Enhanced pneumocystis carinii activity of new primaquine analogues

New analogues of the venerable antimalarial drug primaquine have been synthesized and bioassayed in vivo against Pneumocystis carinii, a life-threatening infection common among immunosuppressed patients. Two of these new compounds are significantly more active than primaquine itself, and provide new information for future drug design and development in this area.     

In vitro efficiency of 9-(N-cinnamoylbutyl)aminoacridines against blood- and liver-stage malaria parasites

Novel 9-aminoacridine derivatives were synthesized by linking the heteroaromatic core to different cinnamic acids through an aminobutyl chain. The test compounds demonstrated mid-nanomolar in vitro activity against erythrocytic stages of the chloroquine-resistant W2 strain of the human malaria parasite Plasmodium falciparum. Two of the most active derivatives also showed in vitro activity against liver-stage Plasmodium berghei, with activity greater than that of the reference liver-stage antimalarial primaquine. The compounds were not toxic to human hepatoma cells at concentrations up to 5 μM. Hence, 9-(N-cinnamoylbutyl)aminoacridines are a new class of leads for prevention and treatment of malaria.     

Effects of novel triple-stage antimalarial ionic liquids on lipid membrane models

Primaquine-based ionic liquids, obtained by acid-base reaction between parent primaquine and cinnamic acids, were recently found as triple-stage antimalarial hits. These ionic compounds displayed significant activity against both liver- and blood-stage Plasmodium parasites, as well as against stage V P. falciparum parasites. Remarkably, blood-stage activity of the ionic liquids against both chloroquine-sensitive (3D7) and resistant (Dd2) P. falciparum strains was clearly superior to those of the respective covalent (amide) analogues and of parent primaquine. Having hypothesized that such behaviour might be ascribed to an enhanced ability of the ionic compounds to permeate into Plasmodium-infected erythrocytes, we have carried out a differential scanning calorimetry-based study of the interactions between the ionic liquids and membrane models. Results provide evidence, at the molecular level, that the primaquine-derived ionic liquids may contribute to an increased permeation of the parent drug into malaria-infected erythrocytes, which has relevant implications towards novel antimalarial approaches based on ionic liquids.     

Synthesis and evaluation of febrifugine analogues as potential antimalarial agents

Febrifugine is an alkaloid isolated from Dichroa febrifuga Lour as the active component against Plasmodium falciparum. Strong liver toxicity has precluded febrifugine as a potential clinical drug. In this study novel febrifugine analogues were designed and synthesized. Lower toxicity was achieved by reducing or eliminating the tendency of forming chemically reactive and toxic intermediates and metabolites. Synthesized compounds were evaluated in vitro against chloroquine sensitive (D6) and chloroquine resistant (W2) P. falciparum strains for efficacy and in freshly isolated rat hepatocytes for potential cytotoxicity. The IC(50)'s of the best compounds were superior to their parent compound febrifugine. Noticeably, these compounds were also over 100 times less toxic than febrifugine. These compounds, as well as the underlying design rationale, may find usefulness in the discovery and development of new antimalarial drugs.     

Hydroxylated metabolites of the antimalarial drug primaquine. Oxidation and redox cycling.

Oxidation and redox cycling of the hydroxylated metabolites of the antimalarial drug primaquine (i.e. 5-hydroxyprimaquine, 5-hydroxydemethylprimaquine, and 5,6-dihydroxy-8-aminoquinoline) were studied. The three metabolites readily oxidized under physiological conditions, forming hydrogen peroxide and the corresponding quinone-imine derivatives as the main products. The latter compounds were characterized by visible, NMR, and infrared spectroscopy. Concomitant formation of drug-derived radicals and hydroxyl radicals was attested by direct and spin-trapping EPR experiments, respectively. The use of the spin stabilization method indicated that the radicals derived from 5-hydroxydemethylprimaquine and 5,6-dihydroxy-8-aminoquinoline are of the o-semiquinone type. Tentative structures are proposed for the radicals based on product identification and computer simulation of the experimental EPR spectra. The quinone-imines obtained from the reduced metabolites did not react at appreciable rates with NADPH but underwent redox cycling upon addition of ferredoxin:NADP+ oxidoreductase, forming hydrogen peroxide and hydroxyl radicals. The effect of antioxidant enzymes on hydroxyl radical yield obtained during oxidation and redox cycling indicates that the main route for hydroxyl radical formation is the metal ion-catalyzed reaction between the drug-derived radicals and hydrogen peroxide. Taken together, the results indicate that hydrogen peroxide is the potential toxic product formed from the primaquine metabolites.     

Febrifugine analogue compounds: synthesis and antimalarial evaluation

Febrifugine is an alkaloid isolated from Dichroa febrifuga Lour as the active component against Plasmodium falciparum, but exhibits toxic side effects. In this study novel febrifugine analogues were designed and efficiently synthesized. New compounds underwent efficacy and toxicity evaluation. Some compounds are much less toxic than the natural product febrifugine and existing antimalarial drugs and are expected to possess wide therapeutic windows. In Aotus monkeys infected with the chloroquine resistant FVO strain of P. falciparum, one interesting compound possesses a 50% curative dose of 2mg/kg/day and a 100% curative dose of 8 mg/kg/day. These compounds, as well as the underlying design rationale, may find usefulness in the discovery and development of new antimalarial drugs.     

Synthesis, antimalarial, antileishmanial, antimicrobial, cytotoxicity, and methemoglobin (MetHB) formation activities of new 8-quinolinamines

We report the synthesis, in vitro antiprotozoal (against Plasmodium and Leishmania), antimicrobial, cytotoxicity (Vero and MetHb-producing properties), and in vivo antimalarial activities of two series of 8-quinolinamines. N1-{4-[2-(tert-Butyl)-6-methoxy-8-quinolylamino]pentyl}-(2S/2R)-2-aminosubstitutedamides (21-33) and N1-[4-(4-ethyl-6-methoxy-5-pentyloxy-8-quinolylamino)pentyl]-(2S/2R)-2-aminosubstitutedamides (51-63) were synthesized in six steps from 6-methoxy-8-nitroquinoline and 4-methoxy-2-nitro-5-pentyloxyaniline, respectively. Several analogs displayed promising antimalarial activity in vitro against Plasmodium falciparum D6 (chloroquine-sensitive) and W2 (chloroquine-resistant) clones with high selectivity indices versus mammalian cells. The most promising analogs (21-24) also displayed potent antimalarial activity in vivo in a Plasmodium berghei-infected mouse model. Most interestingly, many analogs exhibited promising in vitro antileishmanial activity against Leishmania donovani promastigotes, and antimicrobial activities against a panel of pathogenic bacteria and fungi. Several analogs, notably 21-24, 26-32, and 60, showed less MetHb formation compared to primaquine indicating the potential of these compounds in 8-quinolinamine-based antimalarial drug development.     

Primaquine-induced superoxide production by beta-thalassemic red blood cells.

Primaquine, a prooxidant antimalarial drug, incubated with human red blood cells (RBC) induced marked superoxide generation in the cells as detected by exogenous cytochrome c reduction. In the presence of primaquine, beta-thalassemic RBC produced significantly more superoxide than normal RBC, thus reflecting the vulnerability of beta-thalassemic cells to oxidative stress.     

Pyronaridine: A new antimalarial drug

The worldwide spread of strains of Plasmodium falciparum that are resistant to chloroquine has highlighted the urgent need for new antimalarial drugs, particularly in less developed tropical countries. However, in the current economic climate the pharmaceutical giants in the developed world are withdrawing from tropical disease research. Consequently, the following article from Fu Sui and Xiao Shuhuo is of particular interest, not only because it summarizes work on on alternative antimalarial drug that is efficacious against multiply resistant Plasmodium but also because this drug has been developed primarily from Chinese research efforts, the results of which have largely only been published in the Chinese scientific literature. The drug under scrutiny is pyronaridine, and is the product of 30 years of chemistry that began with the mepacrine nucleus. This nucleus was selected as the starting point in the search for a chloroquine alternative because the various derivatives synthesized were active against chloroquine-resistant parasites. However, mepacrine itself also needed replacing as it is too toxic for mass use. After synthesizing and screening a huge series of substitutions, the addition of an amodiaquine side-chain to this nucleus was found to give the greatest activity for fewest adverse effects. Being aware of the rapid selection of pyronaridine-resistant Plasmodium strains that occurs in the laboratory, the Chinese efforts have also investigated the use of drug combinations to circumvent or delay the development of drug resistance. In addition to the triple combination described here, pyronaridine and primaquine combinations are under trial against both P. vivax and P. falciparum. Pyronaridine is a highly active blood schizonticide like chloroquine and amodiaquine. It has already undergone extensive trials in humans against both P. falciparum and P. vivax. However, nothing is known of its mode of action, nor the basis for the development of resistance and although it is active against chloroquine-resistant strains of parasite, paradoxically, pyronaridine-resistant Plasmodium is resistant to chloroquine.     

Primaquine-induced superoxde production by β-thalassemic red blood cells

Primaquine, a prooxidant antimalarial drug, incubated with human red blood cells (RBC) induced marked superoxide generation in the cells as detected by exogenous cytochrome c reduction. In the presence of primaquine, β-thalassemic RBC produced significantly more superoxide than normal RBC, thus reflecting the vulnerability of β-thalassemic cells to oxidative stress.     

A carbamate-based approach to primaquine prodrugs: antimalarial activity, chemical stability and enzymatic activation

O-Alkyl and O-aryl carbamate derivatives of the antimalarial drug primaquine were synthesised as potential prodrugs that prevent oxidative deamination to the inactive metabolite carboxyprimaquine. Both O-alkyl and O-aryl carbamates undergo hydrolysis in alkaline and pH 7.4 phosphate buffers to the parent drug, with O-aryl carbamates being ca. 10(6)-10(10) more reactive than their O-alkyl counterparts. In human plasma O-alkyl carbamates were stable, whereas in contrast their O-aryl counterparts rapidly released the corresponding phenol product, with primaquine being released only slowly over longer incubation periods. Activation of the O-aryl carbamates in human plasma appears to be catalysed by butyrylcholinesterase (BuChE), which leads to carbamoylation of the catalytic serine of the enzyme followed by subsequent slow enzyme reactivation and release of parent drug. Most of the O-aryl and O-alkyl carbamates are activated in rat liver homogenates with half-lives ranging from 9 to 15 h, while the 4-nitrophenyl carbamate was hydrolysed too rapidly to determine an accurate rate constant. Antimalarial activity was studied using a model consisting of Plasmodium berghei, Balb C mice and Anopheles stephensi mosquitoes. When compared to controls, ethyl and n-hexyl carbamates were able to significantly reduce the percentage of infected mosquitos as well as the mean number of oocysts per infected mosquito, thus indicating that O-alkyl carbamates of primaquine have the potential to be developed as transmission-blocking antimalarial agents.     

Synthesis, antiprotozoal, antimicrobial, β-hematin inhibition, cytotoxicity and methemoglobin (MetHb) formation activities of bis(8-aminoquinolines)

In continuing our search of potent antimalarials based on 8-aminoquinoline structural framework, three series of novel bis(8-aminoquinolines) using convenient one to four steps synthetic procedures were synthesized. The bisquinolines were evaluated for in vitro antimalarial (Plasmodiumfalciparum), antileishmanial (Leishmaniadonovani), antimicrobial (a panel of pathogenic bacteria and fungi), cytotoxicity, ??-hematin inhibitory and methemoglobin (MetHb) formation activities. Several compounds exhibited superior antimalarial activities compared to parent drug primaquine. Selected compounds (44, 61 and 79) when tested for in vivo blood-schizontocidal antimalarial activity (Plasmodiumberghei) displayed potent blood-schizontocial activities. The bisquinolines showed negligible MetHb formation (0.2-1.2%) underlining their potential in the treatment of glucose-6-phosphate dehydrogenase deficient patients. The bisquinoline analogues (36, 73 and 79) also exhibited promising in vitro antileishmanial activity, and antimicrobial activities (43, 44 and 76) against a panel of pathogenic bacteria and fungi. The results of this study provide evidence that bis(8-aminoquinolines), like their bis(4-aminoquinolines) and artemisinin dimers counterparts, are a promising class of antimalarial agents.     

Radical curative efficacy of five-day regimen of primaquine for treatment of Plasmodium vivax malaria in India

For over 4 decades the antimalarial program in India has been prescribing a 5-day primaquine regimen as an antirelapse therapy to treat Plasmodium vivax malaria. In view of conflicting reports on the effectiveness of this regimen in the Indian subcontinent, and the varying prevalence of P. vivax in various ecosystems in India, the antirelapse efficacy of this regimen was evaluated in Orissa, a malaria endemic state in eastern India where P. falciparum predominates. In 723 cases of P. vivax infection treated with chloroquine alone and followed up weekly for 1 yr, the prevalence of recurrence of parasitaemia with fever was 8.6%. Among another 759 P. vivax cases treated with chloroquine and a 5-day regimen of primaquine at 15 mg/day (adult dose), the recurrence of infection was 6.5%. The difference in recurrence was not significant (P = 0.53). It is important to note that in a great majority of cases of P. vivax in this area, infection did not recur even without treatment with primaquine. This finding, that the use of the 5-day primaquine regimen with chloroquine had no significant advantage over the use of chloroquine alone, undermines the rationale of using primaquine as an antirelapse drug in forested areas with a high prevalence of P. falciparum.     

Oxidant stress in malaria as probed by stable nitroxide radicals in erythrocytes infected with Plasmodium berghei. The effects of primaquine and chloroquine

Erythrocytes from normal mice and mice infected with the malarial parasite Plasmodium berghei reduce the water-soluble spin probes 2,2,6,6-tetramethylpiperidine-4-hydroxy-N-oxyl (TEMPOL), 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) and 2,2,6,6-tetramethylpiperidine-4-keto-N-oxyl (TEMPONE) at similar rates under both air and N2 atmospheres. The ESR signal of the lipid-soluble spin probe 5-doxyl-stearate is stable on incorporation into erythrocytes from normal mice. In contrast, parasitized red cells reduce this nitroxide probe, at a rate which increases with the level of parasitemia. Inhibitors of electron transport such as KCN and NaN3, increase the rate of reduction. We propose that nitroxide reduction occurs via the electron transport chain in the parasite. The antimalarial drug primaquine causes reduction of both water-soluble and lipid-soluble spin probes. This action of primaquine is independent of its ability to release H2O2 from oxyhemoglobin, and is ascribed to the ability of primaquine to accelerate flux through the hexose monophosphate shunt. The increased production of NADPH results in increased rates of reduction of the nitroxide radicals. Methylene blue, which also increases flux through the shunt, is even more effective than primaquine at reducing the nitroxides. Chloroquine has no such effect. Parasitized mice treated with chloroquine six hours prior to ESR measurements show less nitroxide reducing capacity than do untreated mice. Chloroquine is known to decrease flux through the hexose monophosphate shunt. The metabolic influences of the two antimalarial drugs are, thus, quite different.     

Clinical efficacy of chloroquine versus artemether-lumefantrine for Plasmodium vivax treatment in Thailand

Chloroquine remains the drug of choice for the treatment of vivax malaria in Thailand. Mixed infections of falciparum and vivax malaria are also common in South-East Asia. Laboratory confirmation of malaria species is not generally available. This study aimed to find alternative regimens for treating both malaria species by using falciparum antimalarial drugs. From June 2004 to May 2005, 98 patients with Plasmodium vivax were randomly treated with either artemether-lumefantrine (n = 47) or chloroquine (n = 51). Both treatments were followed by 15 mg of primaquine over 14 days. Adverse events and clinical and parasitological outcomes were recorded and revealed similar in both groups. The cure rate was 97.4% for the artemether-lumefantrine treated group and 100% for the chloroquine treated group. We concluded that the combination of artemether-lumefantrine and primaquine was well tolerated, as effective as chloroquine and primaquine, and can be an alternative regimen for treatment of vivax malaria especially in the event that a mixed infection of falciparum and vivax malaria could not be ruled out.     

Plasmodium falciparum malaria: rosettes are disrupted by quinine, artemisinin, mefloquine, primaquine, pyrimethamine, chloroquine and proguanil.

An assay was developed measuring the disruption of rosettes between Plasmodium falciparuminfected (trophozoites) and uninfected erythrocytes by the antimalarial drugs quinine, artemisinin mefloquine, primaquine, pyrimethamine, chloroquine and proguanil. At 4 hr incubation rosettes were disrupted by all the drugs in a dose dependent manner. Artemisinin and quinine were the most effective anti-malarials at disrupting rosettes at their therapeutic concentrations with South African RSA 14, 15, 17 and The Gambian FCR-3 P. falciparum strains. The least effective drugs were proguanil and chloroquine. A combination of artemisinin and mefloquine was more effective than each drug alone. The combinations of pyrimethamine or primaquine, with quinine disrupted more rosettes than quinine alone. Quinine may be an effective drug in the treatment of severe malaria because the drug efficiently reduces the number of rosettes.     

Synthesis, antimalarial, antileishmanial, and antimicrobial activities of some 8-quinolinamine analogues

In the present communication, newly synthesized 8-quinolinamines (25-27) related to previously reported 2-tert-butylprimaquine (2) were evaluated for their in vitro antimalarial activity against chloroquine sensitive and resistant Plasmodium falciparum strains, in vivo antimalarial activity against P. berghei infected mice, in vitro antileishmanial activity against Leishmania donovani, in vitro antimicrobial activity against various fungi and bacteria, and cytotoxicity in a panel of mammalian cell lines. No promising cytotoxicities were observed for compounds reported herein. Analogue 25 was found to exhibit curative antimalarial activity at a dose of 25 mg/kg/dayx4 in a P. berghei infected mice model, and produced suppressive activity at a lower dose of 10 mg/kg/dayx4. In vitro antileishmanial activities (IC50 and IC90) comparable to standard drug pentamidine were exhibited by all synthesized 8-quinolinamines 25-27. At the same time, promising antibacterial and antifungal activities were also observed for synthesized compounds against a panel consisting of several bacteria and fungi.     

Antimalarial activity and mechanisms of action of two novel 4-aminoquinolines against chloroquine-resistant parasites

Chloroquine (CQ) is a cost effective antimalarial drug with a relatively good safety profile (or therapeutic index). However, CQ is no longer used alone to treat patients with Plasmodium falciparum due to the emergence and spread of CQ-resistant strains, also reported for P. vivax. Despite CQ resistance, novel drug candidates based on the structure of CQ continue to be considered, as in the present work. One CQ analog was synthesized as monoquinoline (MAQ) and compared with a previously synthesized bisquinoline (BAQ), both tested against P. falciparum in vitro and against P. berghei in mice, then evaluated in vitro for their cytotoxicity and ability to inhibit hemozoin formation. Their interactions with residues present in the NADH binding site of P falciparum lactate dehydrogenase were evaluated using docking analysis software. Both compounds were active in the nanomolar range evaluated through the HRPII and hypoxanthine tests. MAQ and BAQ derivatives were not toxic, and both compounds significantly inhibited hemozoin formation, in a dose-dependent manner. MAQ had a higher selectivity index than BAQ and both compounds were weak PfLDH inhibitors, a result previously reported also for CQ. Taken together, the two CQ analogues represent promising molecules which seem to act in a crucial point for the parasite, inhibiting hemozoin formation.     

Docking studies and development of novel 5-heteroarylamino-2,4-diamino-8-chloropyrimido-[4,5-b]quinolines as potential antimalarials

MOE-Dock (Docking software) was used to predict the binding modes of 10 novel and potent 5-substituted amino-2,4-diamino-8-chloropyrimido-[4,5-b]quinolines (compounds I-X) as part of our antimalarial drug development programme. This was done by analyzing the interaction of these compounds with the active sites of 11 enzymes present in Plasmodium falciparum and based on this, effective binding was observed to enzyme P. falciparum glutathione reductase (PfGR). The binding scores for compounds I-X with PfGR were also congruent with their antimalarial activity. Three additional analogs were then designed and synthesized based on the above docking study and the pharmacophoric requirements for this class.     

Isolation and antimalarial activity of peroxydisulfate oxidation products of primaquine

Five compounds formed by peroxydisulfate oxidation of primaquine were isolated using chromatographic methods and evaluated for antimalarial activity in vitro. One compound 6-methoxy-5,8 bis(4′-amino-1′-methylbutylamino)quinoline [P₁] was found to have good gametocytocidal activity against Plasmodium yoelli infected mice at 10 mg kg⁻¹ dose in vivo.