3616 2010, 3616-3631 Potential Therapeutic Applications of ... · Potential Therapeutic...

3616 Current Medicinal Chemistry, 2010, 17, 3616-3631

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Potential Therapeutic Applications of Metal Compounds Directed

Towards Hypoxic Tissues

D. Gambino*

Cátedra de Química Inorgánica, Departamento Estrella Campos, Facultad de Química, Universidad de La República, CC 1157, 11800 Montevideo, Uruguay

Abstract: Medicinal Inorganic Chemistry offers the high diversity of metal coordination chemistry for the development of bioactive compounds for therapeutic or diagnostic medicinal purposes. The design of novel metal-based antitumor agents occupies a privileged position in this discovery process. On the other hand, the research on metal-based radiophar-maceuticals for therapy and imaging is a subarea of high priority and development. This review describes therapeutic ap-plications of metal compounds directed towards hypoxic tissues. Strategies in the search for new bioreductive metal-based prodrugs will be discussed. In addition, approaches for the imaging of hypoxic tissues by using metal radionuclides will be exemplified.

Keywords: Hypoxia selectivity, metal-based compounds, bioreduction, bioreductive metal-based prodrugs, nitrogen mustards, aromatic N-oxides, quinoxalines and thiosemicarbazones metal complexes.

INTRODUCTION

Oxygen has a fundamental importance in all tissues due to its principal role in cellular processes, particularly in all those pathways involved in the cellular growth cycle. There-fore, adequate oxygen supply and tension are regulated in vivo by different mechanisms. In normal tissue partial oxy-gen pressure shows a typical Gaussian distribution with val-ues between 20 and 80 Torr, a median value around 50 Torr and values none less than 10 Torr [1-4]. Hypoxia, reduced oxygenation of tissue, is a sign of disease. For instance, it occurs in stroke, heart disfunction, some types of cancer and bowel diseases and arthritis. The detection of regions of hy-poxic tissue has a significant diagnostic value for several diseases or disorders, for example after interruption of blood flow in brain or myocardial infarction. The localization of hypoxic tissue and the determination of its extent could have significant incidence in the proper clinical treatment of these disorders. On the other hand, hypoxia plays a significant role in low response to radiotherapy and low sensitivity to che-motherapy of certain types of tumors but offers an opportu-nity for the selective treatment of cancer by taking advantage of the differential oxygenation of normal and hypoxic tumor tissues.

In this review the use of the “activation by bioreduction” strategy in the search for novel metal-based hypoxia selec-tive cytotoxins for cancer treatment and hypoxic tissue imag-ing agents is discussed. The development of this kind of metal compounds has been previously only partially re-viewed in manuscripts involving broader themes [5-12].

METAL COMPOUNDS AND INORGANIC MEDICI-NAL CHEMISTRY

Inorganic Medicinal Chemistry offers the high diversity of metal coordination chemistry for the development of novel bioactive compounds for therapeutic or diagnostic

*Address correspondence to this author at the Cátedra de Química Inor-

gánica, Facultad de Química, Universidad de la República, Montevideo,

Uruguay; Tel: +5982-9249739; Fax: +5982-9241906;

E-mail: [email protected]

medicinal purposes. Coordination compounds show a wide range of coordination numbers and geometries, in vivo accessible metal oxidation states and reduction potentials and interesting thermodynamic and kinetic characteristics that can be fine-tuned. In addition, they offer the potential syner-gism or additive effect of intrinsic therapeutic properties of the metal ion and the ligands included in the coordination sphere [13,14]. Coordination to metals could significantly change biological properties of the organic ligands, such as solubility, lipophilicity, stability and charge among others, which results in modifications in biodistribution, in vivo transformation and pharmacokinetics. Accordingly, coordi-nation to metal ions could substantially modify bioavailabil-ity and bioactivity of organic compounds leading to desirable changes in their biological behavior [14].

Research in different design subareas is being extensively performed, like chelation therapy, metal ions supplementa-tion, development of diagnostic and therapeutic radiophar-maceuticals, magnetic resonance imaging (MRI) and design of therapeutic agents, among others [15-22]. Developments achieved in some of these subareas have been reviewed by other authors in this Hot Topic issue.

Different metal compounds have been used as therapeutic agents since ancient times. Nevertheless, organic drugs have dominated pharmacology and medicinal chemistry. After the fortuitous discovery of the antitumoral activity of cisplatin, Medicinal Inorganic Chemistry became a rapidly growing field of research with numerous applications in many branches of medicine. The most significant part of this re-search has been directed to the development of drugs active against different kinds of cancer. Although cisplatin is con-sidered one of the top pharmaceuticals that changed the world and is still today, 40 years after its serendipitous dis-covery, one of the most widely prescribed for many cancer diagnoses [23], other platinum(II) compounds are also cur-rently employed in clinical practice, like carboplatin and oxaliplatin. Extensive research searching for improved therapeutic indexes and wider activity spectra has lead to coordination compounds and organometallics of different metal ions (Pt, Ru, Sn, Ga, V, Ti, Au, among others) that show antitumor activity, being DNA their most common target [13,24,25]. In particular, some mononuclear and

Potential Therapeutic Applications of Metal Compounds Directed Current Medicinal Chemistry, 2010 Vol. 17, No. 31 3617

polinuclear platinum(IV) compounds are currently under clinical trials [14,15,25]. Among others, two ruthenium compounds, NAMI-A and KP1019, and two gallium com-pounds, tris(8-quinolinato)gallium(III) (KP46) and gallium maltolate, are also being evaluated in clinical trials [20,26-28].

Current research for the development of metal-based anticancer drugs focuses on new potential targets and thera-peutic strategies based on the knowledge on cellular proc-esses that are altered in tumor cells.

CANCER AND HYPOXIA

The biological knowledge and the development of drugs for the efficient chemotherapy of the different types of can-cer belong to the most important current challenges of medi-cine and medicinal chemistry. The anticancer chemotherapy began in the 1940s with the first uses of nitrogen mustards, as chemical weapons during the Second World War. The discovery of compounds pharmacologically active against cancer has depended largely on serendipity and on the screening of natural products and their analogs. Neverthe-less, medicinal chemistry is currently involved in the rational design of organic chemotherapeutics. Most of the antitu-moral drugs that are currently used in clinical practice are cytotoxins that kill rapidly dividing tumor cells, most com-monly by producing DNA damage. Due to their mechanism of action they are usually not selective for cancer cells and they simultaneously produce damage on proliferating normal cells. Indeed, they show narrow therapeutic indexes. Another unsolved problem is the high incidence of resistance phe-nomena.

In the late 1980s, scientists began searching for selective anticancer agents that lacked the side effects associated with conventional chemotherapeutic drugs and could target ‘can-cer-specific’ molecules to kill cancer cells without affecting normal cells.

The rapid growth of different types of cancers leads to solid tumors where cancerous cells are relatively isolated

from the blood supply, turning increasingly difficult the dif-fusion of oxygen and resulting, frequently, in hypoxia (Fig. 1). There is evidence that viable hypoxic cells may contrib-ute up to 20 % of the tumor mass [29]. Common anticancer drugs show low therapeutic efficacy for the treatment of solid tumors, since the majority of these tumor cells are not rapidly dividing ones. Furthermore, currently used cytotoxic drugs typically have to diffuse through many layers of nor-mally-oxygenated cells to access hypoxic tumor cells. Hy-poxic cells are indeed not reached by conventional cytotoxic drugs in adequate concentrations due to slow growing and low irrigation. As a result, the concentration of the drugs in the hypoxic regions is often inadequate to kill the tumor cells. Hypoxic tumor cells are also more resistant to ionising radiation therapies than well-oxygenated ones since oxygen is needed to form the free radicals which kill tumor cells and helps to stabilize radiation damage in DNA [30]. In addition to contribute to treatment resistance, hypoxia could also con-tribute to adverse malignant effects by promoting metastasis and angiogenesis [31].

Therefore, different strategies must be applied for the de-velopment of cytotoxic drugs for the selective treatment of these slow-growing hypoxic solid tumors. The physiology of solid tumors at the microenvironmental level is sufficiently different from that of the normal tissues to provide a selec-tive target for cancer treatment [2,3,32]. The difference in oxygenation between solid tumor cells and normal healthy tissue could be exploited to get a selective and, there-fore,almost non toxic treatment. In addition, it has been pro-posed that tumor hypoxia can occur in a second way by tem-porary obstruction of tumor blood flow. This acute hypoxia arises from fluctuating blood flow and could be also present in common human tumors.

Among other strategies, the development of non active prodrugs that are almost non toxic for normal cells but capa-ble of being bioreduced by cellular reductases under physio-logical conditions forming one or more active cytotoxins has lead to hypoxia-selective cytotoxins. These prodrugs would be selectively bioactivated in hypoxic tissue to release the actual cytotoxic drug or drugs, capable of killing these cells

Fig. (1). Main features of hypoxic tissues.

3618 Current Medicinal Chemistry, 2010 Vol. 17, No. 31 D. Gambino

and the surrounding oxygenated ones. Therefore, these hy-poxic cytotoxins would show a different therapeutic profile than radiation and conventional chemotherapy and are pro-posed for performing combined therapeutic schemes together with radiation and conventional antitumoral drugs [3].

Obviously, the bioactivation depends on the redox prop-erties of the compounds, among other physicochemical properties. Useful organic prodrugs for this purpose include different families of compounds, mainly nitroaromatics, qui-nones and N-oxides of aromatic and aliphatic amines. The chemistry and biological properties of these organic com-pounds have been extensively reviewed [5,7,8,33,34]. Never-theless, inorganic chemistry contributions to this approach have been less developed.

BIOREDUCTION AND SELECTIVITY

The mechanism of activation of bioreductive prodrugs (Fig. 2) is the reduction by reductases (typically flavoen-zymes) within tumor-tissue hypoxic cells, leading to active cytotoxic species. Although the enzymes that catalyze the reductive activation occur in all cells, selectivity is achieved by back-oxidation of the transient reduction product in nor-mal cells by molecular oxygen. The reduction product or diffusible cytotoxins formed after further metabolism may also be responsible of killing the surrounding more oxygen-ated cells within the tumor [35,36].

METAL COMPOUNDS AND HYPOXIA

Several attempts towards selectively targeting transition metal-organic complexes to hypoxic cells for imaging or therapy purposes have been described. These attempts can be mainly grouped around the following four main strategies:

I. Metal coordination of common organic cytotoxins

II. Metal coordination of hypoxia-selective cytotoxins

III. Bioreductive metal-radionuclide radiopharmaceuti-cals

IV. Other metals compounds having suitable reduction potential to be bioreduced in biological media

I. Metal Coordination of Common Organic Cytotoxins

Ruthenium, platinum, cobalt and other metals form inert complexes in aqueous media in their high oxidation states, i.e. Ru(III), Pt(IV), Co(III), that can be reduced in biological media to more labile complexes of their low oxidation states metal ions (Ru(II), Pt(II), Co(II)). Having this in mind, some years ago Denny et al hypothesized that the development of cobalt(III) coordination compounds with organic cytotoxins bearing nitrogen donor centers as ligands could mask their high cytotoxicity leading to a selective cytotoxic action of these organic compounds in hypoxic tumor cells [35]. For instance, Co(III), a d

6 ion, forms low-spin hexacoordinated

octahedral complexes that are highly kinetically inert to ligand substitution under oxia and that have appropriate Co(III)/Co(II) reduction potentials to be reduced in vivo by cellular reductants. Therefore, their reduction within hypoxic cells could lead to the labile high-spin Co(II) octahedral complexes that quickly substitute ligands by water mole-cules. As a result, the cytotoxic organic ligand would be se-lectively released inside hypoxic cells together with the sta-ble Co(II) hexaaquo species, [Co(H2O)6]

2+ (Fig. 3). In nor-

mally oxygenated cells back-oxidation of the Co(II) com-plexes should compete efficiently with hydrolysis to avoid release of the cytotoxic ligand thus assuring selectivity through a futile cycle. To assure hypoxia selectivity the Co(II)-cytotoxic ligand coordination species must be stable enough to allow its re-oxidation in normal cells by free oxy-gen before being hydrolyzed. The metal accomplishes two biological effects: on one hand it is a carrier of the active drug and on the other hand it temporarily deactivates the anticancer drug to avoid general cytotoxicity. Having this proposal in mind, Denny´s group developed some series of

Fig. (2). Scheme of the mechanism of activation of a bioreductive prodrug showing the basis of selectivity: a) bioactivation in hypoxic tis-

sue. b) bioreduction and back-oxidation by molecular oxygen in normal oxygenated tissue.


Co(III) coordination compounds synthesizing them by con-ventional coordination chemistry synthetic methods. The selected cytotoxic ligands were well-known non selective DNA alkylating agents, like aziridine [37] and bidentate ali-phatic nitrogen mustards [38] and, more recently, pyr-rolo[3,2-f]quinoline analogues [39].

The action of nitrogen mustard alkylating agents is non selective. They show very low IC50 values together with un-desirable cytotoxicity in healthy cells by covalent interaction with biomolecules, especially DNA. The selected DNA alky-lating ligands would lose their cytotoxicities due to the coor-dination of the metal ion on the nitrogen atom which blocks the electron pair needed for the nucleophilic attack involved in the alkylation process. Under this hypothesis metal coor-dination should lead to almost non aerobic cytotoxicity on normally oxygenated cells. Therefore, all the complexes de-veloped were tested for aerobic cytotoxicity (compared to the parent nitrogen mustard cytotoxin in the same condi-tions) and for air vs. nitrogen differential cytotoxicity to de-termine hypoxia selectivity.

In the early attempts with aziridine ligand (Fig. 4a), chromium was also tested as central metal for the develop-ment of hypoxia-selective cytotoxins since it shares some favorable features possessed by cobalt. Nevertheless, no fur-ther results of this metal complex were reported [37]. Several mixed-ligand Co(III) complexes including aziridine (Az) in the coordination sphere were synthesized and characterized by different techniques: cis-[CoCl(Az)(en)2]Cl2, cis-[Co(NH3) (Az)(en)2]Br3, cis-[CoCl(Az)(trien)]Cl2, trans-[Co(NO2)2(Az)4] Br·2H20·LiBr, cis-[Co(NH3)4(Az)2]Cl3, [Co(NH3)5(Az)]CI3 and cis-[Co(Az)2(en)2]Br3. Since the compounds were de-signed as prodrugs to be activated in hypoxic tumor cells undergoing reduction of the cobalt center by biological re-ductants (-200 - -400 mV vs. NHE), cyclic voltammetry in aqueous solution of the Co(III) aziridine complexes was studied. As expected, a one-electron irreversible electro-chemical reduction process was detected, ascribed to the reduction to Co(II) complexes and subsequent fast ligand substitution on the Co(II) centers. Free aziridine release of the compounds by common biological reductants was de-tected by colorimetric methods. Preliminary in vitro tests under hypoxia showed similar cytotoxicities for the com-plexes and free aziridine, indicating biological reduction and release of the cytotoxic ligand.

Authors suggested that the lack of cytotoxic selectivity for hypoxic cells shown by Co(III) complexes of monoden-tate aziridine could be due to the low stability of the Co(II) species, that avoided its back-oxidation by free oxygen in

normal cells. Therefore, further studies were developed but using bidentate nitrogen mustards ligands (Fig. 4b) instead of monodentate ones (aziridine) to improve the stability of the intermediate Co(II) complexes [38,40]. Two families of octahedral mixed-ligands cationic Co(III) complexes, [Co(Racac)2L]

+, including one bidentate alkylating nitrogen

mustard ligand L (dce or bce) (Fig. 4b) and two acetylaceto-nate derivatives (Racac, 3-alkylpentane-2,4-dionato anion, where R = H, Me, Et, n-Pr and Cl) (Fig. 5) were synthesized, characterized and evaluated as hypoxia selective cytotoxins on CHO-derived cell line AA8 and the sub line UV4 (DNA-repair deficient Chinese hamster ovary fibroblast mutant). In both series, the patterns of cytotoxicities of the cobalt com-plexes were broadly similar to those of the respective free ligands, suggesting that the cytotoxicity of these compounds is due to release of the free cytotoxic ligands. The dce com-plexes resulted more potent than their bce analogues. The Co(III)/Co(II) reduction potentials fall in the range usually estimated as suitable for reduction by biological reductants. The values clearly vary with the nature of the nitrogen ligand. In addition, the nature of R on the acac residue modi-fied the Co(III)/Co(II) reduction potential; the more electron-donating the group R was, the more stabilized resulted Co(III) with respect to Co(II) species. A correlation between biological activity and reduction potentials was observed, suggesting that a narrow potential window may exist to as-sure cytotoxicity. Selected compounds showed hypoxia-selective cytotoxicity on mammalian cells and results sug-gested that the complexes acted under hypoxia by the rapid release of the highly reactive cytotoxic ligand that could dif-fuse to neighbor tissues with higher oxygen tensions. The selected bidentate mustard ligands seemed to provide enough stability to the intermediate Co(II) complexes allowing their back-oxidation under oxia through efficient competition with hydrolysis. Among these complexes, the lead compound SNN24771, [Co(Meacac)2dce]

+, exhibited a 20-fold selectiv-

ity for hypoxic UV4 cells in culture and high activity against hypoxic cells inside multicellular spheroids developed as solid tumor models [41]. Its mechanism of inactivation under oxia was studied in detail by pulse and steady state radiolysis methods which showed a more complicated process than expected demonstrating that initial redox cycling is not in-volved in the hypoxic selectivity [42]. Nevertheless, as pre-viously discussed bidentate nitrogen mustards showed greater hypoxic selectivity than monodentate ones, indicating the importance of the stability of the Co(II) intermediate. To further examine this aspect similar compounds but including tridentate nitrogen mustards were designed and evaluated [43].

Fig. (3). Scheme showing the bioreduction of an octahedral Co(III) complex, the hydrolysis of the resultant Co(II) complex and the subse-

quent release of the cytotoxic drug in the hypoxic cell.


HN

a) aziridine

ClNH

HN

Cl

ClN

NH2

Cl

b) aliphatic mustards

N N

NN

ClCl

ClCl

N N

NN

ClCl

ClCl

c) polyazamacrocyclic nitrogen mustards

NN

N

Cl Cl

Cl

dce

bce

ClN

HN

Cl

dcd

NH2

Fig. (4). Nitrogen mustard ligands.

O- O

R

Racac

NS

S-

dedtc

O

OH

trop

Fig. (5). Some selected coligands.

The complex [Co(acac)(NO2)(dcd)]+ with dcd acting as

tridentate ligand showed a 5-fold selectivity for hypoxic conditions on UV4 cells, which is lower than that of [Co(Meacac)2(dce)]

+ (20-fold). This behavior could be re-

lated to the higher potential shown by [Co(acac)(NO2) (dcd)]

+, which could reduce the ability of oxygen to compete

for reducing equivalents.

The same group designed other mixed-ligand Co(III)-nitrogen mustards compounds by including bis(dialkyl)dith-iocarbamates, tropolonate, carbonate or oxalate as co-ligands instead of acetylacetonates (Fig. 5) [44-46]. Neither the bis(dialkyl)dithiocarbamato nor the tropolonate complexes, like [Co(dedtc)2(dce)]

+ and [Co(trop)2(dce)]

+, showed hy-

poxia selectivity. The complexes [Co(CO3)2(dce)]- and

[Co(ox)2(dce)]- showed attenuated aerobic cytotoxicity in

respect to dce and differential toxicity in air vs. nitrogen at-mosphere but less significant than the lead compound [Co(Meacac)2(dce)]

+ (Table 1).

Similar attempts to manipulate nitrogen mustards selec-tivity via coordination through the nitrogen atoms were per-formed with Cu(II) and polyamine mustards [47]. Selected nitrogen mustards were chloro derivatives of 1,4,7-triazacyclononane (tacn), 1,4,7,10-tetraazacyclododecane (cyclen) and 1,4,8,11-tetraazacyclotetradecane (cyclam) (Fig. 4c). Previous attempts to develop bioreductive prodrugs by coordination of Cu(II) to linear nitrogen mustards en-countered some problems, like insolubility of the compounds and relatively high cytotoxicity to aerobic cells probably due to the low stability of the reduced Cu(I) intermediate [9]. As previously explained, proper selection of the metal centre has to be done on the basis of its redox properties and the lability of the resulting in vivo reduced species. The complexes may have one-electron reduction potentials within the cellular potential range. So, the reduced species formed could release the ligand, i.e. the bioactive drug. Having this in mind, three water soluble Cu(II) cationic complexes of the tetradentate polyazamacrocyclic nitrogen mustards derivatives were de-veloped and assessed in vitro as hypoxia-selective cytotoxins

Table 1. Selected Biological Data for Nitrogen Mustards Cobalt(III) Mixed Ligand Complexes (UV4 Cells)

Compound IC50/ M CT10 air / M h-1

CT10 air/N2 ratio

dce 0.055 [43] 0.18 [45] -

dcd 2.67 [43]

[Co(Meacac)2(dce)](ClO4) 0.31 [45] 2.34 [43] 20.3 [43]

[Co(acac)(NO2)(dcd)](ClO4) 35.4 [43] 262 [43] 5.2 [43]

K[Co(CO3)2(dce)] 0.36 [45] 90 [45] 4.5 [45]

K[Co(ox)2(dce)] 0.04 [45] 32 [45] 2.7 [45]

IC50: determined against aerobic cells in growth inhibition assay. CT10: drug concentration ( M) x time (h) required to reduce cell survival to 10% of controls under specified condi-tions (air or N2) by clonogenic assay using UV4 cells.


on lung-derived human tumor cell line A549 by comparing their cytotoxicities under hypoxic and oxic (oxygenated) conditions. Their IC50 values under air were similar to those of the free ligands. The complexes with the tacn and cyclen derivatives showed no selectivity for hypoxia. But the square pyramidal complex of the cyclam tetra chloro derivative, [CuCl(tetrachlorocyclam)]Cl, showed promising hypoxia-selective cytotoxicity (IC50 (air) 53.4 M; IC50(N2) 2.2 M; HCR (hypoxic cytototoxicity ratio IC50(air)/IC50(N2) 24). It was 24 times more cytotoxic under hypoxia then under oxia on the selected cell line and its aerobic cytotoxicity was al-most 10 times lower than that reported for the most promis-ing Co(III) complex of linear nitrogen mustards previously reported by Denny´s group [38]. Although all these com-pounds showed low stability avoiding its use in vivo, the potentiality of the metal complexes as bioreductive prodrugs was demonstrated.

This approach was further extended by including other cytotoxic ligands, phenazine-1-carboxamides DNA intercala-tors, in the coordination sphere of mixed-ligand Co(III) complexes [48]. The resulting complexes [Co(Racac)2(L)]

+,

with L = N-[2-[(aminoethyl)amino]ethyl]-phenazine-1-car-boxamide, N-[5-[(aminoethyl)amino]pentyl]-phenaz-ine-1-carboxamide or bis[2-(phenazine-1-carboxamido)ethyl]-1,2-diaminoethane, showed only low hypoxic selectivities.

More recently, Denny´s group extended the previous re-search by proposing radiation activated prodrugs, i.e. the use of therapeutic ionizing radiation for reductive activation of metal prodrugs in hypoxic regions of tumors [39,49,50]. This approach is based on the absorption of radiation by water generating the strongly reducing aquated electron (e

-aq)

which is long-lived under hypoxia. If prodrugs could be acti-vated exclusively by radiation and not by reductases, the selectivity to hypoxic tissues would be improved avoiding toxicity in non target tissues and, in addition, hypoxic ne-crotic regions lacking reductases would be also targeted.

In an initial work 8-hydroxyquinoline (8-HQ) was used as model cytotoxic ligand for the DNA alkylator azachloro-methylbenzindoline (azaCBI) [49]. [Co(8-HQ)(cyclen)]

2+

released 8-HQ when reduced by ionizing radiation under hypoxia. Further work has been performed with pyrrolo[3,2-f]quinoline analogues. Three mixed-ligand Co(III) com-plexes, [CoL(cyclen)]

2+, and a Cr(III) complex, [Cr(acac)2

L1], of pyrrolo[3,2-f]quinoline analogues (L) (Fig. 6) were synthesized, characterized and evaluated. The chromium complex did not show hypoxia selectivity probably due to its reduction potential that resulted non suitable for being re-duced by cellular reductants. The cobalt complexes were stable and showed the desired attenuation of cytotoxicity in the prodrug form on two different cell lines, with IC50 values 50 to 150 fold higher than those of the free pyrrolo[3,2-f]quinoline cytotoxins. In addition, they showed significant hypoxic cell cytotoxicity (Hypoxic cytotoxicity ratio IC50aerobic/IC50hypoxic 7.5 to 41- fold). Their aerobic cytotox-icities followed the order of their corresponding cytotoxic ligands, suggesting that they acted through slow release of these ligands. [CoL1(cyclen)]

2+ and [CoL3(cyclen)]

2+ could

act as prodrugs releasing the cytotoxic ligand both by bioreduction and also by irradiation with ionizing radiation. They showed almost quantitative radiolytic release of the cytotoxic ligands.

N

OH

N

OH

pyrrolo[3,2-f]quinoline analogues

N

Cl

O

N

N

OCH3

OCH3

OCH3

O(CH2)2N(CH3)2

ON(CH3)2

L1

L2

L3

[CoL(cyclen)]2+

2+

8-HQ

NH

NH

NH

NH

Co

O

N

Fig. (6). Cytotoxic ligands and Co(III) complexes.

Recently a similar approach has been investigated by Hambley et al through Co(III) and Fe(III) coordination of the matrix metalloproteinase inhibitor marimastat [14,51-53]. Matrix metalloproteinases (MMPs) are ubiquitous zinc-dependent enzymes involved in the degradation of the ex-tracellular

matrix, particularly in connective-tissue break-

down. The MMPs have been implicated in the processes of tumor

growth, invasion, and metastasis and are frequently

over expressed in malignant tumors. Certain types of MMPs

are expressed on the surface of solid tumors. Therefore, they have been recognized as attractive targets for the develop-ment of antitumor drugs bearing antimetastatic activity and recent research focused on the development of suitable in-hibitors of these enzymes for therapeutic purposes. A de-tailed review on MMPs is included in this issue.

Most developed MMPs inhibitors include hydroxamate functional groups capable of chelating the zinc ion present in the active site of these enzymes leading to the inhibition of their proteinase function [54,55]. Among them, marimastat is a synthetic broad-spectrum inhibitor that reduced number and size of animal metastatic foci (Fig. 7). Although it reached phase III clinical trials, it showed no advantages in respect to current drugs to afford further development. Reac-tions suffered by the highly reactive hydroxamate functional-ity may be responsible of its low efficacy in vivo. In particu-lar, deactivation of the hydroxamate group by metals ions may have contributed to the lack of success of this com-pound. Therefore, metal complexes were developed with the aim of providing stability to marimastat in vivo until the ex-tracellular target (MMP) is reached. In this way metal coor-


dination would provide a mechanism for transporting intact marimastat to the target site where it could be released from the metal complex by selective activation by bioreduction in the reductive environment of hypoxic tumors. A model study involving Co(III) complexes with the tripodal tetradentate ligand tris(2-methylpyridyl)amine (tpa) and simple bidentate hydroxamate co-ligands was investigated to provide insight into the potentiality of this Co(III)-tpa system for the devel-opment of Co-MMP inhibitor prodrugs [56]. The complexes showed to be irreversible reduced, releasing the tpa ligand at reduction potentials in the potential window of cellular re-ductases. Having in mind this model results, Co(III)-marimastat mixed-ligand complex (Fig. 7), [Co(mmst)tpa] ClO4·4H2O, with mmstH = marimastat, has been synthesized and preliminary biologically evaluated [51,53]. Results showed that coordination to Co was a suitable strategy for deactivating marimastat by protection of the hydroxamate moiety until reaching the tumor site. Electrochemical results showed that it could be activated by bioreduction releasing free marimastat. The Co-marimastat prodrug showed in-creased tumor growth inhibitory effect in vivo on 4T1.2 mur-ine mammary tumor in respect to free marimastat [51]. Nev-ertheless, both marimastat and the Co complex potentiated tumor metastasis relative to a control. Furthermore, the octa-hedral Fe(III) marimastat complex [Fe(mmst)(salen)]·2H2O with the tetradentate co-ligand salen occupying the four re-maining coordination positions (salen = N,N-bis(salicylidene)-ethane-1,2-diimine), was developed as a stable redox inert carrier system for marimastat that would lead to the labile Fe(II) reduction product and deliver the intact drug at hypoxic tumor sites [52]. It showed an IC50 value for the in vitro inhibition of matrix metalloproteinase MMP-9 one order of magnitude higher than that of free ma-rimastat, demonstrating the effectiveness of the complex as chaperoning system for marimastat. It showed lower stability than the Co(III)-marimastat complex and consequently higher cytotoxicity. Further biological studies are needed to assess the potentiality of this kind of systems.

II. Metal Coordination of Hypoxia Selective Cytotoxins

Among the main different structural classes of organic compounds that are bioactivated under hypoxic conditions and can be considered as hypoxia-selective bioreductive pro-drugs, several N-oxides have been described [5,7,8]. Aro-matic N-oxide moiety is suitable to be bioreduced by cellular reductases within cellular systems through a single-electron reduction process generating a cytotoxic radical that initiates radical-mediated oxidative DNA strand cleavage and leads to

hypoxic cell death. In presence of molecular oxygen back-oxidation and a redox cycling process (superoxide forma-tion) occur. The prototype of this kind of compounds is Tira-pazamine (3-amino-1,2,4-benzotriazine-1,4-dioxide) which is currently in Phase III clinical trials (Fig. 8a). Having this compound as a structural antecedent, the capacity of a large series of quinoxaline N

1,N

4-dioxide derivatives to act as

bioreductive drugs has been described by Monge et al. Many of these derivatives resulted potent hypoxia selective bioreductive prodrugs [8,33]. For instance, good in vitro biological results were obtained with 3-amino-2-carbonitrile derivatives (Fig. 8b). Structure–activity relationship studies showed that the cyano moiety in the 2 position seemed to be necessary for the cytotoxic activity, electron-withdrawing substituents, such as Cl or F, in the 6(7) position improved the potency under hypoxic conditions, and electron-donating substituents, such as CH3 and OCH3, in the 6 and/or 7 posi-tions decreased the potency. The nature of the amine in the 3 position had a very strong influence on the potency and hy-poxic cytotoxicity ratio HCR [8,57-59]. In order to improve the delivery properties, 3-alkylamino derivatives were also developed. Although several of them showed excellent activ-ity and selectivity in vitro and low toxicity in vitro and in vivo, they were not useful for therapy owing to too short in vivo half lives and/or low solubility in physiological media [60-62].

Trying to improve the bioavailability and pharmacologi-cal properties of the quinoxaline N

1,N

4-dioxide derivatives

and to assess the effect of metal coordination on cytotoxicity and selectivity towards hypoxic tissue, novel copper(II), va-nadium(IV) and palladium(II) complexes with 3-aminoquinoxaline-2-carbonitrile N

1,N

4-dioxide derivatives

have been synthesized, characterized and in vitro biologi-cally evaluated by Gambino, Torre, González et al (Fig. 8b) [6,62-68]. Metal ion could play a role on chaperoning or delivery of the active ligand in the desired site of action. As discussed in the previous section, copper is especially attrac-tive for the development of hypoxic selective metal com-plexes. This metal has two common oxidation states and the reduction potential of the Cu(II) complexes in biological conditions (E0´(Cu(II)/Cu(I)) is accessible within the cellular potential range [9]. In addition, both copper cations prefer different donor atoms according to the soft and hard Pearson classification. Cu(II) coordination compounds with bioreduc-tive prodrugs as ligands may be reduced by cellular reduc-tases leading to Cu(I) complexes of low stability, that could release the bioreductive prodrugs of their coordination sphere inside the cell. As an antecedent, the inclusion in the same molecule of a radioactive copper isotope and Tira-

NH

HO

HN

NH

O

O

O

OH

Marimastat (mmstH)

both oxygen

donor atomsM

M = Co(III), Fe(III)

O

OmmstH

Fig. (7). Marimastat structure and schematic representation of the heteroleptic metal complexes including marimastat as bidentate ligand

coordinated through both oxygen donor atoms of the hydroxamate functional group. The tetradentate chelating ligand (tpa or salen) occupy-

ing the four remaining coordination positions of the metal ion was not included for simplicity.


pazamine lead to a potential agent for the treatment of he-patic tumors using combined therapeutic strategies (bioreduction and radiotherapy) [69]. On the other hand, tumor growth inhibition and prophylaxis against carcino-genesis due to selected vanadium compounds are well known. Chemo preventive and anti-tumor effects due to se-lected vanadium compounds and their mechanism of action have been widely investigated on malignant cell lines as well as on experimental animal models [70-72]. V(IV) species usually have accessible reduction potentials in physiological conditions [73]. Therefore, a vanadium-prodrug complex could enter the cell, and, directly act affecting important cell processes, and/or be reduced and, thereafter, undergo com-plete or partial ligand exchange with a variety of biogenic ligands available in the cell, releasing the organic bioreduc-tive prodrug. Once inside the cell the free bioreductive pro-drug would lead to cytotoxic species. Under this hypothesis, coordination to vanadium could lead to a synergistic or addi-tive effect or, at least, improve the bioavailability of the qui-noxaline derivative. Furthermore, palladium was selected as metal center although it just shows a single oxidation state since it has been extensively studied for the development of novel metal-based therapeutic agents [74]. Neutral [M(L-H)2] complexes, where L-H are deprotonated 3-amino-quinoxaline-2-carbonitrile N

1,N

4-dioxide derivatives and M

= Cu(II), V(IV)O2+ or Pd(II), were synthesized and charac-

terized by conventional inorganic chemistry techniques [63-65,68]. The seven selected ligands were prepared with excel-lent yields from the corresponding benzofuroxan and maloni-trile and were generated as a mixture of 6- and 7-substituted isomers that were not possible to separate neither by chroma-tography nor by crystallization (Fig. 8b). They showed dif-ferent substituents in the 6(7) position, resulting in interest-ing tools for the development of the desired complexes due to their different electronic and lipophilic properties that could lead to different biological responses. The complexes were subjected to cytotoxic evaluation on V79 cells (Chinese hamster lung fibroblasts obtained from ECACC, European Collection of Animal Cell Cultures) under hypoxic and aero-

bic conditions using a well established cloning assay. Hy- poxic and oxic cytotoxicity was preliminarily evaluated using 2 h exposure time and initial 20 M drug concentration. The survival fraction values in both conditions (SFair and SFhypox) were determined. Those complexes showing selectivity at this dose were tested at different doses to obtain dose-response curves in air and hypoxia. Potencies (P) and hypoxic cytotoxicity relationship (HCR) values were determined, being P the dose which gives 1% of cell survival with respect to the control (7-chloro-3-[3-(N,N-dimethylamino) propyl amino]-2-quinoxaline carbonitrile N

1,N

4-dioxide hy-

drochloride) in hypoxia, and HCR the relationship between concentration of drug in air and concentration of drug in hypoxia that produce the same level of cell killing (1%) (Table 2).

Survival fractions at single dose (20 μM, 2 h incubation) in normoxia and hypoxia showed that the three vanadyl complexes [65], four of the copper complexes [63,64] and only one of the palladium complexes tested [68] were highly toxic in hypoxia being poorly cytotoxic under well oxygenated conditions. Three of the complexes showed non selective cytotoxicity, being cytotoxic either in hypoxic or in oxic conditions. Two of the complexes showed cytotoxicity neither in oxia nor in hypoxia. So, the biological behavior of these structurally related metal compounds depended not only on the substituent on the quinoxaline moiety but also on the metal ion nature. Four [Cu

II(L-H)2] complexes resulted

excellent bioreductive species in vitro, being their cytotoxicities in hypoxia similar to or higher than those of the free ligands. Nevertheless, structural and electronic modifications due to coordination with copper did not improve the poor solubility of the ligands in physiological conditions. Al- though lipophilicity and superoxide dismutase activity of the copper complexes were studied, no clear correlation could be found that could explain the different behavior of the copper complexes. On the other hand, being DNA a possible target of palladium compounds a study on interaction with plasmid DNA under normoxia was performed [68]. The complexes interacted with DNA in aerobic conditions producing dose

N

N

O

O

CN

NH

M

N

N

O

HN

NC

O

R1

R2

R2

R1N

N

O

O

R1

R2

CN

NH2

M = Cu, Pd, VO2+

[M(L-H)2]

N

N

O

O

NH2

a)

b)

R1 R2

L1 Cl H

L2 Br H

L3 CH3 H

L4 H H

L5 F Cl

L6 Cl OCH3

L7 Cl Cl

Fig. (8). a) Tirapazamine b) Structural formulae of 3-amino-2-carbonitrile quinoxaline N1,N

4-dioxides and their metal complexes.


dependent effects via different mechanisms, but results did not allow explaining the different biological behavior under normoxia. Although coordination to vanadium did not improve selectivity of the methyl derivative (L3), the complexes of the bromo and chloro derivatives (L1 and L2) showed improved potencies in respect to the free ligands, higher potencies than Tirapazamine and excellent selective cytotoxicity in hypoxic conditions. In particular, [VO(L1- H)2] showed to be 3-fold more potent than the free quinoxaline dioxide ligand L1, 10-fold more potent than tirapazamine with a hypoxia selectivity of more than 15 [65]. In addition, the hypoxia cytotoxicity selectivity relationship values, HCR, for the new vanadium compounds were of the same order than those of other hypoxia selective cytotoxins (i.e. Mitomycine C, Misonidazole) [33]. Vanadium complexes were also better hypoxia-selective cytotoxins than copper complexes with the same ligands, showing lower potency values (Table 2). Trying to further understand the reasons for the improvement of potency and hypoxia selectivity due to vanadium complexation, electrochemical behavior of the vanadyl complexes and the free ligands was studied in detail. Previous electrochemical studies of the free ligands showed that as the electron - withdrawing nature of the 6(7)-substituent increased, the reduction potential became less negative, the compounds were more readily reduced and the hypoxia cytotoxicity increased [62]. Taking into account the described mechanism of action for aromatic N-oxide bioreductive prodrugs, their efficacy will depend significantly on the initial single electron reductive activa-

tion. Therefore, redox properties of bioreductive prodrugs and of the enzymes involved in the activation process are fundamental for determining activity and selectivity. As previously hypothesized, the complexes may release in vivo the bioactive prodrug by direct ligand substitution or reduction by cellular reductases and subsequent substitution. For the latter possibility the complexes may have reduction potentials in the appropriate range. A detailed study of the electrochemical behavior of the free ligands and the complexes by cyclic voltammetry showed that the first reduction process of the ligand, that involved the one electron reductive activation significant for the selective bioactivity in hypoxia, did not vary substantially by coordination to vanadium. Since the complexes exhibited a very similar redox behavior, no ap- parent correlation could be demonstrated between reduction potential of the complexes and their potency and hypoxic selectivity [6]. The vanadyl complexes showed improved solubility in hydrophilic solvents, like light alcohols, in comparison to the free ligands. These could be of particular biological significance and indicative that coordination of the quinoxaline-2-carbonitrile N

1,N

4-dioxide derivatives to va-

nadium could improve their bioavailability by modifying their solubility properties [65].

III. Bioreductive Metal-Radionuclide Radiopharmaceuti-cals

As previously discussed hypoxia is often associated with tumors and heart disease and can affect the effectiveness of anti-cancer treatments. Identifying viable versus necrotic

Table 2. Hypoxic and Oxic Cytotoxicity on V79 Cells at 20 M, Potency (P) in Hypoxia and Hypoxic Cytotoxicity Relationship

(HCR) for the Metal Complexes and the Ligands

Compound SFair SFhypox P( M) HCR

L1 [63] 100 0 9.0 150

L2 [63,64] 90 0 7.2 >10

L3 [63] 82 22 nd nd

L5 [64] 2.0 10

[Cu(L1-H)2]·H2O [63] 100 1 35.1 nd

[Cu(L2-H)2] [63] 91 5 27.9 nd

[Cu(L3-H)2]·2H2O [63] 100 10 nd nd

[Cu(L4-H)2] [64] 18 1 nd nd

[Cu(L5-H)2] [64] 77 1 20.0 >2

[Cu(L6-H)2] [64] 100 100

[VO(L1-H)2] [65] 57 0 3.0 15

[VO(L2-H)2] [65] 36 0 3.0 >15

[VO(L3-H)2] [65] 60 0 nd nd

[Pd(L1-H)2] [68] 92 0 5.0 >8

[Pd(L3-H)2] [68] 2 0 nd nd

[Pd(L4-H)2] [68] 0 0 nd nd

[Pd(L7-H)2] [68] 62 48 nd nd

Tirapazamine [62] Nd nd 30.0 75

L1 = 3-amino-6(7)-chloroquinoxaline-2-carbonitrile N1,N4-dioxide, L2 = 3-amino-6(7)-bromoquinoxaline-2-carbonitrile N1,N4-dioxide and L3 = 3-amino-6(7)-methylquinoxaline-2-carbonitrile N1,N4-dioxide, L4 = 3-amino-6(7)-chloroquinoxaline-2-carbonitrile N1,N4-dioxide, L2 = 3-amino-2-carbonitrile N1,N4-dioxide, L5 = 3-amino-6(7)-chloro-7(6)-fluoroquinoxaline-2-carbonitrile N1,N4-dioxide, L6 = 3-amino-6(7)-chloro-7(6)-methoxyquinoxaline-2-carbonitrile N1,N4-dioxide, L7 = 3-amino-6,7-dichloroquinoxaline-2-

carbonitrile N1,N4-dioxide.


tissue following myocardial infarction and strokes and in solid tumors is very important information for the physician for determining the treatment regime for a patient. The de-lineation of hypoxia in tissue has also direct implications for the treatment of other diseases, like diabetic retinopathy and arthritis. Therefore, it is of great clinical utility to develop compounds that allow the non-invasive imaging of hypoxia [75,76]. In addition, therapeutic applications of radiophar-maceuticals bearing suitable radionuclides could help in the treatment of hypoxic tumors. The initial idea by Denny, de-lineated in a previous section of this review, led to a related approach by other researchers (Welch, and Dilworth, among others) towards using redox properties of transition metal center for targeting its complexes to hypoxic tissues, in par-ticular to afford the selective delivery of a radionuclide (Cu-64, Re-188/186, Tc-99m) for radiodiagnosis or radiotherapy purposes [9,11,12,36,77].

Radioactive copper complexes have been utilized for many years for therapy and also for imaging purposes using the noninvasive medical techniques gamma scintigraphy and positron emission tomography (PET) [78]. Copper center is attractive for the development of hypoxia-selective radio-pharmaceuticals since it has a number of radioactive isotopes which decay with combinations of beta, positron and gamma emission processes that are useful for imaging or therapeutic applications in medicine [9,11,12,77,79]. In particular,

64Cu

(t1/2 = 12.7 h) decays by a number of pathways (electron cap-ture, 41%;

- decay, 0.573 MeV, 40%; + decay, 0.656

MeV, 19%, annihilation radiation, 0.511 MeV, 38%; and photons,1.34 MeV, 0.5%), allowing for both diagnostic im-aging via PET as well as therapy via

- particle emission.

This radionuclide has been identified as an emerging PET (positron emission tomography) isotope (see chapter by Ana Rey in this issue for more details). Its decay characteristics allow getting PET images that are comparable in quality to those obtained using 18F labeled compounds. Given its longer half-life compared with

18F and the versatility of copper

chemistry, copper is an attractive alternative for PET imag-ing when longer circulation times are required [11,12]. In addition and as previously discussed, copper is mainly re-stricted to two oxidation states, Cu(II) and Cu(I), being the Cu(II)/Cu(I) reduction accessible within the cellular potential window. For instance, Cu-bis(thiosemicarbazone) complexes have been extensively studied since it has been demonstrated that these complexes can show high selectivity for hypoxic cells depending on the nature of the substituents on the thiosemicarbazone moiety (Fig. 9) [11,12,80-93]. Sele-nosemicarbazone analogues have been also investigated [94,95]. The topic will be only briefly discussed since it has been previously reviewed [11,12]. The mechanism by which Cu(II)-bis(thiosemicarbazone) complexes accumulate in hy-poxic tissue is believed to involve a bioreductive process, similar to that postulated by Denny´s group for Co(III)-nitrogen mustards complexes, where the neutral Cu(II) spe-cies enters the cells by passive diffusion and is subsequently reduced to Cu(I) by intracellular enzymes. Cu(II) and Cu(I) show different coordination geometries, different Pearson´s acid-base behaviors and, therefore, affinities for different donor atoms. Cu(I) is only weakly chelated by the bis(thiosemicarbazone) ligand which is therefore substituted by macromolecular bioligands present inside the cell. In this

way the radionuclide is trapped in the hypoxic tissue cells. Bis(thiosemicarbazones) act just as delivery vehicles for radioactive copper. In normal cells the Cu(I) complex is quickly reoxidized and washed out of the cell. Cu-ATSM complex (ATSM = diacetyl-bis(N

4-methylthiosemicar-

bazone; Fig. (9): R1 = R2 = R3 = R5 = CH3, R4 = R6 = H) re-sulted the most hypoxia selective compound being selec-tively trapped in hypoxic tissue, in both myocardium and tumors, and has been clinically tested as a positron emission tomography (PET) tracer for the delineation of hypoxia and as a potential radiotherapeutic agent [96-98]. Electrochemi-cal, spectroscopic and computational calculations showed that hypoxic selectivity of this derivative is based on the high relative stability of the Cu(I) species [82,90]. This species is trapped in the hypoxic cells and interacts with cellular bio-molecules. Due to its negative charge it is trapped in oxy-genated cells and being enough stable it is back-oxidized before decomposition occurs. The hypoxic selectivity of a significant number of different Cu(II)-bis(thiosemicar-bazonato) complexes was correlated to their reduction poten-tials, the stability of the Cu(I) species and the pKa values [87,99]. Chemical and electrochemical results support the hypothesis that intracellular reduction of the Cu(II) com-plexes to Cu(I) species can lead to two distinct patterns of chemical behavior: rapid acid-catalyzed dissociation for non hypoxia-selective complexes or resistance to dissociation allowing subsequent back-oxidation by molecular oxygen in normal tissue for those hypoxia-selective complexes, like CuATSM (Fig. 9). Reduction has been shown to generate the copper(I) anionic species that may undergo back-oxidation, protonation or ligand dissociation. The hypoxia selectivity arises from a delicate balance between enzyme-mediated one-electron reduction and subsequent back-oxidation by dioxygen in normal tissue and protonation (dependent on intracellular pH) and ligand dissociation in hypoxic tissue [82].

Further research is being currently performed to develop novel

64Cu compounds of modified bifunctional bis(thio-

semicarbazone) ligands for the PET imaging of hypoxia [100]. Functionalization of the bis(thiosemicarbazonato) moiety through formation of imines via condensation be-tween ketones and the hydrazinic nitrogen provided im-proved hypoxia-selective compounds but their high liver uptake determined that they were not viable as PET tracers. Several promising pipeline tracers based on the chelation of 64

Cu with these and other ligands are being currently evalu-ated [11].

Another approach for hypoxic tissue imaging involves Tc-99m as metal radionuclide. Tc-99m is the most widely used radionuclide in nuclear medicine due to its suitable de-cay properties (short half-life of 6 h, decay of 140 keV), broad accessibility from a radionuclide generator system, low cost and versatile chemistry. Almost ten years ago a great effort has been done to develop Tc-99m compounds for selective imaging of hypoxic tissues in vivo through accurate non invasive nuclear medicine techniques (gamma scintigra-phy, SPECT single photon emission tomography). An ideal hypoxia-Tc-99m imaging agent must have a simple, low-cost preparation and show adequate stability, rapid accumulation and sufficient retention times in tumors and rapid clearance from other tissues to provide good images having suitable


Fig. (9). Mechanism of hypoxia selectivity of Cu(II)-bis(thiosemicarbazonato) radiopharmaceuticals (BTSC: bis(thiosemicarbazonato)).

contrast between lesion and background. To function as a hypoxic tissue marker, a radiopharmaceutical must enter the cell and possess an accessible reduction potential. Once the compound entered the hypoxic cells, it should be reduced and trapped, allowing in this way the acquisition of an exter-nal gamma camera image. On the other hand, hypoxia mark-ers do not enter necrotic tissue and remain unaffected in normal oxygenated tissues, diffusing out of the cells. Early developed Tc(V) small-molecule hypoxia targeting mole-cules have been previously reviewed [101]. Mainly, coordi-nation compounds with hypoxia-selective nitroimidazoles as ligands have been tested as hypoxic tissue markers. Their radio halogenated compounds had been previously studied but they had shown the main disadvantage of the non-ready availability of these radionuclides. As previously discussed the nitro moiety is able to undergo an enzyme-mediated one-electron reduction leading to a radical anion that undergoes further reduction to products which bind macromolecules, keeping the associated radionuclide selectively trapped in cells under hypoxic conditions. First attempts to label them with Tc-99m failed due to poor biological properties of the hypoxia markers, like high lipophilicity which rendered clearance via the gastrointestinal or hepatobiliary tract or poor permeability through lipophilic membranes, among others. In recent years published work on Tc compounds has substantially diminished. Nevertheless, some further work on potential Tc-99m hypoxia markers has been reported. Com-pounds with different pharmacophores, mainly 2-nitroimidazole derivatives and amineoxime-type chelators, attached to the cores TcO

3+, TcN

2+ or fac-[Tc(CO)3]

+ have

been proposed (Fig. 10) [102-111]. Among them, the 2-nitroimidazole derivatives BMS 181321, [TcO(PnAO-1-(2-nitroimidazole))], and BRU59-21, [TcO(PnAO-5-oxa-6-(2-nitroimidazole))], showed to be the most promising ones (Fig. 10) [112,113]. Another potential agent, the

99mTc-HL91

(Prognox, GE-Healthcare), the first non nitro-aryl-based ra-diotracer for evaluating hypoxic fraction in neoplasm, stroke and myocardium infarction regions, includes an amine-oxime-type chelator (Fig. 10). It does not involve a bioreducible organic group and its mechanism of hypoxia selectivity remains unclear. Nevertheless, this kind of com-

pounds showed that the redox properties of a metal complex by itself can be utilized to provide hypoxic selectivity. Fur-thermore, it is possible to develop hypoxia markers that in-cluding the TcO

3+ core or other Tc cores may be selectively

reduced in hypoxic cells by making use of the redox charac-teristics of the metal centre [114]. The properties of the tech-netium complexes being yet synthesized are still far from ideal. In this sense, further research on novel potential hy-poxic-tissue tracers has to be developed.

IV. Other Metals Compounds Having Suitable Reduction Potential to be Bioreduced in Biological Media

It has been proposed that bioactive metal compounds of some transition metal ions act as prodrugs being “activated by bioreduction” [115]. As previously mentioned, Ru(III) and Pt(IV) coordination compounds are considered kineti-cally inert in aqueous media. On the other hand, Ru(II) and Pt(II) complexes are more labile. Therefore, ruthenium and platinum compounds in their higher oxidation states could have potential as hypoxia-selective agents if they were pref-erentially activated by bioreduction under the hypoxic condi-tions shown by solid tumors. Accordingly, Ru(III) and Pt(IV) coordination compounds have been extensively stud-ied trying to exploit the “activation by bioreduction” strategy and have been reviewed with regard to further develop drug design strategies [7,10,14]. Therefore, only a brief considera-tion will be included in this review work.

Square planar Pt(II) bioactive compounds are usually la-bile to substitution by ligands present in biological media, like small chelating bioligands, proteins and water. This la-bility is considered the main reason for the toxic side effects shown by Pt(II) drugs widely used in clinical practice as antitumor agents (cisplatin, carboplatin, oxaliplatin). One of the strategies to circumvent this problem has been to convert them in more kinetically inert octahedral hexacoordinated Pt(IV) compounds that could therefore be stable in the blood stream. These compounds would be activated near the target through bioreduction by endogenous reductants, like glu-tathione, ascorbate, proteins or enzymes. Bioreduction of


N

N

NO2

R

2-methyl-5-nitro-imidazole derivatives

N

NH

OH

HN

N

OH

HL91

N

NH

OH

HN

N

OH

PnAO -1-(2-nitroimidazole)

NN

NO2

N

NH

OH

HN

N

OH

O

PnAO-5-0xa-6-(2-nitroimidazole)

N

N

NO2

TcOC

OC

CO

Tc

O3+

[99mTc(V)O]3+ [99mTc(V)N]2+

Tc

N2+a)

b)

fac - [99mTc(I)(CO)3]+

+

Fig. (10). a) 99m

Tc common cores b) Some ligands used for the development of TcO3+

hypoxia markers.

these Pt(IV) compounds could finally lead to the tetracoordi-

nated square-planar Pt(II) active species by losing both axial

ligands (Fig. 11). The Pt(II) more labile resulting species

could easily substitute some of its ligands when interacting

with the biological target, like cisplatin does, leading to the

activity [13,14,116-118]. For instance, it has been confirmed

that some Pt(IV) species, like [PtIV

Cl4(NH3)2], enter the cell

before being reduced [10]. Besides the enhancement of sta-

bility, the possibility of changing the nature of the two extra

ligands would allow improving cellular uptake, selectivity

for a target and toxicity, among other relevant biological

properties [13-15]. These prodrugs could provide better ways

of delivering the actual drug to the target tumor cells and

offer the possibility of targeting it to specific types of cancer

cells by inclusion of selected bioactive ligands in the axial

positions, specifically recognized by receptors present in

certain kinds of tumors. In addition, Pt(IV) compounds

might be selectively reduced in the hypoxic environment of

solid tumors, releasing the actual drug. A series of novel

Pt(IV) complexes with different axial ligands was tested by

Hambley et al. on a 3D multicellular in vitro model of solid

tumor showing none hypoxia selectivity. Nevertheless, it is

interesting to note that although not selective they were ac-

tive in hypoxic environment. Consequently, they could have

the potential to effectively treat solid tumors. Their probable

mechanism of action is still being extensively studied since

even Pt(IV) complexes having reduction potentials outside

the cellular potential window, are active anticancer agents.

Although Pt(IV) complexes reduction potentials can be fine-

tuned by modifying the Pt coordination sphere, it is not

known yet if they could be adequately tuned to be selectively reduced in hypoxic tissues [119-122].

A similar approach could be considered for octahedral

Ru(III) kinetically inert prodrugs converted in vivo in the

more reactive octahedral Ru(II) active species. The selective

activation by reduction in the tumor might contribute to

lower side effects in vivo [20,115,123]. The effect of hypoxia

in increasing the toxicity of certain anticancer agents (trans-

[RuCl4(Im)2]- and cis-[RuCl2(NH3)4]

+) against HeLa cells in

tissue culture was studied several years ago. These studies

showed that decreasing oxygen tensions lead to lower IC50

values [123]. Only two Ru(III) are currently under clinical

trials: (H2im)-[trans-RuIII

Cl4(Him)(S-dmso)], where Him =

imidazole, and (H2Ind)[trans-RuIII

Cl4(Hind)2], where Hind =

indazole. Although intracellular bioreduction is postulated as

part of their mechanism of action and explains their selectiv-

ity for tumor cells none of them showed clear selectivity

towards hypoxic solid tumors. Nevertheless, local hypoxia in

tumor could favor bioreduction and trapping of the reduced species in tumor cells [10,20].

PtIV

L

L

PtII + 2L+2e-

RuIII RuII+e-

Fig. (11). Activation by bioreduction.


CONCLUSIONS

Medicinal Inorganic Chemistry research has lead to sev-eral metal compounds showing high potentiality for therapy of hypoxic tumors or for imaging of hypoxic tissues. Further research through either of the strategies outlined in this re-view is needed in order to design more efficient hypoxia-selective metal compounds. In addition, this area of drug design could be also suitable for the development of metal compounds that although not selective for hypoxia could be able to adequately reach hypoxic tissue exerting there cyto-toxicity. In particular, the strategy based on the design of platinum and ruthenium compounds having suitable reduc-tion potential to be bioreduced in biological media is being extensively investigated and could finally lead to metal-based compounds showing cytotoxicity on hypoxic tumor cells.

ACKNOWLEDGEMENTS

The author wishes to thank the CYTED network 209RT0380 “Red Iberoamericana de Investigación y De-sarrollo de Fármacos basados en Compuestos Metálicos” for supporting collaborative research on metal-based potential drugs, performed by Latin-American laboratories of eight countries. The author wishes also to thank the other re-searchers involved in the published manuscripts on metal compounds of quinoxaline N-oxide derivatives, developed as potential hypoxia selective cytotoxins. In particular, the author is especially grateful to Prof. Dr. Antonio Monge-Vega (Unidad en I+D de Medicamentos, CIFA, University of Navarra, Pamplona, Spain).

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Received: April 26, 2010 Revised: August 22, 2010 Accepted: August 23, 2010

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