Iron-clearing Efficiency Research Articles

Metabolically programmed iron chelators

Extensive structure activity relationship (SAR) studies focused on the desferrithiocin [DFT, (S)-4,5-dihydro-2-(3-hydroxy-2-pyridinyl)-4-methyl-4-thiazolecarboxylic acid] pharmacophore have led to three different DFT analogs being evaluated clinically for the treatment of iron overload diseases, for example, thalassemia. The SAR work revealed that the lipophilicity of a ligand, as determined by its partition between octanol and water, logPapp, could have a profound effect on the drug’s iron clearing efficiency (ICE), organ distribution, and toxicity profile. While within a given structural family the more lipophilic a chelator the better the ICE, unfortunately, the more lipophilic ligands are often more toxic. Thus, a balance between lipophilicity, ICE, and toxicity must be achieved. In the current study, we introduce the concept of ‘metabolically programmed’ iron chelators, that is, highly lipophilic, orally absorbable, effective deferration agents which, once absorbed, are quickly converted to their nontoxic, hydrophilic counterparts.

Substituent effects on desferrithiocin and desferrithiocin analogue iron-clearing and toxicity profiles.

Desferrithiocin (DFT, 1) is a very efficient iron chelator when given orally. However, it is severely nephrotoxic. Structure-activity studies with 1 demonstrated that removal of the aromatic nitrogen to provide desazadesferrithiocin (DADFT, 2) and introduction of either a hydroxyl group or a polyether fragment onto the aromatic ring resulted in orally active iron chelators that were much less toxic than 1. The purpose of the current study was to determine if a comparable reduction in renal toxicity could be achieved by performing the same structural manipulations on 1 itself. Accordingly, three DFT analogues were synthesized. The iron-clearing efficiency and ferrokinetics were evaluated in rats and primates; toxicity assessments were carried out in rodents. The resulting DFT ligands demonstrated a reduction in toxicity that was equivalent to that of the DADFT analogues and presented with excellent iron-clearing properties.

Desferrithiocin analogue iron chelators: iron clearing efficiency, tissue distribution, and renal toxicity.

The current solution to iron-mediated damage in transfusional iron overload disorders is decorporation of excess unmanaged metal, chelation therapy. The clinical development of the tridentate chelator deferitrin (1, Table1) was halted due to nephrotoxicity. It was then shown by replacing the 4'-(HO) of 1 with a 3,6,9-trioxadecyloxy group, the nephrotoxicity could be ameliorated. Further structure-activity relationship studies have established that the length and the position of the polyether backbone controlled: (1) the ligand's iron clearing efficiency (ICE), (2) chelator tissue distribution, (3) biliary ferrokinetics, and (4) tissue iron reduction. The current investigation compares the ICE and tissue distribution of a series of (S)-4,5-dihydro-2-[2-hydroxy-4-(polyether)phenyl]-4-methyl-4-thiazolecarboxylic acids (Table1, 3-5) and the (S)-4,5-dihydro-2-[2-hydroxy-3-(polyether)phenyl]-4-methyl-4-thiazolecarboxylic acids (Table1, 8-10). The three most effective polyether analogues, in terms of performance ratio (PR), defined as mean ICE(primate)/ICE(rodent), are 3 (PR 1.1), 8, (PR 1.5), and 9, now in human trials, (PR 2.2). At the onset of the clinical trial on 9, no data were available for ligand 3 or 8. This is unfortunate, as 3 has many advantages over 9, e.g., the ICE of 3 in rats is 2.5-fold greater than that of 9 and analogue 3 achieves very high levels in the liver, pancreas, and heart, the organs most affected by iron overload. Finally, the impact of 3 on the urinary excretion of kidney injury molecule-1 (Kim-1), an early diagnostic biomarker for monitoring acute kidney toxicity, has been carried out in rats; no evidence of nephrotoxicity was found. Overall, the results suggest that 3 would be a far superior clinical candidate to 9.

The Impact of Polyether Chain Length on the Iron Clearing Efficiency and Physiochemical Properties of Desferrithiocin Analogues

(S)-2-(2,4-Dihydroxyphenyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylic acid (2) was abandoned in clinical trials as an iron chelator for the treatment of iron overload disease because of its nephrotoxicity. However, subsequent investigations revealed that replacing the 4'-(HO) of 2 with a 3,6,9-trioxadecyloxy group, ligand 4, increased iron clearing efficiency (ICE) and ameliorated the renal toxicity of 2. This compelled a closer look at additional polyether analogues, the subject of this work. The 3,6,9,12-tetraoxatridecyloxy analogue of 4, chelator 5, an oil, had twice the ICE in rodents of 4, although its ICE in primates was reduced relative to 4. The corresponding 3,6-dioxaheptyloxy analogue of 2, 6 (a crystalline solid), had high ICEs in both the rodent and primate models. It significantly decorporated hepatic, renal, and cardiac iron, with no obvious histopathologies. These findings suggest that polyether chain length has a profound effect on ICE, tissue iron decorporation, and ligand physiochemical properties.

Desferrithiocin analogues and nephrotoxicity.

The syntheses of a series of 4'-O-alkylated ( S)-4,5-dihydro-2-(2,4-dihydroxyphenyl)-4-methyl-4-thiazole-carboxylic acid and 5'-O-alkylated ( S)-4,5-dihydro-2-(2,5-dihydroxyphenyl)-4-methyl-4-thiazolecarboxylic acid ligands are described. Their partition between octanol and water, log P(app), is determined, along with their iron-clearing efficiency (ICE) in both non-iron-overloaded, bile duct-cannulated rodents and in iron-overloaded primates. The ligand-promoted biliary ferrokinetics in rats are described for each of the chelators. Plots of log P(app) versus ICE in a rodent model for both the 4'-O-alkylated 2,4-dihydroxy and 5'-O-alkylated 2,5-dihydroxy series produced an inverse parabola plot with r(2) values of 0.97 and 0.81, respectively. The plots indicate an optimum log P(app)/ICE relationship. Because of the nature of the data spread in the 4'-O-alkylated 2,4-dihydroxy series, it will be used to help assess the origin of nephrotoxicity in desferrithiocin analogues: is toxicity simply related to lipophilicity, ICE, or a combination of these properties?

Impact of the 3,6,9-trioxadecyloxy group on desazadesferrithiocin analogue iron clearance and organ distribution.

The impact of introducing a 3,6,9-trioxadecyloxyl group at various positions of the desazadesferrithiocin (DADFT) aromatic ring on iron clearance and organ distribution is described. Three DADFT polyethers are evaluated: (S)-4,5-dihydro-2-[2-hydroxy-4-(3,6,9-trioxadecyloxy)phenyl]-4-methyl-4-thiazolecarboxylic acid [(S)-4'-(HO)-DADFT-PE, 3], (S)-4,5-dihydro-2-[2-hydroxy-5-(3,6,9-trioxadecyloxy)phenyl]-4-methyl-4-thiazolecarboxylic acid [(S)-5'-(HO)-DADFT-PE, 6], and (S)-4,5-dihydro-2-[2-hydroxy-3-(3,6,9-trioxadecyloxy)phenyl]-4-methyl-4-thiazolecarboxylic acid [(S)-3'-(HO)-DADFT-PE, 9]. The iron-clearing efficiency (ICE) in rodents and primates is shown to be very sensitive to which positional isomer is evaluated, as is the organ distribution in rodents. The polyethers had uniformly higher ICEs than their corresponding parent ligands in rodents, consistent with in vivo ligand-serum albumin binding studies. Ligand 9 is the most active polyether analogue in rodents and is also very effective in primates, suggesting a higher index of success in humans. In addition, this analogue is also shown to clear more iron in the urine of the primates than many of the other chelators. If this trend were also observed in patients, it would facilitate iron-balance studies in a clinical setting.

The design, synthesis, and evaluation of organ-specific iron chelators.

A series of iron chelators, three (S)-4,5-dihydro-2-(2-hydroxyphenyl)-4-methyl-4-thiazolecarboxylic acid (DADFT) and three (S)-4,5-dihydro-2-(2-hydroxyphenyl)-4-thiazolecarboxylic acid (DADMDFT) analogues are synthesized and assessed for their lipophilicity (log Papp), iron-clearing efficiency (ICE) in rodents and iron-loaded primates (Cebus apella), toxicity in rodents, and organ distribution in rodents. The results lead to a number of generalizations useful in chelator design strategies. In rodents, while log Papp is a good predictor of a chelator's ICE, chelator liver concentration is a better tool. In primates, log Papp is a good predictor of ICE, but only when comparing structurally very similar chelators. There is a profound difference in toxicity between the DADMDFT and DADFT series: DADMDFTs are less toxic. Within the DADFT family of ligands, the more lipophilic ligands are generally more toxic. Lipophilicity can have a profound effect on ligand organ distribution, and ligands can thus be targeted to organs compromised in iron overload disease, for example, the heart.

(S)-4,5-Dihydro-2-(2-hydroxy-4-hydroxyphenyl)-4-methyl-4-thiazolecarboxylic Acid Polyethers: A Solution to Nephrotoxicity

Previous studies revealed that within a family of ligands the more lipophilic chelators have better iron-clearing efficiency. The larger the log P(app) value of the compound, the better the iron-clearing efficiency. What is also clear from the data is that although the relative effects of log P(app) changes are essentially the same through different families, there are differences in absolute value between families. However, there also exists a second, albeit somewhat more disturbing, relationship. In all sets of ligands, the most lipophilic chelator is always the most toxic. The current study focuses on designing ligands that balance the lipophilicity/toxicity problem while iron-clearing efficiency is maintained. Earlier studies with (S)-4,5-dihydro-2-(2-hydroxy-4-methoxyphenyl)-4-methyl-4-thiazolecarboxylic acid [(S)-4'-(CH(3)O)-DADFT, 6] indicated that this methyl ether was a ligand with excellent iron-clearing efficiency in both rodents and primates; however, it was too toxic. On the basis of this finding, a less lipophilic, more water-soluble ligand than 6 was assembled, (S)-4,5-dihydro-2-[2-hydroxy-4-(3,6,9-trioxadecyloxy)phenyl]-4-methyl-4-thiazolecarboxylic acid [(S)-4'-(HO)-DADFT-PE, 11], a polyether analogue, along with its ethyl and isopropyl esters. The parent polyether and its isopropyl and ethyl esters were all shown to be highly efficient iron chelators in both rodents and primates. A comparison of 11 in rodents with the desferrithiocin analogue (S)-4,5-dihydro-2-(2,4-dihydroxyphenyl)-4-methyl-4-thiazolecarboxylic acid [(S)-4'-(HO)-DADFT, 1] revealed the polyether to be more tolerable, achieving higher concentrations in the liver and significantly lower concentrations in the kidney. The lower renal drug levels are in keeping with the profound difference in the architectural changes seen in the kidney of rodents given 1 versus those treated with 11.

Polyamine-Vectored Iron Chelators: The Role of Charge

The utility of polyamines as vectors for the intracellular transport of iron chelators is further described. Consistent with earlier results with polyamine analogues, these studies underscore the importance of charge in the design of polyamine-vectored chelators. Four polyamine conjugates are synthesized, two of terephthalic acid [N(1)-(4-carboxy)benzoylspermine (7) and its methyl ester (6)] and two of (S)-2-(2,4-dihydroxyphenyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylic acid [(S)-4'-(HO)-DADFT] [(S)-4,5-dihydro-2-[2-hydroxy-4-(12-amino-5,9-diazadodecyl-oxy)phenyl]-4-methyl-4-thiazolecarboxylic acid (10) and its ethyl ester (9)]. These four molecules were evaluated in murine leukemia L1210 cells for their impact on cell proliferation (48- and 96-h IC(50) values), their ability to compete with spermidine for the polyamine transport apparatus (K(i)), and their intracellular accumulation. The data revealed that when neutral molecules (cargo fragments) were fixed to the polyamine vector, the conjugates competed well with spermidine for transport and were accumulated intracellularly to millimolar levels. However, this was not the case when the cargo fragments were negatively charged. Metabolic studies of the polyamine-vectored (S)-4'-(HO)-DADFTs in rodents indicated that not only did the expected deaminopropylation step occur, but also a surprisingly high level of oxidative deamination at the terminal primary nitrogens took place. Finally, the iron-clearing efficiency of the (S)-4'-(HO)-DADFT conjugates was determined in a bile-duct-cannulated rodent model. Attaching the ligand to a polyamine vector had a profound effect on increasing the iron-clearing efficiency of this chelator relative to its parent drug.

Partition-Variant Desferrithiocin Analogues: Organ Targeting and Increased Iron Clearance

Altering the lipophilicity (log P(app)) of desferrithiocin analogues can change the organ distribution of the chelators and lead to enhanced iron clearance. For example, alkylation of (S)-2-(2,4-dihydroxyphenyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylic acid [(S)-4'-(HO)-DADFT] and its analogues to more lipophilic compounds, such as (S)-4,5-dihydro-2-(2-hydroxy-4-methoxyphenyl)-4-methyl-4-thiazolecarboxylic acid [(S)-4'-(CH3O)-DADFT], provides ligands that achieved between a 3- and 8-fold increase in chelator concentrations in the heart, liver, and pancreas (the organs most at risk in iron-overload disease) of treated rodents. The 4'-O-methylated compounds are demethylated to their hydroxylated counterparts in rodents; furthermore, this O-demethylation takes place in both rodent and human liver microsomes. The relationship between chelator lipophilicity and iron-clearing efficacy in the iron-overloaded Cebus apella primate is further underscored by a comparison of the iron-clearing efficiency of (S)-2-(2,3-dihydroxyphenyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylic acid [(S)-3'-(HO)-DADFT] and its 3'-(CH3O) counterpart. Finally, these DFT analogues are shown to be both inhibitors of the iron-mediated oxidation of ascorbate as well as effective radical scavengers.

Structure-activity relationships among desazadesferrithiocin analogues.

Desferrithiocin, a natural product iron chelator (siderophore), offers an excellent platform from which to construct orally active iron chelators which have a good therapeutic window. A systematic structure-activity study on desferrithiocin identified the structural fragments necessary for the compound's oral iron-clearing activity. There are strict requirements regarding the distance between the ligating centers; they cannot be altered without loss of efficacy. The thiazoline ring must remain intact. Benz-fusions, which were designed to improve the ligands' tissue residence time and possibly iron-clearing efficiency, are ineffective. The maintenance of an (S)-configured C-4 carbon is optimal in the design of desferrithiocin-based iron chelators. With this information in hand, alteration of the redox potential of the aromatic ring was initiated. Introduction of a hydroxy in the 4'-position of at least three different desazadesferrithiocin analogues resulted in moderate to small changes in iron clearing efficacy yet dramatic reductions in the toxicity of the compounds were observed. Although the toxicity studies of these desferrithiocin analogues are continuing, it is clear that it is possible to alter a siderophore in such a way as to ameliorate its toxicity profile while maintaining its iron-clearing properties.

Structural alterations in desferrioxamine compatible with iron clearance in animals.

The design, synthesis, and biological evaluation of amideless desferrioxamine analogues are described. The design concept is predicated on the idea that a low molecular weight desferrioxamine analogue would represent a better pharmacophore from which to construct an orally effective or more efficient trihydroxamate than the parent chelator. The study demonstrates that (1) the monohydroxamate units of desferrioxamine must be linked to promote iron clearance, (2) the N-propanoyl-N-pentyl fragments of desferrioxamine can be replaced with smaller, e.g., C-5, methylene units without compromising the analogue's iron-clearing properties, and (3) a delicate balance exists between the molecule's iron-clearing efficiency and its lipophilicity.