The iron-binding properties of aminochelin, the mono(catecholamide) siderophore of Azotobacter vinelandii.

Azotobacter vinelandii produces siderophores with different metal-binding properties, depending on the concentration of Fe(III) and molybdate in the growth medium. The three protonation constants of the mono(catecholamide) siderophore aminochelin were determined by simultaneous spectrophotometric and potentiometric titrations as log K(1)=12.1, log K(2)=10.22 and log K(3)=7.04. Based on the two catechol protonation constants, log K(1) and log K(3), the overall stability constant of the aminochelin iron 3:1 complex was found to be log beta(3)=41.3, resulting in a pFe(3+) value of 17.6 at pH 7.45. In order to further investigate the properties of the siderophore, the solubilization of Fe(III) hydroxide by a 8x10(-4) M solution of aminochelin at pH 7 and 25 degrees C was followed spectrophotometrically in the absence and in the presence of molybdate. It was observed that the addition of molybdate resulted in a significant delay in the solubilization.

Synthesis of 2-amido-3-hydroxypyridin-4(1H)-ones: novel iron chelators with enhanced pFe3+ values.

The synthesis of a range of 2-amido-3-hydroxypyridin-4-ones as bidentate iron(III) chelators with potential for oral administration is described. The pKa values of the ligands together with the stability constants of their iron(III) complexes have been determined. Results indicate that the introduction of an amido substituent at the 2-position leads to an appreciable enhancement of the pFe3+ values. The ability of these novel 3-hydroxypyridin-4-ones to facilitate the iron excretion in bile was investigated using a 59Fe-ferritin loaded rat model. The optimal effect was observed with the N-methyl amido derivative 15b, which has an associated pFe3+ value of 21.7, more than two orders of magnitude higher than that of deferiprone (1,2-dimethyl-3-hydroxypyridin-4-one) 1a (pFe3+ = 19.4). Dose response studies suggest that chelators with high pFe3+ values scavenge iron more effectively at lower doses when compared with simple dialkyl substituted hydroxypyridinones.

Hydroxylated phytosiderophore species possess an enhanced chelate stability and affinity for iron(III).

Graminaceous plant species acquire soil iron by the release of phytosiderophores and subsequent uptake of iron(III)-phytosiderophore complexes. As plant species differ in their ability for phytosiderophore hydroxylation prior to release, an electrophoretic method was set up to determine whether hydroxylation affects the net charge of iron(III)-phytosiderophore complexes, and thus chelate stability. At pH 7.0, non-hydroxylated (deoxymugineic acid) and hydroxylated (mugineic acid; epi-hydroxymugineic acid) phytosiderophores form single negatively charged iron(III) complexes, in contrast to iron(III)-nicotianamine. As the degree of phytosiderophore hydroxylation increases, the corresponding iron(III) complex was found to be less readily protonated. Measured pKa values of the amino groups and calculated free iron(III) concentrations in presence of a 10-fold chelator excess were also found to decrease with increasing degree of hydroxylation, confirming that phytosiderophore hydroxylation protects against acid-induced protonation of the iron(III)-phytosiderophore complex. These effects are almost certainly associated with intramolecular hydrogen bonding between the hydroxyl and amino functions. We conclude that introduction of hydroxyl groups into the phytosiderophore skeleton increases iron(III)-chelate stability in acid environments such as those found in the rhizosphere or the root apoplasm and may contribute to an enhanced iron acquisition.

Design, synthesis and evaluation of N-basic substituted 3-hydroxypyridin-4-ones: orally active iron chelators with lysosomotrophic potential.

To investigate the possibility of targeting chelators into the lysosomal iron pool, nine bidentate 3-hydroxypyridin-4-ones with basic chains have been synthesized. As the turnover of ferritin iron is centred in the lysosome, such strategy is predicted to increase chelator efficacy of bidentate ligands. The pKa values of the ligands together with their distribution coefficients between 1-octanol and 4-morpholinepropane sulphonic acid (MOPS) buffer pH 7.4 have been determined. The in-vivo iron mobilization efficacy of these basic 3-hydroxypyridin-4-ones has been investigated in a 59Fe-ferritin-loaded rat model. No obvious correlation was observed between efficacy and the pKa value of the side chain, although those with pKa > 7.0 tended to be more efficient than those with pKa < 7.0. The imidazole-containing molecules are much less effective than the tertiary amine derivatives. A dose-response study suggested that basic pyridinones are relatively more effective at lower doses when compared with N-alkyl hydroxypyridinones. Optimal effects were observed with the piperidine derivatives 4h and 4i. The derivative 4i at a dose of 150 micromol kg(-1) was more effective than 450 micromol kg(-1) deferiprone, the widely adopted clinical dose.

Synthesis, physicochemical characterization, and biological evaluation of 2-(1'-hydroxyalkyl)-3-hydroxypyridin-4-ones: novel iron chelators with enhanced pFe(3+) values.

The synthesis of a range of 2-(1'-hydroxyalkyl)-3-hydroxypyridin-4-ones as bidentate iron(III) chelators with potential for oral administration is described. The pK(a) values of the ligands and the stability constants of their iron(III) complexes have been determined. Results indicate that the introduction of a 1'-hydroxyalkyl group at the 2-position leads to a significant improvement in the pFe(3+) values. Such an effect was found to be greater with the hydroxyethyl substituent than with the hydroxymethyl substituent, particularly in the cases of 1-ethyl-2-(1'-hydroxyethyl)-3-hydroxypyridin-4-one (pFe(3+) = 21.4) and 1,6-dimethyl-2-(1'-hydroxyethyl)-3-hydroxypyridin-4-one (pFe(3+) = 21.5) where an enhancement on pFe(3+) values in the region of two orders of magnitude is observed, as compared with Deferiprone (1, 2-dimethyl-3-hydroxypyridin-4-one) (pFe(3+) = 19.4). The ability of these novel 3-hydroxypyridin-4-ones to facilitate the iron excretion in bile was investigated using a [(59)Fe]ferritin-loaded rat model. Chelators and prodrug chelators possessing high pFe(3+) values show great promise in their ability to remove iron under in vivo conditions.

Investigation into the correlation between the structure of hydroxypyridinones and blood-brain barrier permeability.

Bidentate hydroxypyridinones are under active development as orally active iron chelators. With applications for the treatment of general body iron overload, for instance with thalassaemia, the distribution of the chelators should be limited to peripheral tissue and they should not enter the central nervous system. This study compares the predictive abilities of LogPoctanol and LogPcyclohexane and reports the existence of good correlations between blood-brain barrier (BBB) permeability and both values for N-alkylpyridinones. 1,omega-Hydroxyalkyl hydroxypyridinones penetrate the BBB much more slowly than the simple 1-alkyl derivatives. This observation is likely to have important toxicological implications.

Nicotianamine chelates both FeIII and FeII. Implications for metal transport in plants

Nicotianamine (NA) occurs in all plants and chelates metal cations, including FeII, but reportedly not FeIII. However, a comparison of the FeII and ZnII affinity constants of NA and various FeIII-chelating aminocarboxylates suggested that NA should chelate FeIII. High-voltage electrophoresis of the FeNA complex formed in the presence of FeIII showed that the complex had a net charge of 0, consistent with the hexadentate chelation of FeIII. Measurement of the affinity constant for FeIII yielded a value of 10(20.6), which is greater than that for the association of NA with FeII (10(12.8)). However, capillary electrophoresis showed that in the presence of FeII and FeIII, NA preferentially chelates FeII, indicating that the FeIINA complex is kinetically stable under aerobic conditions. Furthermore, Fe complexes of NA are relatively poor Fenton reagents, as measured by their ability to mediate H2O2-dependent oxidation of deoxyribose. This suggests that NA will have an important role in scavenging Fe and protecting the cell from oxidative damage. The pH dependence of metal ion chelation by NA and a typical phytosiderophore, 2'-deoxymugineic acid, indicated that although both have the ability to chelate Fe, when both are present, 2'-deoxymugineic acid dominates the chelation process at acidic pH values, whereas NA dominates at alkaline pH values. The consequences for the role of NA in the long-distance transport of metals in the xylem and phloem are discussed.

Comparative radical scavenging ability of bidentate iron (III) chelators.

Iron chelators can reduce radical damage inflicted on cells by two mechanisms, either direct scavenging of the radicals or by scavenging loosely bound iron which under aerobic conditions can generate radicals. Frequently it is not possible to distinguish between these two modes of action. 3-Hydroxypyridin-4-ones, in contrast to many iron(III) chelators are poor radical scavengers and therefore have potential in analysing mechanisms involved in biochemical and physiological processes which are centered on radical-induced cell injury.

Synthesis, physicochemical properties, and evaluation of N-substituted-2-alkyl-3-hydroxy-4(1H)-pyridinones.

The synthesis of a range of 3-hydroxy-4(1H)-pyridinones with potential for the chelation of iron(III) is described. The pKa values of respective ligands and the stability constants of their iron(III) complexes are presented. The distribution coefficient values of a range of 48 hydroxypyridinones and their corresponding iron(III) complexes between 1-octanol and MOPS buffer (pH 7.4) are reported. The range of log Dcomplex values covers 7 orders of magnitude. The results suggest the existence of a biphasic relationship between the distribution coefficient values of the chelator and the corresponding iron(III) complexes. For ligands with a log Dligand = -1, a linear relationship exists with a value of the slope 2.53, whereas with ligands with a log Dligand < -1, a linear relationship exists with a slope of 0.49. The reduced slope for the more hydrophilic molecules of the series offers some advantage for this type of hydroxypyridinone as the distribution coefficients for such complexes do not change so rapidly with increasing ligand hydrophilicity. The ability of selected 3-hydroxypyridinones to facilitate the excretion of iron in bile was investigated in non-iron-overloaded, bile duct-cannulated rats and in a [59Fe]ferritin-loaded rat model. Both systems compare the ability of chelators to remove iron from the liver, the prime target organ in thalassemia. The N-(hydroxyalkyl)-3-hydroxypyridin-4-ones are demonstrated to be orally active under the in vivo conditions adopted. Thus both 1-(hydroxyalkyl)- and 1-(carboxyalkyl)pyridinones are able to remove iron from the liver. Although 1-(carboxyalkyl)hydroxypyridinones are active, they do not demonstrate any clear advantage over Deferiprone (1,2-dimethyl-3-hydroxypyridin-4-one). Indeed 1-(hydroxyalkyl)hydroxypyridinones which are known to be rapidly converted to 1-(carboxyalkyl)hydroxypyridinones are also marginally superior to Deferiprone. In contrast, 2-ethyl-1-(2'-hydroxyethyl)-3-hydroxypyridin-4-one, which is not metabolized to the corresponding (carboxyalkyl)hydroxypyridinone, was found to be superior to Deferiprone and therefore deserves further consideration as an orally active iron chelator with potential for the treatment of iron overload associated with transfusion-dependent thalassemia.

Structural dependence of flavonoid interactions with Cu2+ ions: implications for their antioxidant properties.

The flavonoids constitute a large group of polyphenolic phytochemicals with antioxidant properties in vitro. The interactions of four structurally related flavonoids (quercetin, kaempferol, rutin and luteolin) with Cu2+ ions were investigated in terms of the extent to which they undergo complex formation through chelation or modification through oxidation, as well as in their structural dependence. The ortho 3',4'-dihydroxy substitution in the B ring is shown to be important for Cu2+-chelate formation, thereby influencing the antioxidant activity. The presence of a 3-hydroxy group in the flavonoid structure enhances the oxidation of quercetin and kaempferol, whereas luteolin and rutin, each lacking the 3-hydroxy group, do not oxidize as readily in the presence of Cu2+ ions. The results also demonstrate that the reactivities of the flavonoids in protecting low-density lipoprotein (LDL) against Cu2+ ion-induced oxidation are dependent on their structural properties in terms of the response of the particular flavonoid to Cu2+ ions, whether chelation or oxidation, their partitioning abilities between the aqueous compartment and the lipophilic environment within the LDL particle, and their hydrogen-donating antioxidant properties.

Therapeutic iron chelators and their potential side-effects.

A number of iron-chelating agents are currently being considered as orally active alternatives to desferrioxamine (DFO), the therapeutic agent for the treatment of body iron overload that is available at present. These include bidentate hydroxypyridinones (HPO), tridentate desferrithiocin (DFT) analogues and hexadentate aminocarboxylate (HBED) chelators. All chelating agents have the potential to induce toxic effects when iron homoeostasis is affected within the body. This can arise when the absorption, distribution and utilization of iron is affected. Alternatively, chelating agents can induce toxicity by directly interfering with iron-dependent metalloenzymes located within the body. These effects are, however, mainly localized to non-haem enzymes such as ribonucleotide reductase and lipoxygenase. The resultant iron complexes also have the ability to induce toxicity. Depending on the coordination geometry and donor atoms associated with the metal centre, redox cycling of the iron centre with the corresponding generation of free radicals can result.