Antiprotozoal activity of drimane and coloratane sesquiterpenes towards Trypanosoma brucei rhodesiense and Plasmodium falciparum in vitro.

The extracts and 12 sesquiterpenes obtained from the East African medicinal plant Warburgia ugandensis Sprague (Canellaceae) were assessed for their antiplasmodial activity against the chloroquine-sensitive (3D7) and chloroquine-resistant (K1) strains of Plasmodium falciparum and antitrypanosomal activity against Trypanosoma brucei rhodesiense. The dichloromethane extract displayed strong antiplasmodial and antitrypanosomal activities with IC(50) values of 8.10 and 1.10 µg/mL against K1 strain of the malaria parasite and STlB900 strain of T. b. rhodesiense, respectively. Among the compounds evaluated for inhibition of trypomastigotes, both drimane and coloratane sesquiterpenes possessing aldehyde groups at positions 8 and 9 were found to show most antitrypanosomal activity with IC(50) values in the range 0.56-6.4 µM. The antiplasmodial assays also revealed that the six coloratane and six drimane sesquiterpenes isolated from this extract exhibited significant antitrypanosomal activity with IC(50) values ranged from 0.45 to ?114 µM. Among the compounds tested against the malarial parasite P. falciparum 11?-hydroxymuzigadiolide (3) was most active with an IC(50) value of 6.40 µM.

Anti-African trypanocidal and antimalarial activity of natural flavonoids, dibenzoylmethanes and synthetic analogues.

OBJECTIVES: The known anti-protozoal activity of flavonoids has stimulated the testing of other derivatives from natural and synthetic sources. METHODS: As part of our efforts to find potential lead compounds, a number of flavonoids isolated from Neoraputia paraensis, N. magnifica, Murraya paniculata, (Rutaceae), Lonchocarpus montanus, L. latifolius, L. subglaucescens, L. atropurpureus, L. campestris, Deguelia hatschbachii (Leguminosae), dibenzoylmethanes from L. subglaucescens and synthetic analogues were tested for in-vitro activity against chloroquine-sensitive Plasmodium falciparum and Trypanosoma brucei rhodesiense bloodstream form trypomastigotes. An assay with KB cells has been developed in order to compare in-vitro cytotoxicity of flavonoids with a selective action on the parasites. KEY FINDINGS: Thirteen of the compounds tested had IC50 values ranging from 4.6 to 9.9 microm against T. brucei rhodesiense. In contrast, a small number of compounds showed significant activity against P. falciparum; seven of those tested had IC50 values ranging from 2.7 to 9.5 microm. Among the flavones only one had IC50 < 10 microm (7.6 microm), whereas against T. brucei rhodesiense seven had IC50 < 10 microm. Synthetic dibenzoylmethanes were the most active in terms of number (five) of compounds and the IC50 values (2.7-9.5 microm) against P. falciparum. CONCLUSIONS: Dibenzoylmethanes represent a novel class of compounds tested for the first time as antimalarial and trypanocidal agents.

Anti-malarial efficacy of pyronaridine and artesunate in combination in vitro and in vivo.

Pyronaridine is a Mannich base anti-malarial with demonstrated efficacy against drug resistant Plasmodium falciparum, P. vivax, P. ovale and P. malariae. However, resistance to pyronaridine can develop quickly when it is used alone but can be considerably delayed when it is administered with artesunate in rodent malaria models. The aim of this study was to evaluate the efficacy of pyronaridine in combination with artesunate against P. falciparum in vitro and in rodent malaria models in vivo to support its clinical application. Pyronaridine showed consistently high levels of in vitro activity against a panel of six P. falciparum drug-sensitive and resistant strains (Geometric Mean IC50=2.24 nM, 95% CI=1.20-3.27). In vitro interactions between pyronaridine and artesunate showed a slight antagonistic trend, but in vivo compared to pyronaridine and artesunate administered alone, the 3:1 ratio of the combination, reduced the ED90 of artesunate by approximately 15.6-fold in a pyronaridine-resistant P. berghei line and by approximately 200-fold in an artesunate-resistant line of P. berghei. Complete cure rates were achieved with doses of the combination above or equal to 8 mg/kg per day against P. chabaudi AS. These results indicate that the combination had an enhanced effect over monotherapy and lower daily doses of artesunate could be used to obtain a curative effect. The data suggest that the combination of pyronaridine and artesunate should have potential in areas of multi-drug resistant malaria.

Preparation of N-sulfonyl- and N-carbonyl-11-azaartemisinins with greatly enhanced thermal stabilities: in vitro antimalarial activities.

As the clinically used artemisinins do not withstand the thermal stress testing required to evaluate shelf life for storage in tropical countries where malaria is prevalent, there is a need to develop thermally more robust artemisinin derivatives. Herein we describe the attachment of electron-withdrawing arene- and alkanesulfonyl and -carbonyl groups to the nitrogen atom of the readily accessible Ziffer 11-azaartemisinin to provide the corresponding N-sulfonyl- and -carbonylazaartemisinins. Two acylurea analogues were also prepared by treatment of the 11-azaartemisinin with arylisocyanates. Several of the N-sulfonylazaartemisinins have melting points above 200 degrees C and possess substantially greater thermal stabilities than the artemisinins in current clinical use, with the antimalarial activities of several of the arylsulfonyl derivatives being similar to that of artesunate against the drug-sensitive 3D7 clone of the NF54 isolate and the multidrug-resistant K1 strain of P. falciparum. The compounds possess relatively low cytotoxicities. The carbonyl derivatives are less crystalline than the N-sulfonyl derivatives, but are generally more active as antimalarials. The N-nitroarylcarbonyl and arylurea derivatives possess sub-ng ml(-1) activities. Although several of the azaartemisinins possess log P values below 3.5, the compounds have poor aqueous solubility (<1 mg L(-1) at pH 7). The greatly enhanced thermal stability of our artemisinins suggests that strategic incorporation of electron-withdrawing polar groups into both new artemisinin derivatives and totally synthetic trioxanes or trioxolanes may assist in the generation of practical new antimalarial drugs which will be stable to storage conditions in the field, while retaining favorable physicochemical properties.

Evaluation of azasterols as anti-parasitics.

In this article, the design and synthesis of some novel azasterols is described, followed by their evaluation against Trypanosoma brucei rhodesiense, T. cruzi, Leishmania donovani, and Plasmodium falciparum, the causative agents of human African trypanosomiasis, Chagas disease, leishmaniasis, and malaria, respectively. Some of the compounds showed anti-parasitic activity. In particular, a number of compounds appeared to very potently inhibit the growth of the blood stream form T. b. rhodesiense, with one compound giving an IC50 value of 12 nM. Clear structure activity relationships could be discerned. These compounds represent important leads for further optimization. Azasterols have previously been shown to inhibit sterol biosynthesis in T. cruzi and L. donovani by the inhibition of the enzyme sterol 24-methyltransferase. However, in this case, none of the compounds showed inhibition of the enzyme. Therefore, these compounds have an unknown mode of action.

New azasterols against Trypanosoma brucei: role of 24-sterol methyltransferase in inhibitor action.

A series of azasterol derivatives, designed as potential inhibitors of the Delta(24)-sterol methyltransferase enzyme (24-SMT), were synthesized and evaluated for their activities against parasitic protozoa. Values in the nanomolar range were obtained for 50% effective dose against the Trypanosoma brucei subsp. rhodesiense bloodstream form cultured in vitro. In order to investigate the mode of action, Trypanosoma brucei subsp. brucei 24-SMT was cloned and overexpressed and compounds were assayed for inhibitory activity. None of the inhibitors tested appeared to be active against the enzyme. Sterol composition analysis showed that only cholestane type sterols are present in membranes of bloodstream forms while ergosterol is a major component of procyclic sterol extracts. Interestingly, Northern blot analysis showed the presence of 24-SMT mRNA in both the procyclic and the bloodstream forms of the parasite, although levels of mRNA were threefold lower in the latter. Likewise, Western blot analysis and activity determinations evidenced the existence of active enzyme in both forms of the parasite. We conclude that the designed compounds act at sites other than 24-SMT in Trypanosoma brucei.

Irreversible inactivation of trypanothione reductase by unsaturated Mannich bases: a divinyl ketone as key intermediate.

Trypanothione reductase is a flavoenzyme unique to trypanosomatid parasites. Here we show that unsaturated Mannich bases irreversibly inactivate trypanothione reductase from Trypanosoma cruzi, the causative agent of Chagas' disease. The inhibitory potency of the compounds strongly increased upon storage of the DMSO stock solutions. HPLC, NMR, and mass spectrometry data of potential intermediates revealed a divinyl ketone as the active compound inactivating the enzyme. ESI- and MALDI-TOF mass spectrometry of trypanothione reductase modified by the Mannich base or the divinyl ketone showed specific alkylation of the active site Cys52 by a 5-(2'chlorophenyl)-3-oxo-4-pentenyl substituent. The reaction mechanism and the site of alkylation differ from those in Plasmodium falciparum thioredoxin reductase where the C-terminal redox active dithiol is modified. After deamination, unsaturated Mannich bases are highly reactive in polycondensation with trypanothione. Interaction of these compounds with both trypanothione and trypanothione reductase could account for their potent trypanocidal effect against Trypanosoma brucei.

Antimalarial compounds from Kniphofia foliosa roots.

During the course of screening Ethiopian medicinal plants for their antimalarial properties, it was found that the dichloromethane extract of the roots of Kniphofia foliosa Hochst. (Asphodelaceae), which have long been used in the traditional medicine of Ethiopia for the treatment of abdominal cramps and wound healing, displayed strong in vitro antiplasmodial activity against the chloroquine-sensitive 3D7 strain of Plasmodium falciparum with an ED50 value of 3.8 microg/mL and weak cytotoxic activity against KB cells with an ED50 value of 35.2 microg/mL. Five compounds were isolated from the roots and evaluated for their in vitro antimalarial activity. Among the compounds tested, 10-(chrysophanol-7'-yl)-10-(xi)-hydroxychrysopanol-9-anthrone and chryslandicin, showed a high inhibition of the growth of the malaria parasite, P. falciparum with ED50 values of 0.260 and 0.537 microg/mL, respectively, while the naphthalene derivative, 2-acetyl-1-hydroxy-8-methoxy-3-methylnaphthalene, exhibited a less significant antimalarial activity with an ED50 value of 15.4 microg/mL. To compare the effect on the parasite with toxicity to mammalian cells, the cytotoxic activities of the isolated compounds against the KB cell line were evaluated and 10-(chrysophanol-7'-yl)-10-(xi)-hydroxychrysopanol-9-anthrone and chryslandicin displayed very low toxicity with ED50 values of 104 and 90 microg/mL, respectively. This is the first report of the inhibition of the growth of P. falciparum by anthraquinone-anthrone dimers and establishes them as a new class of potential antimalarial compounds with very little host cell toxicity.