INTRODUCTION

Malaria is still one of the world's most devastating infectious diseases, afflicting several hundred million people and killing close to two million children each year (1). Plasmodium falciparum, the most deadly species, has become widely resistant to most available antimalarial therapies (2, 3). The mainstays of treatment and prophylaxis, the quinolines, are excellent parasitocidal agents for susceptible organisms but are becoming less and less efficacious. New antimalarials are desperately needed, and an agent that attacked the same vital target as chloroquine, but was not subject to the same resistance modes, would be highly desirable (4). Chloroquine and related drugs appear to work by accumulating to high concentrations in the parasite digestive vacuole (5, 6), the organelle in which hemoglobin is degraded and free heme is released (4). This heme is toxic to eukaryotic cells and is capable of killing parasites by lysing membranes (7) and inactivating critical proteases (8, 9). To counter this, the parasite polymerizes heme into a crystalline matrix called hemozoin, in which heme units are coordinated via a unique iron-carboxylate linkage (10). A family of molecules called histidine-rich proteins has been implicated in the polymerization reaction (11), and lipids may also contribute to the process (12). It is postulated that once hemozoin formation has been initiated, extension of the polymer is able to proceed nonenzymatically (11). Chloroquine can block this extension reaction, resulting in accumulation of toxic heme (11, 13, 14).

Given the importance attributed to iron metabolites in parasite toxicity, a variety of metal chelating agents such as deferoxamine and reversed siderophores has been explored as potential antimalarial chemotherapeutics (15-20). When administered as free ligands, they have been shown to possess antimalarial activity, perhaps by disrupting ferric iron (Fe(III)) metabolism within the digestive vacuole, but none is ideal in its pharmacological properties (18). Other multidentate ligands, some containing an N_mO_n donor core (m, n = 2 to 4), have also been previously explored as therapeutic chelating agents (21, 22) and their corresponding metal complexes evaluated as diagnostic agents or medicinals (23-30). We recently reported that selected hexadentate (N₄O₂) ethylenediamine-bis[propyl((R)-benzylimino)]Fe(III) complexes are hydrolytically stable and possess pharmacological potential resulting from a favorable balance of hydrophobicity and delocalized monocationic charge which enhance cell membrane permeability (31). In mammalian cells, these compounds exhibit cytotoxic activity that is modulated by expression of the multidrug resistance (MDR1)¹ P-glycoprotein in the plasma membranes (31). Human MDR1 P-glycoprotein is a homolog of the P. falciparum pfmdr1 gene product, Pgh1, both being integral membrane proteins of the ATP-binding cassette superfamily of membrane transporters (32-35). While P-glycoprotein is an outwardly directed drug transporter (35, 36), Pgh1 has been localized to the digestive vacuole membrane and implicated in chloroquine sequestration (37), possibly as an inwardly directed transporter (38). Pgh1 has been proposed to contribute to chloroquine resistance in various clinical isolates of P. falciparum (33), although pfmdr1 has been unlinked from chloroquine resistance in a genetic cross (39).

Because members of the N₄O₂ class of metal(III) complexes are known to interact with at least one ATP-binding cassette transporter, we originally conceived that these compounds could be designed to target ATP-binding cassette homologs in P. falciparum. As potential antimalarial agents, the hexadentate metal(III) complexes with the general structure shown in Fig. 1 offer tremendous flexibility, since their metal binding affinities can be varied by inserting appropriate donor atoms to match the requirements of various coordinated metals, including Fe(III), thereby enhancing stability of the intact complex (31, 40). We report that these compounds comprise a new class of readily synthesized antimalarials that blocks hemozoin formation. Additionally, while several agents were effective against chloroquine-sensitive and -resistant clones, one agent showed paradoxically selective toxicity solely against chloroquine-resistant clones in a non-pfmdr1-dependent manner.

MATERIALS AND METHODS

All reagents, solvents, and metal salts (nitrates and acetylacetonates of Al(III), Fe(III), Ga(III), and In(III)) were obtained from Aldrich or Alpha Chemical Co. as analytical grade materials. Heptadentate precursors of the desired Schiff base hexadentate ligands (1, Fig. 1) were obtained by condensation of the appropriate linear tetramine and three equivalents of substituted salicylaldehyde in ethanol or in dry methylene chloride as described previously (31). A bis-salicylaldamine analog of 1 was obtained by condensation of the appropriate linear tetramine and two equivalents of 3-methoxy-substituted salicylaldehyde followed by in situ reduction of the Schiff base as described (40).² Heptadentate Schiff base precursors and Schiff base reduced analogs were characterized by ¹H and ¹³C NMR, infrared (IR), and mass spectrometry (fast atom bombardment-high resolution mass spectrometry) at the Washington University Resource for Biomedical and Bioorganic Mass Spectrometry Facility (31).

Metal complexes were obtained through the reactions of Schiff base precursors and reduced analogs with the appropriate metal salts in ethanol as described previously (31). Resulting metal(III) complexes were characterized by ¹H NMR (except for Fe(III) complexes), IR, fast atom bombardment-low resolution spectrometry and elemental analysis establishing structural identity and confirming purity.

Plasmodium falciparum lines (HB3, FCR-3, Indochina-1/CDC (Indo-1), Dd2 and progeny of the HB3xDd2 cross (39)) were grown in intra-erythrocytic culture by the method of Trager and Jensen (41). Cultures were maintained at 5% parasitemia, 2% hematocrit using human serum and erythrocytes, in a 3% oxygen, 3% carbon dioxide atmosphere. Synchronization of developmental stage was achieved by sorbitol treatment (42). Parasite growth inhibition and half-maximal inhibitory concentration values (IC₅₀) of antagonists were determined by measuring [³H]hypoxanthine incorporation (43). Parasites were incubated with antagonist for 24 h starting at the late ring stage, and then [³H]hypoxanthine was added for 4 h at the mid-trophozoite stage before harvesting parasites and assaying for incorporated radioactivity. To assay for verapamil reversal, parasites were incubated with antagonist ± verapamil for 72 h, and then [³H]hypoxanthine was added. All drugs were added as 1:1000 dilutions of a 10 mM dimethyl sulfoxide stock. Vehicle alone had no effect on [³H]hypoxanthine incorporation.

The procedure used was modified from those of Slater and Cerami (14) and Dorn et al. (44). Hemin (50 µM) was incubated with parasite-derived hemozoin (2 µg/ml) in 100 mM sodium acetate, pH 5.0 (final volume 0.5 ml). After 16 h at 37 °C, product was harvested by centrifugation at 4 °C, 15,000 × g for 30 min. The pellet was resuspended by brief sonication in 1 ml of 2.5% sodium dodecyl sulfate (SDS) in 0.1 M sodium bicarbonate, pH 9.1, with incubation at 37 °C for 30 min. Centrifugation was repeated and the resultant pellet washed in 1 ml of 2.5% SDS with sonication. After centrifugation, the pellet was solubilized with 2.5% SDS, 50 mN NaOH (1 h at room temperature with intermittent mixing). Product was measured spectrophotometrically (A) at 400 nm. Control incubations had no added hemin or hemin alone, and the sum of their adsorption values was subtracted. Individual inhibitors or chloroquine was added directly to the incubations and was compared with incubations containing drug vehicle (dimethyl sulfoxide) alone. The histidine-rich protein II (HRP II)-mediated polymerization assay was performed as described previously (11) using 500 pmol of protein incubated at 37 °C for 16 h in a 1-ml reaction with 50 µM hematin.

The digestive vacuole proteases plasmepsins I and II and falcipain were purified and assayed using [¹⁴C]globin substrate as described previously (9). Inhibitors were added as 1:1000 dilutions of a 10 mM dimethyl sulfoxide stock.

RESULTS

All reagents and starting materials were inexpensive and commercially available, favorable practical considerations for the development of these agents. The synthesis and characterization of these compounds are described elsewhere (31, 40). An appealing property of the ethylenediamine-N,N-bis[propyl(2-hydroxy-(R)-benzylimino)] ligand 1 ((R)-ENBPI; Fig. 1) and the corresponding Schiff base reduced amino analog ((R)-ENBPA; Fig. 1) is their ability to efficiently form stable "holo-complexes" with a wide range of metal ions (31, 40). Specifically, we synthesized complexes of the general structure 2 coordinating Al(III), Fe(III), Ga(III), and In(III) from the reactions of heptadentate Schiff base precursors and Schiff base amino derivatives with corresponding metal(III) acetylacetonates or hydrated metal(III) salts in refluxing ethanol or methanol, respectively. Formation of these metal(III) complexes from the ligands 1 (Fig. 1) results in formation of racemic mixtures of stereoisomers (31, 45).

Because of the importance of Fe(III) in malarial metabolism and relative biocompatibility of this metal with the host, Fe(III) compounds were the target leads tested against P. falciparum trophozoites in intra-erythrocytic culture. P. falciparum killing curves for the 4,6-dimethoxy-ENBPI Fe(III) derivative are shown in Fig. 2A. IC₅₀ values of 1 to 1.5 µM were obtained against chloroquine-sensitive HB3 as well as against resistant FCR-3, Indo-1, and Dd2 lines. Control experiments with chloroquine confirmed that the HB3 line was sensitive, whereas the others were chloroquine-resistant (Table I, bottom). While the 3-methoxy-ENBPI Fe(III) analog was as potent as the 4,6-dimethoxy analog against the chloroquine-sensitive line, the agent was less effective against the resistant FCR-3 and Indo-1 lines (Table I). Parasite death as measured by the [³H]hypoxanthine incorporation assay correlated well with direct blood smear counts. The drugs were also observed to be most effective at the mid-trophozoite stage. For example, using the 4,6-dimethoxy derivative, more mature parasites were resistant and less mature parasites grew normally until they developed into trophozoites, at which point they were killed by the treatment (Fig. 2B). Parasites cultured in the presence of drug showed greatly diminished hemozoin formation. In contrast, control parasites matured normally, developing a large digestive vacuole filled with hemozoin pigment, and then underwent normal schizogony.

Table I. Antimalarial activity of (R)-ENBPI metal(III) complexes against chloroquine-sensitive (CQS) and -resistant (CQR) P. falciparum clones in vitro Effect of various (R)-ENBPI metal(III) complexes and chloroquine (bottom) on intraerythrocytic P. falciparum growth in culture. The substituent nomenclature is referenced to Fig. 1. Agents were tested at the indicated concentrations against chloroquine-sensitive and -resistant lines by the [3H]hypoxanthine incorporation method; values are presented as percent growth inhibition relative to control cultures without drug. Data are presented as the mean of triplicate determinations from representative experiments. NAA, no antimalarial activity; R5 = H.  Metal (R)-ENBPI complexes Concentration Clones Substitutions HB3 (CQS) FCR-3 (CQR) Indo-1 (CQR) R3 R4 R6 µM % growth inhibition Fe(III) OMe H H 5 90.4 31.1 51.6 Fe(III) H OMe OMe 5 99.9 96.1 96.1 In(III) H OMe OMe 5 NAA NAA NAA Chloroquine 0.02 74.5 NAA NAA

We therefore assessed the ability of drug to block heme polymerization in an in vitro assay. Heme was incubated with pre-formed hemozoin under acidic conditions in the presence of varying concentrations of the 4,6-dimethoxy-ENBPI Fe(III) complex. The compound was a potent polymerization inhibitor with an IC₅₀ of ~4 µM (Table II). Control experiments confirmed an identical IC₅₀ for chloroquine. Recent data suggest that the parasite's HRP II can initiate heme polymerization in the Plasmodium digestive vacuole (11). The 4,6-dimethoxy-ENBPI Fe(III) complex showed equivalent potency in blocking polymerization using the HRP II assay (IC₅₀ ~ 4 µM).

Table II. Inhibition of Plasmodium culture (HB3) and hemozoin formation by (R)-ENBPI metal(III) complexes correlates with MDR1mediated drug resistance IC50 values for inhibition of parasite growth and hemozoin formation are tabulated from this study and compared with MDR1-mediated drug resistance values recalculated from data (31). Derived from studies in human KB cells, the MDR1 data indicate the fold resistance (IC50 in MDR cells/IC50 in drug-sensitive cells; (34)) that expression of MDR1 P-glycoprotein conferred on the cytotoxic potency of the indicated metal(III) complexes. ENBPI ligand Metal Parasite culture IC50 Hemozoin IC50 MDR1-mediated fold resistance µM µM (R) = 3-MeO Fe(III) 2 4 77 Ga(III) >20 50 2.6 Al(III) >20 30 2.6 In(III) >20 >50 1.4 (R) = 4,6-Di-MeO Fe(III) 1 4 >200 Ga(III) 3 2 >200 Al(III) 3 3 >200 In(III) >20 40 1.0

To determine whether coordination of Fe(III) per se was critical for antimalarial activity or whether overall conformations of the intact metallopharmaceutical conferred the desired effect, parasite culture and heme polymerization inhibition curves were also generated for 3-methoxy- and 4,6-dimethoxy-ENBPI complexes using a variety of coordinated metals including Ga(III), Al(III), and In(III) (Table II). An excellent correlation was found between inhibition of hemozoin formation and ability to kill the Plasmodium culture for all complexed metal species. In particular, note that the 3-methoxy-ENBPI Ga(III) complex was a relatively poor inhibitor of both HB3 culture growth and heme polymerization, whereas the 4,6-dimethoxy-ENBPI Ga(III) analog was a potent inhibitor in both assays. Furthermore, the 3-methoxy-ENBPI Ga(III) complex was equally ineffective against the chloroquine-resistant FCR-3 line, whereas the 4,6-dimethoxy-ENBPI Ga(III) analog was equally potent (data not shown). Interestingly, the potency of inhibition of both heme polymerization and HB3 Plasmodium culture correlated well with the fold resistance to the agents induced by expression of the human MDR1 P-glycoprotein in a cancer cell cytotoxicity assay (31) (Table II).

To further determine if demetallation reactions were potentially involved in the mechanism of inhibition of heme polymerization, a variety of metal salts were directly tested in the hemozoin assay (Table III). Although ferric Fe(III) and Al(III) cations were potent inhibitors of the process (IC₅₀ values of 0.7 µM and 1.5 µM, respectively), Ga(III) had little effect (IC₅₀ = 100 µM), despite the substantial antagonist activity of (R)-ENBPI Ga(III) complexes. Conversely, while the In(III) cation showed modest inhibition (IC₅₀ = 25 µM), the (R)-ENBPI In(III) complexes were completely ineffective. With the exception of modest activity of Fe(II), a variety of other physiological dicationic metals were ineffective (Table III). Control experiments demonstrated that the counter anions (iodide, nitrate, and perchlorate) were also without effect (data not shown). Therefore, the lack of correlation between potency of metal(III) salts and their corresponding (R)-ENBPI metal(III) complexes for inhibition of heme polymerization again pointed toward the intact holo-complex as the biochemically active component.

Table III. Effect of metal salts (10 µM) on heme polymerization Inhibition of heme polymerization by metal salts. Heme polymerization was assayed by the preformed hemozoin nucleation reaction (see "Materials and Methods") in the absence or presence of 10 µM concentrations of the indicated metal salts. Data are mean values ± S.E. of duplicate determinations from representative experiments. Metal % inhibition of heme polymerization Fe2+ 54  ± 2 Fe3+ 100  ± 0 Al3+ 94  ± 12 In3+ 22  ± 1 Ga3+ 0  ± 0 Co2+  1  ± 12 Cu2+  3  ± 2 Ni2+  9  ± 2 Zn2+ 5  ± 4 Ca2+  6  ± 5 Mn2+ 2  ± 1 Mg2+ 0  ± 1

Because of the association between (R)-ENBPI metal(III) complexes and P-glycoprotein transport, and since the agents appeared to target the digestive vacuole in Plasmodium culture, assays were performed to examine possible inhibition of vacuolar protease action. The 4,6-dimethoxy-ENBPI Fe(III) complex at 10 µM had no effect on the globin cleavage activities of aspartic proteases plasmepsins I and II nor of the cysteine protease falcipain (data not shown), each purified from cultured parasites.

To explore additional aspects of structure-activity relationships, the reduced amine 3-methoxy-ENBPA Ga(III) complex (MR045) was synthesized. As with its imine analog (Table II), there was relatively poor activity against the chloroquine-sensitive HB3 clone in culture (Fig. 3; IC₅₀  20 µM). However, and quite remarkably, the compound was active against the chloroquine-resistant Dd2 clone (Fig. 3) as well as other resistant lines (IC₅₀ = 0.5-1 µM). Similarly, the agent was a potent inhibitor of hemozoin formation in vitro (IC₅₀ ~1 µM; data not shown). Control experiments confirmed that verapamil, a known reversal agent (46), indeed reversed chloroquine resistance in Dd2, while showing no effect on HB3 (Dd2: chloroquine (40 nM), 10% growth inhibition; verapamil (0.6 µM), 25%; chloroquine (40 nM) plus verapamil (0.6 µM), 87%. HB3: chloroquine (40 nM), 99% growth inhibition; verapamil (0.6 µM), 6%; chloroquine (40 nM) plus verapamil (0.6 µM), 99%. S.E. ± 2%; n = 3 each). However, verapamil did not alter MR045 susceptibility in chloroquine-resistant Dd2 nor reverse MR045 resistance of HB3 (Dd2: MR045 (1 µM), 99% growth inhibition; MR045 (1 µM) plus verapamil (0.6 µM), 99%. HB3: MR045 (1 µM), 11% growth inhibition; verapamil (0.6 µM), 12%; MR045 (1 µM) plus verapamil (0.6 µM), 34%. S.E. ± 2%; n = 3 each). To reconcile these hemozoin and verapamil data requires that the (R)-ENBPA Ga(III) complex, but not chloroquine, was acted upon by a verapamil-insensitive export or sequestration mechanism in HB3 parasites, whereas chloroquine, but not the Ga(III) complex, was acted upon by a verapamil-sensitive mechanism in Dd2 parasites.

To further characterize the basis of this selectivity, the 3-methoxy-ENBPA Ga(III) complex was tested against 21 independent recombinant progeny of a genetic cross between sensitive HB3 and chloroquine-resistant Dd2 lines (39). The pfmdr1 gene, which encodes Pgh1, has been shown not to map with the chloroquine resistance phenotype in this cross. In particular, a BamHI polymorphism at the 3 end of pfmdr1 that uniquely identifies the inherited Dd2 parental gene is found in four clones (TC-05, QC-23, SC-01, and 3B-D5) of eight chloroquine-resistant progeny and in three clones (3B-B1, B1-SD, and TC-08) of eight chloroquine-sensitive progeny indicating that a gene separate from pfmdr1 is the major determinant of chloroquine resistance (39). In addition to these 16 published clones, included herein are five more progeny from this same cross (C188, CH3-61, 116, C408, and D43) in which the inheritance linkage of the chloroquine resistance phenotype has been further refined. Chloroquine-resistant clone C408 and chloroquine-sensitive clone C188 contain genetic crossover markers that localize the chloroquine resistance determinant to a 36-kilobase segment of chromosome 7.³ Control culture growth experiments confirmed chloroquine sensitivity for three clones (C188, CH3-61, 116; chloroquine (40 nM): >97 ± 1% growth inhibition) and chloroquine resistance for the remaining two clones (C408 and D43; chloroquine (40 nM): 3 ± 2% and 14 ± 5% growth inhibition, respectively). As shown in Fig. 4, all of the chloroquine-sensitive progeny were resistant to the ENBPA Ga(III) complex, whereas all of the chloroquineresistant progeny were susceptible to the new agent at 5 µM. This evidence indicated that the chloroquine resistance locus and the MR045 susceptibility locus of chromosome 7 were exclusively co-inherited in these haploid progeny.

DISCUSSION

Disruption of hemozoin formation within the digestive vacuole requires that agents such as chloroquine be transported across several membrane bilayers, including the erythrocyte plasma membrane, the parasitophorous vacuolar membrane, the parasite plasma membrane, and finally, the digestive vacuole membrane. Detailed mechanisms of chloroquine transport and their contributions to drug resistance have yet to be fully understood. Chloroquine is modestly lipophilic and possesses titratable protons that confer a net positive charge in acid environments. Thus, it has been proposed that diffusive transmembrane transport and drug trapping may account for concentrative accumulation of chloroquine within the digestive vacuole (reviewed in Ref. 47). The net cationic charge provided by protonation of the agent or binding to high affinity sites within the vacuole (ferriprotoporphyrins or hemozoin) may prevent back diffusion of the compound (13, 48). Other contributions to chloroquine uptake and antimalarial action may involve its properties as an amphiphilic cation which enables adsorption onto phospholipid bilayers (49) and, as with many hydrophobic compounds possessing a delocalized monocationic charge (50, 51), may allow permeation across membranes and concentrative accumulation within cell interiors in response to the negative transmembrane potentials generated by living cells (52). Additionally, membrane transporters may contribute either directly or indirectly as has been proposed, for example, with pfmdr1 (38, 53). Thus, interfering with or bypassing any one of the steps that regulate drug permeation or the site of action could confer chloroquine resistance.

We report herein the identification of a new class of antimalarial compounds designed to permeate bilayers that demonstrate potent activity in Plasmodium culture and, significantly, against both chloroquine-sensitive and chloroquine-resistant clones. Antimalarial potency of these compounds in culture correlated with their ability to inhibit heme polymerization, the most potent leads being the 4,6-dimethoxy-ENBPI Fe(III) and the 3-methoxy-ENBPA Ga(III) complexes. For the imino complexes, agents that lack significant parasitocidal activity in Plasmodium culture do not inhibit hemozoin formation in vitro. It is unlikely that this can be attributed to differential demetallation reactions of the agents, since this study demonstrated poor correlation between metal(III) salts and intact ENBPI metal(III) complexes for inhibition of hemozoin formation under acidic conditions, and furthermore, the agents have been documented by ¹H NMR and UV-visible spectroscopy to be hydrolytically stable at neutral pH (37 °C) for 72 h (54). Molecular configuration may be relevant. Crystal structures of (R)-ENBPI Fe(III) and Ga(III) analogs show a trans configuration for the phenolic oxygens around the central coordination sphere (as in Fig. 1) (29, 55). However, preliminary molecular modeling data suggest that the identity of the coordination metal in the central core has a profound influence on conformation of the molecule (54). Given the larger ionic radii of six-coordinated In(III) versus Fe(III) and Ga(III) (0.81 Å versus 0.65 and 0.62 Å, respectively) (56), molecular modeling data suggest that steric constraints favor the cis configuration for the (R)-ENBPI Indium(III) compounds (54). Thus, several compounds preferring the trans configuration are recognized by human MDR1 P-glycoprotein (31), are able to block heme polymerization, and can produce parasite death as shown herein, whereas those favoring the cis configuration cannot, potentially tying together these various biological processes into a common metabolic pathway. While data suggest that conformations of the intact holo-complexes confer pharmacological activity, additional experiments will be required to fully evaluate the potential for acid hydrolysis and demetallation reactions in the digestive vacuole on the overall mechanism of action.

The IC₅₀ values for culture inhibition are in some cases slightly lower than those for in vitro heme polymerization. Several possibilities could explain this observation. First, the conditions for heme polymerization in the digestive vacuole could be sufficiently different from those in the assay to change the IC₅₀ value. Second, the compounds could be accumulated against a concentration gradient in the digestive vacuole. Third, it is possible that blocking only a small amount of heme polymerization leads to build-up of enough toxic free heme to kill the intact parasites.

The (R)-ENBPI metal(III) complexes had equivalent potency in the pre-formed hemozoin-initiated polymerization assay as in the HRP II-mediated assay. These were similar to results obtained for chloroquine. It has been shown that heme polymerization can be initiated by HRP II-mediated bonding of hemes (11). Once nucleation has occurred, polymerization can proceed nonenzymatically. Since both the (R)-ENBPI metal(III) complexes and chloroquine work on the hemozoin-seeded (protein-free) as well as the protein-mediated reactions, this suggests that the blockade is either at the nonenzymatic polymer extension phase of the reaction or a direct effect on the heme substrate. Detailed kinetics will be required to distinguish these possibilities.

An unanticipated result of this study, as shown in Fig. 4, was the observation that one (R)-ENBPA Ga(III) complex (MR045), which possessed poor antimalarial activity against sensitive clones, showed selective and potent activity against all chloroquine-resistant progeny of a genetic cross (39). This suggested that a gene for 3-methoxy-ENBPA Ga(III) susceptibility is located on the same 36-kilobase segment of chromosome 7 as the chloroquine resistance gene and likely is the same gene. Of note, the Schiff base (R)-ENBPI Ga(III) analog of this compound showed relatively poor cytotoxicity against both resistant and sensitive lines. Thus, the data indicate that the mechanism that confers chloroquine resistance in these organisms is selectively targeted by the 3-methoxy-ENBPA Ga(III) complex. One possibility is that a transport system is altered in chloroquine resistance (57), by mutation of either the transporter or an associated transport regulator. Because chloroquine resistance maps away from the pfmdr1 gene in this cross (39), the data further imply that the chloroquine resistance activity cannot be attributed to the Pgh1 transporter. Additionally, unlike chloroquine, selective blockade of the chloroquine-resistant Dd2 line by MR045 was verapamil-insensitive. Thus, the unusual properties of 3-methoxy-ENBPA Ga(III) can be exploited to probe the mechanisms of chloroquine resistance.

Clearly, additional experiments must be done to better understand the targeting of these agents, their mechanism of selectivity, and their bioavailability. There is great potential to vary the tetramine backbone and to substitute in a variety of positions on the aromatic ring in this class of compounds. The agents are relatively easy and inexpensive to synthesize, which is a crucial feature for success of a useful reagent or antimalarial drug. It is encouraging that a group of agents with scaffolds capable of incorporating biologically compatible metals like Fe(III) can act against the same putative molecular target as chloroquine but are not susceptible to the same drug resistance mechanisms.