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Coordination Chemistry Reviews 256 (2012) 240– 259

Contents lists available at ScienceDirect

Coordination Chemistry Reviews

journa l h o me page: www.elsev ier .com/ locate /ccr

eview

ydroxypyridinones as “privileged” chelating structures for the design ofedicinal drugs

. Amélia Santos ∗, Sérgio M. Marques, Sílvia Chavesentro de Química Estrutural, Instituto Superior Técnico, Universidade Técnica de Lisboa, Av. Rovisco Pais 1, 1049-001 Lisboa, Portugal

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2412. Biometals in health and disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241

2.1. Iron overload and misplaced related illnesses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2412.2. Aluminum – an aberrant metal ion in diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242

3. Targeting hard metal ions for decorporation or passivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2424. Functionalized 3-hydroxy-4-pyridinone chelators as DFP successors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243

4.1. Hydroxypyridinone development and evaluation methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2434.2. Improvement of 3,4-HP bioavailability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2444.3. Chelating capacity enhancement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244

4.3.1. Extra-functionalization of bidentate ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2454.3.2. The polydenticity approach on hydroxypyridinones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245

4.4. Hexadentate hydroxypyridinones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2464.5. Tetradentate 3,4-HPs: from ligand-target approach to combined chelating approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248

5. Extra-functionalization of 3,4-HP for further therapeutic roles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2495.1. New developments on iron-chelators as anti-neurodegeneratives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

5.1.1. Modulating the BBB permeability of 3,4-HP chelators: drugs and prodrugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2505.1.2. Strategies for antioxidant/iron chelator dual-action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2505.1.3. Further strategies for dual-action with iron chelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2525.1.4. Nanoparticle hydroxypyridinone chelator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253

5.2. Hydroxypyridinones as matrix metalloproteinase inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2535.3. Hydroxypyridinones as anti-microbial, anti-malarial and antiviral agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253

6. Metallodrugs based on hydroxypyridinone ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2556.1. Metal complexes as diagnostic tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2556.2. Anti-diabetic drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2556.3. Anti-cancer drugs based on metal complexes with hydroxypyridinones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256

7. Summary and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257Appendix A. Supplementary data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257

r t i c l e i n f o

rticle history:eceived 2 May 2011ccepted 8 August 2011

a b s t r a c t

Hydroxypyridinones (HPs) are a family of N-heterocyclic core chelators which, based on their specificmetal-coordination, easy manipulation/derivatization and biocompatibility, have been an attractive tar-get for the development of new pharmaceutical drugs with manifold uses. Herein we describe the most

in the literature on HPs, with a special focus on the metal chelating properties

vailable online 16 August 2011 recent advances reported

∗ Corresponding author. Tel.: +351 218419273; fax: +351 218464455.E-mail address: masantos@ist.utl.pt (M.A. Santos).

010-8545/$ – see front matter © 2011 Published by Elsevier B.V.oi:10.1016/j.ccr.2011.08.008

M.A. Santos et al. / Coordination Chemistry Reviews 256 (2012) 240– 259 241

Keywords:Hydroxypyridinones3-Hydroxy-4-pyridinonesIron chelatorsAnti-neurodegenerativesMetallodrugsM

of the 3-hydroxy-4-pyridinone (3,4-HP) derivatives, and the different approaches used to functionalizethese chelators to improve their biological properties, namely in terms of bioavailability and specific bio-targeting abilities. Representative examples of HPs are included, mostly for applications as chelating drugsfor sequestration or passivation of metal overload or deregulated biometals, but also as metallodrugsfor potential diagnostic/therapeutic purposes. These examples are discussed in terms of the chelatingproperties and structure–activity relationships.

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edicinal chemistry

. Introduction

For more than two decades the hydroxypyridinones (HPs) haveeen described as important heterocyclic bidentate ligands forhard” metal ions, such as Fe3+. Their importance as metal-relatedharmaceutical drugs has increased in relation to their role inequestering/delivering specific metal ions from/into the humanody. In particular, one member (deferiprone) of the 3-hydroxy--pyridinones (3,4-HP) class is under clinical use as iron-chelatinggent. This family of strong chelators can be obtained from naturalroducts and be easily derivatized to modulate specific physic-chemical/biological properties, besides presenting low toxicitynd good biocompatibility. HPs have a 6-membered ring scaf-old that offers many possibilities of functionalization at differenting-positions, enabling the modulation of their bioavailabilitynd interaction with several biological targets. A multitude oferivatives has been developed for numerous purposes and appli-ations in bio-medicine. Thus, HP derivatives are based on a singleeterocyclic motif, appearing quite frequently in many bioactiveompounds as potential pharmaceutical drugs and so they cane considered as “privileged” structures in the rational design ofhelating drugs. A schematic representation of these HP moieties,heir main areas of medicinal interest as well as the metal ionsnvolved, are presented in Fig. 1.

Among the potential applications of HPs, this review focusesostly on those related to the administration of a chelator, (namely

f 3-hydroxy-4-pyridinone (3,4-HP) type), to interact with dys-unctional metal ions or metal-dependent pathways in the body,ather than with those involving the administration of a metal-omplex (metallodrugs).

As representative examples of potential chelating drugs weave included: chelatotherapeutics for iron overload or misplacedetal diseases, anti-neurodegeneratives (hybrid drugs), antimicro-

ial, anti-protozoan and inhibitors of matrix metalloproteinasesoverexpressed in cancer or inflammatory diseases). Concerninghe metallodrugs, we report representative examples of metalomplexes with potential pharmaceutical application in diag-osis (nuclear and MRI probes) and therapy (anti-diabetes andnticancer). For the metallodrugs, emphasis is given to activityependence on the type of metal ion (which can be non-essential),n the type of wrapping ligand that has to guarantee the ade-uacy of the metal-complex thermodynamics and kinetics, butlso on the accessibility to specific targets, while minimizing sideffects.

The rational design of multifunctional HP-based drugs is hereinspecially emphasized due to the number of features that can benclosed in the same molecular entity, such as targeting specificissues or biomolecules, membrane crossing ability, delivery andxcretion capacity.

With the analysis and discussion of the new developments onP-based compounds towards several medicinal applications, wexpect to highlight the fact that these easy-to-functionalize het-

roaromatic rings with strong chelating capacity towards hardetal ions can be considered “privileged” structures for future

rug design, therefore providing fresh stimulus to create new moreotent metal-related drugs.

© 2011 Published by Elsevier B.V.

2. Biometals in health and disease

Biometals (e.g. Fe, Zn, Cu, Mo) are essential trace elements whichare vital for organisms and cells in order to grow and develop.Iron undoubtedly plays a central role in a wide variety of cellu-lar events, because it provides a specific binding site for oxygenin hemoglobin and also it is the cofactor of several enzymes (e.g.cytochrome c and ribonucleotide reductase) [1]. Both zinc and cop-per have catalytic function in superoxide dismutase, an antioxidantenzyme, while there are numerous zinc-dependent enzymes, suchas the matrix metalloproteinases (MMPs) and the carbonic anhy-drases (CAs). Additionally, molybdenum is the cofactor componentof various redox enzymes, such as xanthine oxidase (XO).

In a healthy situation, the body is provided with homeostaticmechanisms and buffers to maintain low concentrations of freemetal ions, thus preventing abnormal decompartmentalization,release and trafficking of metal ions. The main reason for theanomalous accumulation of nonessential metal ions in the bodyis environmental exposure (e.g. Al, actinides) and the administra-tion of metallodrugs for therapy/diagnosis purposes, which maycause competition for biometals, generation of oxidative stress andderegulation of several enzyme systems, causing various patholo-gies [2]. Therefore, medicinal inorganic chemistry has to deal withthe introduction of metal ions in the body, but also with theirremoval or passivation, namely through the design of tailoredchelating agents, which may need a multifunctional drug approach.

2.1. Iron overload and misplaced related illnesses

Iron (Fe) is an essential element for all living organisms, mainlydue to its characteristic redox chemistry as well as high affinityfor oxygen. Usually, iron levels in the body are very tightly con-trolled (normal level is 40–50 mg/kg of body weight [1,3]), becausewhen in excess it is extremely toxic due to iron redox cycle (Fen-ton reaction), with concomitant production of reactive oxygenspecies (ROS), such as the hydroxyl radical. This radical may reactwith several endogenous molecules (sugars, lipids, proteins andnucleic acids), with subsequent lipid peroxidation, sugar and pro-tein oxidation as well as DNA and RNA damage, thus leading tovarious pathological situations [4]. Since there is no physiologicalmechanism for iron elimination, situations of severe iron overloadcan occur, such as in transfusional hemosiderosis (caused by par-enteral administration of iron to treat �-thalassemia major), orhemochromatosis (a genetic deficiency associated with iron over-absorption). In such circumstances, the elevated levels of iron canultimately lead to an increase of transferrin and ferritin iron satura-tion in plasma and intracellularly, respectively. This is accompaniedby a higher level of non-bound-iron (NBI) and its deposition incells and specific organs (liver, pancreas, heart, brain) [5] withthe corresponding organ failure and eventual death, unless treat-ment is applied for excess iron removal from the body [6]. Apartfrom haemoglobinopathic disorders (e.g. haemochromatosis and

�-thalassemia), there are additional pathological states associatedwith misplaced iron, particularly in cells and tissues, such as can-cer [7] and neurodegenerative disorders – Parkinson’s disease (PD),Alzheimer’s disease (AD), or Friedreich’s ataxia (FA) [2,8].

242 M.A. Santos et al. / Coordination Chemistry Reviews 256 (2012) 240– 259

F functb ions.

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ig. 1. Schematic representation of the different potential pharmaceutical targets ofiometals, or as metallodrugs for therapeutic (blue) and diagnostic (violet) applicat

.2. Aluminum – an aberrant metal ion in diseases

Aluminum (Al) is a nonessential element that becomes toxichen accumulated in the organism. In spite of its low bioavail-

bility under normal environmental conditions, Al can becomeio-accessible from diet or other environmental exposure. Then,ue to the absence of efficient excretion mechanisms, it can accu-ulate in certain tissues. Aluminum toxicity has mostly been

ssociated with bone disorders (vitamin-D-resistant osteomalacia)nd neurological diseases, such as dialysis encephalopathy syn-rome, and eventually AD [9]. Although the linkage of Al withhe AD physiopathology is still controversial, there is evidencef Al accumulation in the neurofibrillary tangles (NFT), which isne hallmark of AD [2,10]. Al can contribute to (or promote) theron-induced oxidative damage of neurons. This is due to someompeting capacity of Al3+ over Fe3+ for the same bioligands, thusnfluencing the Fe availability, its redox cycling, and respectiveomeostatic mechanisms [11].

The main Al-binding protein in plasma appears to be transfer-in (Tf, log KAlTf = 13.5 [12]), which is only 30% saturated with Fen normal serum, thus still keeping significant chelating capacityor other trivalent metal ions, such as Al. Therefore, Fe/Al-chelators

ust be able to compete with transferrin for aluminum.

. Targeting hard metal ions for decorporation orassivation

For a large range of pathological situations related with metal

mbalance, the use of chelating agents can potentially correct thenomalous metal metabolism or at least attenuate dysfunctionalffects by passivation, redistribution or decorporation of the mis-laced/aberrant metal ions.

ionalized hydroxypyridinones, acting either as ligands (grey) to chelate or passivate

The first approved iron chelating drug, introduced in the marketin 1962, was Desferrioxamine (DFO, Desferal®, Fig. 2), a microbialtris-hydroxamate siderophore isolated from Streptomyces pilosum[13]. In spite of the high iron-chelating efficacy of this hexaden-tate ligand, it has low oral activity, due to its hydrophilic character,high molecular weight and peptide nature. Its use in iron overloadtreatment requires quite inconvenient administration protocols tooptimize the drug efficacy (8–12 h of slow subcutaneous infusion,5–7 days per week) [14]. Thus, in spite of its recognized benefit onpatient survival, the treatment with DFO presents important draw-backs, namely due to low patient compliance and high cost, besidesbeing associated with some hypersensitivity and systemic allergies[15]. The recognized need for orally active and non-toxic chela-tors have lead, over the last three decades, to intensive researchfor alternative therapeutics [16]. Due to the hard nature of Fe3+,several hard ligands, such as polyaminocarboxylic acids, catecholsand hydroxamates, were studied but they still presented problems,due to low oral bioavailability and lack of chelating selectivity witheventual zinc depletion. The major developments in the design ofnew iron chelators occurred after the disclosure, in 1982, of 1,2-dimethyl-3-hydroxy-4-pyridinone (Deferiprone, DFP, Ferriprox®,Fig. 2) [17], which was later on (1999) approved as a second-line therapy for thalassemia patients [5]. Unlike the hexadentateDFO, that forms very stable 1:1 Fe complexes (pFe = 26.3 [18],pFe = −log[Fe3+]), DFP is a bidentate chelator which forms a quitestable 1:3 iron chelate (pFe = 19.4 [16]) at the physiological pH.Although the bidenticity of this ligand is associated with forma-tion of complexes with higher lability than the hexadentate ones,

with somewhat higher potential metal redistribution and toxicity,the high pFe values at the physiological pH and the concentra-tions used in the administration protocols are a guarantee thatcomplex dissociation is not likely to occur [19]. Several other iron

M.A. Santos et al. / Coordination Chemistry Reviews 256 (2012) 240– 259 243

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helators have been recently reported [2,16,19–24], but besidesesferal® and Ferriprox®, only an orally active iron-chelating drug,eferasirox (ICL670, Exjade®) (see Fig. 2) was approved by FDA2005) for use in transfusional iron overload. This tridentate lig-nd forms quite stable 1:2 complexes with iron, and its bindingffinity lies between those of DFO and DFP (pFe = 22.5 [25]). How-ver, concerns regarding toxicity and efficacy of treatments witheferasirox have recently been reported [26].

The combination of DFO and DFP was first used only in spe-ific cases of iron-overload patients with no response to normalherapies with any of these chelators [27], but it became increas-ngly employed after the discovery of additive/synergistic effects28–30].

Concerning Al chelation, the similarities between Fe3+ and Al3+

ionic radii 64 and 50 pm, respectively) are the main rationalesor the use of DFO or DFP on the mobilization and depletionf aluminum [31–33]. Combined chelation of these two drugshowed also promising results in preclinical tests with Al-loadedats [34,35]. Since Fe and Al are associated with many pathologicalrocesses, DFP and deferasirox are potentially useful oral drugs in aide variety of situations not considered as typical metal overload.

ven though it is possible to achieve clinical benefit from theseompounds, more suitable chelators are needed and the majoresign strategy has been focused on conjugating metal chelatingffinity/selectivity with adequate lipo-hydrophilic balance, thuschieving reactivity and drug bioavailability, respectively. Moreecently, research has been focused on the challenge of engineeringew extra-functionalized ligands meant to be used as metal chelat-

ng agents under molecular-targeting strategies. Therefore, theyre addressed more to the inhibition of anomalous metal-proteinnteractions or remediation of local metal dyshomeostasis than tohe increase of systemic metal excretion. These multifunctionaligands, besides the metal binding group(s), incorporate other moi-ties for extra-targeting roles, namely to promote the interactionsith bio-receptors and other bioactive sites. An overview on func-

ionalized HP chelators will be presented herein.

. Functionalized 3-hydroxy-4-pyridinone chelators as DFPuccessors

After the disclosure of DFP for iron-overload, the functionaliza-ion of HPs appeared as a natural and appealing strategy for theiscovery of new chelating analogues with improved drug features.ased on the main requirements for a drug to be used in chela-otherapy (bioavailability and chelating capacity/selectivity), two

ain types of approaches have been used on HP derivatization: the

xtra-functionalization of bidentate monochelators and the ligandolydenticity. For the bifunctional bidentate ligands, higher effi-iency in gut absorption is expected, due to low molecular weights,hile for the polydentates the higher Fe3+ coordination capacity is

nts in clinical use and relevant data.

counter-balanced by a lower absorption efficacy. In the followingsections we report the recent efforts made on the HP functional-ization in view of discovering new chelating agents with potentialpharmacological applications.

4.1. Hydroxypyridinone development and evaluation methods

HPs are N-heterocycles with an endocyclic alpha-hydroxy-ketone (O,O)-chelating group. Among the three main types ofHPs [1-hydroxy-2-pyridinones (1,2-HP), 3-hydroxy-2-pyridinones(3,2-HP) and 3-hydroxy-4-pyridinones (3,4-HP)], the 3,4-HPs areby far the most studied class, due to their higher iron chelatingcapacity (Fig. 1). This fact is mainly due to the different placementof the (O,O)-donor group around the central pyridine ring which, forthe 3,4-HP, provides the hydroxyl group with the highest basicity(pKa ca. 9–9.5) and the highest electronic density at the coordi-nating atoms. This makes them neutral at the physiological pHand with higher affinity for hard metal ions (e.g. Fe3+, Al3+) thanfor soft biologically relevant bivalent metal ions (e.g. Fe2+, Zn2+,Cu2+). On the other hand, the 3,4-HPs are easily ring-substituted,and so many extra-functionalized compounds are known. Althoughmost of the functionalized 3,4-HP result from attachment of dif-ferent groups at 1-position (ring-N), there is also a reasonablenumber of compounds with substitutions at the C-2, while onlya few with modifications at C-5 and C-6. Most of the functionalized3,4-HPs have been developed aimed at improving their physico-chemical and biological properties, in view of potential applicationas monochelating drugs, but some others have been developed aspodant-type polydentate chelators, by attachment of two or threeHP moieties to specific scaffolds, in order to enhance the metalchelating efficacy [19,20].

Standard organic synthetic procedures are used for the prepara-tion of HPs. Besides some synthetic specificity associated with eachtype of ring-functionalization, the preparation of 3,4-HPs involvein general, the aminolysis of the corresponding O-benzyl protectedhydroxypyrones and the O-deprotection as the last step. The 3,4-HP derivatization at 1-position is easier than at 2-position, whereasthe manipulation of C-5 and C-6 is somewhat more difficult [36].Regarding the polydentate compounds, two or three bidentate 3,4-HP moieties are attached to selected scaffolds, usually aided byactivation with coupling reagents [37–40]. The assessment of thephysicochemical properties in solution, such as acid-base proper-ties (pKa values), lipo-hydrophilic character (log D or log P), metalcomplexation capacity (pM = −log[M], at pH 7.4 for micromolarconcentration of the metal ion and 10-fold ligand excess), is per-formed by spectroscopic and potentiometric techniques. Partition

(P) or distribution (D) coefficients have been used to assess thelipophilic character of the compounds and to anticipate their abilityto penetrate biological or lipid barriers. The partition coefficient isthe ratio of concentrations of a compound between two immisci-

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44 M.A. Santos et al. / Coordination C

le solvents (normally water and the hydrophobic octanol), whilehe distribution coefficient is the ratio of the sum of the concen-rations of all the ionized forms of the compound in each of thewo phases and it is a pH dependent parameter. log P and log Dalues are determined either by experimental or computationalpproaches [41] and can be related to each other [42]. The pM val-es allow comparison of the chelating capacity of the ligands, sincehe effects of different ligand basicity, denticity and complex metal-igand stoichiometry are taken in consideration. Concerning theellular and animal models for metal mobilization, radio-labelingethodologies are widely used, such as the pre-administration

f radioisotope-proteins or -carriers for metal-overloaded micee.g. 55Fe [43], 59Fe [44,45] or 67Ga [40,46,47]), administrationf the chelating agent and subsequent analysis (�-emission) ofadiometal content in the biological samples. For non-radio emit-ing metal ions (e.g. Al-overload of rabbits [31] or rats [33]), the

etal-content evaluation in the biological samples involves muchore laborious sampling and analytical methodologies (e.g. ICP-S, atomic absorption).Many 3,4-HP have been functionalized to improve their oral

ctivity and bioavailability at the disease sites, or at increasing theirhelating capacity.

.2. Improvement of 3,4-HP bioavailability

Because of the restricted membrane permeation requirements,amely the molecular size, lipophilicity and charge [41], one ofhe main concerns assisting the design of compounds for potentialhelation therapy has been the modulation of their bioavailability.his is specifically to provide the chelating agent and the corre-ponding metal-complex with the ability for adequate distributionn biological systems. Thus, for the bidentate 3,4-HP chelators, thettachment of a functional group appears as a first approach foruning physico-chemical and biological properties to enable a pref-rential biodistribution in certain organs or tissues, and/or someapacity for molecular recognition by specific biologic targets. Inact, the molecular size of bidentate HPs is usually low (<500),hile the lipophilicity and charge (or ionization state) at physi-

logical conditions can be modulated by convenient derivatizationf the 3,4-HP. Although the lipophilic character of 3,4-HP has beenssociated with an enhancement on the glucuronidation of the 3-ydroxyl group (a DFP drawback) [48], the hypothetical benefitsf minimizing this metabolization by using more hydrophilic HPsould result in reduction of the membrane permeability of the

igands/complexes as well as the gut absorption.The influence of the N-derivatization of 3,4-HPs on the lipo-

ydrophilic character and in in vivo iron mobilization was studiedor a large number of compounds with different substituent groups,uch as alkyl and alkyl-amine, -carboxylic, -amide, and hydrox-alkyl groups [45]. The set of substitutions enabled a range ofariation on the lipophilicity of the ligands, which was evaluatedn the basis of the corresponding logarithmic distribution coeffi-ient values (log Dligand; 1-octanol/water, MOPS buffer, pH = 7.4),anging from ca. −2.9 to 1.9 for carboxylic, hydroxyalkyl and alkylerivatives. These studies revealed the existence of some relation-hips between the lipophilicity of the ligands (log Dligand) and thatf the corresponding iron complexes (log Dcomplex), allowing a reli-ble estimation of log Dcomplex from experimentally determinedog Dligand. Interestingly, the biphasic linear relationship obtaineduggested that, for lipophilic ligands (log Dligand > −1) the complexan be ca. 3-fold more lipophilic than the ligand (due to the mask-ng effect of the three coordinating ligands over that of the metal

on), while for more hydrophilic compounds (log Dligand < −1) theipophilicity of the complexes accompanies more closely that ofhe ligands suggesting identical solvation effects for the ligands andomplexes.

try Reviews 256 (2012) 240– 259

A set of functionalized 3,4-HPs were also reported involv-ing several N-attached extra-functional non-peptidic groups (e.g.alkylcarboxylic acids, alkyl-arylamines, alkyldiethylamines, alkyl-arylpiperazines, alkylaryl-aminoacids, alkyl-aryl-sulphonamides,alkylaryl-glucoside) [32,46,49–52] (see Fig. 3).

Solution studies and bioassays on this set of 3,4-HP derivativesindicated that the N-functionalization has minor effects on thechelating capacity of the ligands, while considerable differenceswere obtained on their logarithmic partition coefficient values(log P; 1-octanol/water, Tris buffer, pH 7.4), covering ca. 3 ordersof magnitude (−2 < log P < 1), on complex speciation near the phys-iological pH, as well as on the biodistribution and clearance rate ofmetal-loaded animals. Although most of these peptidomimetic 3,4-HPs presented very rapid clearance from most organs, the ornithinederivative (Orn-HP) appeared as the best metal decorporatingagent, and it also exhibited the highest metal chelating efficacy(pFeOrn-HP = 21.9 [46], pFeDFP = 19.4 [16]). Moreover, it has also afavorable speciation profile at pH 7.4, apparently due to the ligandzwitterionic form and the chelate which is neutral and water-soluble. However, its high hydrophilicity (log P < −2) may hamperthe potential application of Orn-HP as iron chelator drug. Inter-estingly, the Pip-HP presented the highest lipophilicity (log P = 1.1[51]) and the concomitantly highest brain retention [46] suggestingthat this N-substituent could provide considerable BBB transportproperties to the compound; on the other hand, arylpiperazinegroups are known to target serotonin (5-hydroxyptrypyamine, 5-HT1A) receptors with an important role in psychiatric disorders[53]. Some extra-functional groups provided specific biodistri-bution such as the hydrophilic Car-HP (log P = −1.89 [50]) withsignificant bone uptake. Concerning the 3,4-HP glucoside deriva-tives, they were developed to improve the bioavailability, and alsothe molecular targeting of carbohydrate transporters in the body.However, these compounds were mostly studied in combined lig-and protocols for Al decorporation [52]; further developmentson the glycobiology approach are presented below in the sectionconcerning 3,4-HP-functionalization with extra-roles such as neu-roprotection in iron-dependent neurodegenerative diseases (videinfra).

The 2-amido derivatization of 3,4-HPs, was first intended toprevent the glucuronidation of the DFP 3-OH group, by close attach-ment of a bulky group [54] but more recently, many patentsappeared covering numerous DFP analogues and including variousamides at C-2, which claimed for improvements on pharma-cokinetics and pharmacodynamics over DFP [55]. In particular,enhancement on log P (−0.98 < log P < 0.34) values, when comparedwith that of DFP (−0.77) [56], were rationalized by the existenceof a highly stable intramolecular hydrogen bond between the 2-amido proton and the 3-hydroxyl group, which reduces H-bondinginteractions with water molecules and increases the hydrophobiccharacter. These intramolecular interactions also revealed someeffect on the chelating affinity (see below).

4.3. Chelating capacity enhancement

The search for compounds with higher chelating affinity is ofvital importance in chelatotherapy in order to reduce the amountof administered ligand, but also the complex dissociation, with thesubsequent lower redistribution of the iron to other parts of thebody, as well as drug-induced toxicity, namely due to eventualinteraction with Fe-dependent enzymes.

Among the numerous design strategies towards new HP chela-tors with pFe enhancements, some approaches are based on the

functionalization of the bidentate HPs (mono-HPs) with differentgroups, but most are based on increasing the ligand denticity (bis-or tris-HPs). Such polydentate ligands, specially the hexadentatederivatives to target M3+, present advantages over the bidentate

M.A. Santos et al. / Coordination Chemistry Reviews 256 (2012) 240– 259 245

Car-H P

R1

(CH2)3NN

Orn-H P

Pip-HP

GluA-HP

N

OOH

R2R1

R6

R2 R6

HCH3

CH3 H

CH3 H

GluB-HP CH3 H

CH2

NH2

OHOHO

HO

NH

OH

OHO

HO

HO

NH

OH

O(CH2)3

HO

O

CH2HO

O

CH3 H

functi

Hamap

4

tGm

piwtwtthaatttlticcdpcl

pataMm

Fig. 3. Selected bi

Ps, namely in terms of kinetic inertness and thermodynamics,lthough the fact that they usually possess high molecular weightay represent a limitation to their membrane crossing ability and

ccessibility for scavenging iron from the normal cytoplasmic ironools [41].

.3.1. Extra-functionalization of bidentate ligandsSeveral authors [20,57] have studied the effect of different func-

ional groups on the binding affinity of bidentate HPs for Fe3+ androup 13 metal ions, although in general only modest pFe enhance-ents were obtained with respect to DFP.Noteworthy is a set of bidentate 3,4-HP substituted at the 2-

osition, with 1′-hydroxyaryl and 1′-hydroxyalkyl groups, withncreased pFe values relative to that of DFP [44,58]. Identical results

ere also recently reported for a set of 2-amide 3,4-HP deriva-ives, where an increase of the lipophilicity (see previous section)as accompained by enhancements on pFe values. Since both

ype of derivatives have O-H or N-H groups at a close distanceo the 3-hydroxyl group, pFe increases (20.4–21.5 [44] for 2-(1′-ydroxyalkyl) derivatives; 20–23 [56] for amide derivatives) werettributed to a reduced competition with protons, due to the neg-tive inductive (electron withdrawal) effect of these groups ando the intramolecular six-member ring H-bond network betweenhe 2-substituent and the 3-hydroxyl function (Fig. S1, Supplemen-ary data). Although the stabilization effect on the deprotonatedigands reduces the overall Fe3+ stability constant, it also decreaseshe pKa values of the ligands and these combined changes resultn the increase of the corresponding pFe values. In fact, in thealculation of the global conditional constants, which are linearlyorrelated with pM, the protonation constants are raised to a powerependent on the stoichiometry of the complexes present at thehysiological pH [59]. Detailed dose-response studies show thathelators with higher pFe values scavenge Fe more efficiently atower doses when compared with simpler dialkyl-HPs [16,56].

The effect of 3,4-HP N-functionalization in a series of so calledeptidomimetic groups (specifically containing amine, carboxylic,mide, amide-amine, sulphonamide and amino acid groups, linked

o the N-ring through different alkyl and alkylaryl spacers) waslso evaluated on their chelating capacity towards a set of hard

3+ ions (M = Fe, Al, Ga), as well as on the 67Ga biodistribution inice as an animal model of metal-overload [46]. All these ligands

O

onalized 3,4-HPs.

form quite stable 1:3 metal complexes (pFe = 17–22, pGa = 15–21,pAl = 14–16), with no involvement of the extra-functional group inthe metal-coordination, the most powerful chelating agent beingthe ornithine derivative (Orn-HP, pFe 21.9). This may be rational-ized in terms of the stabilizing electronic effect created by theamphoteric substituent with consequent decrease of the desta-bilizing Coulombic repulsion between both positive charges ofthe ammonium group and the metal ion. Furthermore, based onthe effect of the chelators on the 67Ga biodistribution patternin mice, Orn-HP appeared as a quite promising chelating agent,due to the fastest blood clearance, highest excretion and in vivostability [46].

4.3.2. The polydenticity approach on hydroxypyridinonesTo increase the chelating efficacy of HP-based ligands, sev-

eral authors have decided to mimic the polydentate naturalsiderophores (e.g. DFO and rhodotorulic acid) by appending threeand two chelating moieties to the same scaffold (see Fig. 4).

Despite the recognized higher biological relevance of the 3,4-HPchelators (due to the approved use of DFP in chelation ther-apy), as compared to the other classes of HPs, the 3,2-HPs werestudied earlier as polydentates, than the 3,4-HP analogues. Thislack was recently overcome and several tris- and bis-chelatingpodants were obtained by attaching three or two 3,4-HP moietiesto different scaffolds, namely aminocarboxylate or cyclic carboxy-late backbones (see Fig. 5), thus affording, respectively, tris- andbis-(3,4-HP), under the so called ligand approach or ligand-targetapproach [23].

Under the denominated ligand approach, emphasis was givento the chelating capacity of the final ligand entity and a seriesof hexadentate tris-(3,4-HP) chelators was obtained. Regardingthe ligand-target approach, the architecture of these chelators wasbased on selecting a special scaffold, which could enable the attach-ment of two 3,4-HP chelating moieties, affording a tetradentatebis-(3,4-HP) with improved ligand efficacy as compared with abidentate unit, while it still retained the ability for further extra-

functionalization namely aimed at biological-targeting roles of thefinal chelating entity. The tetradentate 3,4-HPs were also proposedfor combined chelation protocols with bidentate 3,4-HP ligands[23] (vide infra).

246 M.A. Santos et al. / Coordination Chemistry Reviews 256 (2012) 240– 259

F ns: (a)

4

DiHfiehat

Hsrbc

ig. 4. Coordination modes for complexes of HP derivatives with trivalent metal io

.4. Hexadentate hydroxypyridinones

By mimicking the natural siderophores Enterobactin andFO, several hexadentate chelators have been proposed, namely

n the last decade, with HP ligands [60–70]. Although 3,4-P are recognized as the strongest HP iron chelators, therst hexadentate HPs explored used 3,2-HP chelating moi-ties (see some representative examples in Fig. 6). Theyave tripodal structures with three chelating moieties mostlyppended to a central anchoring atom or a cyclic struc-ure.

The most common design strategy for the reported tris-(3,2-P) chelators involves the coupling of the amines from the central

caffold with the carboxylic substituent groups of the pyridinoneing (alpha-positioned to the chelating moiety). The comparisonetween the chelating capacities based on pFe values, in someases, may be questionable because the corresponding stability

R

C

CC

R

R

X Y

XO

OO

NO

X X

Y

O

HN

N

OOH

R1R2

m

m

m

X = p = 0, 1m = 1, 2n = 3, 4

X = Y

X = Y

n

Fig. 5. Molecular backbones and chelating moieties for po

1:3 M3+/mono-HP and example Fe3+/DFP; (b) 1:1 M3+/bis-HP; (c) M3+/tris-HP.

constants were determined in non-aqueous systems (e.g. TRISPYR,HOPObactin). As expected, these tripodal tris-chelators showedhigher chelating efficacy than the analogues with linear supports[61]. Despite the different design strategies to improve the chelat-ing efficacy, it seems to be quite dependent of the steric hindranceassisting the metal complex formation (e.g. CP130 (26.7) [69],TREN-HOPO (27.6) [62]); the cyclic platform did not bring anyimprovement to the complex stabilization, although the triazo-backbone amine groups enhanced water solubility.

Following the ligand approach cited above [23,70] (see Fig. 5),our group has developed a series of hexadentate tris-(3,4-HP),bearing three 3,4-HP-alkylamino arms attached to triscarboxylicacid backbones, namely a cyclic skeleton (Kemp acid) and

two amino-tricarboxylic acids (nitrilotriacetic acid – NTA – andnitrilotripropionic acid – NTP) [39,40,70,71]. Two further tripo-dal tris-(3,4-HP) compounds with a anchoring scaffold, namely atri-carboxylic acid (CP254) [72] and a tris(2-amino-ethyl)amine

O

p

NN

X

O

OY

O X

O

Y

tris-(3,4-HP) Ligan d app roac h

bis-(3,4-HP) Ligand -ta rge t app roac h

lydentate bis-(3,4-HP) and tris-(3,4-HP) derivatives.

M.A. Santos et al. / Coordination Chemistry Reviews 256 (2012) 240– 259 247

F : CP13a

(fF

cbe

ig. 6. Representative iron 3,2-HP tris-chelators, with the corresponding pFe valuesnd N3(etLH)3 (26.5) [66].

CP262) [73], have also been recently reported. The structuresor this set of hexadentate 3,4-HP are schematically presented inig. 7.

As expected, these hexadentate 3,4-HPs presented stronger iron

helating capacities than the hexadentate 3,2-HPs, as illustratedy the reported pFe values (Fig. 6 and Table 1), where the differ-nces can be up to three units, namely for analogues with the same

Fig. 7. Representative iron tris-(3,4-HP) chelators: NTA(BuHP)3, NTP(PrHP)3,

0 (26.7) [69], TREN-HOPO (27.6) [62], TRISPYR (32.23) [68], HOPObactin (27.6) [67]

TREN skeleton (TRENHOPO and CP262). The tris-(3,4-HP) chelatorspresent pFe values in the range 26.8–30.5, higher than that of DFO(26.3) [18]. The highest pFe values were found for CP262 (30.5)[73] and NTP(PrHP)3 (29.4) [40] although, to our best knowledge,

only NTP(PrHP)3 underwent in vivo assays. Among a set of poly-(3,4-HP) chelators tested in metal overloaded mice (with 67Ga as amodel of Fe or Al), this chelator indicated the best capacity for the

Kemp(PrHP)3, Kemp(BuHP)3, CP262 and CP254 (pFe values in Table 1).

248 M.A. Santos et al. / Coordination Chemis

Table 1pMa values (M = Fe, Al) at physiological pH for selected synthetic and biologicligands.

Compounds pFe pAl

Biological ligandsDFO 26.3 [18] 19.4 [81]

27.6 [80] 21.2 [80]Transferrin 20.3 [18] 14.5 [12]Mono-(3,4-HP)b

DFP 19.4 [16] 16.1 [83]20.7 [82]

Car-HP 21.3 [50] 16.1 [50]Orn-HP 21.9 [46] 15.8 [46]R1 = H, R2 = CON(CH3)2, R6 = CH3 22.8 [56] –R1 = R6 = H, R2 = CONH2 21.5 [56] –R1 = (CH2)3OH, R2 = CH(CH3)OH, R6 = H 21.5 [44] –GluA-HP – 15.5 [52]GluB-HP – 13.8 [52]Bis-(3,4-HP)Kemp(PrHP)2 20.5 [37] 18.1 [37]EDTA(HP)2 26.3 [38] 19.0 [38]IDA(HP)2 25.8 [37] 18.8 [37]IDAPip(HP)2 25.7 [78] 18.0 [78]Tris-(3,4-HP)Kemp(PrHP)3 28.0 [39] 21.2 [39]Kemp(BuHP)3 26.8 [39] 21.8 [39]NTA(BuHP)3

c 27.9 [40] 22.0 [40]NTP(PrHP)3 29.4 [40] 22.4 [40]CP254 27.2 [72] –CP262 30.5 [73] –Other synthetic ligandsHBED 30.9 [84] –EDTA 23.4 [85] 16.2 [85]DTPA 24.6 [85] 15.7 [85]

a pM = −log[M3+] with CL/CM = 10 and CM = 10−6 M at pH 7.4.

m

m[

adeofll

FosK[

b According to Fig. 3.c Admitting no precipitation occurs at the concentration conditions of pM deter-ination.

etal decorporation of hard metal ions (vide infra, Table 1, Fig. 8)40].

After all, we may conclude that the hexadentate 3,4-HPs appears the strongest iron chelators at the physiological pH and theiluted conditions that prevail in biological systems. The differ-

nt strategies used in the chelator design were mainly aimed atptimizing the chelating affinity (complex steric hindrance/ligandexibility) and the lipophilicity of chelator/chelate, although this

ast property appeared as the most limiting feature. In particular,

ig. 8. Biodistribution data in the most relevant organs, expressed as %I.D. perrgan for: intravenous administration (iv) of 67Ga–citrate and 67Ga–citrate withimultaneous intraperitoneal injection of the ligands NTP(PrHP)3, NTA(BuHP)3,emp(PrHP)3, IDA(HP)2 and DFP, 1 and 24 h after iv administration in female mice

40].

try Reviews 256 (2012) 240– 259

although the chelators with NTP and NTA skeletons are posi-tional isomers (just differing on the position of the peptide groupalong the side chain) the NTA derivative presented a strongerinternal hydrogen-bond network which results in higher waterinsolubility problems (due to the higher acidity of the backboneammonium proton) and in a slightly lower flexibility of the chelat-ing arms for wrapping the metal ion. Noteworthy is the recentlyreported extra-functionalization of a tris-(3,4-HP) which, based onthe same approach used in CP254, allowed the attachment of flu-orophores, such as rhodamine B, and provided the chelators withsome sensing role [74]. Identical extra-functionalization has beenproposed for the chelator incorporation in polymers to make solidsupported metal sorbents [72], a subject of currently increasingbiological and environmental interest even with bidentate 3,4-HPs[75,76].

4.5. Tetradentate 3,4-HPs: from ligand-target approach tocombined chelating approach

In an attempt to simultaneously improve metal chelating effi-cacy and introduce an extra-targeting role in chelator development,as part of the already mentioned “ligand-target approach” (Section4.3.2), our group proposed a series of bis-(3,4-HP) derivatives. Thefirst compound was obtained by attaching two 3,4-HP units to acyclohexane backbone (Kemp(PrHP)2 [77]), leaving one carboxy-late group (non-coordinated to metal ion) appended to the cyclicmoiety, for further functionalization and interaction with specificbiological receptors. Even though its chelating capacity towardsM2+ and M3+ metal ions was good [37,77] (see Table 1), some appar-ent strain in metal wrapping and water solubility problems ledto a change in the ligand topology and so aminocarboxylic skele-tons were adopted, namely IDA (iminodiacetic acid), IDA(HP)2 [37],and EDTA (EDTA(HP)2 [38]). Subsequently, IDA(HP)2 was extra-functionalized through N-derivatization of the IDA skeleton withan arylpiperazine group (IDAPip(HP)2 [78,79]), aimed at the ligand(or metal complex) vectorization towards neurotransmitter recep-tors. The pM values obtained for these bis-(3,4-HP) (see Table 1)confirm their stronger chelating-affinity and higher specificity forhard metal ions (e.g. Fe, Al) than other biologically relevant metalions (e.g. Zn), when compared with bidentate chelators such as DFPor aminocarboxylic acids (DTPA, EDTA), which have poor chelatingspecificity and can lead to Zn depletion. Moreover, all the com-pounds but Kemp(PrHP)2 (pFe = 20.5) have similar iron chelatingcapacity (pFe 25.7–26.3) and even though the pFe values of the bis-(3,4-HP) (21–26) are between those of the mono-(3,4-HP) (19–23)and the tris-(3,4-HP) (27–30), they are still able to compete withtransferrin (20.3 [18]). In vivo studies, performed with the IDAderivatives in a 67Ga overloaded animal model [37,78], clearly indi-cated the ability of the tetradentate chelating agents for in vivometal chelation and excretion; hypothetical formation of polymericpolynuclear species with excretion limitations, does not seem rel-evant under the in vivo concentration conditions (see Fig. 8).

To improve the efficacy of the monotherapies in metal overloadsituations, new chelating protocols, involving a combined chelatorapproach, have also been recently proposed. This trend emergedmostly after reports about the proven benefits of a combinedDFO/DFP therapy to treat iron balance and myocardial iron deposi-tion [86]. A synergistic effect involved in the DFO/DFP therapy wasattributed to the improved access to non-transferrin bound iron(NTBI) species, otherwise unavailable to DFO, through the shuttlingof iron by DFP to form feroxamine (FO) [87]. Al-decorporation hasalso been tested with the DFO/DFP pair and other chelator combi-

nations [34], such as ascorbate (AS) and DFO and/or Feralex-G [88](see Fig. 9).

In order to find alternative combined drug therapies, our groupstudied also new chelator combinations, each pair involving one

M.A. Santos et al. / Coordination Chemistry Reviews 256 (2012) 240– 259 249

F rbic ar

tomcimmsIHb

tbw6

e(teHtmtIoplcrcmlfp

ig. 9. (a) Structures of some compounds used in combined chelation therapy: ascoeported chelating pairs studied (see also Figs. 2 and 3).

etradentate bis-(3,4-HP) and one bidentate mono-(3,4-HP), inrder to fulfill the metal ion coordination sphere; the small sizedolecule could have a higher accessibility to specific intracellular

ompartments and metal-storage sites and so the correspond-ng metal complexes could easily get out; the larger sized and

ore potent bis-(3,4-HP) chelator has a co-ligand role, with for-ation of a ternary complex that can be transported to the

ystemic circulation for excretion. The three suggested HP pairs,DAPip(HP)2/Orn-HP [78], IDA(HP)2/GluA-HP and IDA(HP)2/GluB-P [52] (see Fig. 9), include N-extra-functionalization in theis-(3,4-HP) (first pair) or in the mono-(3,4-HP) (last two pairs).

For the pair IDAPip(HP)2/Orn-HP, the complexation studies withhe ternary system revealed similar metal affinity (pFe = 26.5 [78])ut improvements on metal complex solubility when comparedith the tetradentate system alone. Moreover, in vivo studies with

7Ga injected mice showed an eventual coadjuvating/synergisticffect when a large excess of the mono- over the bis-chelator100:1 and 1000:1 molar ratios) is used, probably due to competi-ion between each chelator for the metal ion, with need of a largexcess of the less potent ligand. Concerning the IDA(HP)2/GluA-P and IDA(HP)2/GluB-HP combination pairs, bioassays confirmed

he ability of both combined chelator pairs to improve the in vivoetal complexation and induce its rapid elimination, although

he most pronounced coadjuvation effects were observed forDA(HP)2/GluA-HP, eventually rationalized by the higher efficacyf GluA-HP (alkyl linker between the glycosyl and the 3,4-HP,Al = 15.5) to coordinate in vivo relatively to GluB-HP (phenyl

inker, pAl = 13.8). Though it would be interesting to associate thehelating efficacy of the pair IDA(HP)2/GluA-HP to a potential Alemoval from brain, the radioactive metal 67Ga (administered asitrate) in the bioassays was not retained in the brain of our animal

odel. Nevertheless, based on the preliminary in vivo studies, this

igand combination “cocktail” (IDA(HP)2/GluA-HP) may deserveurther studies to be potentially used as trivalent metal ion decor-orating agent in metal overload or misplaced situations.

cid (AS), Feralex-G (FG) and two bis-(3,4-HP) IDA-based chelators; (b) examples of

5. Extra-functionalization of 3,4-HP for further therapeuticroles

5.1. New developments on iron-chelators asanti-neurodegeneratives

Although the brain needs functional metal ions (e.g. iron, cop-per and zinc) deregulation in their homeostatic mechanisms hasbeen implicated in neurodegenerative diseases [89]. Despite themultiple genetic or non-genetic factors that may deregulate theiron transport and storage pathways in brain, it is believed thatbrain iron dyshomeostasis induced by genetic factors is a primaryrisk factor in oxidative stress and neuronal death leading to seriousneurodegenerative diseases, namely Alzheimer’s disease (AD) andParkinson’s disease (PD) [90].

These two main neurodegenerative disorders have quite differ-ent neuropathological alterations, but they are both characterizedby deposits of abnormal protein aggregations (e.g. �-amyloid pep-tide (A�) in AD and �-synuclein in PD) and they are accompaniedby high levels of brain oxidative stress [89]. The patient brains alsorevealed dyshomeostasis of metal ions, in particular accumulationof iron within deposits of those protein aggregates [91]. Many otherneurodegenerative diseases, such as Huntington’s disease, Friedre-ich’s ataxia (FA), amyotrophic lateral sclerosis and prion disease,are associated with changes in iron metabolism and accumula-tion [89]. Moreover, high concentrations of aluminum have alsobeen found in the senile amyloid plaques and intra-neuronal neu-rofibrillary tangles (NFT) within the brains of AD patients [92].Since this is a redox inactive metal ion, it is believed that itcan act in synergy with iron to increase the free radical damage[93].

AD is, by far, the most devastating age-related neurodegen-erative disease, in which a multitude of processes culminates inserious neuronal and synaptic dysfunctions leading to severe andirreversible cognitive losses. Up to now, there is no cure for AD and

250 M.A. Santos et al. / Coordination Chemis

Fg

ti

brpftstppbhbc

bnmgetwt([

bTch(pom

aiwtda

5d

wce

ig. 10. Representative examples of metal chlelators for the treatment of neurode-enerative diseases: CQ, VK28, M30 and d-penicillamine.

he FDA-approved drugs are only able to ameliorate symptoms bynhibition of acetylcholine esterase (AChE).

Although the etiology of AD is not clearly understood, it haseen recognized that a cascade of events may lead to the aboveeferred main hallmarks of AD. Thus, besides the diversity of thera-eutic strategies to act up-stream the main AD onset, a considerableocus has been placed on the development of other therapeu-ic approaches, including chelation therapy, for the targeting andequestration of excess of redox-active metal ions, or antioxidant-herapy for scavenging reactive oxygen species (ROS) [94]. Inarticular, the iron accumulation in the aggregated proteins (senilelaques in AD, and substantia nigra in PD) evidences an associationetween these aggregates and oxidative stress. Accordingly, thereave been recent developments in therapeutic strategies for ADased on the metal homeostatic therapy [95–97], in particular ironhelation [98,99].

Due to the multifactorial nature of AD, therapeutic trends haveeen based on poly-pharmacology, either through drug combi-ation protocols (e.g. AChE inhibition and antioxidants [100]) or,ore recently, with the new approach “one molecule, multitar-

ets paradigma”, which seems to have advantages in terms ofquitable pharmacokinetics and biodistribution [101–103]. In par-icular, metal chelators have been extra-functionalized with groupshich, beyond a disease site directing role, can combine addi-

ional neuro-rescuing or neuro-protecting roles, enzyme inhibitione.g. AChE, MAO-B), antioxidant activity or anti-A� aggregation104–106].

DFO was the first chelator used to remove intracellular iron fromrain, but it revealed high limitations due to its lack of lipophilicity.hus, several metal chelators have been proposed as AD therapeuti-al agents, either as single or bifunctional chelators [122–124]. Theyave been mainly based on 8-hydroxyquilonine (e.g. ClioquinolCQ), VK28 and M30, see Fig. 10), on amino-acid derivatives (e.g. d-enicillamine), to inhibit Cu- and Fe-mediated oxidative stress, orn HP-derivatives (e.g. DFP, Feralex-G, see Figs. 2 and 9), to removeisplaced Fe(III) and Al (III) from nuclear matrix [107–110].For this purpose 3,4-HP-based chelators have attracted a special

ttention because, as stated above, they can be easily functional-zed to modulate important biological properties, namely related

ith bioavailability or extra-targeting roles. In the following sec-ions (grouped by extra target effect) we will review the recentevelopments regarding the bifunctionalized HPs with prospectivepplications to combat neurodegenerative diseases.

.1.1. Modulating the BBB permeability of 3,4-HP chelators:rugs and prodrugs

The recognized abnormal excess of iron in the brains of patientsith neurodegenerative diseases (e.g. AD and PD) makes metal

helation therapy a new potential strategy to combat these dis-ases. Such chelating agents should scavenge the excess of free

try Reviews 256 (2012) 240– 259

iron (without disturbing other essential metal ions such as Ca2+,Mg2+, K+) and excrete the corresponding metal complex; they alsoshould promote the passivation of iron partially bound to pep-tides or proteins, in order to prevent toxic events due to redoxaction (Fenton activity) or further aggregation processes. For thatpurpose, the 3,4-HPs appeared as lead compounds because, undermost physiological conditions, they have very high affinity for bothFe3+ and Fe2+, albeit higher for Fe3+ (log ˇ3 = 27–37) [111,112], arelatively high affinity for Cu2+ (log ˇ2 = 20–22) [112–114], mod-erate affinity for Zn2+ (log ˇ2 = 11–18) [112] and very little affinityfor common biological electrolyte ions such as sodium, potassium,calcium and magnesium [115]. On the other hand, these biden-tate compounds possess low molecular weight (< 500 Da) and formneutral FeL3 complexes, two important features to guarantee mem-brane crossing and metal-complex efflux from the brain. DFP hasdemonstrated neuroprotective roles against A�-induced neurotox-icity in neuronal cell cultures [116]. This chelator has also provedbeneficial on other neurodegenerative diseases with mitochondrialiron accumulation, such as FA [117].

A number of HP derivatives have recently been designed tomodulate the physicochemical properties aimed at improving theblood-brain barrier (BBB) crossing ability and brain availability, forpotential application in the treatment of AD. In particular, a setof 3,4-HP derivatives have been prepared with different positionalsubstitutions, namely at ring 1-position [44,118] and at the 2-position (e.g. the 2-amido-3-hydroxypyridinones [119]), to enablethe interplay of iron affinity and lipophilicity, two important fea-tures to accomplish the desirable neuroprotective effects.

Due to the importance of carbohydrates as energy sources,glucose-bearing drugs have been exploited in relation with trans-port and metabolic pathways. Feralex-G (FG, Fig. 9) is an exampleof a bifunctionalized 3,4-HP which includes d-glucopyranose as N-substituent [120]. This sugar-bearing chelator proved to be able tocross the BBB, to sequester Fe and Al from AD-related neurofib-rillary tangles and reverse these metal-induced neuropathologicalprocesses. It showed a further capacity to promote the disag-gregation of paired filaments in brain cells, thus with potentialapplication for chelation therapy in AD [121]. It was also sug-gested that it could be used in drug combination to treat otherdiseases associated with iron or aluminum toxicity [88]. In fact,accumulation of toxic amounts of Al was found in the brains ofAD patients, but mostly in patients with dialysis encephalopa-thy syndrome. Other glucose-appended 3,4-HPs (Figs. 3 and 11)have been designed to act as potential chelators, to be used eitheralone or in combination therapy [52], or even as metal complexesfor potential diagnostic imaging probes [122,123], in analogy with[18F]-2-deoxy-2-fluoro-d-glucose (FGD) used in PET imaging [124](vide infra).

To improve the brain access of 3,4-HP-based chelators, a prodrugapproach was recently reported, involving tris-functionalizationof HPs which, in addition to the derivatization at ring 1-positionto modulate the lipophilicity, has O-glucosylation to enhance theBBB permeability [125,126]. That prodrug is expected to easilycross the BBB and then release the active ligand (by �-glucosidase-induced cleavage of the sugar unit) to act as chelating agent. A seriesof derivatives with N-substituted lipophilic groups showed quitepromising results for AD therapy, in reasonable agreement withother independent studies [127].

5.1.2. Strategies for antioxidant/iron chelator dual-actionSince oxidative stress has a recognized main role in the

pathogenesis of neurodegenerative diseases, treatments targetingsources of ROS have attracted particular attention [128]. In fact,both anti-oxidants and iron chelating agents have shown neuro-protective efficacy in animal models of AD and PD [129]

M.A. Santos et al. / Coordination Chemistry Reviews 256 (2012) 240– 259 251

f 3,4-

aoiadTattNi(

A

Fig. 11. Some representative examples o

In particular, the HPs, by chelating the labile iron, can act asntioxidants and thus reduce oxidative stress [130]. The effectsf the N-aryl-alkyl functionalization of 3,4-HPs on their antiox-dant capacity have been recently reported in comparison withntioxidant standards, such as �-tocopherol (�-Toc) and butylhy-roxytoluene (BHT) [126] (Fig. 12). The results, expressed as therolox Equivalent Antioxidant Capacity (TEAC), indicated the highbility of these HP derivatives to scavenge free radicals (even higherhan those of known antioxidants such as �-Toc and BHT); whereashe N-alkyl showed lower values than the N-aryl derivatives, the

-phenol derivative presented the highest capacity. These results

ndicate that, besides the antioxidant effect due to iron chelationprevention of free iron redox activity), the N-aryl groups must have

Fig. 12. TEAC values at 6 min for known antioxidants (�-tocopherol (�-Todapted from [126].

HP-Glucoside conjugates [122,125,127].

a further effect, eventually due to the aromatic conjugation andformation of quinonoid species, which certainly contributes to theelectronic stabilization of the radical scavenging products.

Based on the amenability of 3,4-HPs for synthetic manipulation,they were further used in a “pharmacophore conjugation” strat-egy to get hybrid compounds which combine the iron-chelatingand radical scavenging properties. This approach was pioneeredby incorporating in the same molecule an antioxidant food addi-tive, BHT, and a DFP chelator linked with a simple alkyl- oralkyl-thio-chains (Fig. 13) [131]. The novel multifunctional 3,4-HPs

indicated a marked enhancement in neuroprotection, via inhibi-tion of oxidative stress, over the combination of DFP and BHT,with synergistic actions, as indicated by IC50 (inhibition of lipid

c) and 2,6-di-tert-butylhydroxytoluene (BHT)) and various 3,4-HPs.

252 M.A. Santos et al. / Coordination Chemistry Reviews 256 (2012) 240– 259

OH

BHT

OH

OHO2C

Trolo x

OH

LY23161 7

HN

OHR1

L R1NHO

O

a: L = - ; R1 = tBub: L = CH2 ; R1 = tBuc: L = CH2CH2 ; R1 = tBud: L = - ; R1 = OCH3e: L = CH2 ; R1 = OCH3f: L = S ; R1 = tBug: L = NH CH2 ; R1 = tBu1

ZN

HO

O

OH

O

n n = 0 ; 1Z = CH2 ; (CH2)4NHR2 = H ; CH3R2

P-ant

puSitspAbf

5

ddaonttTta[cmsACpu

atP(Fptw

chelating efficacy of this set of compounds, it can be anticipatedto be similar to that of DFP and other bifunctional HP derivatives[46]. The reduced number of these new aminopropargyl 3,4-HP

2

Fig. 13. Structure of some 3,4-H

eroxidation) ca. two order of magnitude lower than additive val-es corresponding to both separated molecular units (see Table1, in Supplementary Data). This strategy was further explored tomprove the water solubility of these hybrid compounds, namely inerms of variations on the linkers with poly-ethyleneglycol (PEG)egments, and on the anti-oxidant phenolic unit, besides somerodrug approaches (esterification of the hydroxyl groups) [132].lthough these strategies lead to promising hydroxypyridinone-ased hybrid drugs/prodrugs against neurodegenerative diseases,urther assays on disease models still need to be performed.

.1.3. Further strategies for dual-action with iron chelationBecause of the complex etiology of the neurodegenerative

iseases, besides the above-mentioned bifunctionalized drug can-idates, many other new approaches have been recently reportedimed at effectively treating AD, through a simultaneous hittingf the multiple targets involved [106]. Among the potential anti-eurodegeneratives based on metal-chelators extra-functionalizedo gain additional properties, one representative example involveshe combination of a DPTA unit with the main scaffold of thioflavin-

(ThT), a known amyloid-binder. This compound proved to reducehe Zn2+-induced A� aggregation in solution and to attenuate themyloid pathology in mice (XH1, Fig. S2, Supplementary data)2,133,134]. Identical approach has also been recently reported, byombining the main features of ThT and CQ (M1, Fig. S2, Supple-entary data), which showed potential as AD therapeutics [135]. In

pite of the indicated interest on conjugating metal chelation with�-intercalating properties, these compounds are more specific foru2+ and Zn2+ and, to our best knowledge, there are only studies inrogress with analogous functionalized 3,4-HPs (M.A. Santos et al.,npublished results).

Other representative multi-targeting drugs recently reportedre based on the aminopropargyl derivatization of chelators forhe treatment of AD and other neurodegenerative diseases such asD [136]. Specifically the orally active bifunctional Fe-chelator, 5-N-methyl-N-propargylamine-methyl)-8-hydroxyquinoline (M30,

ig. 10) and several other analogues have been studied also under arochelator approach [104]. That extra-functionalization provideshe compounds with extra neuroprotective properties, namelyith inhibitory activity towards monoamine oxidase B (MAO-B),

ioxidant conjugates [131,132].

an important therapeutic target both for AD and PD. This outermitochondrial membrane-bound isozyme catalyzes the oxidativedeamination of important amine neurotransmitters (e.g. dopamine,serotonine), with concomitant elevation of brain levels of neuro-toxic free radicals and oxidative stress, its activity increasing withage and being particularly localized around the senile plaques ofAD patients [137].

There is a recent report on analogous aminopropargylation ofHPs to account for potential extra activity with MAO-B inhibition.In particular, 3,4-HPs were extra-functionalized with at least onepropargylamine group (compounds 3a, 3b and 3c, Fig. 14) [138].These two pharmacophore units were linked by spacers of slightlydifferent size and type, to account for some differentiation in thelipophilicity and membrane crossing abilities, as well as in thepotential interaction with biological targets (e.g. piperazine seg-ment, also found in VK28). This set of novel compounds indicatedan improved in vitro neuro-protective efficacy, as compared withDFP, in both models of PD and AD induced neuronal cell deathas well as the suppression of caspases-mediated apoptotic celldeath. Although no solution studies were performed to assess iron-

Fig. 14. Hybrid propargylated 3,4-HP chelators [138].

hemis

dsomtac

fadtap(ntndb

5

ecapuFr

otrtnltlDbmagcpacis(pbt

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emaaomMd

M.A. Santos et al. / Coordination C

erivatives hampered conclusions on structure–activity relation-hips, although the lipophilicity seems to exert an apparent benefitn their bioavailability and BBB crossing ability. 3a appeared as theost efficient compound, acting on several disease targets, but fur-

her studies need to be performed, before these compounds can bedmitted as potential neuro-protective and neuro-restorative drugandidates for PD or AD.

Besides the herein described strategies for extra-unctionalization of 3,4-HP chelators, aimed at potentialnti-neurodegeneratives, further steps can be expected in futurerug design strategies, namely to increase the drug accessibilityhrough the BBB, to improve the chelator/anti-oxidant dual actionnd also to obtain additional extra targeting roles with thera-eutical relevance, such as: (1) inhibition of nitric oxide synthaseNOS) to decrease oxidative damage and inflammatory-relatedeurodegenerative processes; (2) inhibition of AChE, to decreasehe symptoms of AD memory impairment due to loss of theeurotransmitter acetylcholine; (3) drug vectorization to theisease active site (A� plaques) in order to improve drug efficacy,y promoting anti-aggregation and/or solubilization processes.

.1.4. Nanoparticle hydroxypyridinone chelatorNano-scale drug delivery systems, using nanoparticle carri-

rs, are emerging technologies which have appealed medicinalhemists to engineer new drug capacities, namely to increase theirccessibility to cellular and intracellular targets, including trans-ort across the BBB. Although nanoparticles have found widespreadse in drug delivery, up to now only a small number has beenDA-approved for various uses from cancer to infections and neu-ological diseases [139].

After the discovery that certain polymeric nanoparticles basedn biocompatible materials could enable the drug transport acrosshe BBB [140], some challenging therapeutic options have beenecently suggested based on nanoparticle-aided chelator delivery,o modulate the toxic effects of metal ion (such as iron) excess ineurodegeneration. Such approaches could overcome some prob-

ems associated with the administration of free chelators, such ashose related to the inadequacy of lipo-hydrophilic character of theigands/complexes and their difficulty for membrane crossing (e.g.FO, DFP). In particular, it was recently reported that covalently-onded 3,4-HP nanoparticles could cross the BBB and attenuateembrane-diffusion limitations, thus potentially providing safer

nd more effective chelation therapeutics for AD or other neurode-enerative diseases [141,142]. This work involved nanoparticlesontaining carboxylic groups on their surface, which were cou-led with N-functionalized bidentate 3,4-HPs via formation ofmide bonds. To avoid toxicity associated with iron incompleteoordination by the bidentate 3,4-HPs, it can be anticipated thatdentical approach could be used with hexadentate tris-(3,4-HP),ince high molecular weight limitations on membrane-crossingLipinsky rules) are not relevant in nanoparticle-mediated trans-ort. These results seem quite interesting, although they are onlyased on in vitro studies, and so further studies are needed to assessheir actual chelating efficacy, toxicity and BBB permeability.

.2. Hydroxypyridinones as matrix metalloproteinase inhibitors

Matrix metalloproteinases (MMPs) are a class of zinc-dependentndopeptidases present in the extracellular matrix, which degradeost of its components. There are a number of pathologies

ssociated with the overexpression of MMPs, namely degener-tive inflammatory diseases, such as rheumatoid arthritis and

steoarthritis, neuroinflammatory dysfunctions, cancer develop-ent and tumor invasion [143,144]. Therefore the development ofMP inhibitors (MPIs) has been an important target in new drug

evelopments. The catalytic site of the MMPs includes a zinc ion

try Reviews 256 (2012) 240– 259 253

bound to three histidine residues and one water molecule, whichare fundamental for the hydrolytic cleavage of the scissile peptidebond. For this reason, the great majority of MPIs have in their scaf-fold a chelating moiety (zinc-binding group, ZBG), in order to bindthe catalytic Zn and thus inactivate the MMP proteolytic action. Tra-ditionally, the most explored ZBG was the hydroxamic acid (HA,CONHOH), because it binds the zinc in a very favorable geome-try, allowing the formation of two extra H-bonds with the protein(Fig. S3, Supplementary data) [143]. Nevertheless, because of thefrustrating results from the clinical trials with hydroxamate-basedMPIs [145,146], in the last decade we have assisted to an increasingsearch for new ZBGs with more favorable pharmacokinetic prop-erties than the HAs [146–148]. Several chelating moieties bindthe catalytic zinc ion within the MMPs, and therefore might besuitable as ZBG to be used in the scaffolds of new MPIs. Hydroxypy-rones and hydroxypyridinones (HPs) are among the set of chelatorsthat proved capacity for binding the zinc within the MMP catalyticsite [149–153]. In fact, these simple chelating moieties revealedhigher inhibitory activities than acetohydroxamic acid (compare4–6 with acetohydroxamic acid, AHA, Fig. 15) [149], which repre-sents a very promising result. Special relevance has been given tothe 3,4-hydroxypyrones and 3,4-HPs, as well as to their sulfur ana-logues, since they are stronger Zn2+ chelators, while being muchless toxic than HAs, and they can easily be extra-functionalized.Moreover, most of these HP-based ZBGs proved to be non-toxic toneonatal rat fibroblast cardiac cells at 100 �M [149,154].

By attaching extra-functional groups to the ZBG rings, itbecomes possible to form favorable interactions with specific cavi-ties of the catalytic site, in order to increase the inhibitory activity.Several authors have shown that through structure optimizationit is possible to enhance the inhibitory activities of the HP-basedcompounds, in some cases even reaching activities close to thoseof known potent HA-based MPIs (compare AM2 and CGS27023A,Fig. 15). Modeling studies showed that the inhibitor AM2 seemsto have the biphenylmethylene (PhPhCH2) group well insertedinto the hydrophobic S1′ cavity of MMP-3, while the 6-methylmay interact with the hydrophobic S1 subsite (Fig. S3, Supplemen-tary data). There are also reports on 1,2-HP-based inhibitors, withsub-nanomolar inhibitory activities, even more potent than mostof HA-based MPIs (e.g. compound 7) [157].

Overall, hydroxypyrones, hydroxypyridinone and their ana-logues, when properly functionalized, have demonstrated to besuitable for MMP inhibition, and thus biological and therapeuticalapplications can be envisaged.

5.3. Hydroxypyridinones as anti-microbial, anti-malarial andantiviral agents

It is well known that iron is vital for all the microorganisms,which need this element for their cellular functions, includingenergy production, DNA replication and protection from the ROS[158]. They acquire iron from the environment by secreting strongand selective iron-sequestering agents, generally tetra- or hexa-dentate low-molecular weight compounds, called siderophores[159]. A therapeutic strategy to combat microbial infectionsinvolves the treatment with strong chelating agents, in order toeffectively compete with the natural siderophores released bythose pathogens, inducing iron-limitation/starvation. There aremany reports of the antimicrobial action of chelating agents againsta number of infectious diseases, including malaria [160], trypanoso-miasis, hepatitis C, tuberculosis and mycobacteria infections [74].HPs are among these chelating agents, and have already proved

to inhibit the growth of several bacteria and fungus, such asEscherichia coli, Listeria inocua, Staphylococcus aureus, Pseudomonasaeruginosa [158], Candida albicans and Mycobacterium avium [74].The early reports of 3,4-HP functionalization, aimed at developing

254 M.A. Santos et al. / Coordination Chemistry Reviews 256 (2012) 240– 259

F [154,i

ao4dibhw�csrrspfsmpsitaicoi

m

ig. 15. Examples of HA-based MPIs (acetohydroxamic acid (AHA) and CGS27023Anhibitory activities (IC50 values) towards representative MMPs [156,157].

ntimicrobials with potential use in the treatment of several casesf infections, involved N-derivatization of 3-hydroxy-2-methyl--pyridinones with alkylamines and alkylamides. Some of theseerivatives achieved 90–100% growth inhibition of E. coli and L.

nocua, at 20 mM of ligand [161]. Later on, several 3,4-HPs haveeen derivatized at position 2 or 6 of the heterocyclic ring withydrophobic aromatic groups, such as phenyl, pyridyl or naphthyl,hich resulted in activities in the order of few hundreds to units ofg/mL of minimum inhibitory concentration (MIC, the lowest con-

entration to completely inhibit microorganisms growth) towardseveral bacteria and fungus [162]. Quantitative structure–activityelationship (QSAR) studies with 3,4-HP derivatives have beeneported, in which, by making use of different chemometric tools,everal descriptors (e.g. geometrical, functional and topologicalarameters) play important roles in the activity of these extra-unctionalized 3,4-HPs against S. aureus and C. albicans [158]. Morepecific functionalization seemed to be necessary in cases of certainycobacteria, such as M. avium. This mycobacterium resides and

roliferates inside the macrophages of their hosts, and it is not veryusceptible to traditional iron-chelation therapy [74]. Noteworthys the fact that the above referred hexadentate 3,4-HP chela-ors, containing lipophilic fluorescent moieties (Section 4.4), havelso displayed anti-microbial activity, namely against M. avium,n low micromolar range. Therefore, these extra-functional groupsombine the anti-microbial activity with that of cell-permeant flu-

rescent probes allowing the spectroscopic investigation of thentracellular distribution of the compounds [74,163].

Malaria is also a major global health problem, which is esti-ated to affect more than 2 billion people. It is caused by the

155]), and several hydroxypyrone- and hydroxypyridinone-based MPIs, with their

protozoan parasite Plasmodium, which is transmitted to humansby the bite of an infected female Anopheles mosquito. The mostsevere variation of the disease is caused by P. falciparum. The globalincreasing resistance of this parasite to insecticides and traditionalantimalarial drugs made the demand for alternative therapeuticagents an urgent issue. Similarly to other pathogens, also P. fal-ciparum requires iron for its development and proliferation, andseems to extract it from the hemoglobin present in the host ery-throcytes [160]. Treatment with strong iron chelators, such as DFO,proved to inhibit the growth of the parasite, apparently involvingintracellular chelation of the iron pools within the parasitized cells,and many authors have later on investigated chelating agents forantimalarial activity [160,164]. Orally active HP derivatives havealso been evaluated with this purpose, namely DFP and other HPswith different lipo-hydrophilic properties. The results showed thatsome HPs are able to suppress malaria in vitro and in vivo (ID50values in the range15–45 �M) and a correlation has been foundbetween activity and the lipophilic character, probably due to ahigher capacity for crossing the cell membranes and enter into theparasitized cells [165]. Despite the lack of conclusive clinical results[166], a number of 3,4-HPs have been functionalized at the posi-tions 1, 2 and 6, in order to modulate their lipo-hydrophilic andacid-base properties. Some of these compounds showed strongerinhibitory activities against P. falciparum (IC50 values down to8 �M), probably because of their slightly basic properties, which

drove them to be kept in the iron pools after entering the cells, dueto the existing pH gradient [167].

HPs and other iron chelators have also proved to inhibit thereplication of several viruses, such as human immunodeficiency

hemis

td3HidcfspwSdlt1

Hce

6

6

geirmtftmMoweG[sdeceiaa[thcc

acltbepitl

M.A. Santos et al. / Coordination C

ype 1 (HIV-1) and herpes simplex (HSV-1 and -2) viruses, throughifferent possible mechanisms [168,169]. DFP and mimosine, two,4-HP-based compounds, showed antiretroviral activity againstIV-1 (IC50 in the range 50–100 �M) [170]. On the other hand,

ron is an essential microelement for the virus replication and itsepletion through chelating agents necessarily inhibits this pro-ess. Thus, these compounds appeared as potential good candidatesor use in antiretroviral combination therapy [168,171]. 2-amide-ubstituted 3,4-HP derivatives have been reported to inhibit HIVroliferation, with IC50 values down to 20 �M, and a correlationas found between the pFe values and the antiviral activities [172].

ince nucleoside analogues are one of the major classes of antiviralrugs in use, some 3,4-HP derivatives containing nucleoside ana-

ogues have been developed, revealing moderate activities againsthe herpes viruses HSV-1 and -2 (EC50 down to 32 �M against HSV-) [173].

After all, besides the vast diversity of potential applications ofP chelators, we should be aware of some eventual toxicity thatan be associated with the inhibition of non-haem iron-dependentnzymes, such as lipoxygenase and hydroxylase families [19].

. Metallodrugs based on hydroxypyridinone ligands

.1. Metal complexes as diagnostic tools

Although several imaging reporters have already been investi-ated as diagnostic tools, along the past 20 years much researchffort has been devoted to Gd-chelates for magnetic resonancemaging (MRI) and also 68Ga chelates for positron emission tomog-aphy (PET) [174]. The most common commercially availableetal-based MRI and PET imaging probes are based on polyden-

ate ligands, namely poly(amino-carboxylate) chelators, derivedrom DTPA, NOTA or DOTA, some of them bearing molecularargeting moieties. Nevertheless, they are not specific for those

etal ions and their Gd-complexes have some limitations asRI contrast agents, because their multidenticity usually leaves

nly one coordination site for one inner-sphere water moleculehich results on low water exchange rates and low contrast

nhancements [175]. To overcome this problem, a new class ofd-chelates, tris-HPs (1,2-HP, 3,2-HP) has been recently developed

176–181]. The hexadenticity of these compounds guarantees atrong chelating efficacy, leaving a higher number (2–3) of coor-ination sites available for the water ligands, with concomitantnhancements on relaxivity imaging sensitivity. Moreover, theiromplexes present high thermodynamic stability to avoid toxicffects, due to demetallation or even transmetallation with phys-ologically available cations (Ca and Zn) [181]. Fig. 16a includes

schematic structure of one of the first Gd3+-tris-HP complexess a new generation of contrast agents for MRI applications182]. The three bidentate chelating moieties of the hexaden-ate tris-3,2-HP-based ligand (TREN(1-Me-3,2-HOPO)3) provideexacoordination while two extra water molecules can stilloordinate to the metal ion, thus guaranteeing the contrast effi-acy.

To optimize the Gd-complex properties, namely in terms of lig-nd and complex aqueous solubility, as well as complex overallharge, stability and relaxivity, modifications have been made oninkers [180] or even by replacement of HP moieties by tereph-halamide groups [177] and some of these complexes proved toe successful from in vivo assays. Although, to our best knowl-dge, there are no reports of analogous 3,4-HP-based Gd complexes,

reliminary studies in our group with a tris-3,4-HP (NTP(PrHP)3)

n aqueous medium revealed interesting results (pGd = 14.1 andwo inner-sphere water molecules, M A. Santos et al., unpub-ished results), even though the Gd chelating capacity of this

try Reviews 256 (2012) 240– 259 255

ligand is lower than that of approved contrast agents for MRI,such as Gd(DTPA-BMA) (pGd = 15.8 [181]), GdDTPA (pGd = 18.8) orGdDOTA (pGd = 19.3) [183].

Concerning the nuclear imaging probes based on galliumradionuclide, namely 68Ga complexes for PET, there has been recentresearch on new ligands and protocols [184]. Based on the analo-gies between Fe3+ and Ga3+, including the high affinity for 3,4-HPligands, there are some reports about the potential use of radioiso-tope gallium complexes with 3,4-HP ligands [46], namely withcarbohydrate bearing derivatives for diagnostic imaging probes[122,123] (see Fig. 11), in analogy with [18F]-2-deoxy-2-fluoro-d-glucose (FGD), used in PET imaging of glucose metabolism inseveral neurological pathologies [124]. However, these galliumcomplexes have 1:3 (M:L) stoichiometry, with eventual demetalla-tion problems. Therefore, a further strategy was recently followed,based on using a tris-3,4-HP ligand, NTP(PrHP)3, which forms anextremely stable 1:1 Fe3+ complex (pFe = 29.4) [40]. Studies indi-cated that NTP(PrHP)3 forms a hexa-coordinated 1:1 complex witha pseudo-octahedral geometry and its high stability (pGa = 27.5) is aguarantee of reduced risk of toxicity due to Ga hydrolytic demetal-lation or transmetallation by competitive blood serum compounds[71]. On the other hand, biodistribution and scintigraphic assaysof the corresponding 67Ga-labeled chelate in an animal model,demonstrated its high in vivo stability and good biodistributionand excretion profiles. Thus, the 68Ga-NTP(PrHP)3 appeared as apotential lead probe for nuclear imaging (PET), although it may stillbe optimized with extra-functionalization of the ligand scaffold toprovide specific targeting ability.

Noteworthy are the recently emerged reports on the use ofluminescent lanthanide (e.g. Eu3+, Tb3+) complexes to exploreand manipulate cell biology [185]. In particular, a number ofnon-macrocyclic luminescent lanthanide complexes, based onoctadentate HP chelating systems (e.g. 1,2-HP, 3,2-HP), under sin-gle or molecular conjugation approaches, showed highly efficientemission with exceptional stabilities (pEu = 20.7–21.8) in neutralbiological conditions [186]. These lanthanide complex systems arenot usually assumed as metallodrugs but, as luminescent molecularprobes, they are very relevant tools for biomedical research.

6.2. Anti-diabetic drugs

Diabetes mellitus (DM) is a very complex metabolic disease,with the number of patients increasing worldwide. In the absenceof insulin production (DM1) or insulin resistance (DM2), highblood glucose level (hyperglycemia) occurs and its control isreached by daily injection of insulin (DM1) or by administra-tion of drugs with insulin enhancing properties (DM2). Afterthe discovery of the pharmacological potential role of vanadiumcompounds in DM2 treatment, there was an increasing inter-est for even more effective drugs, which lead to the search fornew small organic ligands for orally active vanadyl [VO]2+ com-plexes. Both bis(maltolato)oxovanadium(IV) and correspondingethylmaltol analogue, bis(ethylmaltolato)oxovanadium(IV), weredeveloped and undergone on pre-clinical testing for safety andefficacy [187].

In addition to the 3-hydroxy-4-pyrones, a series of HP ana-logues, such as DFP, and the corresponding (O,S)-donor analogues(hydroxythiopyridinones) were also explored as vanadyl com-plexes and proved insulin mimetic properties [188].

Due to the non-essentiality/toxicity of vanadium, other bis-charged metal ions (e.g. Zn, MoO2) have also been studied as thecorresponding hydroxy(thio)-pyridinonato and –pyronato com-

plexes (see examples in Fig. 16b). Concerning the MoO2 complexes,though only a moderate activity as insulin mimetics was foundfor the thio-maltolato complex [189], this ligand indicated a highactivity as xanthine oxidase inhibitor [190], which may be signif-

256 M.A. Santos et al. / Coordination Chemistry Reviews 256 (2012) 240– 259

F P) asa

izwpSt

dafb[

6h

3cemcp

oHrdtnFb(qfbshcfa

ig. 16. Representative examples of HP-containing metallodrugs: (a) Gd-tris(3,2-Hctivity; (c) dinuclear M(II)-arene complexes with antitumor activity [193].

cant for the development of multimodal therapies. Regarding theinc chelates, besides the high activity presented by the complexesith O4 coordination modes (e.g. 3,4-HP and pyrones), the mostromising in vivo properties were found for the complexes with2O2 coordination provided by thio-analogue ligands, in particularhe thiopyronate ligands [189,191].

The results have been mostly rationalized in terms of coor-ination environments, namely according to the Pearson’s hardnd soft acids and bases. Nevertheless, some effects of extra-unctionalization induced changes on the hydrophilic/lipophilicalance, which also exerted some structure–activity relationship191,192].

.3. Anti-cancer drugs based on metal complexes withydroxypyridinones

The side effects of cisplatin anticancer drug (launched over0 years ago) have lead to the search of novel metallodrugs forhemotherapy with improved effectiveness. The antitumor prop-rties of complexes with a number of non-essential non-platinumetal ions have been evaluated, but only very few followed in

linical studies. Some promising HP-containing Ru2+ and Ga3+ com-lexes have recently been reported [193,194].

Most of the recently reported Ga-complexes have been devel-ped for radiopharmaceutical imaging purposes, namely involvingP-based ligands (vide supra [71]). The application of non-

adioactive gallium compounds in the treatment of importantisorders (e.g. cancer and infectious diseases) makes attractivehe design of novel gallium-based drugs, to substitute the pio-eering use of naked Ga3+ ions (chloride or nitrate salts) [195].or this purpose, many in vivo studies of gallium complexes withidentate O-donor ligands have been reported, including maltolGa-trismaltol went in clinical trials) [196], thiomaltol [197] anduite a number of 3,4-HPs. In fact, as already reported, 3,4-HPsorm stable, water-soluble complexes and their lipophilicity andiological properties can easily be modulated by appropriate ring-ubstitutions [46]. To avoid problems of transferrin transchelation,

exadentate frameworks have been also explored, but the con-omitant increase in its in vivo inertness let to address them mostlyor coordination with the radioactive Ga isotopes in view of imagingpplications [71].

contrast MRI imaging agent; (b) 3,4-HP metallocomplexes with insulin-mimetic

Concerning the Ru-complexes, their interest as anticancer met-allodrugs has been widely recognized [198], namely after the reportof the “half-sandwich” organometallic Ru2+-arene complexes [(�6-arene)Ru(X)(Y)(Z)], with a multitude of possible derivatizations onthe arene or on the monodentate (X, Y, Z) or the bi-dentate (YZ)ligands to attach Ru(II) [199]. Thus, it was possible to improve theirphysicochemical or biological properties, namely their reactivitywith biological targets (e.g. proteins and DNA), while keeping ade-quate inertness for use as anticancer drug. Due to the similaritywith Ru2+-arene complexes, the Os2+-analogues have also beenrecently reported as potential anticancer agents. The chelating lig-ands, by providing additional stability to the complex, can play acrucial role in anticancer activity. The use of pyrone derivatives asbidentate (YZ) ligands in [M(cymene)Cl(pyronate)] (M = Ru2+, Os2+)showed only mild anticancer activity, although it was improvedby introduction of substituents at the pyrone scaffold [200,201]and specially by replacement of the carbonyl oxygen by a sul-fur atom [202], which leads to increased complex lipophilicityand stability. Since HPs can easily be manipulated and form morestable metal complexes than pyrones (e.g. for Ru2+-(cym) com-plexes, log ˇmaltol = 9.05 and log ˇDFP = 11.86 [203]), several 3,4-HPshave been used as bidentate ligands for ruthenium and osmiumcomplexes. These studies revealed that aminoacids can com-pete with the HPs for the metal ion, which may be a rationalefor the low in vitro anticancer activity [204]. Thus similar com-plexes of Ru(arene) with 3,4-HP were recently studied, under amono- and polynuclear approach [205]. The mononuclear com-plexes presented higher activity than the pyrone derivatives (IC50 inlow micromolar range). However, the binuclear Ru(cymene) com-plexes, containing a bis(3,4-HP) with an alkane linker (see Fig. 16c),revealed the highest in vitro anticancer activity, namely for the com-pound with dodecane spacer (IC50 = 0.28 �M), which is one order ofmagnitude more potent than cisplatin (IC50 = 4.5 �M). The activityof these dinuclear compounds seems to be quite dependent on thespacer length, which may also be correlated with their lipophilicity.

7. Summary and outlook

This review is focused on the most recent developments involv-ing hydroxypyridinone (HP) chelating agents and, in particular, thediverse extra-functionalization approaches that can provide thesemetal-related compounds with targeting ability towards different

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athological sites or mechanisms. The manifold applications of HPsre based on their specific metal-binding ability, which enableshem either to chelate/passivate specific deregulated metal ionsr to deliver metal ions for therapeutic and diagnostic purposes.he HPs, and specially the 3-hydroxy-4-pyridinones (3,4-HP), wererst reported for chelatotherapy in metal overload diseases andheir extra-functionalization was mostly addressed to improvehe metal sequestering efficacy and excretion profiles. Concerningheir use in metal modulation, namely in some neuronal metal-nbalanced pathologies (e.g. AD and PD), incorporation of specifichemical structures on HP-based compounds allowed the manage-ent of drug activity through several determinant mechanisms

metal chelation, antioxidant protection, anti-A� aggregation andnzyme inhibition), which may have important results in futuretrategies for effectively treating the complex AD. Another impor-ant approach related with the HP-chelating function is the ironequestration in microorganisms. Extra-functionalization of 3,4-Ps has enabled the access to special cellular compartments of

everal microorganisms (bacteria or protozoan), with their sub-equent iron starvation and growth inhibition. HP derivativesave also been reported as promising scaffolds to be included

n the design of potent metalloproteinase inhibitors, namely theatrix metalloproteinases (MMP). Also in this case, the extra-

unctionalization was crucial for the emergence of a new generationf MMP inhibitors with potential therapeutical applications iniverse pathologies.

Although the main employment of HPs has been the targetingf biometals in specific organs or proteins, their use as ligandsn metallodrugs has also been reported, namely as: complexesf Ga radioisotopes and Gd with hexadentate HP-based ligands,s nuclear or MRI diagnostic probes; complexes with Zn, VO andoO2, as insulin-mimetics; organometallic Ru- and Os-arene com-

lexes, as anticancer agents.We hope that the final outcome of clinical trials with some extra-

unctionalized HPs and metallodrugs presented herein will providenderstanding of their mechanism of action towards several metal-elated diseases and ultimately delineate the best therapeutictrategies.

cknowledgements

The authors thank our present and past collaborators thatave important contributions to part of the work reported herein.e also thank PEst-OE/QUI/UI0100/2011 and the Portuguese

oundation for Science and Technology (FCT) for financial sup-ort.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.ccr.2011.08.008.

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