Artemisinin and its derivatives dihydroartemisinin (DHA), artemether and artesunate are used for chemotherapy of malaria in combination with a long half-life partner drug in so-called artemisinin combination therapy.

Artemisinin is not a cheap compound, and its supply is somewhat limited because of reliance on extraction from the natural source, the plant Artemisia annua. If totally synthetic analogues can be prepared easily by a cheap route, this will remove the reliance on a plant source. Examples are already provided by 1,2,4-trioxanes, 1,2,4-trioxolanes and 1,2,4,5-tetraoxanes, some of which are being developed as clinical candidates, or are regarded as useful lead compounds. A relatively little known class of compound called 1,2,4-dioxazolidines comprising a five membered...[ Read more ]

Artemisinin and its derivatives dihydroartemisinin (DHA), artemether and artesunate are used for chemotherapy of malaria in combination with a long half-life partner drug in so-called artemisinin combination therapy.

Artemisinin is not a cheap compound, and its supply is somewhat limited because of reliance on extraction from the natural source, the plant Artemisia annua. If totally synthetic analogues can be prepared easily by a cheap route, this will remove the reliance on a plant source. Examples are already provided by 1,2,4-trioxanes, 1,2,4-trioxolanes and 1,2,4,5-tetraoxanes, some of which are being developed as clinical candidates, or are regarded as useful lead compounds. A relatively little known class of compound called 1,2,4-dioxazolidines comprising a five membered ring containing a peroxide group and a nitrogen atom are very easily prepared from ketones by using aqueous ammonia and dilute hydrogen peroxide. It is therefore worthwhile to evaluate the suitability of these compounds as antimalarial agents. In the first project, the preparation of 1,2,4-dioxazolidines and their urea adducts is reexamined. Attempts have been made to optimize the preparation by use of selected metal catalysts. In addition, efforts to isolate and fully characterize the α-hydroperoxy-amine intermediates are made. The antimalarial activities of the dioxazolidines and their thermally stable urea derivatives are assessed.

The second part of this thesis focusses on the mechanism of action of artemisinin antimalarials. The popular interpretation is that the peroxide is cleaved in a Fenton reaction by the ferrous iron inside the malaria parasite to provide C-centered radicals as the cytotoxic agents. However, there are problems with the generation of the C-radicals and their biological relevance. Recently, our group has shown that artemisinins rapidly oxidize leucomethylene blue (LMB) to methylene blue (MB), and oxidize reduced flavin cofactors to the corresponding flavins. At the same time, the artemisinins undergo two-electron reduction to the deoxygenated products. These results indicate that artemisinins are able to perturb the redox balance in the parasite by interfering with the parasite flavoenzymes such as glutathione reductase (GR), thioredoxin reductase (TrxR) and others. This proposal is called the 'cofactor model'.

In order to better mimic the biological environment, E. coli flavin reductase (Fre) is introduced here to generate reduced cofactors such as FADH₂ in presence of NAD(P)H in aqueous buffer at physiological pH. Oxidation of FADH₂ generated in situ by NAD(P)H-Fre by artemisinins can be monitored by UV spectroscopy. Together with the biomimetic BNAH-riboflavin (RF) system developed previously, a study of the decomposition of both of known artemisinin antimalarial drugs and peroxide analogues is carried out. This project is divided into five parts. In the first part, the ability of 1,2,4,5-tetraoxanes, 1.2.4-trioxolanes and 1,2,4-dioxazolidine to oxidize the reduced flavin models is first examined.

In the second part of the mechanism project, the effects of 4-aminoquinoline and arylmethanol antimalarial drugs on the rate of reduction of the reduced flavins by artemisinins in the aqueous buffer are studied. As artemisinins are used in combination with 4-aminoquinolines and arylmethanols, any antagonistic or synergistic interaction of the drugs with artemisinins is important for the selection of partner drugs in combination therapies. An attempt is made to correlate the effects of the quinolines and arylmethanols on the rate of oxidation of the reduced flavins by artemisinins with the antagonism or synergism of antimalarial activities already reported for the drug pairs in the literature.

In the third part of the mechanism project, the effect of pH on the rate of oxidation of the reduced flavin cofactors by the artemisinin is examined. This is also extended to an evaluation of the inhibiting effects of the quinolines either as the free bases or as their salts. The Hendersen-Hasselbalch equation is used to calculate the relative amounts of protonated quinolines present at designated pH values of the reaction mixture, and how the protonated quinoline itself may affect the rate of reduction of the reduced cofactor.

In the fourth part of the mechanism project, it is found that 1,2,4-dioxazolidine urea adducts do not oxidize the reduced flavin cofactors, whereas the parent dioxazolidines do so. Attempts are made to establish the reason for this, including use of model ureas added to reaction mixtures containing peroxides that oxidize the reduced cofactors. The new artemisinin urea derivative RW177, that is approximately twice as active against the malaria parasite as artemisone, the new generation artemisinin drug, is also examined.

Artemisone has undergone clinical trials, where it turns out to be three times more active than artesunate. However, artemisone decomposes in aqueous solution below pH 2 into DHA. The methylene homologues of artemisone were previously prepared in our group. These were expected to be more stable than artemisone, but were substantially less active in vitro against the malaria parasite. In the fifth part of the mechanism project, the mode of action of the methylene homologues is studied in terms of the co-factor model in order to establish if there is a correlation between the rate of oxidation of reduced flavin cofactors with the inferior in vitro antimalarial activity.