia (for reviews, see Richardson, 1997, 2002; Lovejoy and Richardson, 2003; Buss et al., 2003c, 2004a). However, this hexadentate chelator has many disadvantages owing to it being expensive and orally inactive and having a short serum half-life (Aouad et al., 2002; Chaston et al., 2004). Indeed, the high hydrophilicity of the ligand results in it being poorly absorbed through the gastrointestinal tract (Franchini et al., 2000). As a consequence, DFO treatment requires long periods of subcutaneous infusion, resulting in swelling and pain at the site of injection in a third of patients (Wong and Richardson, 2003). Hence, more orally effective, economical, specific, and efficient iron chelators have been developed as alternatives to DFO for the treatment of iron overload disease and cancer. The evolution of iron chelators from a range of primordial siderophores and aromatic heterocyclic ligands has lead to the formation of a new generation of potent and efficient iron chelators. This review will discuss the evolution of novel iron chelators from their initial lead compounds with regards to their pharmacological actions and structure-activity relationships for the treatment of iron overload disease and cancer.
Iron is essential for life as it plays an important role in many cellular processes, including energy generation, oxygen transport, and DNA synthesis (for reviews, see Richardson and Ponka, 1997; Andrews, 1999; Lieu et al., 2001; Richardson, 2002; Hentze et al., 2004). The dependence of life upon this transition metal is due to the fact that iron acts as a cofactor within the active site of key enzymes involved in these critical biochemical pathways. This catalytic ability lies in the unique redox activity of iron, which is able to cycle between two stable configurations, the ferric [Fe(III)] and ferrous [Fe(II)] states, allowing it to act as an electron donor and acceptor.
The biological importance of iron and particularly its pathological consequences are due to its participation in the oxidation-reduction process known as the Haber-Weiss reaction (Fig. 1, eq. 3) (Halliwell and Gutteridge, 1989; Lieu et al., 2001). During the iron-catalyzed Haber-Weiss reaction, hydrogen peroxide (H2O2) reacts to form the hydroxyl radical (OH·) and the hydroxide anion (OH-) (Halliwell and Gutteridge, 1989).
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Although iron is crucial for life, an excess of iron is toxic. This is due to the deleterious effects of reactive oxygen species (ROS), such as the hydroxyl radical (OH·) which is produced via the Fenton reaction (Fig. 1, eq. 2). The highly reactive OH· radical is able to induce cell death through initiating a series of chemical reactions with many significant biomolecules, resulting in DNA oxidation, mitochondrial damage, and the peroxidation of membrane lipids (Bergeron et al., 2003c; Barnham et al., 2004). In addition, excess free iron can also react with unsaturated lipids to produce alkoxyl and peroxyl radicals (Lieu et al., 2001). These oxidative reactions result in the impairment of cellular functions and lead to damage of cells, tissues, and organs, which is evident in the iron-loading diseases, -thalassemia and Friedreich's ataxia (FA) (Shinar and Rachmilewitz, 1990; Wong et al., 1999; for reviews, see Alper and Narayanan, 2003; Schrier et al., 2003; Wong and Richardson, 2003). Consequently, iron levels are tightly regulated by specialized proteins, which transport and store iron in a soluble and nontoxic form, thus preventing the deleterious side effects of the Haber-Weiss reaction (Richardson and Ponka, 1997).
III. Iron Metabolism in Normal Cells
A. Iron Absorption from the Gut
The absorption of iron from dietary sources occurs within the small intestine, where enterocytes take up iron in two forms, being inorganic iron and heme (for review, see Conrad and Umbreit, 2002). Inorganic iron is transported across the enterocyte cell membrane via the divalent metal transporter 1 (DMT1), which is also known as the divalent cation transporter, the natural resistance-associated macrophage protein 2 (Nramp2) or solute carrier family 11a member 2 (Slc11a2) (Fleming et al., 1997; Gunshin et al., 1997; Napier et al., 2005). However, as its name suggests, DMT1 can only transport metals in a divalent state, and thus, iron must be in its ferrous form to be absorbed (Hentze et al., 2004). As most dietary nonheme iron is found as ferric complexes, iron must be reduced to the ferrous form prior to absorption. This role is thought to be carried out by the membrane-bound duodenal cytochrome b enzyme, which has been found to possess ferric reductase activity (McKie et al., 2001). However, to date, there has been no direct demonstration that duodenal cytochrome b enzyme activity facilitates iron uptake, and further research is required to confirm the role of this protein in iron transport within enterocytes.
Despite heme being a major source of iron in the human diet, the pathway involved in heme uptake by enterocytes remains unclear. Initial studies suggested that the uptake of heme was believed to occur through passive diffusion, although recent investigations have suggested that a receptor system may be involved (Uc et al., 2004). Considering this, a heme transporter, known as the human feline leukemia virus subgroup C receptor, was recently identified, but its role in heme transportation in the gut remains unknown (Quigley et al., 2004). Heme, once within the enterocyte, is then metabolized by heme oxygenase to liberate iron (Watts et al., 2003).
The precise mechanism of iron transport within enterocytes and its release into the bloodstream is yet to be fully determined. Several proteins have been implicated in the trafficking and release of iron, including hephaestin (Vulpe et al., 1999) and ferroportin 1 (also known as metal transporter 1 or iron-regulated transporter 1; Abboud and Haile, 2000; Donovan et al., 2000; McKie et al., 2000). Hephaestin is a transmembrane ceruloplasmin homolog that is highly expressed in the intestine and was first identified in the sla mouse (Vulpe et al., 1999). A mutation in this protein was found to reduce iron release into circulation, resulting in iron accumulation within enterocytes of the sla mouse (Vulpe et al., 1999). Therefore, it has been suggested that hephaestin may play a role in facilitating iron release in cooperation with the iron transporter, ferroportin 1 (Chen et al., 2004). Mammalian ferroportin 1 has been found to be expressed on the basolateral surface of duodenal enterocytes and is believed to be responsible for iron release from enterocytes into the bloodstream (Donovan et al., 2000).
More recently, a peptide hormone secreted by the liver known as hepcidin has been identified to be critical in iron homeostasis by acting as an iron-regulatory hormone (Nicolas et al., 2001; for review, see Ganz, 2003), regulating enterocyte ferroportin 1 expression (Nemeth et al., 2004). Under conditions of iron overload, hepcidin is highly expressed in the liver (Pigeon et al., 2001). It is thought that hepcidin negatively regulates intestinal iron absorption, maternal-fetal iron transport across the placenta, and iron release from hepatic stores and macrophages (Ganz, 2003). Once in the circulation, hepcidin may bind to ferroportin 1 on cell membranes, leading to its internalization and degradation (Nemeth et al., 2004). This results in reduced iron efflux from enterocytes and completes a homeostatic loop, whereby iron regulates hepcidin secretion that then affects ferroportin-1 expression on the cell surface (Nemeth et al., 2004). In addition to these molecules, the serum protein ceruloplasmin probably is also involved in iron efflux (Lee et al., 1968; Owen, 1973; Richardson, 1999). Patients suffering from aceruloplasminemia were found to have a genetic defect within the ceruloplasmin gene, resulting in iron loading (Harris et al., 1995). Therefore, it is possible that the ferroxidase activity of ceruloplasmin is necessary for the removal of iron from transport elements on the cell surface (Lee et al., 1968; Oshiro et al., 1993; Richardson, 1999).
In serum, iron is found in a highly soluble form, bound to the iron transport protein, transferrin (Tf) (for reviews, see Morgan, 1981; Richardson and Ponka, 1997). Tf is able to bind two atoms of Fe(III) with high affinity at sites located in the N and C termini of the protein (Morgan, 1981; Richardson and Ponka, 1997). Two molecules of diferric Tf then avidly bind to the transferrin receptor 1 (TfR1) homodimer located on the cell surface, whereas iron-free Tf (apo-Tf) cannot (Fig. 2). A second Tf receptor known as TfR2 has been discovered, although its role in iron metabolism remains unclear (Kawabata et al., 1999, 2000). The Tf-TfR1 complex formed is internalized into an endosome via receptor-mediated endocytosis (Morgan, 1981; Klausner et al., 1983). Within the endosome, iron is released from Tf following the decrease in intravesicular pH mediated by an endosomal membrane-bound proton pump (Morgan, 1981). Recently, Cheng et al. (2004) observed that a conformational change occurs in Tf after binding to TfR1, which is thought to promote iron release where the lobes open, possibly allowing for the access of other proteins to iron (Cheng et al., 2004; Richardson, 2004). Such a direct transfer of iron from Tf to other proteins including DMT1 would prevent iron precipitation within the endosome. Although Fe(III) binds to Tf, it is released in the Fe(II) state, but it is not known whether Fe(III) is reduced before or after its release from Tf. It is speculated that perhaps a membrane-bound ferrireductase is able to associate with the Tf-TfR1 complex, subsequently reducing iron to its Fe(II) state (Cheng et al., 2004; Richardson, 2004). DMT1 then transports this reduced iron across the endosomal membrane and into the cytosol. Another structural change is believed to occur in Tf after the release of its iron, thus resulting in a decreased affinity for TfR1 (Cheng et al., 2004; Richardson, 2004). This freed apo-Tf is released into the plasma via exocytosis, whereby TfR1 is returned to the cell surface (Fig. 2) (Morgan, 1981; Klausner et al., 1983).
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; Kaplan et al., 1991; Richardson and Baker, 1991; Richardson and Ponka, 1995). However, under physiological conditions, most iron is bound to Tf, and the presence of low molecular weight iron is only appreciable in patients with iron overload disease (Hershko et al., 1978; Grootveld et al., 1989; Hider, 2002a). Under these conditions, iron uptake from low molecular weight iron complexes may be significant and could contribute to the pathological iron-loading observed (Gutteridge et al., 1985; Halliwell and Gutteridge, 1986).
C. The Labile Iron Pool and Iron Storage
Upon release of iron from the endosome, this transition metal then enters the labile iron pool (LIP), a poorly characterized compartment believed to comprise small molecular weight iron complexes or high molecular weight intermediates. Although the identity of LIP substituents remains elusive, it has been suggested that iron in this pool is bound to amino acids, nucleotides, and sugars (Jacobs, 1977). However, little low molecular weight iron is present within cells, suggesting that such a pool must exist in very low concentrations or may not be present (Vyoral et al., 1992; Richardson et al., 1996). In fact, several models of intracellular iron transport have been proposed that do not involve the presence of low molecular weight species of iron (Zhang et al., 2005).
Iron chelators are believed to bind iron from the LIP, and, hence, it is also known as the chelatable iron pool (Konijn et al., 1999). Iron is distributed from the LIP to specific compartments within the cell, implying that a defined iron-trafficking system exists, used for iron storage or for the inclusion of iron into the active sites of proteins (Richardson, 2002; Buss et al., 2003c). For example, iron is targeted to the mitochondrion in erythroid cells for heme synthesis (Richardson et al., 1996; Ponka, 1997; Zhang et al., 2005).
Iron in the LIP can be targeted to the iron storage protein, ferritin, which is composed of two subunits, heavy and light, forming a protein able to accommodate >4500 iron atoms (Harrison and Arosio, 1996; Buss et al., 2003c). The synthesis of this protein is up-regulated under conditions of iron abundance, whereas the expression of ferritin is reduced when iron availability is low, contributing to overall iron homeostasis (Lieu et al., 2001). Storage of iron within ferritin is critical for the prevention of ROS formation, protecting the cell from oxidative damage.
As iron is required for a variety of cellular processes, a balance between iron uptake, usage, and storage must be maintained. This is achieved by regulating the expression of proteins at the post-transcriptional level via the intracellular iron concentration (for reviews, see Hentze and Kühn, 1996; Hentze et al., 2004). Two mRNA-binding molecules, iron-regulatory proteins 1 and 2 (IRP1 and IRP2), regulate the expression of crucial proteins involved in iron homeostasis. This is attained by the binding of the IRPs to hairpin-loop structures known as iron-responsive elements (IREs) located in the 5'- or 3'-untranslated regions of their mRNAs (Hentze and Kühn, 1996; Hentze et al., 2004). The binding of IRP1 and IRP2 to the IRE is controlled by intracellular iron levels. This iron-mediated regulatory feedback mechanism allows for cells to achieve and maintain a desired intracellular iron level (Hentze and Kühn, 1996; Hentze et al., 2004).
1. Iron-Regulatory Proteins 1 and 2.
Sequence analysis of IRP1 has shown that this protein has high homology to mitochondrial aconitase, being the cytosolic equivalent (Hentze and Kühn, 1996; Hentze et al., 2004). Under high intracellular iron levels, IRP1 contains a [4Fe-4S] cluster, which results in the loss of IRE-binding ability, imparting aconitase activity (Hentze and Kühn, 1996; Hentze et al., 2004). In contrast, IRP1 of iron-depleted cells does not contain this [4Fe-4S] cluster and hence is able to bind to IREs.
IRP2 has binding affinity to IREs similar to that of IRP1, although this protein does not have a [4Fe-4S] cluster. This protein is rapidly degraded in iron-depleted cells via the proteosome (Hentze et al., 2004).
2. Iron-Regulatory Protein Binding: Control of Iron Homeostasis.
As mentioned previously, IRPs are able to bind to IREs located at the 3' or 5' end of mRNA, either increasing mRNA stability or inhibiting translation and consequently regulate protein expression (Hentze and Kühn, 1996; Richardson and Ponka, 1997; Hentze et al., 2004). Under conditions of iron deficiency, IRPs are able to bind to IREs located at the 3' end of mRNA-encoding iron-uptake proteins, protecting the molecule from exonuclease activity and hence improving mRNA stability (Hentze and Kühn, 1996; Richardson and Ponka, 1997; Hentze et al., 2004). This increases the expression of TfR1 and other proteins involved in iron uptake, thus elevating intracellular iron levels. In iron-replete cells, IRPs bind to IRE within the 5' untranslated region of ferritin mRNA, sterically hindering translation, which allows the cell to use the iron that is present (Hentze and Kühn, 1996; Hentze et al., 2004).
On the other hand, when iron is abundant, IRPs cannot bind to IREs located at the 3' end of mRNA of iron-uptake proteins, allowing for mRNA degradation and subsequently decreases in intracellular iron levels (Hentze and Kühn, 1996; Hentze et al., 2004). Simultaneously, under high iron levels, IRP can no longer bind to the 5' end of ferritin mRNA, increasing ferritin expression and levels of iron in storage.
Although a vast network of proteins are involved in the metabolism of iron, no efficient mechanism of iron excretion exists, and, hence, problems can arise when errors occur within this metabolic pathway. Excess iron, which cannot be bound by ferritin, is able to participate in the Haber-Weiss reaction and in turn induces oxidative stress. This production of free radicals can result in severe damage to tissue and organs and can be fatal if left untreated (Bergeron et al., 2003a). Iron chelation therapy represents an avenue of treatment for these conditions, preventing oxidative effects by removing catalytically active iron (for review, see Chaston and Richardson, 2003a).
Two 
- and -globin chains combine to form the essential heme-containing protein, hemoglobin, that is vital for oxygen transport (Nick et al., 2003). Mutations affecting the gene result in a lack of effective hemoglobin synthesis and leads to -thalassemia (for reviews, see Cao, 1995; Olivieri and Brittenham, 1997; Wong and Richardson, 2003). Approximately 200 point mutations present within patients are responsible for the reduction in or lack of -chain synthesis (Wong and Richardson, 2003). The most prevalent point mutation leads to an alteration in the splice junction, creating aberrant mRNA. Others occur within the promoter region, resulting in a decrease in RNA polymerase binding, and within exons, leading to shifts in the mRNA reading frame which creates a premature stop codon (Wong and Richardson, 2003). The subsequent imbalance between - and -chain production creates unstable -chain aggregates that precipitate, affecting erythrocyte membrane plasticity. This causes ineffective erythropoiesis and leads to anemia (Wong and Richardson, 2003).
The first step in treatment of -thalassemia involves repeated blood transfusions to reverse the anemic state, and this, in combination with iron absorption from the gastrointestinal tract, leads to iron overload (Nick et al., 2003). Potentially fatal tissue damage and fibrosis are seen in the iron-overloaded heart and liver, which is typical of this condition, due to oxidative reactions initiated by the redox activity of iron (Olivieri and Brittenham, 1997; Schrier et al., 2003). Iron chelation therapy offers a secondary route of treatment to relieve the iron burden of these cells.
FA is an inherited condition characterized by neurodegeneration and cardiomyopathy (for reviews, see Pandolfo, 2002, 2003; Napier et al., 2005). In this condition, iron overload affects the central nervous system, which results in a gradual loss of motor skills leading to paralysis and death (Puccio and Koenig, 2000; Lieu et al., 2001). This form of iron overload disease is due to the decreased expression of frataxin, a small, mitochondrial protein believed to play a role in mitochondrial iron homeostasis (Puccio et al., 2001; for reviews, see Pandolfo, 2002; Alper and Narayanan, 2003). Frataxin is encoded within a gene known as FRDA, located on chromosome 9q13 (Lodi et al., 2002). In the majority of FA patients, a GAA triplet expansion occurs within the first intron of the FRDA gene, resulting in a reduction of mature frataxin mRNA which in turn decreases frataxin levels (Campuzano et al., 1996; Lodi et al., 2002; Alper and Narayanan, 2003).
The mitochondrion is of great importance to the cell as it plays a pivotal role in energy production through oxidative phosphorylation. This organelle is also responsible for heme and [Fe-S] cluster biosynthesis, and hence, any mitochondrial damage has a large impact on the function of a cell (Cooper and Schapira, 2003). Although the exact function of frataxin has not been determined, it is thought to take part in mitochondrial iron metabolism and [Fe-S] cluster synthesis (Puccio et al., 2001; Becker et al., 2002; Muhlenhoff et al., 2002; for reviews, see Pandolfo, 2002; Alper and Narayanan, 2003; Napier et al., 2005). This correlates with iron accumulation seen in the liver, heart, and spleen of FA patients in a pattern consistent with mitochondria (Sachez-Casis et al., 1976; Lamarche et al., 1993), whereas a decrease in respiratory chain activities of the [Fe-S] cluster-containing complexes I, II, and III is also observed (Rotig et al., 1997; Puccio et al., 2001; for reviews, see Becker and Richardson, 2001; Lodi et al., 2002; Cooper and Schapira, 2003). The lack of productive oxidative phosphorylation is thought to lead to a decrease in intracellular ATP levels, resulting in cellular dysfunction and death (Ristow et al., 2000).
Alternatively, the reduction in activity of these respiratory chain complexes may also be, in part, due to their sensitivity to free radical damage induced by excess iron accumulated within the mitochondrion (Cooper and Schapira, 2003). Excess iron can generate free radicals within the mitochondria, creating oxidative damage that can be at least partially inhibited by antioxidants (Rustin et al., 1999; for reviews, see Alper and Narayanan, 2003; Richardson, 2003). Iron-mediated oxidation leads to the loss of mitochondrial DNA, greatly impairing the function of this organelle (Rotig et al., 1997; Lodi et al., 2002). This free radical damage is believed to be a secondary event after the disruption of iron homeostasis within the mitochondria. Therefore, iron chelators that are able to penetrate the mitochondria and facilitate iron removal provide a potential route of treatment for this iron overload condition (Richardson et al., 2001; Richardson, 2003).
V. Targeting Iron in Cancer Therapy: Iron Metabolism in Neoplastic Cells
The iron chelation field also extends to the treatment of cancer by targeting the enzyme involved in the rate-limiting step of DNA synthesis, ribonucleotide reductase (RR) (Kicic et al., 2001a; for reviews, see Richardson, 1997; Buss et al., 2003c, 2004a; Dayani et al., 2004; Richardson, 2005). This enzyme mediates the conversion of all four ribonucleotides to their deoxyribonucleotide counterparts, providing the precursors necessary for DNA synthesis (Thelander and Reichard, 1979; Kolberg et al., 2004). RR comprises two subunits, R1 and R2, of which R1 is responsible for the binding of ribonucleotides and allosteric factors (Shao et al., 2004). Iron is essential for the catalytic activity of RR and stabilizes the tyrosyl radical located within the R2 subunit (Thelander et al., 1983), making iron an obvious target for chemotherapeutic agents (Thelander and Gräslund, 1983).
Cancer cells have a higher requirement for iron than normal cells as they rapidly proliferate. Hence, iron metabolism is altered within these cells. This is reflected by the fact that tumor cells have higher numbers of Tf receptors on their cell surface, mediating a high rate of iron uptake (Richardson and Baker, 1990, 1992; Trinder et al., 1996; for reviews, see Kwok and Richardson, 2002; Le and Richardson, 2002). Production of the iron storage protein, ferritin, is also increased in tumor tissue (Vaughn et al., 1987). In addition, RR is up-regulated in cancerous cells, facilitating the production of deoxyribonucleotides for DNA replication and subsequent cell division (Elford et al., 1970). Therefore, depleting iron from rapidly dividing cancer cells through the implementation of iron chelators deprives these cells of the DNA precursors necessary for replication.
A. Antiproliferative Activity and Lipophilicity
Chelators with high lipophilicity theoretically possess advantages over less lipid-soluble ligands as these can easily enter cells and subsequently deplete iron from intracellular pools necessary for iron incorporation into the R2 subunit. Alternatively, these ligands can interact directly with the hydrophobic iron center of RR (Hodges et al., 2004). Previous evidence has demonstrated an increase in antiproliferative activity with the elevation in chelator hydrophobicity (Johnson et al., 1982; Baker et al., 1985; Richardson et al., 1995; Hodges et al., 2004). This trend is attributed to the ability of lipophilic ligands to pass through membranes and thus gain access to intracellular iron pools critical for cellular proliferation (Baker et al., 1985; Richardson et al., 1995).
B. Antiproliferative Activity and Redox Activity
In addition, chelators with the highest antiproliferative activity not only chelate iron, decreasing the available iron in the LIP, but may also enhance its redox activity (Fig. 3). Examples of these ligand types include thiosemicarbazones such as Triapine (Chaston et al., 2003; Yuan et al., 2004) and some aroylhydrazones such as those of the 2-pyridylketone isonicotinoyl hydrazone (PKIH) class (Chaston et al., 2004). Ligands that are able to bind both Fe(II) and Fe(III) have the potential to redox cycle (Hider, 2002b; Liu and Hider, 2002a; Bernhardt et al., 2003). For instance, chelators containing "soft" donor atoms, such as nitrogen, possess lower redox potentials and as such the iron core can be enzymatically reduced under biological conditions (Fig. 3) (Liu and Hider, 2002a). The resulting Fe(II) can catalyze the generation of damaging oxygen radicals, leading to oxidative damage and subsequent cell death. In contrast, "hard" coordinating groups such as the hydroxamate oxygens of the hexadentate siderophore, DFO (Fig. 4), have a large affinity for Fe(III) over Fe(II) (Hider, 2002b). With such hexadentate ligands, the coordination sphere is fully occupied, preventing direct access of hydrogen peroxide or oxygen with the complexed iron center. On the other hand, bidentate and tridentate complexes are kinetically labile and are able to form partially dissociated complexes, producing 2:1 and 1:1 complexes, respectively. These partial complexes render the cation surface exposed and as a consequence can facilitate the production of damaging radical species. Such properties enable iron chelators to promote the redox activity of this metal ion, initiating ROS production and consequently leading to cell death. This provides an alternative form of treatment in the fight against cancer in which tumor resistance to standard chemotherapeutics remains an ongoing problem.
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VI. The Evolution of Iron Chelators
In the quest to develop effective iron chelators, three main considerations must be taken into account, including the route of administration, the iron chelation efficiency, and the toxicity profile of the ligand. Ideally a drug should be orally active, economical and highly specific, while exhibiting no adverse side effects. Although a large variety of chelators designed to be orally active have been created, further research is being conducted to increase the efficiency and reduce the toxicity associated with these drugs. These parent molecules act as a starting point for the development of a new generation of potent iron chelators. Below we describe the evolution of some of the most effective lead compounds through to the formation of a novel range of efficient iron chelators.
Siderophores are natural product iron chelators, secreted by microbes in response to the insoluble nature of iron in the environment (for review, see Neilands, 1995). These low molecular weight ligands were selectively evolved to sequester this transition metal in a soluble form, allowing microorganisms to take up these complexes via specific transport systems. These molecules were created specifically for chelating iron; thus, siderophores represent a range of selective lead compounds. Some of these have been examined and structurally altered in the search for novel, specific, and nontoxic chelators for clinical use.
1. Desferrioxamine.
DFO (Fig. 4), a hexadentate siderophore isolated from Streptomyces pilosus, is the current clinical chelator of choice for the treatment of iron overload diseases such as -thalassemia (Aouad et al., 2002; Brittenham, 2003). This ligand has validated iron chelation therapy as an effective form of treatment by showing an improvement in the survival and well-being of iron-overloaded patients (Olivieri and Brittenham, 1997; Hershko et al., 2003). The high affinity of this siderophore for Fe(III) renders iron bound in the resulting 1:1 complex metabolically inactive, preventing the production of ROS. Consequently, DFO is able to decrease the oxidative stress in iron-overloaded cells, alleviating the symptoms associated with iron overload disease (Olivieri and Brittenham, 1997). However, the highly hydrophilic nature of DFO limits the efficiency of this ligand, imparting poor absorption from the gastrointestinal tract and a short plasma half-life of 12 min due to rapid drug metabolism (Aouad et al., 2002). As a result, DFO must be administered via subcutaneous infusion for extensive periods to achieve a negative iron balance, ranging from 8 to 12 h, five to seven times per week at a daily dosage of 20 to 60 mg/kg (Hershko et al., 2003). Apart from the cumbersome administration route and high cost, a third of patients treated with DFO experience pain and swelling at the injection site, cumulatively leading to poor patient compliance (Olivieri and Brittenham, 1997; Wong and Richardson, 2003).
Previous studies and clinical trials have illustrated that aggressive tumors, including NB and leukemia, are sensitive to iron chelation therapy with DFO (for review, see Buss et al., 2003c). After a 72-h exposure to 60 µM DFO, a >80% reduction in viability of NB cell lines was evident, demonstrating some cytotoxic effect (Blatt and Stitely, 1987). In addition, DFO has been shown to inhibit DNA synthesis in a NB cell line after a 4-h incubation (Blatt et al., 1988). The cytotoxic effects of DFO were prevented upon the addition of iron or iron-saturated DFO (Blatt and Stitely, 1987), suggesting that the antiproliferative action of this siderophore can be attributed to iron depletion and subsequent inhibition of RR.
Apart from in vitro assessment of antitumor activity, an Italian clinical study of nine NB patients given a single course of DFO, administered by an 8-h continuous infusion at 150 mg/kg for 5 days, resulted in a response in seven of nine patients (Donfrancesco et al., 1990). A decrease in bone marrow infiltration was observed as well as a 48% decrease of tumor size in one patient. At this dosage level, no significant side effects were evident (Donfrancesco et al., 1990).
Although DFO has shown pronounced antiproliferative activity during in vitro studies and clinical trials, the high hydrophilicity of this ligand limits its membrane permeability and efficiency. There have been numerous attempts to improve upon the efficacy of DFO. These include the generation of a high molecular weight form of the chelator coupled to hydroxyethyl starch. In a clinical trial, high plasma concentrations of hydroxyethyl starch-DFO were obtained (3 mM) after a 4-h intravenous infusion (Dragsten et al., 2000), and this treatment was well tolerated and led to substantial urinary iron excretion (Dragsten et al., 2000). However, the major problem with DFO was not overcome, that is, the need for long infusions. More lipophilic DFO analogs have been prepared by reacting the terminal primary amino group with fatty and aromatic acid chlorides or anhydrides (Ihnat et al., 2000). However, to date, none of these have been reported to be more effective than DFO administered via the subcutaneous route.
More recently, combinatorial approaches have been used to generate libraries of structural DFO analogs including nonamide analogs, C-terminal modified analogs, reverse-amide analogs, and hybrid analogs (Poreddy et al., 2004). However, none of these compounds have been evaluated in animal models. To the present day, the evolution of DFO analogs has not resulted in chelators with advantages over the original siderophore. Hence, other orally active alternatives have been sought in the quest to develop effective and specific iron chelators, which are discussed in detail below.
2. Desferrithiocin.
Desferrithiocin (DFT) [2-(3-hydroxypyridin-2-yl)-4-methyl-4,5-dihyrothiazole-4-carboxylic acid; Fig. 4] is a tridentate siderophore from the bacterium Streptomyces antibioticus DSM 1865 (for reviews, see Bergeron et al., 2002; Brittenham, 2003; Nick et al., 2003). This ligand binds iron through its phenolic oxygen, carboxylate oxygen, and thiazole nitrogen donor atoms (Brittenham, 2003). Desferrithiocin has a formation constant of 4 x 10-29 M with Fe(III) and was among the first orally active siderophores to be discovered (for review, see Tam et al., 2003). DFT was found to be orally active in a bile duct-cannulated rodent study (Bergeron et al., 1991). In an iron-overloaded Cebus apella primate model, when orally administered at 150 µmol/kg, DFT was 3 times more active than subcutaneously administered DFO (Bergeron et al., 1993).
Although DFT showed much promise as a chelating agent for the treatment of iron overload disease, a major drawback was that it exhibited severe nephrotoxicity (Bergeron et al., 1993). This is believed to be due to the cytotoxic effects of the desferrithiocin-iron complex (Baker et al., 1992a). However, the high oral activity of DFT made this chemical backbone a promising scaffold warranting further structural investigations, and as such, this siderophore has acted as a lead compound in the pursuit of novel orally active iron chelators. Structure-activity relationship (SAR) studies have been performed to develop less toxic derivatives of this pharmacophore, and these investigations are described below.
a. A structure-activity relationship examination of the desferrithiocin scaffold.
Due to the nephrotoxicity of DFT, studies have been carried out to identify which structural components confer this adverse side effect. These studies lead to the synthesis of (S)-desmethyldesferrithiocin (DMDFT; Fig. 5), an analog discovered to retain iron clearing activity in the bile duct-cannulated rat model (Bergeron et al., 1993). In contrast, a decrease in iron chelation efficacy of DMDFT was observed in the Cebus apella primate model compared with DFT (Bergeron et al., 1993). Although the efficiency of this analog was below that of DFT, this ligand was found to exhibit no toxic side effects when orally administered on a daily basis to rodents in a 30-day trial (Bergeron et al., 1999b). Interestingly, this dramatic shift in toxicity was simply due to the removal of the thiazoline methyl group. However, the fact that this ligand still retained the pyridyl moiety reduced its application as a lead compound, as the pyridine ring does not lend itself to electrophilic aromatic substitution. Hence, other DFT analogs, including (S)-desazadesferrithiocin (DADFT; Fig. 5) and (S)-desazadesmethyldesferrithiocin (DADMDFT; Fig. 5), were also synthesized, containing a carbon in place of the pyridyl nitrogen (Bergeron et al., 1999a,b). Unfortunately, both DADFT and DADMDFT were found to elicit severe gastrointestinal toxicity during rodent trials (Bergeron et al., 1993, 1999b). Despite their adverse side effects, DADFT and DADMDFT have acted as critical intermediate structures during the SAR examination of this series of iron chelators.
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). The addition of a methylene group between the two rings of the scaffold or either one or two methylene groups between the carboxyl and thiazoline ring resulted in poor orally active analogs (Fig. 5). Even when administered subcutaneously, the efficiency was not observed to increase (Bergeron et al., 1999b). From these results it can be hypothesized that the poor activity of these analogs was not due to bioavailability but rather that these modifications altered the optimum spacing needed for chelation, reducing their iron-binding capabilities.
Further structural studies examining the effect of thiazoline ring alterations on iron clearing activity were also conducted on DMDFT and DADMDFT (Fig. 5). Various thiazoline modifications, including oxidation to a thiazole, reduction to a thiazolidine, expansion by a methylene group, and substitution of the thiazoline sulfur with oxygen, nitrogen, or a methylene group resulted in a loss of activity (Bergeron et al., 1994, 1999b). In all cases, these alterations lead to a drastic reduction in iron chelation efficiency (Bergeron et al., 1994, 1999b). Upon modification of the thiazoline ring, it would be expected that the iron-binding properties of these analogs would be altered, but the complete lack of activity was difficult to explain. From these results, it can be concluded that the thiazoline ring must remain intact for iron clearing activity.
Finally, changing the C-4 stereochemistry (Fig. 5) showed an interesting effect on iron clearing efficiency in the primate model. By retaining the same stereochemistry as DFT, the (S)-enantiomers of DMDFT and DADMDFT were more effective than their corresponding (R)-enantiomers in promoting iron clearance (Bergeron et al., 1993, 1999a,b).
b. A structure-activity relationship examination of chemical parameters.
In the hope of increasing residence time and thus efficiency, the lipophilicities of the (R)- and (S)-enantiomers of DMDFT and DADMDFT were increased by incorporating an additional aromatic ring (Fig. 6). However, overall, the addition of lipophilic features did not increase iron clearing efficiency, with these analogs showing less activity during the primate model than the parent compounds, DMDFT and DADMDFT (Bergeron et al., 1996, 1999b).
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Another chemical parameter investigated involved altering the electronic effects within the aromatic ring of the parent compound, DADMDFT, by the addition of electron-withdrawing groups (Fig. 6). For example, a hydroxyl or methoxyl group was added to the 3-position of the aromatic ring to confer an inductive electron-withdrawing effect on the thiazoline nitrogen. Also a carboxylic acid group at the 4-position was used to electronically confer the same effect. Interestingly, these electron-withdrawing analogs were found to have diminished iron clearing efficiency when orally administered in the rodent model (Bergeron et al., 1999b). The methoxy analog was found to have the highest efficiency of these latter analogs, albeit lower then that of the parent compound, DADMDFT (Bergeron et al., 1999b). It seemed that the electron-withdrawing effect induced on the thiazoline nitrogen reduced iron clearing efficiency.
Alternatively, electron-donating groups were also used to alter the redox activity of the aromatic ring. In this case, a hydroxyl group placed at the 4-position was able to induce a donating effect, electronically, to the thiazoline nitrogen. This had a profound effect on both the efficiency and toxicity of the compound. The 4'-hydroxylated analog of DADMDFT (4-hydroxydesazadesferrithiocin [4-(OH)-DADMDFT]; Fig. 6) was found to induce iron clearance using both the rodent and primate models (Bergeron et al., 1999b). Although this was not as active as DADMDFT in the primate model, toxicity trials over 10- and 30-day treatment periods illustrated that the gastrointestinal toxicity of the parent compound was eliminated (Bergeron et al., 1999b). All histopathologies seemed to be normal compared with the control (Bergeron et al., 1999b). In addition, the 4'-hydroxylated analog of the 5,5-dimethyl thiazoline DADMDFT derivative (Fig. 6) was found to have good iron chelation efficacy in the primate model (Bergeron et al., 2003c). The addition of a hydroxyl group at the 4-position of DADFT [4'-(OH)-DADFT; Fig. 6] led to an extremely effective iron chelator in the primate model (Bergeron et al., 1999a). Due to the remarkable level of iron clearance, 4'-(OH)-DADFT was further analyzed for its toxic effects. During a 10-day trial, rats treated with this chelator demonstrated no severe side effects, with the compound being well tolerated and almost all histological assessments were normal (Bergeron et al., 1999a). This analog represents one of the most active and nontoxic iron chelators of this series.
In relation to the electron-donating substituents, it has been speculated that the formation of a quinone structure (Fig. 7) may occur on complexation (Bergeron et al., 1999b). In this process, the electrons are redistributed around the aromatic ring, donating a lone pair of electrons onto the thiazoline nitrogen. As a result, a negative charge is located on the nitrogen, enhancing donation to Fe(III) (Bergeron et al., 1999b). Although the effect of the 4'-hydroxy group on the stability of the complex is yet to be determined, the proposed intermediate structure certainly begins to explain the decrease in activity observed with the electron-withdrawing analogs, which remove electron density from the thiazoline nitrogen.
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). Preclinical animal studies have illustrated no toxic effects at levels to be used clinically, and as such, 4'-(OH)-DADFT is ready for human phase I evaluation.
c. Hexadentate desferrithiocin analog.
Generally, it is known that hexacoordinate ligands form more stable complexes than those of lower denticity (Liu and Hider, 2002a). Due to their additional stability, the majority of natural siderophores are hexadentate. By fully occupying the coordination sphere of iron, hexadentate chelators can prevent the interaction of iron with surrounding molecules, avoiding the formation of ROS through Fenton chemistry (Liu and Hider, 2002a; Bergeron et al., 2003b; Chaston et al., 2004). Owing to these advantages, Bergeron's group synthesized a hexacoordinate chelator based upon the nontoxic tridentate ligand, 4'-(OH)-DADMDFT, named (S,S)-1,11-bis[5-(4-carboxy-4,5-dihydrothiazol-2-yl)-2,4-dihydroxyphenyl]-4,8-dioxaundecane (BDU; Fig. 4). This design was based on linking two tridentate units by a polyether tether and Job's plot of BDU confirmed the hexadentate nature of this ligand (Bergeron et al., 2003b).
In vivo studies in the noniron-overloaded, bile duct-cannulated rodent model demonstrated that the hexadentate chelator had little iron chelation efficiency when administered orally (Bergeron et al., 2003b). This was found to be within the experimental error of the tridentate unit, 4'-(OH)-DADMDFT, at equivalent iron-binding doses. However, when administered subcutaneously, the iron clearing efficiency of BDU was substantially increased, being 3 times that of the tridentate unit when administered via the same route (Bergeron et al., 2003b). Unfortunately, trials in the iron-overloaded Cebus apella monkey showed a decrease in iron clearing efficiency of BDU when subcutaneously administered. This was found to be only half as effective as the tridentate unit given subcutaneously at the equivalent iron-binding capacity (Bergeron et al., 2003b). Thus, no great advantage was found by coupling two 4'-(OH)-DADMDFT units together. The lack of activity of the hexadentate ligand suggests that this large chelator may be less membrane permeable than the smaller tridentate unit, thus decreasing intracellular chelator levels.
d. Desferrithiocin as a selective antiproliferative agent.
Although the studies on DFT described above mainly concentrated on the efficacy of the chelator for the treatment of iron overload disease, several studies have assessed the antiproliferative activity of this ligand in vitro (Kicic et al., 2001b, 2002). These investigations showed that DFT was more effective than DFO at inhibiting proliferation (IC50 = 40 µM) although its activity was far less than that of other chelators in current development, for example, those of the di-2-pyridylketone thiosemicarbazone (DpT) class for which IC50 values of 0.03 µM were reported (Yuan et al., 2004) (see section VI.B.5.c.). Importantly, the antiproliferative effect of DFT on normal cells (hepatocytes and fibroblasts) was far less than that found in hepatoma cells and their antitumor activity could be diminished by saturating the ligand with iron (Kicic et al., 2002).
3. Desferri-exochelin.
Desferri-exochelins (D-Exo; Fig. 4) are a group of hexadentate siderophores released by Mycobacterium tuberculosis, which use oxygen donor atoms to avidly bind iron (Pahl et al., 2000). Their balance of lipophilicity and hydrophilicity allows them to readily enter cells and complex iron, rendering their bound iron redox-inactive (Hodges et al., 2004). A previous study demonstrated the ability of these siderophores to directly remove iron from both human Tf and horse ferritin (Gobin and Horwitz, 1996). Upon incubation of D-Exo with 95% iron-saturated Tf, D-Exo was observed to become iron saturated after just 1 min (Gobin and Horwitz, 1996). Similarly, D-Exo was demonstrated to remove iron from horse ferritin, although at a much slower rate than with Tf, requiring 3 days to reach saturation (Gobin and Horwitz, 1996). These results demonstrate the iron scavenging ability of this siderophore and illustrate its potential application to iron overload disease.
Further research has illustrated the potential use of D-Exo 772SM (Fig. 4) as an antiproliferative agent. An in vitro study examining the effect of 20 µM D-Exo 772SM on MCF-7 breast cancer cells illustrated potent antineoplastic activity where all cells were killed after 4 days of treatment (Pahl et al., 2001). No cytotoxicity was evident when normal human mammary epithelial cells were treated at 5 or 20 µM, although a growth arrest was seen (Pahl et al., 2001). These effects were prevented when either cell line was incubated with the iron-loaded D-Exo, indicating that the iron-binding ability of this ligand was responsible for its antiproliferative activity (Pahl et al., 2001). Further studies examining D-Exo 772SM on DNA synthesis demonstrated a 95% to 97% reduction of [3H]thymidine incorporation in both the normal and cancerous breast cell lines after a 6-h incubation (Pahl et al., 2001). A 40-fold higher concentration of DFO was necessary to inhibit [3H]thymidine incorporation to a similar extent (Pahl et al., 2001). These results indicated that the increased antiproliferative effects of D-Exo 722SM resulted from its greater lipophilicity and enhanced membrane permeability compared with DFO, allowing access to critical intracellular iron pools required for DNA replication (Pahl et al., 2001). Recently, the ability of D-Exo 772SM in inhibiting RR was examined by measuring the tyrosyl radical signal using electron spin resonance spectroscopy in cell lysates (Hodges et al., 2004). After treatment with 50 µM D-Exo 772SM at 37°C, the electron spin resonance signal of RR was shown to rapidly decline after 60 min (Hodges et al., 2004). Further work on the antiproliferative activity of this chelator is required, particularly in relation to the high activity of aroylhydrazones and thiosemicarbazones of the PKIH and DpT series, respectively (Becker et al., 2003; Yuan et al., 2004) (see sections VI.B.4.e. and VI.B.5.c., respectively).
In contrast to the naturally occurring siderophores described above, a vast array of chelators have been artificially designed and synthesized in the search for an ideal iron chelator. To attain maximum iron clearance or antiproliferative activity, a large range of chelators have been synthesized, forming a generation of lead compounds with great potential. Below we discuss the development of novel synthetic iron chelators from lead compounds to potent ligands.
1. ICL670A.
ICL670A (Fig. 8) is a tridentate chelator that uses a triazolyl nitrogen and two phenolic oxygens as donor groups. This ligand, representing one of >700 compounds designed through computer modeling, belongs to the bis-hydroxyphenyl-triazole class of iron chelators possessing oral activity intended for the treatment of -thalassemia (Nick et al., 2003). Of central importance is the ability of these chelators to selectively bind Fe(II) and/or Fe(III) and not other metal ions of biological importance such as Cu(II) and Zn(II). By using hard donor atoms such as oxygen, ICL670A can bind Fe(III) specifically, avoiding disturbances to other significant transition metals (Heinz et al., 1999).
). Equation 2 is the Fenton reaction that results in the production of the hydroxyl radical (OH·) and the hydroxide anion (OH-) from hydrogen peroxide (H2O2). Equation 3 shows the iron-catalyzed Haber-Weiss reaction.
