Novel phenothiazine antimalarials: synthesis, antimalarial activity, and inhibition of the formation of beta-haematin

We report the synthesis of a series of novel phenothiazine compounds that inhibit the growth of both chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum. We found that the antimalarial activity of these phenothiazines increased with an increase in the number of basic groups in the alkylamino side chain, which may reflect increased uptake into the parasite food vacuole or differences in the toxicities of individual FP-drug complexes. We have examined the ability of the parent phenothiazine, chlorpromazine, and some novel phenothiazines to inhibit the formation of beta-haematin. The degree of antimalarial potency was loosely correlated with the efficacy of inhibition of beta-haematin formation, suggesting that these phenothiazines exert their antimalarial activities in a manner similar to that of chloroquine, i.e. by antagonizing the sequestration of toxic haem (ferriprotoporphyrin IX) moieties within the malaria parasite. Chlorpromazine is an effective modulator of chloroquine resistance; however, the more potent phenothiazine derivatives were more active against chloroquine-sensitive parasites than against chloroquine-resistant parasites and showed little synergy of action when used in combination with chloroquine. These studies point to structural features that may determine the antimalarial activity and resistance modulating potential of weakly basic amphipaths.     

Vacuolar acidification and chloroquine sensitivity in Plasmodium falciparum.

The antimalarial chloroquine concentrates in the acid vesicles of Plasmodium falciparum partially as a result of its properties as a weak base. Chloroquine-resistant parasites accumulate less drug than sensitive parasites. A simple hypothesis is that the intravacuolar pH of resistant strains is higher than that for sensitive strains, as a consequence of a weakened proton pump in the vacuoles of resistant strains, thereby explaining the resistance mechanism. We have attempted to test this hypothesis by the use of bafilomycin A1, a specific inhibitor of vacuolar proton pumping ATPase systems in plant cells, animal cells and microorganisms. Bafilomycin A1 significantly reduces uptake of [3H]chloroquine into both chloroquine-sensitive and -resistant strains of P. falciparum, at concentrations of inhibitor which have no antimalarial effect. Additionally, chloroquine-resistant strains of P. falciparum are more sensitive to bafilomycin A1 than chloroquine-sensitive strains. The use of bafilomycin A1 in combination with chloroquine in the standard in vitro sensitivity assay, produced an apparent reduction in sensitivity of both strains to chloroquine. The reported data support the hypothesis that chloroquine resistance in P. falciparum is associated with increased vacuolar pH, possibly due to a weakened vacuolar proton pumping ATPase.     

Synthesis, cytotoxicity and antimalarial activity of ferrocenyl amides of 4-aminoquinolines

Series of 4-aminoquinolines bearing an amino side chain linked to the ferrocene moiety through an amide bond were synthesized and evaluated for their antimalarial activity against both chloroquine-sensitive (D10, CQ-S) and chloroquine-resistant (Dd2, CQ-R) strains of Plasmodium falciparum. They were also tested for cytotoxicity against Chinese Hamster Ovarian (CHO) cells. Amide 12 featuring propyl side chain linked to the ferrocene ring was the most active of all tested compounds. With an IC50 value of 0.08 microg/mL, this amide showed 1.5-fold higher activity than chloroquine diphosphate (IC50 = 0.12 microg/mL) against the resistant strain, with a selectivity index of 550 indicating its high selectivity towards the parasite. Derivatives which were equipotent against both strains also showed up to ten-fold increase in activity compared to primaquine.     

In vitro metabolism of ferroquine (SSR97193) in animal and human hepatic models and antimalarial activity of major metabolites on Plasmodium falciparum.

Ferroquine (SSR97193) has been shown to be a promising antimalarial, both on laboratory clones and on field isolates. So far, no resistance was documented in Plasmodium falciparum. In the present work, the metabolic pathway of ferroquine, based on experiments using animal and human hepatic models, is proposed. Ferroquine is metabolized mainly via an oxidative pathway into the major metabolite mono-N-demethyl ferroquine and then into di-N,N-demethyl ferroquine. Some other minor metabolic pathways were also identified. Cytochrome P450 isoforms 2C9, 2C19, and 3A4 and, possibly in some patients, isoform 2D6, are mainly involved in ferroquine oxidation. The metabolites were synthesized and tested against the 3D7 (chloroquine-sensitive) and W2 (chloroquine-resistant) P. falciparum strains. According to the results, the activity of the two main metabolites decreased compared with that of ferroquine; however, the activity of the mono-N-demethyl derivative is significantly higher than that of chloroquine on both strains, and the di-N-demethyl derivative remains more active than chloroquine on the chloroquine-resistant strain. These results further support the potential use of ferroquine against human malaria.     

Synthesis of aminoquinoline-based aminoalcohols and oxazolidinones and their antiplasmodial activity

Novel aminoquinoline β-aminoalcohol and oxazolidinone derivatives were designed, synthesized, and evaluated for in vitro antiplasmodial activity against a chloroquine-sensitive (3D7) and chloroquine-resistant (K1) strains of Plasmodium falciparum. A few β-aminoalcohol derivatives were more potent than chloroquine against chloroquine-sensetive Plasmodiums. The potency of these derivatives decreased against chloroquine-resistant species in all cases (higher resistance indices), suggesting a possible cross-resistance between this group of compounds and chloroquine which could be due to their structural similarity. Although changing β-aminoalcohols to their oxazolidinone counterparts decreased the potency in all the cases, the compounds were still active and the resistance indices for these compounds improved significantly in comparison with those of β-aminoalcohols. This may indicate the absence of cross-resistance between these new derivatives and chloroquine.     

Antimalarial drugs. An update

Over the last decade, chloroquine-resistant falciparum malaria has spread to other areas from its original foci in Southeast Asia and South America. Additionally, new knowledge about the life-cycle of the malaria parasite, and about the pharmacokinetic properties of antimalarial drugs, has emerged. It is appropriate to reassess our approach to prevention and management of malaria with these factors in mind. Antimalarial drugs can be classified in two ways: biologically as tissue schizontocides, hypnozoitocides, blood schizontocides, gametocytocides or sporontocides; or by a mixed chemical/biological classification as 8-aminoquinolines, antimetabolites and (again) blood schizontocides. Chloroquine resistance in P. falciparum can now be found in most areas where malaria occurs. Malarial strains moderately resistant to the chloroquine group of drugs (chloroquine and mepacrine) are generally susceptible to the aryl amino alcohols such as quinine. Indeed, quinine is the most widely used drug for treating malaria due to chloroquine-resistant strains, followed by a 7-day course of tetracycline where some resistance to quinine is also found. Alternatively, the course of quinine may be followed by sulfadoxine/pyrimethamine or the newer quinoline derivative, mefloquine. Quinidine has also shown activity against quinine-resistant strains. Prophylaxis of chloroquine-resistant strains is best undertaken with daily proguanil (chloroguanide), and weekly chloroquine. In severe malaria, including cerebral malaria, an intravenous loading dose of quinine should be considered, and plasma concentration monitoring may be advisable to assist with dosage adjustment. In patients with severe renal insufficiency, there is evidence that the elimination of chloroquine is prolonged, and dosage adjustments may be necessary. Other recent findings on the pharmacodynamic properties, mechanisms of action and toxicity of antimalarial drugs are also discussed.     

Comparison of proteases from chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum

An aminopeptidase and four hemoglobin-degrading acid proteases have been isolated from cloned strains of chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum. Amino-peptidases from both strains showed similar properties including molecular weights of 63,000 and non-competitive inhibition by chloroquine; Ki = 535 and 410 microM for enzymes from the sensitive and resistant strains respectively. The acid proteases from the chloroquine-sensitive strain included a low molecular weight enzyme in the soluble fraction (protease S), an enzyme weakly associated with membrane (protease M2), and two enzymes strongly associated with membrane (proteases M3 and M4). The acid proteases from the chloroquine-resistant strain included protease S, protease M2, a second enzyme weakly associated with membrane (protease M1), and protease M3. All of the acid proteases were inhibited by ferriprotoporphyrin IX and by the chloroquine-ferriprotoporphyrin IX complex, I50 = 5-25 microM. The data were consistent with a model for chloroquine action wherein chloroquine acts to divert ferriprotoporphyrin IX from sequestration into malarial pigment, leaving ferriprotoporphyrin IX (or its chloroquine complex) to interfere with digestion of host cytosol by inhibiting hemoglobin-degrading proteases. However, the similarities among the proteases from chloroquine-sensitive and chloroquine-resistant strains of parasites suggest that chloroquine resistance does not result from changes in parasite proteases.     

Mechanism-based design of parasite-targeted artemisinin derivatives: synthesis and antimalarial activity of new diamine containing analogues

The potent antimalarial activity of chloroquine against chloroquine-sensitive strains can be attributed, in part, to its high accumulation in the acidic environment of the heme-rich parasite food vacuole. A key component of this intraparasitic chloroquine accumulation mechanism is a weak base "ion-trapping" effect whereupon the basic drug is concentrated in the acidic food vacuole in its membrane-impermeable diprotonated form. By the incorporation of amino functionality into target artemisinin analogues, we hoped to prepare a new series of analogues that, by virtue of increased accumulation into the ferrous-rich vacuole, would display enhanced antimalarial potency. The initial part of the project focused on the preparation of piperazine-linked analogues (series 1 (7-16)). Antimalarial evaluation of these derivatives demonstrated potent activity versus both chloroquine-sensitive and chloroquine-resistant parasites. On the basis of these observations, we then set about preparing a series of C-10 carba-linked amino derivatives. Optimization of the key synthetic step using a newly developed coupling protocol provided a key intermediate, allyldeoxoartemisinin (17) in 90% yield. Further elaboration, in three steps, provided nine target C-10 carba analogues (series 2 (21-29)) in good overall yields. Antimalarial assessment demonstrated that these compounds were 4-fold more potent than artemisinin and about twice as active as artemether in vitro versus chloroquine-resistant parasites. On the basis of the products obtained from biomimetic Fe(II) degradation of the C-10 carba analogue (23), we propose that these analogues may have a mode of action subtly different from that of the parent drug artemisinin (series 1 (7-16)) and other C-10 ether derivatives such as artemether. Preliminary in vivo testing by the WHO demonstrated that four of these compounds are active orally at doses of less than 10 mg/kg. Since these analogues are available as water-soluble salts and cannot form dihydroartemisinin by P450-catalyzed oxidation, they represent useful leads that might prove to be superior to the currently used derivatives, artemether and artesunate.     

Primaquine synergises the activity of chloroquine against chloroquine-resistant P. falciparum

In recent years, resistance to the antimalarial drug, chloroquine, has become widespread. It is, therefore, imperative to find compounds that could replace chloroquine or work synergistically with this drug to overcome chloroquine resistance. We have examined the interaction between chloroquine, a 4-aminoquinoline, and a number of 8-aminoquinolines, including primaquine, a drug that is widely used to treat Plasmodium vivax infections. We find that primaquine is a potent synergiser of the activity of chloroquine against chloroquine-resistant Plasmodium falciparum. Analysis of matched transfectants expressing mutant and wild-type alleles of the P. falciparum chloroquine resistance transporter (PfCRT) indicate that primaquine exerts its activity by blocking PfCRT, and thus enhancing chloroquine accumulation. Our data suggest that a novel formulation of two antimalarial drugs already licensed for use in humans could be used to treat chloroquine-resistant parasites.     

Quinoline antimalarials containing a dibemethin group are active against chloroquinone-resistant Plasmodium falciparum and inhibit chloroquine transport via the P. falciparum chloroquine-resistance transporter (PfCRT)

A series of 4-amino-7-chloroquinolines with dibenzylmethylamine (dibemethin) side chains were shown to inhibit synthetic hemozoin formation. These compounds were equally active against cultures of chloroquine-sensitive (D10) and chloroquine-resistant (K1) Plasmodium falciparum. The most active compound had an IC(50) value comparable to that of chloroquine, and its potency was undiminished when tested in three additional chloroquine-resistant strains. The three most active compounds exhibited little or no cytotoxicity in a mammalian cell line. When tested in vivo against mouse malaria via oral administration, two of the dibemethin derivatives reduced parasitemia by over 99%, with mice treated at 100 mg/kg surviving the full length of the experiment. Three of the compounds were also shown to inhibit chloroquine transport via the parasite's chloroquine-resistance transporter (PfCRT) in a Xenopus oocyte expression system. This constitutes the first example of a dual-function antimalarial for which the ability to inhibit both hemozoin formation and PfCRT has been demonstrated directly.     

Evaluation of Diarylureas for Activity Against Plasmodium falciparum

A library of diarylurea IGFR inhibitors was screened for activity against chloroquine-sensitive (3D7) and chloroquine-resistant (K1) strains of Plasmodium falciparum. The 4-aminoquinaldine-derived diarylureas displayed promising antimalarial potency. Further exploration of the B ring of 4-aminoquinaldinyl ureas allowed identification of several quinaldin-4-yl ureas 4{13, 39} and 4{13, 58} sufficiently potent against both 3D7 and K1 strains to qualify as bone fide leads.     

Antimalarial dual drugs based on potent inhibitors of glutathione reductase from Plasmodium falciparum

Plasmodium parasites are exposed to higher fluxes of reactive oxygen species and need high activities of intracellular antioxidant systems providing a steady glutathione flux. As a future generation of dual drugs, 18 naphthoquinones and phenols (or their reduced forms) containing three different linkers between the 4-aminoquinoline core and the redox active component were synthesized. Their antimalarial effects have been characterized in parasite assays using chloroquine-sensitive and -resistant strains of Plasmodium, alone or in drug combination, and in the Plasmodium berghei rodent model. In particular, two tertiary amides 34 and 36 showed potent antimalarial activity in the low nanomolar range against CQ-resistant parasites. The ability to compete both for (Fe (III))protoporphyrin and for chloroquine transporter was determined. The data are consistent with the presence of a carrier for uptake of the short chloroquine analogue 2 but not for the potent antimalarial amide 34, suggesting a mode of action distinct from chloroquine mechanism.     

Biologically active derivatives of gossypol: synthesis and antimalarial activities of peri-acylated gossylic nitriles

A series of peri-acylated gossylic nitriles were synthesized from gossypol dioxime by treatment of the dioxime with the appropriate acid anhydride and its salt. The reaction pathway was elucidated by isolation and characterization of intermediates. Peri-acylated gossylic nitriles (acyl = acetyl, propionyl, butyryl, and valeryl) were compared with gossypol for activity against both chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum. The gossylic nitriles all retain activity, with activity increasing with the length of the peri-acyl group. Gossylic nitrile 1,1'-divalerate shows antimalarial activity comparable to gossypol itself. The peri-acylated gossylic nitriles are strong inhibitors of parasite lactate dehydrogenase.     

Synthesis of 4-aminoquinoline-1,2,3-triazole and 4-aminoquinoline-1,2,3-triazole-1,3,5-triazine hybrids as potential antimalarial agents

We report herein synthesis of a series of 4-aminoquinoline-1,2,3-triazole and 4-aminoquinoline-1,2,3-triazole-1,3,5-triazine hybrids and evaluate their antimalarial activity against D6 and W2 strains of Plasmodium falciparum. To study the structure-activity relationship of substituted 4-aminoquinoline-based hybrids, 34 structurally diverse compounds were synthesized and tested against D6 and W2 strains of P. falciparum. Some of the compounds have shown promising antimalarial activity without toxicity against Vero cells.     

Antagonism of serum of mice infected with chloroquine-resistant 'NS' line to the antimalarial action of chloroquine

Chloroquine (CQ) solution was separately mixed with the serum of mice infected with chloroquine-resistant 'NS' line (SMNS), the serum of mice infected with chloroquine-sensitive Plasmodium berghei ANKA strain (SMCS), and the serum of normal mice (SM). These mixtures were then used in treating mice inoculated with P. berghei ANKA strain. The results obtained on d 5 after drug-serum administration showed that the erythrocyte infection rates in the SMNS + CQ, SMCS + CQ, and SM + CQ groups were 32, 16, and 14% respectively. There were significant differences with respect to parasitemia between the SMNS + CQ group and the SMCS + CQ or SM + CQ group (P less than 0.05), suggesting that SMNS may be antagonistic to the antimalarial action of chloroquine. Further study showed that when the 'NS' line lost resistance to chloroquine, the antagonism of SMNS to chloroquine disappeared. The antagonism rates of SMNS to chloroquine, piperaquine, hydroxypiperaquine and pyronaridine were 75, 56, -5, and -17% respectively, a trend similar to that of the results from cross-resistance tests on chloroquine-resistant P. berghei ANKA strain. The results indicate that drug-resistant malaria parasites may produce and release a certain 'specific anti-drug substance'.     

Pharmacophore-based predictive model generation for potent antimalarials targeting haem detoxification pathway

Pharmacophore hypotheses were developed for molecules having antimalarial activities targeting the haem detoxification pathway of the malaria parasite. A training set consisting of 23 compounds was selected to generate these hypotheses, and their activities were evaluated for haem polymerization inhibition and against chloroquine-sensitive (3D7) as well as chloroquine-resistant (K1) strains of p. falciparum. The models were cross-validated by Fischer’s randomization test at a 95% confidence level. The model developed against chloroquine-resistant malaria parasites was found to yield the best predictions among the three models.     

Oxygenated chalcones and bischalcones as a new class of inhibitors of DNA topoisomerase II of malarial parasites

The DNA topoisomerase (topo) II of chloroquine-sensitive and chloroquine-resistant strains of the rodent malaria parasite P. berghei were utilized as a target for testing of antimalarial compounds. Compounds belonging to the bischalcone and chalcone series significantly inhibited enzyme activity and percentage parasitaemia of chloroquine-sensitive and chloroquine-resistant strains of P. berghei. Compounds 1a, 1b, 2a, 2b, and 2c showed 100% inhibition while compounds 2h and 2i showed 60% and 63% inhibition of topoisomerase II activity of the chloroquine-sensitive strain, respectively. Compounds 2a, 2b, and 2d significantly inhibited the topo II activity of chloroquine-resistant strain. Compounds 2g and 2e specifically inhibited the topo II activity of the chloroquine-resistant strain of P. berghei with no effect on the chloroquine-sensitive strain. The in vitro topo II inhibition by chalcone and bischalcone analogs can be correlated with their in vivo antimalarial activity, as compounds 2c and 2h inhibited both in vitro activity of topo II and in vivo parasitaemia of the chloroquine-sensitive strain of P. berghei. In the chloroquine-resistant strain, compounds 2c, 2e, 2g, and 2i inhibited activity against both in vitro topo II and parasitaemia in vivo. The significant inhibition of topo II in the chloroquine-resistant strain by some of the analogs suggests the utilization of these structures for the synthesis of compounds active against chloroquine-resistant malarial parasites.     

Design, synthesis and biological evaluation of some novel 3-cinnamoyl-4-hydroxy-2H-chromen-2-ones as antimalarial agents

A novel series of 3-cinnamoyl-4-hydroxy-2H-chromen-2-ones were designed, synthesized and screened for antiplasmodial activity. Eleven compounds of the series exhibited micromolar potency against chloroquine sensitive and chloroquine resistant strains. The most potent compound 4-hydroxy-3-(3-(4-nitrophenyl)acryloyl)-2H-chromen-2-one showed inhibitory potency (IC₅₀) of 3.1 and 4?μg/ml against chloroquine sensitive and chloroquine resistant strains, respectively. A structure activity relationship study was performed by correlating the effect of substituents with the antimalarial activity of the title compounds. The novel 3-cinnamoyl-4-hydroxy-2H-chromen-2-ones reported here should be good lead for further development of antimalarial agents that can overcome resistance.     

A prodrug form of a Plasmodium falciparum glutathione reductase inhibitor conjugated with a 4-anilinoquinoline.

Glutathione (GSH), which is known to guard Plasmodium falciparum from oxidative damage, may have an additional protective role by promoting heme catabolism. An elevation of GSH content in parasites leads to increased resistance to chloroquine (CQ), while GSH depletion in resistant P. falciparum strains is expected to restore the sensitivity to CQ. High intracellular GSH levels depend inter alia on the efficient reduction of GSSG by glutathione reductase (GR). On the basis of this hypothesis, we have developed a new strategy for overcoming glutathione-dependent 4-aminoquinoline resistance. To direct both a 4-aminoquinoline and a GR inhibitor to the parasite, double-drugs were designed and synthesized. Quinoline-based alcohols (with known antimalarial activity) were combined with a GR inhibitor via a metabolically labile ester bond to give double-headed prodrugs. The biochemically most active double-drug 7 of this series was then evaluated as a growth inhibitor against six Plasmodium falciparum strains that differed in their degree of resistance to CQ; the ED(50) values for CQ ranged from 14 to 183 nM. While the inhibitory activity of the original 4-aminoquinoline-based alcohol followed that of CQ in these tests, the double-drug exhibited similar efficiency against all strains, the ED(50) being as low as 28 nM. For the ester 7, a dose-dependent decrease in glutathione content and GR activity and an increase in glutathione-S-transferase activity were determined in treated parasites. The drug was subsequently tested for its antimalarial action in vivo using murine malaria models infected with P. berghei. A 178% excess mean survival time was determined for the animals treated with 40 mg/kg 7 for 4 days. No cytotoxicity due to this compound was observed. Work is in progress to extend and validate the strategy outlined here.     

Bisquinolines. 1. N,N-bis(7-chloroquinolin-4-yl)alkanediamines with potential against chloroquine-resistant malaria.

On the basis of observations that several bisquinolines such as piperaquine possess notable activity against chloroquine-resistant malaria, 13 N,N-bis-(7-chloroquinolin-4-yl)alkanediamines were synthesized and screened against Plasmodium falciparum in vitro and Plasmodium berghei in vivo. Twelve of the thirteen bisquinolines had a significantly lower resistance index than did chloroquine; the resistance index was apparently unrelated to either in vitro or in vivo activity. Except for two compounds, there was a reasonable correlation between in vitro and in vivo activities. Seven of the thirteen bisquinolines had IC50's of less than 6 nM against both chloroquine-sensitive (D-6) and -resistant (W-2) clones of P. falciparum and were curative against P. berghei at doses of 640 mg/kg. In contrast to chloroquine, these bisquinolines did not show any toxic deaths at curative dose levels. Four bisquinolines, however, caused skin lesions at the site of injection. Maximum activity was seen in bisquinolines with a connecting bridge of two carbon atoms where decreased conformational mobility seemed to increase activity. Bisquinoline 3 (+/-)-trans-N1,N2-bis(7-chloroquinolin-4-yl)cyclohexane-1,2-diamin e was not only the most potent bisquinoline in vitro, but was clearly unique in its in vivo activity--80% and 100% cure rates were achieved at doses of 160 and 320 mg/kg, respectively. In summary, these preliminary results support the premise that bisquinolines may be useful agents against chloroquine-resistant malaria.