Accumulation of artemisinin trioxane derivatives within neutral lipids of Plasmodium falciparum malaria parasites is endoperoxide-dependent

The antimalarial trioxanes, exemplified by the naturally occurring sesquiterpene lactone artemisinin and its semi-synthetic derivatives, contain an endoperoxide pharmacophore that lends tremendous potency against Plasmodium parasites. Despite decades of research, their mechanism of action remains unresolved. A leading model of anti-plasmodial activity hypothesizes that iron-mediated cleavage of the endoperoxide bridge generates cytotoxic drug metabolites capable of damaging cellular macromolecules. To probe the malarial targets of the endoperoxide drugs, we studied the distribution of fluorescent dansyl trioxane derivatives in living, intraerythrocytic-stage Plasmodium falciparum parasites using microscopic imaging. The fluorescent trioxanes rapidly accumulated in parasitized erythrocytes, localizing within digestive vacuole-associated neutral lipid bodies of trophozoites and schizonts, and surrounding the developing merozoite membranes. Artemisinin pre-treatment significantly reduced fluorescent labeling of neutral lipid bodies, while iron chelation increased non-specific cytoplasmic localization. To further explore the effects of endoperoxides on cellular lipids, we used an oxidation-sensitive BODIPY lipid probe to show the presence of artemisinin-induced peroxyl radicals in parasite membranes. Lipid extracts from artemisinin-exposed parasites contained increased amounts of free fatty acids and a novel cholesteryl ester. The cellular accumulation patterns and effects on lipids were entirely endoperoxide-dependent, as inactive dioxolane analogs lacking the endoperoxide moiety failed to label neutral lipid bodies or induce oxidative membrane damage. In the parasite digestive vacuole, neutral lipids closely associate with heme and promote hemozoin formation. We propose that the trioxane artemisinin and its derivatives are activated by heme-iron within the neutral lipid environment where they initiate oxidation reactions that damage parasite membranes.     

Extraordinarily potent antimalarial compounds: new, structurally simple, easily synthesized, tricyclic 1,2,4-trioxanes.

New, racemic, tricyclic trioxane alcohol 3 was designed and synthesized as a structurally simple analog of clinically useful, tetracyclic, antimalarial artemisinin. A series of 20 ester and ether derivatives of alcohol 3 were prepared easily, without destruction of the essential trioxane system. Chemical structure-antimalarial activity for each derivative was evaluated in vitro against chloroquine-resistant and chloroquine-sensitive Plasmodium falciparum parasites. Many of these derivatives were highly efficacious; carboxylate ester 9f, carbamate ester 10a, and sulfonate ester 12a had antimalarial potency similar to that of artemisinin, and carboxylate esters 9b and 9d, carbamate esters 10b and 10c, and phosphate esters 11a-c had antimalarial potency up to 7 times higher than that of artemisinin. Several of these most active analogs (e.g., carboxylate 9b and carbamates 10a and 10c) are stable crystalline solids, a feature of considerable practical value for any new drug candidate.     

Identification of intermediates and enzymes involved in the early steps of artemisinin biosynthesis in Artemisia annua

An important group of antimalarial drugs consists of the endoperoxide sesquiterpene lactone artemisinin and its derivatives. Only little is known about the biosynthesis of artemisinin in Artemisia annua L., particularly about the early enzymatic steps between amorpha-4,11-diene and dihydroartemisinic acid. Analyses of the terpenoids from A. annua leaves and gland secretory cells revealed the presence of the oxygenated amorpha-4,11-diene derivatives artemisinic alcohol, dihydroartemisinic alcohol, artemisinic aldehyde, dihydroartemisinic aldehyde and dihydroartemisinic acid. We also demonstrated the presence of a number of biosynthetic enzymes such as the amorpha-4,11-diene synthase and the--so far unknown--amorpha-4,11-diene hydroxylase as well as artemisinic alcohol and dihydroartemisinic aldehyde dehydrogenase activities in both leaves and glandular trichomes. From these results, we hypothesise that the early steps in artemisinin biosynthesis involve amorpha-4,11-diene hydroxylation to artemisinic alcohol, followed by oxidation to artemisinic aldehyde, reduction of the C11-C13 double bond to dihydroartemisinic aldehyde and oxidation to dihydroartemisinic acid.     

Kinetics of iron-mediated artemisinin degradation: effect of solvent composition and iron salt

The antimalarial endoperoxides, such as artemisinin, are postulated to exert their potent parasiticidal activity via the formation of reactive intermediates in the iron-rich infected erythrocyte. The in vitro chemical reaction profile of putative endoperoxide antimalarials and ferrous iron is often qualitatively used to assess their potential antimalarial activity and to develop a structure-reactivity relationship. This study utilized LCMS to monitor the kinetics of artemisinin degradation and product formation in the presence of iron. A second order degradation reaction (k = 18 M(-1) h(-1)) was observed from the reaction of artemisinin with ferrous sulphate in aqueous acetonitrile to produce a number of stable isomeric rearrangement products. A systematic study of the effect of a number of solvent systems and different iron salts showed pronounced changes in reaction rate and product distribution. The significant effects observed in the current study highlight the need to carefully control reaction conditions when studying peroxide antimalarial stability or attempting to develop in vitro/in vivo correlations of endoperoxide antimalarials and their reactivity with iron.     

Comparison of the reactivity of antimalarial 1,2,4,5-tetraoxanes with 1,2,4-trioxolanes in the presence of ferrous iron salts, heme, and ferrous iron salts/phosphatidylcholine

Dispiro-1,2,4,5-tetraoxanes and 1,2,4-trioxolanes represent attractive classes of synthetic antimalarial peroxides due to their structural simplicity, good stability, and impressive antimalarial activity. We investigated the reactivity of a series of potent amide functionalized tetraoxanes with Fe(II)gluconate, FeSO(4), FeSO(4)/TEMPO, FeSO(4)/phosphatidylcholine, and heme to gain knowledge of their potential mechanism of bioactivation and to compare the results with the corresponding 1,2,4-trioxolanes. Spin-trapping experiments demonstrate that Fe(II)-mediated peroxide activation of tetraoxanes produces primary and secondary C-radical intermediates. Reaction of tetraoxanes and trioxolanes with phosphatidylcholine, a predominant unsaturated lipid present in the parasite digestive vacuole membrane, under Fenton reaction conditions showed that both endoperoxides share a common reactivity in terms of phospholipid oxidation that differs with that of artemisinin. Significantly, when tetraoxanes undergo bioactivation in the presence of heme, only the secondary C-centered radical is observed, which smoothly produces regioisomeric drug derived-heme adducts. The ability of these tetraoxanes to alkylate the porphyrin ring was also confirmed with Fe(II)TPP and Mn(II)TPP, and docking studies were performed to rationalize the regioselectivity observed in the alkylation process. The efficient process of heme alkylation and extensive lipid peroxidation observed here may play a role in the mechanism of action of these two important classes of synthetic endoperoxide antimalarial.     

Kinetic parameters for the reaction of 1,2,4‐trioxolanes and artemisinin with iron(II): New evidence for the source of antimalarial activity

The Fenton‐like reductive cleavage of antimalarial peroxides like artemisinin by iron(II) species is a chemical reaction whose mechanistic pathway has not been yet fully understood; it is, however, known that there is considerable production of radical species centered at both the oxygen and carbon, which are important to the therapeutical effects of those compounds. This article reports kinetic data for the reaction of artemisinin and two model 1,2,4‐trioxolanes with iron(II) species and also a mechanistic interpretation of this reductive cleavage from transition state thermodynamics. The suggestion of the presence of an enhancing specific factor inside the plasmodium is made.     

Antimalarial and antitumor evaluation of novel C-10 non-acetal dimers of 10beta-(2-hydroxyethyl)deoxoartemisinin

Four series of C-10 non-acetal dimers were prepared from key trioxane alcohol 10beta-(2-hydroxyethyl)deoxoartemisinin (9b). All of the dimers prepared displayed potent low nanomolar antimalarial activity versus the K1 and HB3 strains of Plasmodium falciparum. The most potent compound assayed was phosphate dimer 14a, which was greater than 50 times more potent than the parent drug artemisinin and about 15 times more potent than the clinically used acetal artemether. In contrast to their potent activity versus malaria parasites, virtually all of the dimers expressed poor anticancer activity apart from the trioxane phosphate ester dimers 14a and 14b, which expressed nanomolar growth inhibitory (GI50) values versus a range of cancer cell lines in the NCI 60 human cell line screen. Further detailed studies on these dimers in vitro in HL60 cells demonstrate that both phosphate ester dimers (14a and 14b) are more potent than the anticancer agent doxorubicin. Interestingly, phosphate ester monomers 9c and 9d, antimalarially active in the low nanomolar region versus P. falciparum, are inactive as anticancer agents even at concentrations in the millimolar region. This observation emphasizes the importance of two trioxane units for high antiproliferative activity, and we propose that the nature of the linker in dimers of this type plays a crucial role in imparting potent anticancer activity.     

Comparative embryotoxicity of different antimalarial peroxides: In vitro study using the rat whole embryo culture model (WEC)

Three groups of compounds: (i) active peroxides (artemisinin and arterolene), (ii) inactive non-peroxidic derivatives (deoxyartemisinin and carbaOZ277) and (iii) inactive peroxide (OZ381) were tested by WEC system to provide insights into the relationship between chemical structure and embryotoxic potential, and to assess the relationship between embryotoxicity and antimalarial activity.Deoxyartemisinin, OZ381 and carbaOZ277 did not affect rat embryonic development. Artemisinin and arterolane affected primarily nucleated red blood cells (RBCs), inducing anemia and subsequent tissue damage in rat embryos, with NOELs for RBC damage at 0.1 and 0.175 μg/mL, respectively. These data support the idea that only active antimalarial peroxides are able to interfere with normal embryonic development. In an attempt to establish whether and to what extent activity as antimalarials and embryotoxicity can be divorced, IC₅₀s for activity in Plasmodium falciparum strains and the NOELs for RBCs were compared. From this comparison, arterolane showed a better safety margin than artemisinin.     

Reactions of artemisinin and arteether with acid: implications for stability and mode of antimalarial action

The currently accepted mechanism of trioxane antimalarial action involves generation of free radicals within or near susceptible sites probably arising from the production of distonic radical anions. An alternative mechanistic proposal involving the ionic scission of the peroxide group and consequent generation of a carbocation at C-4 has been suggested to account for antimalarial activity. We have investigated this latter mechanism using DFT (B3LYP/6-31+G* level) and established the preferred Lewis acid protonation sites (artemisinin O5a>O4a approximately O3a>O2a>O1a; arteether O4a>or=O3a>O5b>O2a>O1a; Figure 3) and the consequent decomposition pathways and hydrolysis sites. In neither molecule is protonation likely to occur on the peroxide bond O1-O2 and therefore lead to scission. Therefore, the alternative radical pathway remains the likeliest explanation for antimalarial action.     

Spiro- and dispiro-1,2-dioxolanes: contribution of iron(II)-mediated one-electron vs two-electron reduction to the activity of antimalarial peroxides

Fourteen spiro- and dispiro-1,2-dioxolanes were synthesized by peroxycarbenium ion annulations with alkenes in yields ranging from 30% to 94%. Peroxycarbenium ion precursors included triethylsilyldiperoxyketals and -acetals derived from geminal dihydroperoxides and from a new method employing triethylsilylperoxyketals and -acetals derived from ozonolysis of alkenes. The 1,2-dioxolanes were either inactive or orders of magnitude less potent than the corresponding 1,2,4-trioxolanes or artemisinin against P. falciparum in vitro and P. berghei in vivo. In reactions with iron(II), the predominant reaction course for 1,2-dioxolane 3a was two-electron reduction. In contrast, the corresponding 1,2,4-trioxolane 1 and the 1,2,4-trioxane artemisinin undergo primarily one-electron iron(II)-mediated reductions. The key structural element in the latter peroxides appears to be an oxygen atom attached to one or both of the peroxide-bearing carbon atoms that permits rapid beta-scission reactions (or H shifts) to form primary or secondary carbon-centered radicals rather than further reduction of the initially formed Fe(III) complexed oxy radicals.     

Site-specific DNA damage by phenylhydrazine and phenelzine in the presence of Cu(II) ion or Fe(III) complexes: roles of active oxygen species and carbon radicals.

Phenylhydrazine cleaved isolated DNA in the presence of Cu(II), Mn(III), hemin, Fe(III)-EDTA, or peroxidase/H2O2, while phenelzine cleaved in the presence of Cu(II). DNA cleavage by phenylhydrazine in the presence of Mn(III), hemin, or Fe(III)-EDTA occurred without marked site specificity. Inhibitory effects of scavengers of hydroxyl free radical (.OH) on the DNA damage suggest the involvement of .OH. On the other hand, Cu(II)-mediated DNA cleavage by phenylhydrazine or phenelzine was inhibited by catalase and bathocuproine, a Cu(I)-specific chelator, but not by .OH scavengers. The predominant cleavage site was the thymine residue of 5'-GTC-3' sequence. Since the cleavage pattern was similar to that induced by Cu(I) plus H2O2 but not to that induced by Cu(II) plus H2O2, it is speculated that the copper-oxygen complex derived from the reaction of H2O2 with Cu(I) participates in DNA damage by phenylhydrazine or phenelzine in the presence of Cu(II). A comparison between scavenger effects on the DNA damage and those on radical production detected with ESR suggests that carbon-centered radicals (phenyl radical, 2-phenylethyl radical) do not play an important role in Cu(II)-, hemin-, or Fe(III)-EDTA-mediated DNA damage by phenylhydrazine or phenelzine of relatively low concentrations (less than 0.5 mM). However, during the oxidation of a high concentration (10 mM) of phenylhydrazine by ferricyanide, phenyl radical seemed to cause DNA damage, especially the breakage of the deoxyribose phosphate backbone. The possibility that active oxygen species (copper-oxygen complex, .OH) are more important in DNA damage induced by hydrazines in vivo than carbon-centered radicals is discussed.     

Second generation, orally active, antimalarial, artemisinin-derived trioxane dimers with high stability, efficacy, and anticancer activity

In only two steps and in 63% overall yield, naturally occurring 1,2,4-trioxane artemisinin (1) was converted into C-10-carba trioxane conjugated diene dimer 4. This new dimer was then transformed easily in one additional 4 + 2-cycloaddition step into phthalate dimer 5, and further modification led to bis-benzyl alcohol dimer 7 and its phosphorylated analogues 8 and 9. Bis-benzyl alcohol dimer 7 is the most antimalarially active in vitro, 10 times more potent than artemisinin (1). Bis-benzyl alcohol dimer 7 is approximately 1.5 times more orally efficacious in rodents than the antimalarial drug sodium artesunate and is about 37 times more efficacious than sodium artesunate via subcutaneous administration. Both dimers 5 and 7 are thermally stable neat even at 60 degrees C for 24 h. Phthalate dimer 5 is very highly growth inhibitory but not cytotoxic toward several human cancer cell lines; both dimers 5 and 7 very efficiently and selectively kill human cervical cancer cells in vitro in a dose-dependent manner with no cytotoxic effects on normal cervical cells.     

Iron-mediated degradation kinetics of substituted dispiro-1,2,4-trioxolane antimalarials

The iron-mediated reactivity of various dispiro-1,2,4-trioxolanes was determined by automated kinetic analysis under standard reaction conditions. The active antimalarial compounds underwent peroxide bond cleavage by Fe(II) resulting in products indicative of carbon-centered radical formation. The rate of reaction was heavily influenced by the presence of spiro-substituted adamantane and cyclohexane rings, and was also significantly affected by cyclohexane ring substitution. Steric hindrance around the peroxide oxygen atoms appeared to be the major determinant of reaction rate, however polar substituents also affected reactivity by an independent mechanism. A wide range of reaction rates was observed within this class of peroxide antimalarials, however iron-mediated reactivity did not directly correlate with in vitro antimalarial activity.     

Simultaneous determination of Fe(II) and Fe(III) in pharmaceutical formulations with chromogenic mixed reagent by using principal component artificial neural network and multivariate calibration

The use of chemometric approaches for the simultaneous determination of Fe(II) and Fe(III) ions has been explored by means of a two component reagent. Mixed reagents of 1,10-phenanthroline and thiocyanate were used as a selective chromogenic system for speciation of Fe(II) and Fe(III). Although the complexes of Fe(II) and Fe(III) with mixed reagent show a spectral overlap, they have been simultaneously determined with chemometric approaches, such as principal component artificial neural network (PC-ANN), principal component regression (PCR) and partial least squares (PLS). A set of synthetic mixtures of Fe(II) and Fe(III) was evaluated and the results obtained by the applications of these chemometric approaches were discussed and compared. It was found that the PC-ANN and PLS methods afforded better precision relatively than its of PCR. PC-ANN and PLS methods were also applied satisfactorily in determination of Fe(II) and Fe(III) in pharmaceutical samples.     

Self-inactivation of Fe(II)-bleomycin

Fe(II)-Bleomycin is activated in air to form an electron paramagnetic resonance (EPR)-active species, termed "activated bleomycin", that cleaves DNA, when present. When DNA is absent, the potential DNA cleavage activity is lost and the drug becomes self inactivated. A method is described for the preparation and purification of this self-inactivated product from bleomycin A2, together with some of its physical properties. It is shown that the loss of DNA cleavage activity parallels an alteration of bithiazole fluorescence, attributed to chemical change at this residue. EPR evidence is brought forth that the Cu(II) binding site of inactivated bleomycin in not altered, nor is the ability to form a species with Fe(II) and O2 having the identical spectroscopic signature as activated bleomycin.     

Differential effects of clinically used derivatives and metabolites of artemisinin in the activation of constitutive androstane receptor isoforms

BACKGROUND AND PURPOSE Widespread resistance to antimalarial drugs requires combination therapies with increasing risk of pharmacokinetic drug-drug interactions. Here, we explore the capacity of antimalarial drugs to induce drug metabolism via activation of constitutive androstane receptors (CAR) by ligand binding. EXPERIMENTAL APPROACH A total of 21 selected antimalarials and 11 major metabolites were screened for binding to CAR isoforms using cellular and in vitro CAR-coactivator interaction assays, combined with in silico molecular docking. Identified ligands were further characterized by cell-based assays and primary human hepatocytes were used to elucidate induction of gene expression. KEY RESULTS Only two artemisinin derivatives arteether and artemether, the metabolite deoxyartemisinin and artemisinin itself demonstrated agonist binding to the major isoforms CAR1 and CAR3, while arteether and artemether were also inverse agonists of CAR2. Dihydroartemisinin and artesunate acted as weak inverse agonists of CAR1. While arteether showed the highest activities in vitro, it was less active than artemisinin in inducing hepatic CYP3A4 gene expression in hepatocytes. CONCLUSIONS AND IMPLICATIONS Artemisinin derivatives and metabolites differentially affect the activities of CAR isoforms and of the pregnane X receptor (PXR). This negates a common effect of these drugs on CAR/PXR-dependent induction of drug metabolism and further provides an explanation for artemisinin consistently inducing cytochrome P450 genes in vivo, whereas arteether and artemether do not. All these drugs are metabolized very rapidly, but only artemisinin is converted to an enzyme-inducing metabolite. For better understanding of pharmacokinetic drug-drug interaction possibilities, the inducing properties of artemisinin metabolites should be considered.     

Cross-linking of gelatin capsules with formaldehyde and other aldehydes: an FTIR spectroscopy study

Attenuated total reflection Fourier transform infrared spectroscopy (FTIR) has been used to study cross-linking in hard gelatin capsules induced by exposure to formaldehyde, acetaldehyde, and propionaldehyde. These aldehydes are known to cause cross-linking between the amino acid chains of gelatin. Using FTIR spectroscopy, it is possible to analyze the cross-linking mechanisms by studying changes in the vibrational bands of the gelatin spectrum. The FTIR spectrum changes over time when the capsules are left in an aldehyde-rich environment. Analysis of the spectra shows that the early observed spectral changes conform to reaction intermediates proposed in previous work based on nuclear magnetic resonance experiments, specifically, the formation of amine methyl alcohol of arginine and lysine residues. Further spectral changes appear to be mostly from unreacted aldehydes absorbed to the gelatin, although a minor shift of the amide II peak is attributed to cross-link formation.     

Artesunate inhibits osteoclast differentiation by inducing ferroptosis and prevents iron overload-induced bone loss

Artemisinin compounds have been demonstrated to have anti-osteoporosis effects by inhibiting bone resorption. During osteoclast differentiation, osteoclasts take up a large amount of iron through transferrin receptor 1 (TfR1) mediated endocytosis of transferrin (Tf). Since iron-dependent cleavage of endoperoxide bridge is of great importance for the antimalarial effects of artemisinin compounds, we raised a hypothesis that the cytotoxic effects of artemisinin compounds on osteoclasts were associated with enhanced iron uptake. In the present study, we found that Tf aggravated the inhibitory effects of artesunate (ART) on osteoclast viability and differentiation. ART induced the production of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) in a dose-dependent manner and led to the appearance of mitochondrial features of ferroptotic cells. TfR1 knockdown alleviated these cytotoxic effects of ART on osteoclasts. In addition, ART effectively prevented bone loss induced by iron overload. Our results indicate that ART inhibits iron-uptake stimulated osteoclast differentiation by inducing ferroptosis. Artemisinin compounds are potential drugs for treating iron overload-induced osteoporosis.     

Antimalarial, antiproliferative, and antitumor activities of artemisinin-derived, chemically robust, trioxane dimers.

Nine C-10 non-acetal derivatives of the natural trioxane artemisinin (1) were prepared as dimers using some novel chemistry. As designed, each dimer was stable chemically. C-10 Olefinic dimers 7 and C-10 saturated dimers 8-13 all showed good to excellent antimalarial and antiproliferative activities in vitro. Dimers 8, 10, and 12 were especially potent and selective at inhibiting growth of some human cancer cell lines in the NCI in vitro 60-cell line assay.     

Stereoelectronic properties of antimalarial artemisinin analogues in relation to neurotoxicity.

Quantum chemical calculations on the molecular electronic structure of artemisinin (qinghaosu) and eight of its derivatives have resulted in stereoelectronic discriminators that differentiate between analogues with higher and lower neurotoxicities. Detailed ab initio quantum chemical calculations leading to complete optimization of geometry of each of the molecules were followed by calculation of their stereoelectronic properties using the 3-21G split valence basis sets and comparison of the stereoelectronic properties to in vitro neurotoxicity. The least neurotoxic compounds are more polar with an electric field pointing away from the endoperoxide bond and have a higher positive potential on the van der Waals surface of the all carbon-containing ring C, a more stable peroxide bond to cleavage, a less negative electrostatic potential by the endoperoxide, and a single negative potential region extending beyond the van der Waals surface of the molecule. In general, higher intrinsic lipophilicity is associated with greater neurotoxicity.