Diego Monti

The Fe2+-mediated decomposition, PfATP6 binding, and antimalarial activities of artemisone and other artemisinins: the unlikelihood of C-centered radicals as bioactive intermediates.

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.

A combinatorial approach to 2,4,6-trisubstituted triazines with potent antimalarial activity: combining conventional synthesis and microwave-assistance.

Malaria nowadays remains one of the world’s greatest public health problems. It is responsible for two million deaths per year, mostly African children under five years old, particularly affecting peoples in developing countries. Among the four malaria species that infect humans, the parasite P. falciparum is universally considered the most aggressive. Particularly impressive is its ability in mutating forms in response to administered antiplasmodial treatment, rapidly giving rise to adaptation and resistance. Hence, it is extremely urgent to find an effective combination of antimalarial drugs, not only to improve the efficacy of the therapy, but also to prevent further development of resistance. The discovery of the great potential of artemisinin has significantly encouraged the search in this area of medicinal chemistry. However, artemisinin and its active derivatives are ideal for rapid parasite clearance and clinical recovery, but they need to be combined with longer-acting drugs to prevent recrudescence. Continuous efforts in the search for new drugs together with modeling and analytical investigations on the plasmodia mechanism of invasion have highlighted two possible important targets. The dihydrofolate reductase (DHFR) of P. falciparum is one of the few well-defined targets in antimalarial therapy. This enzyme catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate. DHFR is considered the target of cycloguanil 1 and of other antifolates in use for anti-

Facile oxidation of leucomethylene blue and dihydroflavins by artemisinins: relationship with flavoenzyme function and antimalarial mechanism of action.

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 FADH2 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 FADH2 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 FADH2 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 reactivity described herein reconciles disparate observations that are incompatible with the ferrous iron–carbon radical hypothesis for antimalarial mechanism of action. Finally, the urgent enquiry into the emerging resistance of the malaria parasite to artemisinins may now in one part address the possibilities either of structural changes taking place in parasite flavoenzymes that render the flavin cofactor less accessible to artemisinins or of an enhancement in the ability to use intra‐erythrocytic human disulfide reductases required for maintenance of parasite redox balance.

Resolving the Structure of Ligands Bound to the Surface of Superparamagnetic Iron Oxide Nanoparticles by High-Resolution Magic-Angle Spinning NMR Spectroscopy

A major challenge in magnetic nanoparticle synthesis and (bio)functionalization concerns the precise characterization of the nanoparticle surface ligands. We report the first analytical NMR investigation of organic ligands stably anchored on the surface of superparamagnetic nanoparticles (MNPs) through the development of a new experimental application of high-resolution magic-angle spinning (HRMAS). The conceptual advance here is that the HRMAS technique, already being used for MAS NMR analysis of gels and semisolid matrixes, enables the fine-structure-resolved characterization of even complex organic molecules bound to paramagnetic nanocrystals, such as nanosized iron oxides, by strongly decreasing the effects of paramagnetic disturbances. This method led to detail-rich, well-resolved (1)H NMR spectra, often with highly structured first-order couplings, essential in the interpretation of the data. This HRMAS application was first evaluated and optimized using simple ligands widely used as surfactants in MNP synthesis and conjugation. Next, the methodology was assessed through the structure determination of complex molecular architectures, such as those involved in MNP3 and MNP4. The comparison with conventional probes evidences that HRMAS makes it possible to work with considerably higher concentrations, thus avoiding the loss of structural information. Consistent 2D homonuclear (1)H- (1)H and (1)H- (13)C heteronuclear single-quantum coherence correlation spectra were also obtained, providing reliable elements on proton signal assignments and carbon characterization and opening the way to (13)C NMR determination. Notably, combining the experimental evidence from HRMAS (1)H NMR and diffusion-ordered spectroscopy performed on the hybrid nanoparticle dispersion confirmed that the ligands were tightly bound to the particle surface when they were dispersed in a ligand-free solvent, while they rapidly exchanged when an excess of free ligand was present in solution. In addition to HRMAS NMR, matrix-assisted laser desorption ionization time-of-flight MS analysis of modified MNPs proved very valuable in ligand mass identification, thus giving a sound support to NMR characterization achievements.

Artemisinin Antimalarials Do Not Inhibit Hemozoin Formation

The mechanism of action of artemisinin antimalarials may be ascribed to C-centered radicals that alkylate biomolecules or to the peroxide moiety, which inhibits a specific, as yet undefined, target (4). It is also proposed that artemisinins kill the parasite through inhibition of hemozoin formation, thereby allowing buildup of toxic heme monomer (1, 2, 5). Artemisinin and dihydroartemisinin may be unstable under aqueous conditions used for hemozoin studies; ring-opened products (3) may therefore bind to the heme and inhibit hemozoin formation. 10-Deoxodihydroartemisin (Fig. ​(Fig.1)1) has no oxygen at C-10 and is less able to undergo ring opening under aqueous conditions. Therefore, even though it is an active antimalarial it may not interfere with hemozoin formation. To test this, dihydroartemisinin and 10-deoxodihydroartemisinin were screened for inhibition of β-hematin (hemozoin) formation by using both the HPIA (heme polymerization inhibitory activity) assay (2) (hematin in acetic acid at pH 2.7, 37°C, 18 h) and the BHIA (β-hematin inhibitory activity) assay (hemin in dimethyl sulfoxide-acetate buffer at pH 5.0, 37°C, 18 h). The first identifies ligands that bind axially with the protoporphyrin iron, and the second identifies ligands undergoing π-π interactions with hematin (6). FIG. 1. Structures of artemisinin, dihydroartemisinin, and 10-deoxodihydroartemisinin. Dihydroartemisinin showed a dose-dependent inhibition in the HPIA assay but not in the BHIA assay. The 10-deoxy compound was inactive in both assays (Fig. ​(Fig.2).2). Thus, peroxidic antimalarials do not interfere with hemozoin formation in the parasite and are differentiated from quinoline antimalarials in that they cannot bind via π-π interactions with the heme molecule. This is also evident in their failure to inhibit β-hematin formation in the BHIA assay (6). It is uncertain if dihydroartemisinin undergoes ring opening under the HPIA assay conditions. In this case, it cannot be excluded that in the HPIA assay (pH 2.7), the ring-opened form of dihydroartemisinin will form an axial ligand with the porphyrin iron, thus inhibiting β-hematin formation. 10-Deoxodihydroartemisinin cannot easily undergo ring opening and therefore cannot bind porphyrin—it has no effect on β-hematin formation, yet it displays potent antimalarial activity. FIG. 2. Results of in vitro assays of inhibition of β-hematin formation. DHA, dihydroartemisinin. Thus, inhibition of β-hematin formation in the HPIA assay by artemisinin (2) and dihydroartemisinin (this letter), but not by 10-deoxodihydroartemisinin, reflects a reactivity that is not related to their antimalarial action. Furthermore, binding of artemisinins with Fe(III)PPIX is not necessary for antimalarial activity. These data also confirm that the HPIA and BHIA assays are useful for distinguishing compounds forming π-π interactions with heme from those forming axial ligands.

Synthesis of carboranyl derivatives of alkynyl glycosides as potential BNCT agents

Abstract A series of amphiphilic carbohydrate-carborane hybrids consisting of a lipophilic core (carborane cage) and a glycoside moiety for conferring high-affinity recognition by the cellular lectins have been prepared in a chemically accessible fashion.

Non‐iron porphyrins inhibit β‐haematin (malaria pigment) polymerisation

Infrared spectroscopy was used to evaluate the effect of non‐iron porphyrins (protoporphyrin IX and haematoporphyrin) on haematin polymerisation to β‐haematin at acidic pH. Both molecules effectively inhibited the reaction, with haematoporphyrin 6 times as active as protoporphyrin IX. We postulated that the interaction between the π electron system of porphyrin rings leads to the formation of π–π adducts, which inhibit polymer elongation in the same way as antimalarial drugs (e.g., chloroquine); the presence of hydroxyl groups able to bind haem iron enhances activity.

Evidence that haem iron in the malaria parasite is not needed for the antimalarial effects of artemisinin

The role of haem iron (II) and oxidative stress in the activation and antimalarial activity of artemisinin is unclear. Thus, we submitted malaria parasite to modified culture conditions: artemisinin activity increased by 20–30% under an oxygen‐rich atmosphere (20% O2 instead of “standard” 1% O2), and by 40–50% in the presence of carboxy‐haemoglobin, and 2% carbon monoxide, conditions which inhibit haem iron (II) reactivity. In all cases, parasite growth and chloroquine activity were unaffected. We conclude that in the malaria parasite artemisinin is not activated by haem iron and that free radicals are not needed for its toxicity.

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 FADH2 in aqueous buffer at pH 7.4 under argon. Under the same conditions, FADH2 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 FADH2 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 FADH2 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. 1H NMR spectroscopic studies using the BNAH–riboflavin (RF) model system indicate that the tetraoxane is capable of using both peroxide units in oxidizing the RFH2 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.

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–FeIII results in an enhanced efflux from the DV into the cytosol. In addition, the lipophilic heme–FeIII 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–FeIII. The quinoline or arylmethanol reenters the DV, and so transfers more heme–FeIII out of the DV. In this way, the 4‐aminoquinolines and arylmethanols exert antimalarial activities by enhancing heme–FeIII and thence free FeIII concentrations in the cytosol. The iron species enter into redox cycles through reduction of FeIII to FeII largely mediated by reduced flavin cofactors and likely also by NAD(P)H–Fre. Generation of ROS through oxidation of FeII 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 Ca2+ 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.