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Bioorganometallic Compounds with Antimalarial Targets: Inhibiting
Hemozoin Formation
Maribel Navarro,,†^ William Castro,‡^ and Christophe Biot,§
†School of Chemical and Mathematical Sciences, Murdoch University, Western Australia 6150, Australia
‡Lab. Quı mica Bioinorgá nica, Centro de Qú ı mica, Instituto Venezolano de Investigaciones Cientí́ ficas (IVIC), Caracas 1020-A,
Venezuela §Unité de Glycobiologie Structurale et Fonctionnelle, UMR CNRS 8576, Université Lille 1, 59650 Villeneuve d’Ascq, France
ABSTRACT: About half of the world’s population is at risk of malaria, and in tropical countries it remains a major cause of morbidity and death in children. Drug resistance to current established antimalarial drugs such as chloroquine is driving the rise in malaria-attributed deaths. In the mid- 1990s, two groupsin France and in Venezuelaprobed the potential contribution of organometallic analogues as a means of discovering new antimalarial drugs. In the present review, key topics of organometallic antimalarials are outlined using examples from the literature. The interdisciplinary research environment of bioorganometallics allows researchers to investigate the whole spectrum of the drugs’ mechanisms of action. Targeting the digestive vacuole and inhibiting hemozoin formation is believed to be the main mechanism by which these drugs induce parasite death.
1. INTRODUCTION
Malaria is a major cause of illness and death in children and adults in tropical countries. It currently affects an estimated 500 million people and threatens more than 1 billion people around the world. 1 −^3 An estimated one in every five childhood deaths (20%) in Africa is due to this disease, with a child there dying from malaria every 30 s. It is calculated that an African child has on average 1.6−5.4 episodes of malarial fever each year. 4 The disease is caused by blood parasites of the Plasmodium species; five species of Plasmodium spp. are infectious to humans: P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. P. falciparum is responsible for most of the deaths from malaria. This huge public health problem has been addressed primarily by three different approaches: (1) insecticides, (2) vaccines, and (3) chemotherapy. 5 First, the chlorinated organic insecticide DDT (dichloro-diphenyl-tri- chloroethane) was originally used in order to control the insect population, but this practice was discarded due to environ- mental impact, indiscriminate spraying, and a development of resistance to DDT in some populations of malaria vector Anopheles mosquitoes. However, today there is a renewed interest in the use of indoor residual spraying (IRS) as one of the primary vector control interventions for reducing and interrupting malaria transmission in African countries. How- ever, it has very recently been reported that mosquitoes are developing resistance to the newest long-lasting insecticide nets.^6 Second, immunological methods have been implemented in order to obtain the vaccines, which are one of the most effective modes of treatment available, but despite these efforts 7,8^ there are no available vaccines that effectively target a parasitic infection. The third approach employed is
chemotherapy. A number of organic compounds have been used as antimalarial drugs, such as quinine, chloroquine, hydroxychloroquine, mefloquine, primaquine, proguanil, dox- ycycline, sulfadoxine, pyrimethamine, artemether, lumefantrine, artesunate, and amodiaquine (Figure 1). Current treatments are based on a combination of two or three of these drugs. Historically, chloroquine was almost an ideal drug for antimalarial treatment due to its high efficacy, safety (including during pregnancy), low cost, and ease of use.^9 However, resistance to chloroquine is now widespread, 10 and the loss of this drug has been a major setback to the effective treatment and control of malaria. Additionally, due to the numerous cases of resistance to antimalarial drugs, including chloroquine, there now exists an urgent need to broaden the arsenal of available therapies well beyond the conventional purview of medicinal chemistry within the context of pharmaceutical research. There is compelling evidence that the target of the most important group of antimalarials, the quinoline derivatives, remains useful for the design of new drugs. Currently available data suggest that these drugs inhibit hemozoin formation during the blood stage of the malaria parasite.
2. HEMOZOIN IN THE MALARIA PARASITE
The malarial parasite is pathogenic only during the part of its life cycle when it is present in the host’s bloodstream, invading red blood cells (RBCs). This cycle involves four stages: namely
Special Issue: Organometallics in Biology and Medicine
Received: April 15, 2012 Published: May 21, 2012
Review pubs.acs.org/Organometallics
© 2012 American Chemical Society 5715 dx.doi.org/10.1021/om300296n | Organometallics 2012, 31, 5715− 5727
the merozoite, ring, trophozoite, and blood schizont stages. The merozoite is an extracellular stage of short duration that rapidly invades RBCs. The parasite then develops into a ring stage that lasts for about 26 h before becoming a trophozoite, which is the metabolically most active part of the cycle. At about 38 h, the trophozoite matures into a schizont that begins to divide into daughter cells. After 48 h, eruption of the resulting merozoites from the RBC completes the cycle. 11 −^13 The P. falciparum trophozoite ingests and degrades large quantities of host cell hemoglobin (Hb). Studies indicate that 60 −80% of the Hb originally present in the RBC is digested
and degrades in an acidic compartment within the parasite called a digestive vacuole, which has a pH in the range 5.2− 5.6. 14,15^ This process involves the four aspartic endopeptidases plasmepsins I, 16 II, 17 III (also called histoaspartic protease, HAP), and IV, the three cysteine endopeptidases falcipain 1, 2, and 3,^18 and the metalloprotease falcilysin. Heme is derived from hemoglobin; the iron center is oxidized, probably through spontaneous auto-oxidation by O 2 , to produce hematin (aquaferriprotoporphyrin IX or H 2 O−Fe III^ PPIX). A large body of evidence has demonstrated that, once in a “free” state, heme is able to induce oxygen-derived free radical
Figure 1. Organic compounds used as antimalarials.
Figure 2. Process of hemoglobin degradation and heme detoxification by intraerythrocytic malaria parasite.
carboxylate bonds. Dimers are then also linked with each other through hydrogen bonds between remaining carboxylate groups, leading to chain extensions (Figure 3). This structure of hemozoin differentiates it from monomeric heme, heme aggregates, and μ-oxo dimers and provides it with distinct solubility, X-ray diffraction, and FT-IR characteristics. In the same study 43 it was demonstrated that β-hematin (Fe III^ - protoporphyrin-IX) 2 is chemically, spectroscopically, and crystallographically identical with hemozoin. These molecules are linked into dimers through reciprocal iron−carboxylate bonds to one of the propionic side chains of each porphyrin, and the dimers form chains linked by hydrogen bonds in the crystal. These results have implications for understanding the action of current antimalarial drugs and possibly for the design of new therapeutic agents. The hemozoin formation is essential for the survival of the Plasmodium parasite, 44 as it has been suggested that antimalarial drugs such as chloroquine, quinoline methanols, quinine and quinidine, 45 and artemesinin 46 have heme and/or hematin as targets of action in order to avoid hemozoin formation. These compounds exert potent action against the blood stages of Plasmodium in a mechanism that impairs hemozoin formation. It was shown that 4-aminoquinolines interact with “free” hematin, hindering its crystallization into hemozoin. The “free” hematin interacts with membranes and exerts severe toxic effects, ultimately killing the parasite through oxidative stress. 47 An additional theory suggests that heme−quinoline complexes incorporate into a growing crystal face, influencing its external appearance and blocking its growth. 48,
With all these results, many scientists have focused their research on the development of new drugs which are able to fight disease more effectively than those currently used while seeking to evade the resistance developed by the parasites. A line in this field with excellent results has been the synthesis of organometallic complexes, linking transition metals to organic antimalarial drugs and thereby generating a synergistic effect in terms of biological activity.
3. ORGANOMETALLIC COMPOUNDS AS
ANTIMALARIALS
One drug design strategy for new therapies against tropical diseases as malaria was based on the use of organic compounds with known or potential activity through the coordination of a transition metal into the molecular structure. This modification is important within biological systems due to the binding capability and reactivity of the transition metals, which are determined by the d orbitals. Sanchez-Delgado et al. in 1996 proposed the modification of chloroquine through this strategy. RhCl(COD)CQ was the first organometallic complex synthe- sized, characterized, and evaluated against malaria parasites (complex 1 , Figure 4). NMR studies indicated that CQ binds to the metal in 1 through the unsubstituted N(1) atom, which is a good donor site in this molecule, forming a 16-electron complex with square-planar geometry, a typical configuration for Rh(I). The in vitro activities exhibited a ratio of IC 50 (parental compound)/IC 50 (metal complex) ≤ 1, indicating that complex 1 acts at a level similar to that of CQDP (chloroquine diphosphate) against the malaria parasites.
Figure 4. Structures proposed for rhodium and ruthenium complexes with chloroquine.
However, in vivo experiments displayed that complex 1 caused a reduction of the parasitemia of 94% at a concentration equivalent to 1 ED 50 (50% effective levels) of CQDP, demonstrating the potential of the novel metal-based compounds for the development of chemotherapies against malaria and other tropical diseases. 49 In the search to increase the biological activity displayed by complex 1 and other compounds coordinated with chloroquine, a new molecular design was later adopted using ruthenium as the metal center and varying the ancillary ligands. These Ru II^ (η^6 -arene) chloroquine derivatives are shown in Figure 4, where it is possible to notice that complexes 2 − 4 display the chloroquine bound to ruthenium in the η^1 -N mode through the quinoline nitrogen atom, whereas in 5 there is an unprecedented η^6 bonding through the carbocyclic ring. These compounds showed in vitro activity against four CQ-susceptible strains (FcB1, 3D7, PFB, and F32) and three CQ-resistant strains (W2, Dd2, and K1) of P. falciparum. These results have special importance for the susceptible strains, with the potency of all complexes consistently higher than that of chloroquine diphosphate. From these results, the authors proposed that the combination of RuII^ and chloroquine in a single molecule produced an enhanced activity against resistant strains of the parasite. 50 Continuing the search of organometallic molecules that can increase the biological activity, ferrocene (Fc) is presented as an
interesting option for the modeling of new complexes with medicinal purposes. Through its small and rigid sandwich structure, its lipophilic properties allow for the effective penetration of cellular membranes. Additionally, it is stable in aqueous, aerobic media and allows access to a large variety of derivatives. These characteristics together make Fc very attractive for biological applications and especially for drug design. 51 −^53 Among the numerous ferrocene bioconjugates reported, one famous example is the structural variation of the anticancer drug tamoxifen to give ferrocifen. 54,55^ Another example is ferrocerone, which was developed to treat iron deficiency anemias. This used to be marketed in Russia^56 but has recently been employed as an antibacterial agent. 57 The concept of grafting a ferrocenyl moiety inside an established antimalarial molecule was first applied during the mid-1990s, and these investigations are ongoing. A library of ∼150 complexes has been prepared on the basis of ferrocene- conjugate (or metallocene-conjugate) analogues of known antimalarial drugs such as mefloquine (complex 6 , Figure 5), quinine (complex 7 , Figure 5),^58 artemesinin (complex 8 , Figure 5), 59 atovaquone (complex 9 , Figure 5), 60 chalcones (complex 10 , Figure 5), 61 ellagitannin derivatives (complex 11 , Figure 5), 62 ferrocenyl carbohydrate conjugates (complex 12 , Figure 5), 63 ciprofloxacin (complex 13 , Figure 5), 64 −^66 ferrocenyl pyrrolo (complex 14 , Figure 5), 67 ferrocene− strychnobrasiline (complex 15 , Figure 5), 68 mepacrine
Figure 5. Ferrocene analogues of known organic antimalarial agents.
The efficacy of ferroquine was monitored in several rodent malarial strains that are widely used for in vivo tests (e.g., P. berghei N, P. yoelii NS, P. vinckei). Each of these strains showed a very wide range of curative doses for chloroquine, ranging from 50 mg kg−^1 per day to more than 100 mg kg−^1 per day in the standard 4 day test. Subsequently, P. yoelii NS was tested in the absence and presence of chloroquine pressure (effective doses of chloroquine that reduce parasitaemia by 90% (ED 90 ): 2.78 and 8.33 mg kg−^1 per day, respectively). Chloroquine had a curative dose for CQ-susceptible P. vinckei clone of 70 mg kg−^1 per day, while the curative dose for the CQ-resistant clone was up to 400 mg kg−^1 per day. For all strains tested, ED 90 values for ferroquine were measured and ranged from 1.96 to 3.89 mg kg−^1 per day. The curative dose observed was 10 mg kg−^1 per day for all strains tested, irrespective of the route of administration. 80,81^ More recently an in vivo study of the effects of artesunate and ferroquine on the gametocytogenesis of the rodent malaria parasite P. y. yoelii and its vector A. stephensi was performed. The effect of the same drugs on the infectivity of gametocytes was also studied. Subcurative doses (5 and 10 mg kg−^1 ) of both ferroquine and artesunate increased gametocyto- genesis at 90 and 300 min post-treatment, the most effective being 5 mg/kg artesunate. Additionally, a decrease in the infectivity of gametocytes of P. y. yoelii in mosquitoes both at 90 and 300 min post-treatment was also observed. These results should be confirmed with a study on a revised distribution of the different gametocyte stages and the transmission stages from gametocyte to oocyst development in the mosquito. 82 The
current development of FQ is related with the results obtained in phase I and II in clinical trials, 79,83^ through the dose ranging study of ferroquine with artesunate in African adults and children with uncomplicated P. falciparum malaria (FARM) carried out by Sanofi-Aventis Co. The decision after this study was to modify the ferroquine development strategy; it is worth mentioning that the discontinuation was not due to unexpected safety- or activity-related findings. 83 Putting together all results mentioned previously, new synthetic strategies of FQ have been developed in order to make its synthesis more viable, effective, and economical in the production of this molecule in industry. In fact, a synthesis of ferroquine has recently been published 84 which involves a reductive amination reaction. These reactions comprise (1) a condensation step of the aldhehyde-amino ferrocene with the 7-chloroquinolin-4-amine, (2) reduction of the condensation product obtained in the preceding stage, and (3) then a hydrolysis reaction in the presence of an aqueous solution of ammonia or of citric acid. This process involves fewer reaction steps and increases yields, leading to a better and more feasible synthetic strategy in comparison to the original synthetic method. 76 After the first reports of the biological activity of ferroquine, an extensive investigation was carried out in search of analogues with better activities, improved properties as a drug, and better understanding of structure−activity relationships. Changes in the tertiary amines (complex 23, Figure 8) showed strong antimalarial activity, especially against the CQ-resistant Dd
Figure 8. Ferroquine derivatives.
and W2 strains. These compounds were 2- to 10-fold more active than chloroquine and as active as ferroquine. 73 In the search of mimicking the antimalarial drug hydroxychloroquine (HCQ), three ferroquine derivatives were prepared (complex 24 , Figure 8). These complexes differed from ferroquine in their side chains on the tertiary amines. Additionally, the OH group was introduced with the aim of reducing the cytotoxic effects in comparison to ferroquine. The results in strains 3D and W2 showed an increase in activity with respect to chloroquine but a decrease with respect to ferroquine. 85 Changes in the secondary amines also showed antimalarial activity comparable to that of ferroquine (complex 25 , Figure 8). All these compounds exhibited better inhibitory activity against the Dd2 strain than chloroquine itself. 86 These studies showed that the remarkable activity of ferroquine depends on the position of the ferrocenic nucleus in the side chain and that the in vitro antimalarial activity was not disturbed by slight modifications in the lateral basic side chain. A series of analogues was synthesized from the combination of ferroquine with thiosemicarbazones (complex 26 , Figure 8), and a covalent bonding between both active fragments was envisaged by merging the amino groups. In order to compare the contribution from each fragment, analogues without the ferrocenyl moiety and analogues without the 4-aminoquinoline moiety were also synthesized. 87 With this work, the authors concluded that the presence of the aminoquinoline structure, allowing transport of the compounds to the digestive vacuole of the parasite, seems to be the major contributor to the antimalarial activity and that the presence of the ferrocenyl moiety within the lateral chain is the main condition required to retain a strong antimalarial activity on CQ-resistant P. falciparum. In another effort in the search for new active complexes, a series of compounds was synthesized and characterized on the basis of poly(propyleneimine) dendrimers functionalized with derivatized ferrocenylthiosemicarbazones conjugated to the periphery of the scaffold (complex 28 , Figure 8). Metallodendrimers show antimalarial activity in the low micromolar range and show antiplasmodial activity superior to that of the nonconjugated ferrocenyl thioesters and the free dendritic ligand. On the basis of these preliminary observations, the authors propose that the biological properties of these metallodendrimers are worth studying and further investiga- tions into the mechanism of action are ongoing. 88 A new class of antimalarial agents named trioxaquines, based on the concept of hybrid molecules with a dual mode of action, have been reported. 89 These molecules contain two covalently linked pharmacophores: 1,2,4-trioxane, as in artemisinin, and 4- aminoquinoline, as in chloroquine. Such hybrid molecules might be considered a possible response to the recently growing resistance of various parasites to artemisinin, 90,91^ and the first generation of trioxaquines was already highly active against CQ-resistant strains of P. falciparum. 92,93^ From more than 100 trioxaquines, PA1103/SAR116242 was selected as a drug candidate. 94 Recently, the syntheses of complexes that contain a 4-aminoquinoline, a ferrocene, and a trioxane (complex 27 , Figure 8) were reported, as well as their biological studies, which showed that they are active in vitro against CQ-resistant P. falciparum. Also, the in vivo experiments in P. vinckei petteri infected mice displayed that they caused a reduction of the parasitemia below a detectable level. Another strategy to produce new, more active complexes at a more affordable cost for industrial production was the dual drug strategy using an association of ferroquine with glutathione
reductase (GR) inhibitors 95 (complex 29 , Figure 8). The resulting biologics of these complexes show two important aspects; first, a slight modification of the basic side chain does not affect the activity if the substituents are not too large, and second, a decrease in the antimalarial activity of the dual molecules was observed, in comparison with that of ferroquine analogues, which might be explained by the fact that both the amide bond and the side chain of the ferroquine derivative are cleaved following the oxidative metabolism in the digestive vacuole. Other ferrocene and ruthenocene analogues of chloroquine were also reported (complex 30 , Figure 8), exhibiting activities similar to that of chloroquine in susceptible strains (D10), but in most cases remaining active in resistant strains (K1), much like ferroquine. This observation is consistent with the hypothesis that the mechanism for drug resistance in the Plasmodium parasites is compound specific. It has been suggested that ferroquine has reduced affinity for the P. falciparum chloroquine resistance transporter (PfCRT). Addi- tionally, these results are consistent with previous structure− activity relationships performed on aminoquinolines where a hydrophobic group, e.g. an alkyl spacer, and an amino group, for pH trapping, are essential for high antiplasmodial activity. The subsequent substitution of tri-n-butylstannyl groups in bis(tri-n-butylstannyl)ferrocene was used to prepare several ferrocene−chloroquine analogues. Most of the compounds exhibited moderate to strong antiplasmodial activity when tested against both CQ-susceptible (D10) and CQ-resistant (K1) strains of P. falciparum. 96, Some interesting organobimetallic compounds have been synthesized, such as [Au(L)(PPh 3 )]NO 3 , [Au(L)(C 6 F 5 )]NO 3 , and [RhCl(COD)L], where L is ferroquine (complexes 31 − 33 , Figure 8). Complexes 31 − 33 have improved efficacy with respect to chloroquine in both susceptible and resistant strains. However, complexation of the second metal to these compounds at best has little effect on the overall efficacy of the compounds and at worst appears to have a significant antagonistic effect: the presence of the second metal center makes the ferrocenyl moiety far more difficult to oxidize. It should be noted that while the gold and rhodium heterobimetallic compounds do not show additive or synergistic behavior, this does not preclude this possibility with other metal combinations. 98 To investigate the role of the electron-donating ferrocenyl moiety, new organometallic analogues of chloroquine bearing a cyclopentadienyltricarbonylrhenium moiety with an electron- withdrawing effect (complexes 34 and 35 , Figure 8) were prepared and evaluated. The evaluation of antimalarial activity was measured in vitro against a CQ-resistant strain (W2) and a CQ-susceptible strain (3D7) of P. falciparum. This showed that these cyrhetrene conjugates are less active compared to their ferrocene and organic analogues, suggesting an original mode of action of ferroquine and ferrocenyl analogues in relationship with the redox pharmacophore. 99 Following the idea of studying “half sandwich” systems, two new (η^6 -arene- quinoline)Cr(CO) 3 complexes, specifically the [η^6 - N-(7-chlor- oquinolin-4-yl)-N′-(2-dimethylaminomethylbenzyl)ethane-1,2- diamine]tricarbonylchromium compounds (complexes 36 and 37 , Figure 8) were synthesized and evaluated, showing high in vitro activity against CQ-susceptible and CQ-resistant strains of P. falciparum. The activity of this complex against the CQ- resistant parasite strain was 3 times greater than that presented
that all compounds are able to inhibit the heme aggregation process. Furthermore, while the complex [(p-cymene)- RuII^ Cl 2 (CQ)] appears somewhat less active than CQDP in this assay, the other complexes 3 and 4 display an activity very similar to that of CQDP and to each other. The IC 50 of FQ was 0.8 equiv relative to hematin, whereas the IC 50 of CQ was 1.9. 116 An explanation for the higher antimalarial activity of FQ compared to that of the “classical” organic drugs could be due to its (proposed) preferential localization at the lipid−water interface. FQ could prevent the conversion of hematin into hemozoin by maintaining toxic hematin in the aqueous environment. Additionally, molecular docking allowed the demonstration of the noncovalent interactions between FQ and hemozoin, thus blocking crystal growth (Figure 9).^117 These hypotheses would explain why the activity of FQ is steady despite the level of resistance of the strains. 116
In 2009, Sanchez-Delgado proposed a variation of the β- hematin inhibition activity method reported by Egan 118 to measure the abilities of CQDP and complexes to inhibit the formation of β-hematin in a lipid−water interface. When the heme aggregation inhibition assay was conducted in an aqueous/n-octanol mixture at pH 4.9, in which the hematin is carefully introduced close to the interface after the drug has been equilibrated between the two phases, the overall aggregation process is much faster (60 min) and the activity trend changes drastically with respect to the results of the assay in aqueous buffer. Ru-CQ complexes are significantly more potent than CQDP in the inhibition of heme aggregation near the interface. Complex 2 , which appeared the least active in aqueous buffer, displays the highest activity at the interface, twice as active as CQDP. Although no significant differences were observed in the activity of complexes 3 − 5 in the experiments performed in an aqueous medium ( 3 ≈ 4 ≈ 5 ≈ CQDP > 2 ), the values measured at the interface for the four compounds follow a clear trend: 2 > 4 > 5 > 3 > CQDP. This concludes that (a) the main mechanism of action of the ruthenium−chloroquine complex is the inhibition of formation of β-hematin, and so trials in these targets (specifically in a lipid−water interface) are excellent predictors of the in vitro biological activity and (b) the [Ru(η^6 -arene)(CQ)Cl 2 deriva- tives are potential drugs for the development of chemotherapies against malaria. 112 (c). Influence of Lipophilicity and Basicity in Anti- malarial Activity. Diverse studies have been carried out in the search for the targets of action of organometallic compounds. There is strong evidence that the action is linked to its
localization in the digestive vacuole of the parasite and to the inhibition of the hemozoin formation, similar to the case for chloroquine, as well as an increase of partition coefficients, which influences the lipophilicity and thus their biological behavior. Particularly at the vacuolar pH (4.9), the Ru−CQ complexes were more lipophilic than CQDP, with the exception of the complex [(p-cymene)Ru II^ (π-CQDP)][BF 4 ] 2 , which was highly hydrophilic. In contrast, at pH 6.6 only complex 5 accumulates preferentially in the aqueous phase (D < 1), while the other Ru−CQ complexes are less lipophilic than CQDP. Alter- natively, in complex 5 , the basicity of the π-bonded Ru−CQDP is modestly reduced with respect to free CQDP (pKa1 and pKa values of 9.31 and 7.91, respectively). 112 On the other hand, in complexes 2 − 4 , the quinoline nitrogen is blocked by coordination to ruthenium and, as a consequence, only pKa values were measured, and they indicate a lower basicity with respect to the pKa1 of CQDP. The antiplasmodial activities (in diverse strains) of these complexes are higher than that of CQDP, presumably owing to the enhanced lipophilicity of the Ru-CQ derivatives. Although the activities do not correlate linearly with lipophilicity, these results show higher complexity of the antimalarial mechanism. One explanation could be that, with infected RBCs, the drug needs to cross three membranes (of the RBC, the parasite, and the digestive vacuole) to reach the target. Another important factor to consider is that for it to accumulate in the digestive vacuole, the drug must travel down a pH gradient from approximately 7.5 to approximately 4.5 and, therefore, the basicity of each complex becomes an important additional feature to consider. Complexes 2 − 4 are all less basic than CQDP and therefore, in the absence of other effects, they would all be expected to accumulate less in the digestive vacuole, as predicted by the VAR values collected in Table 1. The authors proposed that, in the case of CQ-resistant parasites, the dominant factors for the complexes to reach and remain in the digestive vacuole are their modified molecular structures and their enhanced lipophilicity with respect to free CQ. This explains the higher activity of the complexes in relation to CQ, which would be more effectively expelled from the digestive vacuole by PfCRT. The conclusion of this study was that the overall potency of Ru(η^6 -arene)(CQ) derivatives against resistant malaria parasites results from an important structural modification of the CQ molecule, coupled with a major lipophilicity effect and a more subtle influence of the basicity. Biot et al., on the other hand, almost simultaneously re- evaluated the mechanism of action of ferroquine in relation to its basicity and lipophilicity, 119 finding that in terms of basicity FQ follows the same trend as the compounds of Ru-CQ, with reduced pKa values with respect to free CQDP. Comparison of the apparent partition coefficient (log D) at postulated cytosolic pH 7.4 and vacuolar pH 5.2 showed that, at cytosolic pH, ferroquine was over 100-fold more lipophilic than chloroquine, whereas the difference in lipophilicity is only slight at acidic digestive vacuole pH. This result might be interpreted as a higher concentration of ferroquine in the digestive vacuole, despite its weaker base properties. Knowing how and where ferroquine works can aid the development of new medicines for other pathogens that have become resistant to current treatments. Furthermore, clarifying the mechanisms of action can reveal how the organometallic- based analogue strategy can guide the design of new treatments. The synchrotron-based nanoimaging technique allowed Dubar
Figure 9. Intermolecular contacts between ferroquine and synthetic hemozoin at the {1,0,0} crystal surface (left) and at the {0,0,−1} crystal surface (right).
et al. to locate unlabeled chloroquine and ferroquine in vitro. 120 Ferroquine specifically targeted the digestive vacuole of the infected red blood cells. Molecular modeling suggested that ferroquine, which forms an intramolecular H bond, opens up when it comes into contact with the growing crystal faces of hemozoin and seems to act by its stereospecific interaction with those faces and thus inhibits their growth. 117 The inhibition of ferroquine upon the growth of hemozoin leads to accumulation of toxic heme (free or complexed with ferroquine) and thus to the death of malaria parasite. The chemistry of the ferrocene core may also contribute to the antimalarial activity via its high hydrophobicity 119 and/or via the redox behavior of the ferrocene/ferrocenium 121 couple. Owing to its ferrocenyl moiety, ferroquine is able to generate small amounts of hydroxyl radicals from H 2 O 2 via a Fenton-like reaction. Upon such specific oxidizing conditions (parasitic digestive vacuole), this production of reactive oxygen species appears to be not sufficient enough to affect the stability of ferroquine. On the other hand, it should be sufficient to promote significant damage to the membranes of the parasite digestive vacuole, also suspected to be a site of concentration of ferroquine. By specific in situ production of hydroxyl radicals, ferroquine might induce severe damage to the parasite before intervention of resistance mechanisms. 120,121^ Finally, Dubar et al. suggest that the intramolecular hydrogen bond in the lateral side chain of ferroquine plays a crucial role in the antimalarial activity of the drug. 116,117,120^ These results are in agreement with previous observations where (a) ferroquine analogues and (b) hydroxyferroquines including an intramolecular H bond in their side chain showed better activity than molecules lacking this noncovalent interaction (Figure 10).
Another contribution to the antimalarial activity of ferroquine could also arise from its redox properties. All these studies lead to the conclusion that differences in (i) shape, (ii) volume, (iii) lipophilicity, (iv) effects on basicity, and (v) electronic profile dramatically modify the pharmacological behavior of the parent drug. Moreover, FQ should present reduced affinity for the postulated transporter linked to CQ resistance, a structure which seems to be extremely specific. This may partially explain the remarkable activity of ferroquine against CQ-resistant strains or isolates.
5. CONCLUSION
Since 1979 and the discovery of the antitumor activity of titanocene dichloride, the number of organometallic-based drug candidates in the pipeline (clinical trials phase I−III) has increased constantly, but only few are available on the pharmaceutical market; two representative examples are salvarsan (arsphenamine), a drug used to treat syphilis, and cardiolite (radioisotope technetium-99m with the ligand
methoxyisobutylisonitrile), used in nuclear medicine imaging. Clearly, the great potential of bioorganometallics has not yet been realized. At the moment of writing this review, the antimalarial ferroquine was the most advanced organometallic drug candidate. Deciphering the mode of action of ferroquine should improve the chances of putting this drug on the market in 5 years time. If ferroquine (or any other organometallic- based drug candidates reviewed in this special issue) becomes a marketable drug, we can envisage a “big-bang” in bioorgano- metallic research.
■ AUTHOR INFORMATION
Corresponding Author *M.N.: e-mail, m.navarroacosta@murdoch.edu.au, maribelnava@gmail.com; tel, 61-8-93602856. C.B.: e-mail, christophe.biot@univ-lille1.fr; tel, +33-(0)320434893; fax, +33-(0)320436585. Notes The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
All searches on ferroquine carried out at the University Lille 1 were funded by Pierre Fabre Médicament and Sanofi. C.B. and M.N. are very grateful to all Ph.D. and Master's students who participated in this work and provided an excellent job.
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Figure 10. Flip-flop bewteen the open and folded conformations of ferroquine.
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