CN128: A New Orally Active Hydroxypyridinone Iron Chelator.

Deferoxamine, deferiprone, and deferasirox are used for the treatment of systemic iron overload, although they possess limitations due to lack of oral activity, lower efficacy, and side effects. These limitations led to a search for an orally active iron chelator with an improved therapeutic index. The lower efficacy of deferiprone is due to rapid glucuronidation, leading to the formation of a nonchelating metabolite. Here, we demonstrate that the influence of metabolism can be reduced by introducing a sacrificial site for glucuronidation. A log P-guided investigation of 20 hydroxpyridinones led to the identification of CN128. The Fe(III) affinity and metal selectivity of CN128 are similar to those of deferiprone, the log P value is more lipophilic, and its iron scavenging ability is superior. Overall, CN128 was demonstrated to be safe in a range of toxicity assessments and is now in clinical trials for the treatment of ß-thalassemia after regular blood transfusion.

pH indicator titration: a novel fast pKa determination method.

This study describes a fast spectrophotometic titration method for apparent ionization constant (pKa) determination. In this method, a Universal pH indicator is utilized instead of the conventional pH electrode. An autoburette is set to add HCl at a constant rate to a vigorously stirred 1 cm UV cuvette which contains sample and indicator solution. A spectrophotometer continuously records the spectra. Acquired spectral data are processed by calculating the pH from the indicator spectra in the visible region and extracting sample spectra from the UV region. Five compounds possessing pKa values in the range 2-10 were investigated. These results differed from measurements by conventional spectrophotometric titration by +/-0.05 to +/-0.10 log unit.

Determination of the labile iron pool of human lymphocytes using the fluorescent probe, CP655.

The present study introduces a method for determining the labile iron pool (LIP) in human lymphocytes. It is measured using the probe CP655, the fluorescence of which is stoichiometrically quenched by the addition of iron. The intracellular CP655 fluorescence in lymphocytes was quenched by increasing intracellular iron concentrations using the highly lipophilic 8-hydroxyquinoline iron complex. Intracellular fluorescence quenching, mediated by the physiological intracellular labile iron, can be recovered on the addition of excess membrane-permeable iron chelator, CP94. The intracellular probe concentration was measured using laser scanning microscopy. An ex situ calibration was performed in a "cytosolic" medium based on the determined intracellular CP655 concentration and probe fluorescence quenching in the presence of iron. The concentration of the LIP of healthy human lymphocytes was determined to be 0.57 +/- 0.27 microM. The use of the fluorescent probe CP655 renders it possible to record the time course of iron uptake and iron chelation by CP94 in single intact lymphocytes.

3-Hydroxy-2-(5-hydroxypentyl)-4H-chromen-4-one: a bidentate or tridentate iron(III) ligand?

The pK(a) value and iron affinity constants for 3-hydroxy-2-(5-hydroxypentyl)-4H-chromen-4-one 1 have been determined by spectrophotometry using aqueous methanol solutions. The extrapolated affinity constants beta(1), beta(2), and beta(3)( )()for iron(III) in aqueous solution were 9.95, 18.69, and 26.02, respectively, with a corresponding pFe(3+) value of 14.64. Job plot and MS spectra data demonstrated that the 3:1 species is favored at pH 7.0. These results indicate that 1 acts as a bidentate ligand when coordinated to iron(III).

Chelation and determination of labile iron in primary hepatocytes by pyridinone fluorescent probes.

A series of fluorescent iron chelators has been synthesized such that a fluorescent function is covalently linked to a 3-hydroxypyridin-4-one. In the present study, the fluorescent iron chelators were loaded into isolated rat hepatocytes. The intracellular fluorescence was not only quenched by an addition of a highly lipophilic 8-hydroxyquinoline-iron(III) complex but also was dequenched by the addition of an excess of the membrane-permeable iron chelator CP94 (1,2-diethyl-3-hydroxypyridin-4-one). The time course of uptake of iron and iron chelation in single, intact cells was recorded on-line by using digital fluorescence microscopy. Intracellular concentrations of various fluorescent iron chelators were determined by using a spectrofluorophotometer subsequent to lysis of probe-loaded cells and were found to depend on their partition coefficients; the more hydrophobic the compound, the higher the intracellular concentration. An ex situ calibration method was used to determine the chelatable iron pool of cultured rat hepatocytes. CP655 (7-diethylamino-N-[(5-hydroxy-6-methyl-4-oxo-1,4-dihydropyridin-3-yl)methyl]-N-methyl-2-oxo-2H-chromen-3-carboxamide), which is a moderately lipophilic fluorescent chelator, was found to be the most sensitive probe for monitoring chelatable iron, as determined by the intracellular fluorescence increase induced by the addition of CP94. The concentration of the intracellular chelatable iron pool in hepatocytes was determined by this probe to be 5.4+/-1.3 microM.

Novel iron-specific fluorescent probes.

A series of novel iron-specific fluorescent probes is reported where the chelator function unusually forms part of the fluorescent moiety. The ability of this range of molecules to permeate human erythrocyte ghost membranes was investigated.

Design, synthesis, physicochemical properties, and evaluation of novel iron chelators with fluorescent sensors.

The synthesis of a range of novel 3-hydroxypyridin-4-ones and 3-hydroxypyran-4-ones linked with different coumarin substituents is described. These compounds have been developed in order to provide a series of molecular probes for the quantification of intracellular labile iron pools. An evaluation of the effect of iron(III) on fluorescence intensity was undertaken. Chelation of iron(III) causes quenching of fluorescence. The relationship between iron(III) concentration and the extent of fluorescence quenching indicates that the metal is chelated in a complex with a metal-to-ligand stoichiometry of 1:3. The fluorescence of hydroxypyridinone compounds was found to be more efficiently quenched by iron(III) than were the hydroxypyranones. The metal-to-ligand stoichiometry at which maximum quenching is observed was found to depend on the site at which coumarin is attached. The efficiency of fluorescence quenching by iron(III) is markedly influenced by solvent polarity and pH. The permeability of two representative fluorescent chelators across human erythrocyte ghost membranes was investigated. The rate of permeability for a series of probes was found to be related to the corresponding ClogP values.