Derivatives of a benzoquinone acyl hydrazone with activity against Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite with global incidence. The acute infection, toxoplasmosis, is treatable but current regimens have poor host tolerance and no cure has been found for latent infections. This work builds upon a previous high throughput screen which identified benzoquinone acyl hydrazone (KG8) as the most promising compound; KG8 displayed potent in vitro activity against T. gondii but only marginal in vivo efficacy in a T. gondii animal model. To define the potential of this new lead compound, we now describe a baseline structure-activity relationship for this chemotype. Several derivatives displayed IC50's comparable to that of the control treatment pyrimethamine with little to no cytotoxicity. The best of these, KGW44 and KGW59, had higher metabolic stability than KG8. In an in vivo T. gondii murine model, KGW59 significantly increased survivorship. This work provides new insights for optimization of this novel chemotype.

Novel Toxoplasma gondii inhibitor chemotypes.

We profiled three novel T. gondii inhibitors identified from an antimalarial phenotypic high throughput screen (HTS) campaign: styryl 4-oxo-1,3-benzoxazin-4-one KG3, tetrahydrobenzo[b]pyran KG7, and benzoquinone hydrazone KG8. These compounds inhibit T. gondii in vitro with IC50 values ranging from 0.3 to 2µM, comparable to that of 0.25 to 1.5µM for the control drug pyrimethamine. KG3 had no measurable cytotoxicity against five mammalian cell lines, whereas KG7 and KG8 inhibited the growth of 2 of 5 cell lines with KG8 being the least selective for T. gondii. None of the compounds were mutagenic in an Ames assay. Experimental gLogD7.4 and calculated PSA values for the three compounds were well within the ranges predicted to be favorable for good ADME, even though each compound had relatively low aqueous solubility. All three compounds were metabolically unstable, especially KG3 and KG7. Multiple IP doses of 5mg/kg KG7 and KG8 increased survival in a T. gondii mouse model. Despite their liabilities, we suggest that these compounds are useful starting points for chemical prospecting, scaffold-hopping, and optimization.

Short report: floxacrine analog WR 243251 inhibits hematin polymerization.

Floxacrine was a promising antimalarial compound that led to the identification of WR 243251. On the basis of their structures, we suspected that these compounds might be good inhibitors of hematin polymerization. Indeed, WR 243251 was as potent and floxacrine was only 2-fold less potent than chloroquine as inhibitors of this process. However, this hematin polymerization inhibition did not completely account for the increased antimalarial potency of WR 243251 versus chloroquine. The WR 243251 ketone hydrolysis product WR 243246 was without activity against hematin polymerization. These data also confirm that hematin polymerization inhibition can be quite sensitive to small changes in inhibitor structure.

Structural analysis of chloroquine resistance reversal by imipramine analogs.

For imipramine, desipramine, and eight analogs of these well-known drugs, an N-5-aminoalkyl substitution was a minimum but insufficient structural feature associated with chloroquine resistance reversal. Although a second distal aliphatic nitrogen atom was unnecessary for resistance reversal, the direction of the dipole moment vector was critical.

Synthesis and solution properties of deferoxamine amides.

The poor membrane permeability and oral bioavailability of the iron chelating agent deferoxamine (DFO) mesylate result from the low octanol/water partition coefficient and high aqueous solubility. With the ultimate aim to improve biomembrane permeability while retaining the iron-binding ability of DFO, a series of more lipophilic amides were prepared by reacting the terminal primary amino group with fatty and aromatic acid chlorides or anhydrides. Octanol/water partition coefficients and equilibrium solubilities of these analogs in solvents, chosen to delineate physicochemical interactions, were determined as a function of temperature. Solid-state properties were evaluated by calorimetry. All DFO amide derivatives had higher melting points, indicating that derivatives formed strong intermolecular interactions in the solid phase. Formamidation of the primary amine of deferoxamine resulted in a 200-fold increase in the octanol/water partition coefficient and reduced aqueous solubility at least 2000-fold compared with the parent molecule. The partition coefficient increased and aqueous solubility decreased 2-fold with the addition of each methylene group in the homologous series of aliphatic amides. Solubilities of the derivatives in water-saturated octanol and hexane showed irregular profiles as a function of increasing aliphatic chain length that were attributed to intermolecular packing in the solid state. The temperature dependence of the partition coefficients was interpreted to indicate that interfacial transfer of the deferoxamine amides was, in part, affected by an apparent diminished ability to form energetically favorable interactions in the water-saturated organic phase.

Synthesis and antimalarial activity of sixteen dispiro-1,2,4, 5-tetraoxanes: alkyl-substituted 7,8,15,16-tetraoxadispiro[5.2.5. 2]hexadecanes.

Sixteen alkyl-substituted dispiro-1,2,4,5-tetraoxanes (7,8,15, 16-tetraoxadispiro[5.2.5.2]hexadecanes) were synthesized to explore dispiro-1,2,4,5-tetraoxane SAR and to identify tetraoxanes with better oral antimalarial activity than prototype tetraoxane 1 (WR 148999). The tetraoxanes were prepared either by peroxidation of the corresponding cyclohexanone derivatives in H(2)SO(4)/CH(3)CN or by ozonolysis of the corresponding cyclohexanone methyl oximes. Those tetraoxanes with alkyl substituents at the 1 and 10 positions were formed as single stereoisomers, whereas the five tetraoxanes formed without the stereochemical control provided by alkyl groups at the 1 and 10 positions were isolated as mixtures of diastereomers. Three of the sixteen tetraoxanes were inactive (IC(50)'s > 1000 nM), but five (2, 6, 10, 11, 12) had IC(50)'s between 10 and 30 nM against the chloroquine-sensitive D6 and chloroquine-resistant W2 clones of Plasmodium falciparum compared to corresponding IC(50)'s of 55 and 32 nM for 1 and 8.4 and 7.3 nM for artemisinin. We suggest that tetraoxanes 13, 16, and 17 were inactive and tetraoxanes 4 and 7 were weakly active due to steric effects preventing or hindering peroxide bond access to parasite heme. Tetraoxanes 1, 10, 11, and 14, along with artemisinin and arteether as controls, were administered po b.i.d. (128 mg/kg/day) to P. berghei-infected mice on days 3, 4, and 5 post-infection. At this dose, tetraoxanes 10, 11, and 14 cured between 40% and 60% of the infected animals. In comparison, artemisinin and tetraoxane 1 produced no cures, whereas arteether cured 100% of the infected animals. There was no apparent relationship between tetraoxane structure and in vitro neurotoxicity, nor was there any correlation between antimalarial activity and neurotoxicity for these seventeen tetraoxanes.

Characterization of chloroquine-hematin mu-oxo dimer binding by isothermal titration calorimetry.

Numerous studies indicate that a key feature of chloroquine's (CQ) antimalarial activity is its interaction with hematin. We now characterize this CQ-hematin interaction in detail using isothermal titration calorimetry (ITC). Between pH 5.6 and 9.0, association constants (K(a) values) for enthalpy-driven CQ-hematin mu-oxo dimer binding fell in the narrow range of 2.3-4.4 x 10(5) M(-1). It is therefore probable that CQ-hematin mu-oxo dimer binding affinity does not diminish at the pH range (4.8-5.4) of the parasite food vacuole. The binding affinity was unaffected by high salt concentrations, suggesting that ionic interactions do not contribute significantly to this complexation. With increasing ionic strength, the entropic penalty of CQ-hematin mu-oxo dimer binding decreased accompanied by increased hematin mu-oxo dimer aggregation. A stoichiometry (n) of 1:4 in the pH range 6.5-9.0 indicates that CQ binds to two hematin mu-oxo dimers. At pH 5.6, a stoichiometry of 1:8 suggests that CQ binds to an aggregate of four hematin mu-oxo dimers. This work adds further evidence supporting the hypothesis that CQ impedes hematin monomer incorporation into hemozoin by producing a forward shift in the hematin monomer-hematin mu-oxo dimer equilibrium, contributing to a destructive accumulation of soluble forms of hematin in the parasite and leading to its death by hematin poisoning.

Methyl-substituted dispiro-1,2,4,5-tetraoxanes: correlations of structural studies with antimalarial activity.

Two tetramethyl-substituted dispiro-1,2,4,5-tetraoxanes (7,8,15, 16-tetraoxadispiro[5.2.5.2]hexadecanes) 3 and 4 were designed as metabolically stable analogues of the dimethyl-substituted dispiro-1, 2,4,5-tetraoxane prototype WR 148999 (2). For a positive control we selected the sterically unhindered tetraoxane 5 (7,8,15, 16-tetraoxadispiro[5.2.5.2]hexadecane), devoid of any substituents. Tetraoxanes 3 and 4 were completely inactive in contrast to tetraoxanes 2 and 5. We hypothesize that the two inactive tetraoxanes possess sufficient steric hindrance about the tetraoxane ring due to the two additional axial methyl groups to prevent their activation to presumed parasiticidal carbon radicals by inhibiting electron transfer from heme or other iron(II) species. For each of the tetraoxanes 2-4, the tetraoxane and both spirocyclohexyl rings are in a chair conformation and the bond lengths and angles are all quite normal except for the C1-C2 bond which is slightly lengthened. Comparison of the modeled and X-ray structures for tetraoxanes 2-5 reveals that molecular mechanics (MMX and MM3) and 3-21G calculations each gave accurate structural parameters such as bond lengths, bond angles, and dihedral angles. In contrast, semiempirical methods such as AM1 gave poor results.

Assessment of the antimalarial potential of tetraoxane WR 148999.

The antimalarial peroxide, dispiro-1,2,4,5-tetraoxane WR 148999, was synergistic with chloroquine, quinine, mefloquine, and artemisinin against both D6 and W2 clones of Plasmodium falciparum. In consideration of the contrasting antagonism between artemisinin and chloroquine, these drug combination data imply that WR 148999 and artemisinin may not share a common mechanism of action. For Plasmodium berghei-infected mice given oral, subcutaneous, and intraperitoneal doses of WR 148999 ranging from 2 to 1024 mg/kg in the Thompson test, median survival times were 8.8, 11.8, and 27.5 days, respectively, compared to 8 days for control animals. Using subcutaneous administration, WR 148999 had a considerably longer duration of action than did artemisinin against P. berghei. WR 148999 did not significantly inhibit cytochrome P450 isozymes CYP 2C9, 2C19, 2D6, 2E1, or 3A4 (IC50 >500 microM) but did inhibit CYP 1A2 with an IC50 value of 36 microM, suggesting that WR 148999 may be metabolized by the latter CYP isozyme. These results combined with previous observations that formulation strategies and incorporation of polar functional groups in a series of WR 148999 analogs both failed to enhance tetraoxane oral antimalarial activity suggest that oral bioavailability of tetraoxane WR 148999 is more likely a function of extensive first-pass metabolism rather than solubility-limited dissolution.

Structural specificity of chloroquine-hematin binding related to inhibition of hematin polymerization and parasite growth.

Considerable data now support the hypothesis that chloroquine (CQ)-hematin binding in the parasite food vacuole leads to inhibition of hematin polymerization and parasite death by hematin poisoning. To better understand the structural specificity of CQ-hematin binding, 13 CQ analogues were chosen and their hematin binding affinity, inhibition of hematin polymerization, and inhibition of parasite growth were measured. As determined by isothermal titration calorimetry (ITC), the stoichiometry data and exothermic binding enthalpies indicated that, like CQ, these analogues bind to two or more hematin mu-oxo dimers in a cofacial pi-pi sandwich-type complex. Association constants (K(a)'s) ranged from 0.46 to 2.9 x 10(5) M(-1) compared to 4.0 x 10(5) M(-1) for CQ. Remarkably, we were not able to measure any significant interaction between hematin mu-oxo dimer and 11, the 6-chloro analogue of CQ. This result indicates that the 7-chloro substituent in CQ is a critical structural determinant in its binding affinity to hematin mu-oxo dimer. Molecular modeling experiments reinforce the view that the enthalpically favorable pi-pi interaction observed in the CQ-hematin mu-oxo dimer complex derives from a favorable alignment of the out-of-plane pi-electron density in CQ and hematin mu-oxo dimer at the points of intermolecular contact. For 4-aminoquinolines related to CQ, our data suggest that electron-withdrawing functional groups at the 7-position of the quinoline ring are required for activity against both hematin polymerization and parasite growth and that chlorine substitution at position 7 is optimal. Our results also confirm that the CQ diaminoalkyl side chain, especially the aliphatic tertiary nitrogen atom, is an important structural determinant in CQ drug resistance. For CQ analogues 1-13, the lack of correlation between K(a) and hematin polymerization IC(50) values suggests that other properties of the CQ-hematin mu-oxo dimer complex, rather than its association constant alone, play a role in the inhibition of hematin polymerization. However, there was a modest correlation between inhibition of hematin polymerization and inhibition of parasite growth when hematin polymerization IC(50) values were normalized for hematin mu-oxo dimer binding affinities, adding further evidence that antimalarial 4-aminoquinolines act by this mechanism.

Deferoxamine: stimulation of hematin polymerization and antagonism of its inhibition by chloroquine.

The iron chelator deferoxamine enhances the clearance of Plasmodium falciparum parasitemia and may be useful in drug combinations for the treatment of cerebral malaria. However, the deferoxamine-chloroquine drug combination is antagonistic, or at best additive, against P. falciparum in vitro. As chloroquine is thought to exert its antimalarial activity by interacting with hematin released from the proteolytic degradation of hemoglobin in the parasite food vacuole, we hypothesized that deferoxamine might interfere with the ability of chloroquine to inhibit hematin polymerization, since it was reported that deferoxamine interacts with hematin. Therefore, we assessed deferoxamine-hematin binding in more detail and investigated the effect of deferoxamine on hematin polymerization in the presence and absence of chloroquine. Isothermal titration calorimetry (ITC) experiments demonstrated an enthalpy-driven deferoxamine:hematin mu-oxo dimer binding with an association constant of 2.8 x 10(4) M(-1) at pH 6.5, a binding affinity 14-fold lower than that measured for chloroquine. At least two of the three hydroxamic acid functional groups of deferoxamine must be unionized for effective binding. We also discovered that deferoxamine antagonized chloroquine-mediated inhibition of hematin polymerization. Unexpectedly, deferoxamine increased the concentration of soluble forms of hematin and enhanced the rate of hematin polymerization. Deferoxamine also could initiate hematin polymerization. In contrast, chloroquine decreased the concentration of soluble forms of hematin and inhibited hematin polymerization. This work supports the postulate that initiation of hematin polymerization requires a higher concentration of soluble hematin monomer than does the elongation phase of polymerization and provides one possible explanation for the observed antagonism between deferoxamine and chloroquine against parasites in culture.

Synthesis and antimalarial activity of 11 dispiro-1,2,4,5-tetraoxane analogues of WR 148999. 7,8,15,16-Tetraoxadispiro[5.2.5.2]hexadecanes substituted at the 1 and 10 positions with unsaturated and polar functional groups.

Eleven novel dispiro-1,2,4,5-tetraoxanes 3 bearing unsaturated and polar functional groups were designed to enhance the oral antimalarial activity of the prototype tetraoxane 2 (WR 148999). With the exception of 3g and 3h, tetraoxanes 3 were available via the peroxidation of corresponding cyclohexanone derivatives in H2SO4/CH3CN. Tetraoxanes 3g and 3h were prepared by hydrolysis of ester tetraoxanes 3e and 3i, respectively. Five of the 11 tetraoxanes were inactive, but six tetraoxanes had IC50 values of 6-26 nM against the K1 and NF54 strains of Plasmodium falciparum compared to corresponding IC50 values of 28 and 39 nM for 2, and 10 and 12 nM for artemisinin (1). Ester tetraoxane 3e was the most active in vitro, some 2-fold more potent than 1. However, none of the six tetraoxanes active in vitro were as effective as either 1 or 2 in vivo; at single doses of 100 mg/kg most possessed little to no vivo activity in mice infected with Plasmodium berghei. Unsaturated tetraoxane 3a was uniquely more active when administered per os (po) than subcutan (sc). For this series of tetraoxanes, the discrepancy between vitro and vivo activities underscores the limitations of conclusions drawn solely from in vitro antimalarial data and illustrates a practical benefit of complementary single-dose in vivo antimalarial screens.

8-Aminoquinolines active against blood stage Plasmodium falciparum in vitro inhibit hematin polymerization.

From the Walter Reed Army Institute of Research (WRAIR) inventory, thirteen 8-aminoquinoline analogs of primaquine were selected for screening against a panel of seven Plasmodium falciparum clones and isolates. Six of the 13 8-aminoquinolines had average 50% inhibitory concentrations between 50 and 100 nM against these P. falciparum clones and were thus an order of magnitude more potent than primaquine. However, excluding chloroquine-resistant clones and isolates, these 8-aminoquinolines were all an order of magnitude less potent than chloroquine. None of the 8-aminoquinolines was cross resistant with either chloroquine or mefloquine. In contrast to the inactive primaquine prototype, 8 of the 13 8-aminoquinolines inhibited hematin polymerization more efficiently than did chloroquine. Although alkoxy or aryloxy substituents at position 5 uniquely endowed these 13 8-aminoquinolines with impressive schizontocidal activity, the structural specificity of inhibition of both parasite growth and hematin polymerization was low.

Depentylation of [3H-pentyl]methyl-n-amylnitrosamine by rat esophageal and liver microsomes and by rat and human cytochrome P450 isoforms.

Methyl-n-amylnitrosamine (MNAN) induces esophageal cancer in rats, probably involving activation by cytochromes P450. We studied the metabolic depentylation of MNAN. [3H-4,5-pentyl]MNAN and [3H-2,3-pentyl]-MNAN were synthesized, purified, and incubated with rat esophageal microsomes (REM) or rat liver microsomes (RLM) to give [3H]pentaldehyde (depentylation), an indicator of MNAN activation. [3H]Pentaldehyde was determined by high-performance liquid chromatography of its 2,4-dinitrophenylhydrazone. Adding 5 mM semicarbazide to incubations increased the observed depentylation (except that due to CYP2E1) by >60%. MNAN depentylation by REM and uninduced and induced RLM showed Km values of 64, 610, and 170-330 microM, respectively (Vmax: 20, 220, and 160-1270 pmol/mg protein/min, respectively). The depentylation of 100 microM MNAN by REM was inhibited 98% by CO and 65% by coumarin preincubated for 15 min with REM (Ki, 120 microM) but was unaffected by antibodies inhibitory to various P450s. MNAN inhibited coumarin 7-hydroxylation by RLM and CYP2A6 (Ki, 3000 and 320 microM, respectively). REM showed slight coumarin 7-hydroxylase activity. MNAN depentylation by RLM was 41% inhibited by an antibody to CYP2C11. Km for rat CYP2E1, human CYP2E1, and human CYP2A6 was 210, 115, and 17 microM, respectively (Vmax: 900, 570, and 120 pmol/nmol P450/min, respectively). We conclude that MNAN activation by REM is probably due to a P450 related to CYP2A3, a rodent nasal P450.

Bisquinolines. 2. Antimalarial N,N-bis(7-chloroquinolin-4-yl)heteroalkanediamines.

N,N-Bis(7-chloroquinolin-4-yl)heteroalkanediamines 1-11 were synthesized and screened against Plasmodium falciparum in vitro and Plasmodium berghei in vivo. These bisquinolines had IC50 values from 1 to 100 nM against P. falciparum in vitro. Six of the 11 bisquinolines were significantly more potent against the chloroquine-resistant W2 clone compared to the chloroquine-sensitive D6 clone. For bisquinolines 1-11 there was no relationship between the length of the bisquinoline heteroalkane bridge and antimalarial activity and no correlation between in vitro and in vivo antimalarial activities. Bisquinolines with alkyl ether and piperazine bridges were substantially more effective than bisquinolines with alkylamine bridges against P. berghei in vivo. Bisquinolines 1-10 were potent inhibitors of hematin polymerization with IC50 values falling in the narrow range of 5-20 microM, and there was a correlation between potency of inhibition of hematin polymerization and inhibition of parasite growth. Compared to alkane-bridged bisquinolines (Vennerstrom et al., 1992), none of these heteroalkane-bridged bisquinolines had sufficient antimalarial activity to warrant further investigation of the series.

An assessment of drug-haematin binding as a mechanism for inhibition of haematin polymerisation by quinoline antimalarials.

Chloroquine is thought to exert its antimalarial activity by preventing the polymerisation of toxic haematin released during proteolysis of haemoglobin in the Plasmodium digestive vacuole. However, the molecular mechanisms by which this inhibition occurs and the universality of this mechanism for other quinoline antimalarials remain to be established. We demonstrate here a correlation for eight antimalarial quinolines between inhibition of haematin polymerisation in vitro and inhibition of P. falciparum growth in culture, confirming haematin polymerisation as the likely target of quinoline blood schizonticides. Furthermore, using isothermal titration microcalorimetry, a correlation was observed between the haematin binding constant of these compounds and their ability to inhibit haematin polymerisation, suggesting that these compounds mediate their activity through binding to haematin. It was also observed that the compounds bind primarily to the mu-oxo dimer form of haematin rather than the monomeric form. It is postulated that this binding inhibits haematin polymerisation by shifting the haematin dimerisation equilibrium to the mu-oxo dimer, thus reducing the availability of monomeric haematin for incorporation into haemozoin. These data reconcile the haematin polymerisation theory with the Fitch hypothesis, which states that chloroquine mediates its activity through binding to haematin.

A comparison and analysis of several ways to promote haematin (haem) polymerisation and an assessment of its initiation in vitro.

We compared several methods for producing haematin polymerisation at physiological temperatures (i.e., 37 degrees) and found that a trophozoite lysate-mediated reaction was inappropriate for measuring compound inhibition of haematin polymerisation. Using this method, we obtained significantly higher IC50 values (concentration inhibiting haematin polymerisation by 50%) for certain compounds than when other methods were used, including a food vacuole lysate-mediated reaction. This difference was probably due to the binding of these compounds to cytosolic parasite proteins, as proteinase K treatment of the trophozoite lysate reversed this effect. The initiation of haematin polymerisation was also investigated using several assays. It was found that haematin polymerisation occurred spontaneously, in the absence of preformed haemozoin, over a period of several days, but that the process was more rapid when an acetonitrile extract of malarial trophozoites was added. This extract contained no detectable protein, and its activity could be replicated using an extract from uninfected erythrocytes and by using lipids. We therefore postulate that no protein or parasite-specific material is absolutely required for the initiation of haematin polymerisation. The formation of beta-haematin de novo using the acetonitrile extract is more pH-dependent than the generation of newly synthesised beta-haematin from preformed haemozoin and cannot proceed much above pH = 6. We postulate that the initiation of haematin polymerisation is more sensitive to the equilibrium of haematin between its monomeric and mu-oxo dimer form and requires a higher concentration of monomer than for the elongation phase of polymerisation.

Haematin (haem) polymerization and its inhibition by quinoline antimalarials.

Haematin (ferriprotoporphyrin IX) is released from haemoglobin during its degradation in the malarial parasites' food vacuole and is detoxified by its polymerization into a form of beta-haematin called haemozoin, or malarial pigment. This process is protein independent in vitro. Quinoline antimalarial blood schizonticides accumulate in the food vacuole and may inhibit haematin polymerization by binding to haematin and preventing its incorporation into the growing haemozoin chain. Drug resistance to quinolines is thought to be due to reduced accumulation of the drug in the food vacuole. As some quinolines overcome this resistance, quinolines, as a class, remain a potential source of future antimalarial drugs.

Antimalarial dyes revisited: xanthenes, azines, oxazines, and thiazines.

In 1891 Guttmann and Ehrlich (P. Guttmann and P. Ehrlich, Berlin Klin. Wochenschr. 28:953-956, 1891) were the first to report the antimalarial properties of a synthetic, rather than a natural, material when they described the clinical cure of two patients after oral administration of a thiazine dye, methylene blue. Since that time, sporadic reports of the antimalarial properties of several xanthene and azine dyes related to methylene blue have been noted. We report here the results from a reexamination of the antimalarial properties of methylene blue. Janus green B, and three rhodamine dyes and disclose new antimalarial data for 16 commercially available structural analogs of these dyes. The 50% inhibitory concentrations for the chloroquine-susceptible D6 clone and SN isolate and the chloroquine-resistant W2 clone of Plasmodium falciparum were determined by the recently described parasite lactate dehydrogenase enzyme assay. No cross-resistance to chloroquine was observed for any of the dyes. For the 21 dyes tested, no correlation was observed between antimalarial activity and cytotoxicity against KB cells. No correlation between log P (where P is the octanol/water partition coefficient) or relative catalyst efficiency for glucose oxidation and antimalarial activity or cytotoxicity was observed for the dyes as a whole or for the thiazine dyes. The thiazine dyes were the most uniformly potent structural class tested, and among the dyes in this class, methylene blue was notable for both its high antimalarial potency and selectivity.

In vitro activity of bisquinoline WR268,668 against African clones and isolates of Plasmodium falciparum.

The in vitro activity of a new bisquinoline, WR268,668, was determined against chloroquine-susceptible and chloroquine-resistant African clones and isolates of Plasmodium falciparum using an isotopic semimicro drug susceptibility assay. The chloroquine-resistant clone (mean 50% inhibitory concentration [IC50] = 61.2 nM) was 11 times less susceptible to WR268,668 than the chloroquine-susceptible clone (IC50 = 5.75 nM). A similar result was obtained with fresh clinical isolates, with the chloroquine-susceptible isolates (IC50 = 5.36 nM, n = 11) being significantly (P < 0.05) more susceptible to WR268,668 than the chloroquine-resistant isolates (IC50 = 16.1 nM, n = 18). The compound WR268,668 exhibited a high activity against some moderately chloroquine-resistant isolates. There was a significant positive correlation between the in vitro responses to chloroquine and WR268,668 (r = 0.904, P < 0.05). Combinations of WR268,668 and desipramine, a chloroquine efflux inhibitor, showed that resistance to WR268,668 can be reversed against the chloroquine-resistant clone and that desipramine has no effect on the activity of WR268,668 against the chloroquine-susceptible clone. The results of the study indicate the presence of cross-resistance between chloroquine and WR268,668, and suggest that the basis of resistance to WR268,668 may be similar to that of other 4-aminoquinolines.