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...[
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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 FADH
2 in presence of NAD(P)H in
aqueous buffer at physiological pH. Oxidation of FADH
2 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.
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