Two culture-adapted strains of P. falciparum were used: the chloroquine-sensitive strain FCR-8/West African, and chloroquine-resistant strain FCR-3/Gambia subline F-86 of P. falciparum obtained from ATCC (Nguyen-Dinh and Trager, 1980; Jensen and Trager, 1978). The medium used was RPMI medium containing hypoxanthine and supplemented with HEPES buffer, sodium bicarbonate, human A serum, glutamine, gentamicin, and uninfected human O erythrocytes.

The assays were conducted in vitro by a modification of the semiautomated microdilution technique of Desjardins et al. (1979) and Delhaes et al. (2002) based on radiolabeled [³H]hypoxanthine incorporation. Drug testing was carried out in 96-well, microtiter plates. Stock solutions of each compound were prediluted in complete culture medium (RPMI 1640 supplemented with 10% pooled human A serum), and titrated in duplicate in serial twofold dilutions. The final concentrations ranged from 1.96-250 nmole L^-1 for artemisinin derivatives and artemisinin, and 11.2-1435nmole L^-1 for chloroquine. After the addition of a suspension of parasitized erythrocytes in complete culture medium (200µl per well, 0.7% initial parasitemia with a majority of ring stages, 1.8-2% haematocrit) and [H3]hypoxanthine (Amersham, UK, TRK74, 1µl per well), the test plates were incubated at 37℃ for 24 h in an atmosphere of 5% O₂, 5% CO₂, and 90% N₂. Growth of the parasites was estimated from the incorporation of radiolabeled [H³]hypoxanthine into the parasites` nucleic acids, measured in a liquid scintillation spectrometer (Packard, USA). The 50% inhibitory concentration (IC50) values refer to the molar concentrations of drug causing a 50% reduction in [H³]hypoxanthine incorporation, compared to drug-free control wells. IC50 values were estimated by linear regression analysis of log-dose-response curves.

In the screening of the two standard molecules, chloroquine and artemisinin, against chloroquine-resistance (50005 = FCR-3) and -sensitive (50028 = FCR-8) parasites, we could confirm the biological property of each cell line and the inhibitory activity of each drug (Table 1). At first, two diastereomers, 10α-(Fig. 3-5) and 10β-thiophenyl-dihydroartemisinins (Fig. 3-6), had a similar inhibition activity with artemisinin. Interestingly, 10α- (Fig. 3-7) and 10β-benzenesulfonyl-dihydroartemisinins (Fig. 3-8) showed different inhibitory activity against each cell line according to the change of stereochemistry in the C-10 position of artemisinin. 10β-Diastereomer (Fig. 3-8) was ten times more active than 10α-diastereomer (Fig. 3-7). In particular, 10β-benzenesulfonyl-dihydroartemisinin (Fig. 3-8) was two times more active than artemisinin and ninety times more than chloroquine. This preliminary screening of each thiophenyl- and benzenesulfonyl-dihydroartemisinin derivative indicated that 10β-sulfonyl-dihydroartemisinin derivative can be a potentially promising antimalarial drug against chloroquine-resistance parasites.