Redox reaction of artemisinin with ferrous and ferric ions in aqueous buffer.

Artemisinin, a sesquiterpene with endoperoxide bond, possesses potent antimalarial activity against the ring and late stage of chloroqine-resistant Plasmodium falciparum malaria both in vitro and in vivo. The mode of antimalarial activity of artemisinin is iron-dependent. The aim of this study was to investigate the reactions of artemisinin with ferrous and ferric ions in aqueous buffer. Artemisinin generated a cycle of iron oxidation and reduction. It oxidized ferrous and reduced ferric ions with similar rate of reaction (k=10+/-0.5 M(-1) x s(-1) for ferrous and k=8.5+/-2.0 M(-1) x s(-1) for ferric ion). The major active product was dihydroartemisinin which exhibited antimalarial activity at least 3 times more potent than artemisinin. Dihydroartemisinin preferably binds to ferric ion, forming ferric-dihydroartemisinin complex. The re-oxidation of the complex gives artemisinin and ferric ion. This suggests that in aqueous buffer, the reaction of artemisinin with iron may give rise to the active reaction products, one of them being dihydroartemisinin, which is responsible for antimalarial activity.     

Synthesis and antimalarial activities of structurally simplified 1,2,4-trioxanes related to artemisinin

Eleven derivatives of the clinically useful, antimalarial, 1,2,4-trioxane artemisinin have been synthesized in only several steps from commercial cyclohexanones. Of these simple, tricyclic 1,2,4-trioxanes, 10 showed considerable in vitro antimalarial activity, with one being as potent as artemisinin. Some structure-activity relationship generalizations are made from this series of artemisinin analogs. Triethylsilyl hydrotrioxide (Et₃SiOOOH), prepared in situ from ozone and triethylsilane, is shown to be a mild, fastacting, and effective dioxetane-forming reagent with vinyl ethers and with a vinyl thioether on relatively small (50–100 mg) scale.     

A new library of C-16 modified artemisinin analogs and evaluation of their anti-parasitic activities

A library of C-16 modified artemisinin analogs was prepared and their antimalarial as well as antileishmanial activities were evaluated. Synthesis of these compounds involved the conversion of artemisinin to its phenol derivatives 7 and 12, and subsequent parallel derivatization by introducing new chemical groups through ester, carbamate, sulfate, phosphate and isourea linkages. Comparison of in vitro antimalarial activities showed that C9-beta artemisinin analogs (8a-f) are more potent than the corresponding C9-alpha diastereomers (9a-f); however, their antileishmanial activities were in the same range. Many of the 10-deoxoartemisinin analogs studied here showed promising antiparasitic activities. For example, compounds 13a-e are approximately three times more active against drug resistant W2 strain of P. falciparum, compared to artemisinin (IC(50), approximately 0.2 - 0.6 nM; cf. artemisinin = 1.6 nM). Further, a number of compounds in this series were notably leishmanicidal, with activities comparable to or better than pentamidine (e.g., 13g and 13j). Detailed in vivo studies involving these active compounds are underway to identify lead candidates for further development.     

Synthesis of New 1,2,4-Trioxanes and their Antimalarial Activity

The three dihydronaphtho[1,2,4]trioxines 9 – 11 have been synthesized and two of them converted to the five carbamate and ester derivatives 12 – 16 (Schemes 1 and 2). The resulting new trioxanes together with two already known and ascaridole ( 7 ) were tested for antimalarial activity against the sensitive N strain of Plasmodium berghei in mice. On comparison with artemisinin ( 1 ) and dihydroartemisinin ( 2 ), modest activity was found. The four most active compounds were some 12–18 times less potent than 1 .     

Molecular Basis of Artemisinin Derivatives Inhibition of Myeloid Differentiation Protein 2 by Combined in Silico and Experimental Study

Artemisinin (also known as Qinghaosu), an active component of the Qinghao extract, is widely used as antimalarial drug. Previous studies reveal that artemisinin and its derivatives also have effective anti-inflammatory and immunomodulatory properties, but the direct molecular target remains unknown. Recently, several reports mentioned that myeloid differentiation factor 2 (MD-2, also known as lymphocyte antigen 96) may be the endogenous target of artemisinin in the inhibition of lipopolysaccharide signaling. However, the exact interaction between artemisinin and MD-2 is still not fully understood. Here, experimental and computational methods were employed to elucidate the relationship between the artemisinin and its inhibition mechanism. Experimental results showed that artemether exhibit higher anti-inflammatory activity performance than artemisinin and artesunate. Molecular docking results showed that artemisinin, artesunate, and artemether had similar binding poses, and all complexes remained stable throughout the whole molecular dynamics simulations, whereas the binding of artemisinin and its derivatives to MD-2 decreased the TLR4(Toll-Like Receptor 4)/MD-2 stability. Moreover, artemether exhibited lower binding energy as compared to artemisinin and artesunate, which is in good agreement with the experimental results. Leu61, Leu78, and Ile117 are indeed key residues that contribute to the binding free energy. Binding free energy analysis further confirmed that hydrophobic interactions were critical to maintain the binding mode of artemisinin and its derivatives with MD-2.     

Synthesis of N 11-anchoring biotinylated artemisinin derivatives and their preliminary biological assessment

Unique endoperoxide moiety of artemisinin and its derivatives has been considered the functionality exhibiting highly potent antimalarial and anticancer activities. To investigate the mechanisms of their biological actions, development of suitable molecular probes including biotinylated derivatives is of extreme significance. The synthesis and preliminary biological assessment of four new biotinylated artemisinin derivatives have been reported in this work.     

Epiartemisinin,a Remarkably Poor Antimalarial: Implications for the Mode of Action

Epiartemisinin ( 7 ) was prepared by the base epimerization of artemisinin ( 1 ) and its structure determined by X‐ray analysis. The antimalarial activity of 7 against the chloroquine‐sensitive and resistant strains of Plasmodium berghei and P. yoelii in the mouse was compared with that of the highly effective schizonticide 1 and found to be drastically diminished. It is argued that the mode of action on the intraerythrocytic parasite by 7 is compromised by steric hindrance arising from the α‐disposed Me group. In the initial step, intimate complexation with heme is hindered or biased to favor the formation of a less potent C‐centered radical, the final lethal agent.     

Synthesis of N~(11)-anchoring biotinylated artemisinin derivatives and their preliminary biological assessment

Unique endoperoxide moiety of artemisinin and its derivatives has been considered the functionality exhibiting highly potent antimalarial and anticancer activities.To investigate the mechanisms of their biological actions,development of suitable molecular probes including biotinylated derivatives is of extreme significance.The synthesis and preliminary biological assessment of four new biotinylated artemisinin derivatives have been reported in this work.     

Conversion of antimalarial drug artemisinin to a new series of tricyclic 1,2,4-trioxanes1

A highly efficient route for the conversion of the antimalarial drug artemisinin to a novel hydroxy-functionalized tricyclic 1,2,4-trioxane 6 is reported. Neither the trioxane 6 nor its derivatives 14-16, all of which lack the hydrolytically unstable acetal-lactone linkage, show antimalarial activity comparable with that of artemisinin.     

Artemisinin evaluation in Romanian Artemisia annua wild plants using a new HPLC/MS method

Artemisinin, a sesquiterpene lactone from Artemisia annua L., has received considerable attention in the last few decades as a potent antimalarial drug. Artemisinin has rather low toxicity; it is effective against drug-resistant Plasmodium species and against cerebral malaria. This study reports the development of a rapid and sensitive assay for the quantification of artemisinin in A. annua by reversed phase HPLC/MS. In the selected optimal experimental conditions, artemisinin exhibited a well-defined chromatographic peak with a retention time of 2 ± 0.2 min. The chromatographic signal shows a linear dependence with artemisinin concentration, enabling the use of this signal for artemisinin quantification according to the following regression equation: y = 2665.40x - 14697.61. The correlation coefficient (R(2)) was 0.9989. For every concentration within the range of the standard curve (0.1-2 μg mL(-1)), accuracy was between 95 and 104%. Artemisinin content in Romanian A. annua wild plants varies between 0.17 and 0.21% (dry weight basis).     

The role of charge distribution on the antimalarial activity of artemisinin analogues

In this work the calculated nuclear quadrupole coupling constants (NQCC; chi) of 17O in artemisinin and some of its derivatives and the effects of charge density due to the nature of ligands on NQCC of 17O were investigated. All calculations were performed at the HF/3-21G level using the Gaussian 98 program. The results show that the O-O linkage has a characteristic role in the antimalarial activity of artemisinin. In addition, various substitutions on C4 change the charge density on these oxygens and consequently change the pharmaceutical effect of artemisinin. Our results suggest that due to a larger charge density on O1, the heme iron approaches the endoperoxide moiety at the O1 position with preference to the O2 position.     

Identification and validation of tetracyclic benzothiazepines as Plasmodium falciparum cytochrome bc1 inhibitors

Here we report the discovery of tetracyclic benzothiazepines (BTZs) as highly potent and selective antimalarials along with the identification of the Plasmodium falciparum cytochrome bc₁ complex as the primary functional target of this novel compound class. Investigation of the structure activity relationship within this previously unexplored chemical scaffold has yielded inhibitors with low nanomolar activity. A combined approach employing genetically modified parasites, biochemical profiling, and resistance selection validated inhibition of cytochrome bc₁ activity, an essential component of the parasite respiratory chain and target of the widely used antimalarial drug atovaquone, as the mode of action of this novel compound class. Resistance to atovaquone is eroding the efficacy of this widely used antimalarial drug. Intriguingly, BTZ-based inhibitors retain activity against atovaquone resistant parasites, suggesting this chemical class may provide an alternative to atovaquone in combination therapy.     

Recent Advances in Malaria Chemotherapy

A short review of the currently used antimalarial drugs is reported. The molecular aspects of the different possible mechanisms of action of artemisinin is documented, including recent data on heme alkylation. The preparation and the in vitro antimalarial activity of new modular molecules named “trioxaquines” is also presented.     

Peroxide bond strength of antimalarial drugs containing an endoperoxide cycle. Relation with biological activity

Several endoperoxide compounds are very efficient antimalarial analogues of the natural drug artemisinin. Quantum chemical calculations have been used to correlate the computed free energies of the O-O bond with respect to the total number of oxygen atoms contained in the cycle, and with the size/strain of the cycle (5- or 6-membered cycles). The gas-phase homolysis of the O-O bond has been studied for five- and six-membered oxygenated cycles which are models of the "real" drugs. Our results indicate that, in 6-membered cycles, the stability order is the following: 1,2-dioxane > 1,2,4-trioxane > 1,2,4,5-tetraoxane. In cycles containing 3 oxygen atoms, the 5-membered cycle 1,2,4-trioxolane was found much less stable than its 6-membered counterpart 1,2,4-trioxane. This feature indicates the possible role of the cycle strain for the O-O bond stability, and may also explain the high antimalarial activity of some trioxolane derivatives. Similar trends in the O-O bond strength have been found for the real antimalarial drugs. However, the O-O bond stability is not in itself a decisive argument to anticipate the antimalarial activity of drugs.     

Simultaneous determination of artemisinin and its analogs and flavonoids in Artemisia annua crude extracts with the use of NMR spectroscopy

Artemisia annua is a promising and potent antimalarial herbal drug. This activity has been ascribed to its component artemisinin, a sesquiterpene lactone. The ability to determine artemisinin and its known analogs in plant extracts is an especially difficult task because the compounds are present in low concentrations, are thermolabile, and lack ultraviolet or fluorescent chromophores. We report herein a facile and rapid 1-D ¹H, 1-D total correlation spectroscopy, 2-D ¹H–¹³C heteronuclear single quantum coherence, and ¹H–¹³C heteronuclear multiple bond correlation nuclear magnetic resonance techniques for the simultaneous identification and quantification of artemisinin and five of its analogs along with five flavonoids, an aromatic ketone, and camphor (in total, 13 compounds) in crude diethyl ether A. annua extract without the need of laborious isolation of the individual analytes. The above method was validated in terms of precision, linearity, and limit of detection. The analytical results were found to be in excellent agreement with those obtained with the use of the time consuming high-performance liquid chromatography with diode-array detection and liquid chromatography with tandem mass spectrometry for the compounds that standards were available.     

Characterization of noncovalent complexes of antimalarial agents of the artemisinin-type and Fe(III)-heme by electrospray mass spectrometry and collisional activation tandem mass spectrometry

In this study, we demonstrate, using electrospray ionization mass spectrometry (ESI-MS) and collision-induced dissociation tandem mass spectrometry (ESI-MS/CID/MS), that stable noncovalent complexes can be formed between Fe(III)-heme and antimalarial agents, i.e., quinine, artemisinin, and the artemisinin derivatives, dihydroartemisinin, alpha- and beta-artemether, and beta-arteether. Differences in the binding behavior of the examined drugs with Fe(III)-heme and the stability of the drug-heme complexes are demonstrated. The results show that all tested antimalarial agents form a drug-heme complex with a 1:1 stoichiometry but that quinine also results in a second complex with the heme dimer. ESI-MS performed on mixtures of pairs of various antimalarial agents with heme indicate that quinine binds preferentially to Fe(III)-heme, while ESI-MS/CID/MS shows that the quinine-heme complex is nearly two times more stable than the complexes formed between heme and artemisinin or its derivatives. Moreover, it is found that dihydroartemisinin, the active metabolite of the artemisinin-type drugs in vivo, results in a Na(+)-containing heme-drug complex, which is as stable as the heme-quinine complex. The efficiency of drug-heme binding of artemisinin derivatives is generally lower and the decomposition under CID higher compared with quinine, but these parameters are within the same order of magnitude. These results suggest that the efficiency of antimalarial agents of the artemisinin-type to form noncovalent complexes with Fe(III)-heme is comparable with that of the traditional antimalarial agent, quinine. Our study illustrates that electrospray ionization mass spectrometry and collision-induced dissociation tandem mass spectrometry are suitable tools to probe noncovalent interactions between heme and antimalarial agents. The results obtained provide insights into the underlying molecular modes of action of the traditional antimalarial agent quinine and of the antimalarials of the artemisinin-type which are currently used to treat severe or multidrug-resistant malaria.     

Determination of artemisinin in human serum by high-performance liquid chromatography with on-line UV irradiation and peroxyoxalate chemiluminescence detection

Artemisinin is an antimalarial drug containing an internal endoperoxide linkage in its structure. A simple, selective and sensitive high-performance liquid chromatography (HPLC)-peroxyoxalate chemiluminescence (PO-CL) method for the determination of artemisinin was developed. This method is based on the fact that endoperoxide in artemisinin structure can be converted to hydrogen peroxide (H(2)O(2)) under ultraviolet (UV) irradiation and the generated hydrogen peroxide can be measured using PO-CL detection. The HPLC-PO-CL system was optimized on a mobile phase, post column chemiluminescence reagent, UV source and irradiation time. In addition, the system was combined with simple liquid-liquid extraction using n-hexane that allowed selective and sensitive determination of artemisinin in serum. The limit of detection using 0.5 mL of blood was 0.062 micromol/L (17.5 ng/mL) at a signal-to-noise ratio of 3. Calibration curve obtained for artemisinin in human serum 4-80 micromol/L (1.1-22.6 microg/mL) showed a good linearity (r = 0.999).     

Correlating the molecular electrostatic potentials of some organic peroxides with their antimalarial activities

The molecular electrostatic potentials (MEPs) of artemisinin (also known as qinghaosu), yingzhaosu A, and some synthetic analogues have been calculated and studied as a means of distinguishing between high and low antimalarial activity. To facilitate comparison, the dimensionality of the MEP was reduced by Kohonen Neural Network transforms. The reduction revealed that peroxides exhibiting high antimalarial activity are characterized by a continuous strip of negative electric potential surrounding the molecule, whereas peroxides of lesser activity show a broken strip.     

5-Phenoxy Primaquine Analogs and the Tetraoxane Hybrid as Antimalarial Agents

The rapid emergence of drug resistance to the current antimalarial agents has led to the urgent need for the discovery of new and effective compounds. In this work, a series of 5-phenoxy primaquine analogs with 8-aminoquinoline core (7a–7h) was synthesized and investigated for their antimalarial activity against Plasmodium falciparum. Most analogs showed improved blood antimalarial activity compared to the original primaquine. To further explore a drug hybrid strategy, a conjugate compound between tetraoxane and the representative 5-phenoxy-primaquine analog 7a was synthesized. In our work, the hybrid compound 12 exhibited almost a 30-fold increase in the blood antimalarial activity (IC₅₀ = 0.38 ± 0.11 μM) compared to that of primaquine, with relatively low toxicity against mammalian cells (SI = 45.61). Furthermore, we found that these 5-phenoxy primaquine analogs and the hybrid exhibit significant heme polymerization inhibition, an activity similar to that of chloroquine, which could contribute to their improved antimalarial activity. The 5-phenoxy primaquine analogs and the tetraoxane hybrid could serve as promising candidates for the further development of antimalarial agents.