Evaluation of 4-hydroxy-6-methyl-3-pyridinecarboxylic acid and 2,6-dimethyl-4-hydroxy-3-pyridinecarboxylic acid as chelating agents for iron and aluminium Annalisa Dean a , Maria Grazia Ferlin b , Paola Brun c , Ignazio Castagliuolo c , Robert A. Yokel d , Alfonso Venzo e , G. Giorgio Bombi a , Valerio B. Di Marco a,⇑ a Dipartimento di Scienze Chimiche, Università di Padova, via Marzolo 1, 35131 Padova, Italy b Dipartimento di Scienze Farmaceutiche, Università of Padova, via Marzolo 5, 35131 Padova, Italy c Dipartimento di Istologia, Microbiologia e Biotecnologie Mediche, via Gabelli 63, 35121 Padova, Italy d Pharmaceutical Sciences Department, 511C Multidisciplinary Science Building, University of Kentucky Academic Medical Center, Rose Street, Lexington, KY 40536-0082, USA e CNR, Istituto di Scienze e Tecnologie Molecolari, via Marzolo 1, 35131 Padova, Italy article info Article history: Received 21 December 2010 Received in revised form 4 April 2011 Accepted 8 April 2011 Available online 19 April 2011 Keywords: Iron Aluminium Chelation therapy Potentiometry NMR In vitro toxicity abstract 4-Hydroxy-6-methyl-3-pyridinecarboxylic acid (DQ6) and the new compound 2,6-dimethyl-4-hydroxy- 3-pyridinecarboxylic acid (DQ726) were evaluated for possible application for iron (Fe) and aluminium (Al) chelation therapy. Metal/ligand solution chemistry, cytotoxicity, octanol/water partitioning (D o/w ), and chelation efficiency were studied. The solution chemistry of the two ligands with Fe(III) and Al(III) was investigated in aqueous 0.6 m (Na)Cl at 25 °C by means of potentiometric titrations, UV–Vis spectro- photometry, and 1 H NMR spectroscopy. DQ6 exhibited a high coordination efficiency towards Al(III). Fe(III)/DQ6, Al(III)/DQ726, and Fe(III)/DQ726 complexes were less stable. These results were confirmed by chelation efficiency measurements performed in an octanol/aqueous solution. Accordingly, the effects of the substitution at various ring positions of 4-hydroxy-3-pyridinecarboxylic acid were rationalised. Partitioning experiments at pH 7.4 showed both DQ6 and DQ726, and their Fe(III) and Al(III) complexes, to be hydrophilic. The toxicity of DQ6 and of DQ726 was investigated with human cancer cell lines and normal human primary cells: no cytotoxic effects were observed up to 0.1 mM, following a 3 days expo- sure. According to our results, DQ6 has the favourable properties to be a chelating agent for Al. Ó 2011 Elsevier B.V. All rights reserved. 1. Introduction Hydroxypyridinecarboxylic acids (HPs, Table 1) are new poten- tial chelating agents for iron (Fe) and aluminium (Al) because they display a number of favourable properties. They form strong com- plexes with both Fe(III) and Al(III) [1–6], and have a very low affin- ity towards Zn(II) [7,8], which suggests the absence of essential metal decorporation in vivo [9]. They have a low molecular weight, which is a prerequisite for oral activity [10]. Toxic side effects in- duced by redox activity are unlikely for both the free ligands and the Fe(III)/ligand complexes [3,6]. The HPs investigated so far (Table 1) display negligible toxic effects (IC 50 > 0.1 mM) to cancer cell lines and primary human cells, following a 3 days exposure [3,6]. DT0 is non-toxic towards animals, and it was proposed as an aspirin-like drug [11,12]. An analogue, 4-pyridoxic acid (3- hydroxy-5-hydroxymethyl-2-methyl-4-pyridinecarboxylic acid), the main metabolite of vitamin B6, is also non-toxic [13]. The simplest HPs, the unsubstituted DT0 and DQ0, have a dis- tinct disadvantage: although their affinity towards Fe(III) and Al(III) is very high, it is still much lower than that of chelators available presently, such as deferiprone (L1). This affinity was sig- nificantly increased by methyl substitutions at the pyridinic ring. DT1 and DT2 showed a higher coordination strength than that of DT0. The complexation strength of DQ1 towards Fe(III) was slightly higher than that of DQ0; that towards Al(III) did not change. The 2- methyl substitution of DQ0 (which gives DQ2) decreased signifi- cantly the coordination strength towards Fe(III) and Al(III). The complexes of DQ716 with both Fe(III) and Al(III) are much more stable than those of the other HPs examined so far [6], so that this compound was proposed as a chelating agent for Fe and Al. More than one HP having the proper chemical requirements would be needed, in order to perform a pharmacological screening (e.g. evaluation of chelation efficiency and toxicity in vivo) and eventually individuate at least one compound to be used as drug. Moreover, the effects of the methyl substitution at different ring positions on the metal stability of the complexes are still not clear. The metal–ligand solution chemistry of other derivatives should be 0020-1693/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ica.2011.04.009 ⇑ Corresponding author. Tel.: +39 0498275219; fax: +39 0498275271. E-mail address: valerio.dimarco@unipd.it (V.B. Di Marco). Inorganica Chimica Acta 373 (2011) 179–186 Contents lists available at ScienceDirect Inorganica Chimica Acta journal homepage: www.elsevier.com/locate/ica