Methylene Homologues of Artemisone: An Unexpected Structure-Activity Relationship and a Possible Implication for the Design of C10-Substituted Artemisinins.

We sought to establish if methylene homologues of artemisone are biologically more active and more stable than artemisone. The analogy is drawn with the conversion of natural O- and N-glycosides into more stable C-glycosides that may possess enhanced biological activities and stabilities. Dihydroartemisinin was converted into 10ß-cyano-10-deoxyartemisinin that was hydrolyzed to the α-primary amide. Reduction of the ß-cyanide and the α-amide provided the respective methylamine epimers that upon treatment with divinyl sulfone gave the ß- and α-methylene homologues, respectively, of artemisone. Surprisingly, the compounds were less active in vitro than artemisone against P. falciparum and displayed no appreciable activity against A549, HCT116, and MCF7 tumor cell lines. This loss in activity may be rationalized in terms of one model for the mechanism of action of artemisinins, namely the cofactor model, wherein the presence of a leaving group at C10 assists in driving hydride transfer from reduced flavin cofactors to the peroxide during perturbation of intracellular redox homeostasis by artemisinins. It is noted that the carba analogue of artemether is less active in vitro than the O-glycoside parent toward P. falciparum, although extrapolation of such activity differences to other artemisinins at this stage is not possible. However, literature data coupled with the leaving group rationale suggest that artemisinins bearing an amino group attached directly to C10 are optimal compounds.

Considerations on the mechanism of action of artemisinin antimalarials: part 1--the 'carbon radical' and 'heme' hypotheses.

The isolation of artemisinin from the traditional medicinal herb qing hao (Artemisia annua), its characterization as a peroxide and preparation of the derivatives dihydroartemisinin, artemether and artesunate in the 1970s and 1980s by Chinese scientists under the umbrella of Project 523 collectively represents one of the great events in medicine in the latter third of the 20(th) Century. Artemisinins have become the most important component of chemotherapy of malaria: although used initially in monotherapy, they are now used in combination therapies or ACTs with longer half-life quinolines or arylmethanols. Nevertheless, the recent emergence of artemisinin-tolerant strains of the malaria parasite as reflected in increased clearance times of parasitaemia in patients treated with ACTs represents the greatest threat to control of malaria since resistance to chloroquine was first reported over 55 years ago. Importantly, the event brings into sharp focus the realization that relatively little is precisely understood, as opposed to widely assumed, for the mechanism of drug action of artemisinins and their synthetic peroxide analogues. Thus, we review here their antimalarial activities, the use of artemisinins in combination therapies, drug-drug interactions with the quinolines and arylmethanols, and metabolism of the artemisinins and synthetic peroxides. The mechanism of action of quinolines and arylmethanols, in particular their ability to induce redistribution of heme into the parasite cytosol, is also highlighted. This collective information is then used as a counterpoint to screen the validity of two of the prevailing hypotheses of drug action of artemisinins and synthetic peroxides, namely i. 'the C-radical hypothesis' wherein the peroxide undergoes 'bioactivation' by ferrous iron to generate C-radicals that are held to be the cytotoxic agents and ii. the 'heme hypothesis' wherein ferrous heme may generate either the same type of 'cytotoxic' C-radical, or the peroxide forms heme adducts that apparently inherit the exquisite cytotoxicities of the parent peroxide in one way or another. In a subsequent review, we screen the third and fourth hypotheses: the SERCA hypothesis wherein artemisinins modulate operation of the malaria parasite sarcoendo plasmic reticulum calcium pump SERCA Ca(2+)-ATPase ATP6 and the co-factor hypothesis wherein artemisinins act as oxidant drugs through rapidly oxidizing reduced conjugates of flavin cofactors, or those of flavin cofactor precursors such as riboflavin, and other susceptible endogenous substrates that play a role in maintaining intraparasitic redox homeostasis. For the C-radical hypothesis, details of in vitro chemical studies in the context of established chemistry of C-radicals and their ability to react with radical trapping agents such as nitroso compounds, cyclic nitrones, persistent nitroxyl radicals and atmospheric oxygen (dioxygen) are summarized. Overall, there is no correlation between antimalarial activities and abilities of the derived C-radicals to react with trapping agents in a chemical flask. This applies in particular to the reactions of C-radicals from artemisinins and steroidal tetraoxanes with the trapping agents vis-a-vis those from adamantyl capped systems. In an intraparasitic medium, it is not possible to intercept C-radicals either through use of a vast excess of a nitroxyl radical or dioxygen. The lack of correlation of antimalarial activities also applies to the Fe(2+)-mediated decomposition of artemisinins and synthetic peroxides, where literature data taken as indicating otherwise are critically assessed. The antagonism to antimalarial activities of artemisinins exerted by desferrioxamine (DFO) and related Fe(3+)-chelating agents is due to formation of stable chelates with bioavailable Fe(3+) that shuts down redox cycling through Fe(2+) and the subsequent generation of reactive oxygen species (ROS) via the Fenton reaction. The generation of ROS by Fe(2+) complements the action of artemisinins, to be discussed in Part 2; there is no need to posit a reaction of Fe(2+) with the artemisinins to account for their antimalarial activity. The ability of artemisinins and synthetic peroxides to elicit membrane damage is examined in the light of established processes of autoxidation. The oxidant character of the intraparasitic environment is incompatible with the reducing conditions required for generation of C-radicals, and in contrast to the expectation raised by the C-radical hypothesis, and indeed by the heme hypothesis outlined below, antimalarial activities of artemisinins are enhanced under higher partial pressures of dioxygen. Structure-activity data from a wide variety of artemisinins and synthetic peroxides cannot be accommodated within the bounds of the C-radical hypothesis. Finally, the antimalarial Cradical construct sharply contrasts with that of the potently antitumour-active ene-diyne antibiotics such as neocarzinostatin. In an iron-free process, these compounds generate highly reactive aryl C-radicals that abstract H atoms from deoxyribose units in DNA to generate alkyl C-radicals. The last do react with dioxygen in a normal intracellular environment to initiate DNA strand cleavage. Overall, it must be concluded that the C-radical hypothesis as the basis for antimalarial activities of artemisinins and synthetic peroxides is untenable. Heme has been intensively studied as an 'activator' of artemisinins and other antimalarial peroxides, and indeed the hypothesis seemingly has become firmly embedded in the underlying brickwork of the scientific edifice. The locus of activity of the peroxides interacting with the heme is considered to be the parasite digestive vacuole. The basis for the nanomolar activities of artemisinins and synthetic peroxides is variously ascribed to heme-Fe(2+)-mediated generation of C-radicals from the peroxides, formation of heme-artemisinin adducts that are held either to engage in redox cycling with concomitant generation of ROS or to inhibit formation of hemozoin. In the last case, just like the aminoquinolines and arylmethanols, the peroxides are not the active agents, but exert their parasiticidal effects through allowing the build-up of free heme-Fe(3+), the ultimate cytotoxic entity. We assess the literature relating to generation of heme by hemoglobin digestion, and the stage at which this process becomes significant in the intraerythrocytic parasite. The claims of production of heme and conversion into hemozoin occurring in a lipid environment may have to be put aside based on recent literature data that indicates crystallization of hemozoin must take place an aqueous interface; association of lipids with the heme/hemozoin is likely to be a reflection of attractive van der Waals interactions involving the hydrophobic surface of the heme or hemozoin aggregates. In addition, the observation leading to the claim that hemozoin manufacture commences at the mid-ring stage cannot be independently verified. That the quinoline and arylmethanol antimalarials have essentially no activities on the ring stage parasites and exert greatest efficacy at the trophozoite stage where heme production is maximal is consistent with this. Conversely, artemisinins, and indeed redox active drugs such as methylene blue and others, are highly active against early ring stage parasites. Thus, there is a prominent disconnect between stage specificities of artemisinins vis-a-vis those of 4-aminoquinolines and arylmethanols suggesting that heme is not the target of the former class of drug. Further, the ability of the Fe(3+) chelate DFO to antagonize antimalarial activities of artemisinins, but not the activities of 4-aminoquinolines, cannot be explained by involvement of heme as a target for artemisinins. We critically examine the basis for formation of products obtained from reaction of heme with artemisinins and synthetic peroxides under conditions ranging from biomimetic - reactions employing catalytic reagents under aqueous or semi-aqueous conditions - to those conducted under highly reducing and eminently artificial conditions, usually in the solvent dimethyl sulfoxide (DMSO) that both forms well characterized complexes with heme-Fe(2+) and actually assists in driving single electron transfer processes. It is noted that alkylated products tend to form in high yields under the last conditions, and this aspect is readily explained. Irrespective of product yields obtained under various conditions, an overarching correlation between facility of the reaction of the peroxide with heme and their antimalarial activities does not exist. The is underscored by the reproducible outcomes of reactions conducted under biomimetic conditions indicating adducts cannot form in physiologically meaningful concentrations and that heme is a recalcitrant reaction partner to artemisinins in general. Again, as in the case of the C-radical hypothesis, structure-activity data from a wide variety of artemisinins and synthetic peroxides is difficult to reconcile with the heme hypothesis. This applies in particular to dimeric and trimeric artemisinin derivatives where the ascribing of biological activity to reactions of the derived radicals or to the vastly encumbered artemisinin-heme adducts is physically unrealistic. Finally, the facile metabolism and induction of metabolism of the current clinically used artemisinins by members of the CYP superfamily - heme proteins that require an intimate interaction of the heme with the artemisinin for metabolism to occur - is incompatible with the oft-cited proclivity of the peroxide to associate via complex formation with heme as a prelude to its 'activation' as an antimalarial agent within the malaria parasite. (ABSTRACT TRUNCATED)

Interactions between artemisinins and other antimalarial drugs in relation to the cofactor model--a unifying proposal for drug action.

Artemisinins are proposed to act in the malaria parasite cytosol by oxidizing dihydroflavin cofactors of redox-active flavoenzymes, and under aerobic conditions by inducing their autoxidation. Perturbation of redox homeostasis coupled with the generation of reactive oxygen species (ROS) ensues. Ascorbic acid-methylene blue (MB), N-benzyl-1,4-dihydronicotinamide (BNAH)-MB, BNAH-lumiflavine, BNAH-riboflavin (RF), and NADPH-FAD-E. coli flavin reductase (Fre) systems at pH 7.4 generate leucomethylene blue (LMB) and reduced flavins that are rapidly oxidized in situ by artemisinins. These oxidations are inhibited by the 4-aminoquinolines piperaquine (PPQ), chloroquine (CQ), and others. In contrast, the arylmethanols lumefantrine, mefloquine (MFQ), and quinine (QN) have little or no effect. Inhibition correlates with the antagonism exerted by 4-aminoquinolines on the antimalarial activities of MB, RF, and artemisinins. Lack of inhibition correlates with the additivity/synergism between the arylmethanols and artemisinins. We propose association via π complex formation between the 4-aminoquinolines and LMB or the dihydroflavins; this hinders hydride transfer from the reduced conjugates to the artemisinins. The arylmethanols have a decreased tendency to form π complexes, and so exert no effect. The parallel between chemical reactivity and antagonism or additivity/synergism draws attention to the mechanism of action of all drugs described herein. CQ and QN inhibit the formation of hemozoin in the parasite digestive vacuole (DV). The buildup of heme-Fe(III) results in an enhanced efflux from the DV into the cytosol. In addition, the lipophilic heme-Fe(III) complexes of CQ and QN that form in the DV are proposed to diffuse across the DV membrane. At the higher pH of the cytosol, the complexes decompose to liberate heme-Fe(III) . The quinoline or arylmethanol reenters the DV, and so transfers more heme-Fe(III) out of the DV. In this way, the 4-aminoquinolines and arylmethanols exert antimalarial activities by enhancing heme-Fe(III) and thence free Fe(III) concentrations in the cytosol. The iron species enter into redox cycles through reduction of Fe(III) to Fe(II) largely mediated by reduced flavin cofactors and likely also by NAD(P)H-Fre. Generation of ROS through oxidation of Fe(II) by oxygen will also result. The cytotoxicities of artemisinins are thereby reinforced by the iron. Other aspects of drug action are emphasized. In the cytosol or DV, association by π complex formation between pairs of lipophilic drugs must adversely influence the pharmacokinetics of each drug. This explains the antagonism between PPQ and MFQ, for example. The basis for the antimalarial activity of RF mirrors that of MB, wherein it participates in redox cycling that involves flavoenzymes or Fre, resulting in attrition of NAD(P)H. The generation of ROS by artemisinins and ensuing Fenton chemistry accommodate the ability of artemisinins to induce membrane damage and to affect the parasite SERCA PfATP6 Ca(2+) transporter. Thus, the effect exerted by artemisinins is more likely a downstream event involving ROS that will also be modulated by mutations in PfATP6. Such mutations attenuate, but cannot abrogate, antimalarial activities of artemisinins. Overall, parasite resistance to artemisinins arises through enhancement of antioxidant defense mechanisms.

A partial convergence in action of methylene blue and artemisinins: antagonism with chloroquine, a reversal with verapamil, and an insight into the antimalarial activity of chloroquine.

Artemisinins rapidly oxidize leucomethylene blue (LMB) to methylene blue (MB); they also oxidize dihydroflavins such as the reduced conjugates RFH2 of riboflavin (RF), and FADH2 of the cofactor flavin adenine dinucleotide (FAD), to the corresponding flavins. Like the artemisinins, MB oxidizes FADH2, but unlike artemisinins, it also oxidizes NAD(P)H. Like MB, artemisinins are implicated in the perturbation of redox balance in the malaria parasite by interfering with parasite flavoenzyme disulfide reductases. The oxidation of LMB by artemisinin is inhibited by chloroquine (CQ), an inhibition that is abruptly reversed by verapamil (VP). CQ also inhibits artemisinin-mediated oxidation of RFH2 generated from N-benzyl-1,4-dihydronicotinamide (BNAH)-RF, or FADH2 generated from NADPH or NADPH-Fre, an effect that is also modulated by verapamil. The inhibition likely proceeds by the association of LMB or dihydroflavin with CQ, possibly involving donor-acceptor or π complexes that hinder oxidation by artemisinin. VP competitively associates with CQ, liberating LMB or dihydroflavin from their respective CQ complexes. The observations explain the antagonism between CQ-MB and CQ-artemisinins in vitro, and are reconcilable with CQ perturbing intraparasitic redox homeostasis. They further suggest that a VP-CQ complex is a means by which VP reverses CQ resistance, wherein such a complex is not accessible to the putative CQ-resistance transporter (PfCRT).

Reactions of antimalarial peroxides with each of leucomethylene blue and dihydroflavins: flavin reductase and the cofactor model exemplified.

Flavin adenine dinucleotide (FAD) is reduced by NADPH-E. coli flavin reductase (Fre) to FADH(2) in aqueous buffer at pH 7.4 under argon. Under the same conditions, FADH(2) in turn cleanly reduces the antimalarial drug methylene blue (MB) to leucomethylene blue. The latter is rapidly re-oxidized by artemisinins, thus supporting the proposal that MB exerts its antimalarial activity, and synergizes the antimalarial action of artemisinins, by interfering with redox cycling involving NADPH reduction of flavin cofactors in parasite flavin disulfide reductases. Direct treatment of the FADH(2) generated from NADPH-Fre-FAD by artemisinins and antimalaria-active tetraoxane and trioxolane structural analogues under physiological conditions at pH 7.4 results in rapid reduction of the artemisinins, and efficient conversion of the peroxide structural analogues into ketone products. Comparison of the relative rates of FADH(2) oxidation indicate optimal activity for the trioxolane. Therefore, the rate of intraparastic redox perturbation will be greatest for the trioxolane, and this may be significant in relation to its enhanced in vitro antimalarial activities. (1)H NMR spectroscopic studies using the BNAH-riboflavin (RF) model system indicate that the tetraoxane is capable of using both peroxide units in oxidizing the RFH(2) generated in situ. Use of the NADPH-Fre-FAD catalytic system in the presence of artemisinin or tetraoxane confirms that the latter, in contrast to artemisinin, consumes two reducing equivalents of NADPH. None of the processes described herein requires the presence of ferrous iron. Ferric iron, given its propensity to oxidize reduced flavin cofactors, may play a role in enhancing oxidative stress within the malaria parasite, without requiring interaction with artemisinins or peroxide analogues. The NADPH-Fre-FAD system serves as a convenient mimic of flavin disulfide reductases that maintain redox homeostasis in the malaria parasite.