Antiplasmodial activities versus the chloroquine sensitive D10 strain of Plasmodium falciparum of a series of N(1),N(1)-diethyl-N(2)-(4-quinolinyl)-1,2-ethanediamines with 11 different substituents at the 7-position on the quinoline ring have been investigated in vitro. Electron-withdrawing groups at the 7-position have been shown to lower the pK(a) of both the quinoline ring nitrogen atom and the tertiary amino nitrogen in the alkyl side chain. The quinoline nitrogen pK(a) ranges from 6.28 in the nitro derivative to 8.36 in the amino derivative, while the tertiary amino nitrogen has a pK(a) ranging between 7.65 in the trifluoromethyl derivative and 10.02 in the amino derivative. Calculation suggests that the resulting pH trapping of these compounds in the parasite food vacuole ranges between about 7% of that observed in chloroquine for the NO(2) derivative and 97% in the amino derivative. A direct proportionality between antiplasmodial activity normalized for pH trapping and beta-hematin inhibitory activity was observed. Activity could not be correlated with any other observed physical parameter. The beta-hematin inhibitory activity of these derivatives appears to correlate with both the hematin-quinoline association constant and the electron-withdrawing capacity of the group at the 7-position (Hammett constant). For the compounds under investigation, the hematin association constant is in turn influenced by the lipophilicity of the group at the 7-position.

The antimalarial drug methylene blue (MB) affects the redox behaviour of parasite flavin-dependent disulfide reductases such as glutathione reductase (GR) that control oxidative stress in the malaria parasite. The reduced flavin adenine dinucleotide cofactor FADH(2) initiates reduction to leucomethylene blue (LMB), which is oxidised by oxygen to generate reactive oxygen species (ROS) and MB. MB then acts as a subversive substrate for NADPH normally required to regenerate FADH(2) for enzyme function. The synergism between MB and the peroxidic antimalarial artemisinin derivative artesunate suggests that artemisinins have a complementary mode of action. We find that artemisinins are transformed by LMB generated from MB and ascorbic acid (AA) or N-benzyldihydronicotinamide (BNAH) in situ in aqueous buffer at physiological pH into single electron transfer (SET) rearrangement products or two-electron reduction products, the latter of which dominates with BNAH. Neither AA nor BNAH alone affects the artemisinins. The AA-MB SET reactions are enhanced under aerobic conditions, and the major products obtained here are structurally closely related to one such product already reported to form in an intracellular medium. A ketyl arising via SET with the artemisinin is invoked to explain their formation. Dihydroflavins generated from riboflavin (RF) and FAD by pretreatment with sodium dithionite are rapidly oxidised by artemisinin to the parent flavins. When catalytic amounts of RF, FAD, and other flavins are reduced in situ by excess BNAH or NAD(P)H in the presence of the artemisinins in the aqueous buffer, they are rapidly oxidised to the parent flavins with concomitant formation of two-electron reduction products from the artemisinins; regeneration of the reduced flavin by excess reductant maintains a catalytic cycle until the artemisinin is consumed. In preliminary experiments, we show that NADPH consumption in yeast GR with redox behaviour similar to that of parasite GR is enhanced by artemisinins, especially under aerobic conditions. Recombinant human GR is not affected. Artemisinins thus may act as antimalarial drugs by perturbing the redox balance within the malaria parasite, both by oxidising FADH(2) in parasite GR or other parasite flavoenzymes, and by initiating autoxidation of the dihydroflavin by oxygen with generation of ROS. Reduction of the artemisinin is proposed to occur via hydride transfer from LMB or the dihydroflavin to O1 of the peroxide. This hitherto unrecorded reactivity profile conforms with known structure-activity relationships of artemisinins, is consistent with their known ability to generate ROS in vivo, and explains the synergism between artemisinins and redox-active antimalarial drugs such as MB and doxorubicin. As the artemisinins appear to be relatively inert towards human GR, a putative model that accounts for the selective potency of artemisinins towards the malaria parasite also becomes apparent. Decisively, ferrous iron or carbon-centered free radicals cannot be involved, and the reactivi...

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The results of Fe2+‐induced decomposition of the clinically used artemisinins, artemisone, other aminoartemisinins, 10‐deoxoartemisinin, and the 4‐fluorophenyl derivative have been compared with their antimalarial activities and their ability to inhibit the parasite SERCA PfATP6. The clinical artemisinins and artemisone decompose under aqueous conditions to give mixtures of C radical marker products, carbonyl compounds, and reduction products. The 4‐fluorophenyl derivative and aminoartemisinins tend to be inert to aqueous iron(II) sulfate and anhydrous iron(II) acetate. Anhydrous iron(II) bromide enhances formation of the carbonyl compounds and provides a deoxyglycal from DHA and enamines from the aminoartemisinins. Ascorbic acid (AA) accelerates the aqueous Fe2+‐mediated decompositions, but does not alter product distribution. 4‐Oxo‐TEMPO intercepts C radicals from a mixture of an antimalaria‐active trioxolane, 10‐deoxoartemisinin, and anhydrous iron(II) acetate to give trapped products in 73 % yield from the trioxolane, and 3 % from the artemisinin. Artemisone provides a trapped product in 10 % yield. Thus, in line with its structural rigidity, only the trioxolane provides a C radical eminently suited for intermolecular trapping. In contrast, the structural flexibility of the C radicals from the artemisinins allows facile extrusion of Fe2+ and collapse to benign isomerization products. The propensity towards the formation of radical marker products and intermolecular radical trapping have no relationship with the in vitro antimalarial activities of the artemisinins and trioxolane. Desferrioxamine (DFO) attenuates inhibition of PfATP6 by, and antagonizes antimalarial activity of, the aqueous Fe2+‐susceptible artemisinins, but has no overt effect on the aqueous Fe2+‐inert artemisinins. It is concluded that the C radicals cannot be responsible for antimalarial activity and that the Fe2+‐susceptible artemisinins may be competitively decomposed in aqueous extra‐ and intracellular compartments by labile Fe2+, resulting in some attenuation of their antimalarial activities. Interpretations of the roles of DFO and AA in modulating antimalarial activities of the artemisinins, and a comparison with antimalarial properties of simple hydroperoxides and their behavior towards thapsigargin‐sensitive SERCA ATPases are presented. The general basis for the exceptional antimalarial activities of artemisinins in relation to the intrinsic activity of the peroxide within the uniquely stressed environment of the malaria parasite is thereby adumbrated.

The induction of heme oxygenase-1 (HO-1) is widely recognized as an effective cellular strategy to counteract a variety of stressful events. We have shown that curcumin and caffeic acid phenethyl ester, two naturally occurring phytochemicals that possess antioxidant, anti-inflammatory, and anticarcinogenic activities, induce HO-1 in many cell types. This suggests that stimulation of HO-1 could partly underlie the beneficial effects exerted by these plant-derived constituents. Here we examined the ability of additional plant constituents to up-regulate heme oxygenase activity and HO-1 in aortic endothelial cells. Incubation of endothelial cells with a series of polyphenolic chalcones (5-50 M) resulted in increased heme oxygenase activity; interestingly, the chemical structure dictated the pattern of heme oxygenase induction, which was unique to each particular compound employed. We also found that rosolic acid, a constituent isolated from the rhizome of Plantago asiatica L. dramatically increased HO-1 in a concentration-and time-dependent manner. Severe cytotoxicity was observed after prolonged exposure (24 or 48 h) of cells to curcumin and caffeic acid phenethyl ester, whereas 2Ј-hydroxychalcone and rosolic acid did not affect cell viability. By using different mitogenactivated protein kinase inhibitors, we determined that the extracellular signal-regulated kinase, p38, and c-Jun NH 2 -terminal protein kinase pathways play only a minor role in the induction of HO-1 by rosolic acid and 2Ј-hydroxychalcone. On the other hand, increased intra-and extracellular thiols markedly reduced the rise in heme oxygenase activity elicited by rosolic acid. Thus, this study identified novel plant constituents that highly induce HO-1 in endothelial cells and investigated some of the mechanisms involved in this effect.