Synthesis and Antiplasmodial Activity of Novel Fosmidomycin Derivatives and Conjugates with Artemisinin and Aminochloroquinoline

Malaria, despite many efforts, remains among the most problematic infectious diseases worldwide, mainly due to the development of drug resistance by Plasmodium falciparum. The antibiotic fosmidomycin (FSM) is also known for its antimalarial activity by targeting the non-mevalonate isoprenoid synthesis pathway, which is essential for the malaria parasites but is absent in mammalians. In this study, we synthesized and evaluated against the chloroquine-resistant P. falciparum FcB1/Colombia strain, a series of FSM analogs, derivatives, and conjugates with other antimalarial agents, such as artemisinin (ART) and aminochloroquinoline (ACQ). The biological evaluation revealed four new compounds with higher antimalarial activity than FSM: two FSM-ACQ derivatives and two FSM-ART conjugates, with 3.5–5.4 and 41.5–23.1 times more potent activities than FSM, respectively.


Introduction
Despite worldwide efforts, malaria remains among the most dangerous infectious diseases. Malaria incidence has decreased significantly since 2010, but, unfortunately, after 2014, the number of malaria cases is increasing again, according to the World Health Organization [1]. The main reason is the elevated resistance to artemisinin combination therapy (ACT) [2]. In that respect, there is an urgent need for antimalarial agents with a novel mechanism of action (MOA).
During the last decade of the 20th century, the discovery of the non-mevalonate isoprenoid biosynthesis pathway as an essential target raised hope for potential therapeutic opportunities [3]. The pathway is proved to be essential for malaria parasites, while it is absent in mammalian hosts who generate isoprenoids exclusively via the mevalonate pathway [4,5]. The enzymes of the non-mevalonate pathway are located in the apicoplast organelle of malaria parasites, which only host small numbers of proteins, thus becoming attractive drug targets [6][7][8]. In this respect, the seminal work of Jomaa et al. [9] set the basis of an intense research work on compounds targeting the non-mevalonate pathway of the Plasmodium. These authors reported in 1999 that the antibiotic fosmidomycin (FSM, 1, Figure 1) inhibits the IspC (see Figure 1), which catalyzes the first committed step of the non-mevalonate pathway and is believed to be rate limiting. FSM, which was initially isolated from Streptomyces lavendulae [10,11], is a phosphonate structural analogue of the IspC substrate DOXP (4), which furthermore bears a hydroxamic acid moiety in replacement of the alpha hydroxy ketone of 4 and thus is not subject to hydrolysis by cellular phosphatases [12]. FSM may act as a transition state intermediate, with the hydroxamic acid group chelating the metal ion and the phosphono moiety resembling the phosphate group of 4 [13]. FSM was first developed as an antibacterial agent, but due to its less than ideal pharmacokinetics [10,11,14], it was abandoned and later repositioned as a potential antimalarial, inhibiting a new different target compared to artemisinin (ART). The shortcomings of FSM have prompted attempts by several research groups to improve its activity by performing several chemical modifications, suitable for structure-activity relationship studies [15][16][17][18][19][20][21][22]. One of the most studied FSM's analogues is FR900098 (2), a phosphonic acid antibiotic, where the formyl group of FSM is replaced by an acetyl one. Although it is structurally very close to FSM, it is about twice as active as FSM in vitro and in a mouse model [9,23,24]. In brief, structural modifications of FSM have focused on four main areas: (i) replacement of the phosphonate motif by (bio)isosteres (3, 7, 8, 11), synthesis of phosphonate prodrugs (6, 9) [25]; (ii) replacement or modification of the hydroxamate moiety that chelates the catalytically essential divalent cation, e.g., compounds 10-14 [26,27]; (iii) modulation of the aliphatic linker between the anionic anchor group and the chelating head group, e.g., compounds 14-15 [28]; and (iv) modification of the aliphatic chain by introduction of (typically aromatic) substituents, e.g., compounds 15 [22]. As an example, FR900098-prodrugs, where an acetyl group replaced the formyl one of the FSM, displayed improved in vitro and enhanced in vivo activity [9]. Furthermore, lipophilic phosphonate prodrug derivatives of FSM have previously been reported to display significantly enhanced antiplasmodial [29] and antimicrobial activities [30].
Recent studies have shown that the combination of two antimalarial drugs with different molecular targets in one single entity either as a hybrid or a conjugate proved to be more efficient against diseases, such as malaria and cancer [31]. Hybrid molecules are rather promising since they have less possibilities of developing drug resistance [32].
In this work, a synthesis and antiplasmodial evaluation along with the cytotoxicity of three focused families of compounds possessing the propyl phosphonate frame (ester or acid form) of FSM are reported. The first family contains five compounds: FSM, FR900098, and their phosphonate diesters (16,17), along with a dimeric form of FSM (18). The second family was constructed by attaching active antimalarial pharmacophores like ACQ (19-20) [33] or ART (21-23) [34,35] next to the carbonyl group of the hydroxamate thus preparing five new compounds (hybrids or conjugates). The third family (10 compounds, 26-33) possesses various amines connected to the hydroxamic function. For the last two compounds (34, 35), the hydroxamate moiety was replaced by o-phenylenediamine, thus changing the nature of the groups that might intervene in the chelation process ( Figure 2).

Results and Discussion
2.1. Synthesis of the Key Intermediate 39, FSM, FR9000098, and Dimer 18 The first family includes the known compounds FSM and FR9000098 and their corresponding esters (compounds 16 and 17, Scheme 1), along with the tetraethylphosphonate dimer 18. Synthesis of FSM and FR900098 was conducted as reported in the literature by Suresh et al. [36] and Uh et al. [37]. tert-Butyl N-(benzyloxy)carbamate 36 and the bromophosphonic diester 37 were synthesized in an 89% and 50% total yield, respectively, according to literature procedures [21,25] FSM was obtained in three steps and 72% total yield through formylation of the secondary amine of compound 39, hydrogenolysis of the benzyl protective group affording compound 16, and deprotection of the ethyl ester functions followed by a basic work-up.
Compounds 17 and FR900098 were obtained through a similar sequence of reactions involving acetylation of 39, hydrogenolysis, and ethyl ester groups' elimination. This sequence afforded better yields concerning acetylation and hydrogenolysis; thus compound 17 and FR900098 were obtained in a 68% and 83% yield, respectively. Based on this procedure, the FSM dimer 18 was synthesized (Scheme 1) by introducing a succinic acid spacer between the two fragments. Thus, compound 39 was allowed to react with succinic anhydride in the presence of a catalytic amount of 4-(dimethylamino)pyridine (DMAP), providing the acid 42 in an 88% yield. Then, coupling of 42 with the amine 39 was performed using the system HBTU/Et 3 N in chloroform to give the protected fosmidomycin dimer 43 in a 61% yield. Cleavage of the benzyl groups by cat. hydrogenolysis led to the tetraethyl phosphonate 18 in moderate yield (50%, Scheme 1).

Synthesis of FSM-ACQ 19-20 and FSM-ART Conjugates 21-23
Concerning the second family of compounds, namely the FSM conjugates, the FSM core is attached through a linker to either 4-amino-7chloro-quinoline (ACQ) or artemisinin frames. In this respect, five compounds were synthesized, two of them bearing the ACQ and three the ART moiety (compounds 19-20 (Scheme 2) and 21-23, respectively, Schemes 3 and 4). For the FSM-ACQ conjugates, two different ACQ amine derivatives (44 and 45), bearing a piperazine or an ethylenediamine linker attached in position 4 of the 4,7-dichloroquinoline [38], were prepared. While coupling of O-benzyl compound 42 with the piperazino chloroquinoline 44 worked well, the following cleavage of the benzyl group proved to be harsh, affording a mixture of compounds, where hydrogenolysis of the quinoline group was also observed. By reversing the sequence of reactions, thus first performing hydrogenolysis and then coupling of the hydroxamic acid 47 with piperazine 44, the desired hybrid 19 was obtained in a 58% yield over two steps.
For the second hybrid, possessing an ethylene diamine linker, this sequence of reactions could not be applied. A complex mixture of compounds was obtained that was difficult to purify. In this respect, the Boc group was used as alternative protection for the hydroxamic function; treatment of 47 with the system Boc 2 O/Et 3 N/DMAP afforded acid 48 and then followed a coupling reaction with the primary amino group of compound 45 to give the desired compound 49 in a 50% yield, after FCC purification. Boc-deprotection, by TFA-mediated acidolysis, yielded the FSM-ACQ hybrid 20 in a 60% yield. For FSM-ART conjugates, one artemisinin fragment was first introduced through the acid derivative 50 (Scheme 3), which was prepared as previously described [35]. Reaction of compound 50 with benzyloxyamine 39 under peptide coupling conditions afforded derivative 51, which upon hydrogenolysis reaction led to FSM-ART hybrid 21 in a 45% yield over 2 steps, after FCC purification.
Moreover, based on our recent work, where hybrids possessing two artemisinin moieties linked with polyamine spacers, such as spermidine or homospermidine, present very good activities against Plasmodium falciparum [28], compounds 22 and 2, possessing a FSM moiety attached through a spermidine or homospermidine linkers to two artemisinin moieties, were synthesized. Protected spermidine and homospermidine derivatives 52 and 53, respectively, were prepared according to the procedure described in our previous work [35]. Reaction of 52 and 53 with the acid 42 under different and optimal coupling conditions afforded compounds 54 and 55 in 72% and 96% yields, respectively. One-pot deprotection of both benzyl and trityl groups under catalytic hydrogenation conditions unfortunately afforded a mixture of compounds. We thus proceeded in two steps: first with TFA-mediated acidolysis of the trityl groups, followed by hydrogenation for the benzyl group cleavage. The two-step procedure afforded the valuable intermediates 24 and 25 in 91% and 95% yields, respectively (Scheme 4).
Attempts to couple compounds 24 or 25 with the artemisinin acid derivative 50 as for the ACQ derivatives failed to proceed in a clean manner; a mixture of derivatives bearing two and three artemisinin moieties appeared in the mass spectrum, also indicating coupling on the N-OH function. So, precursors 56 and 57 were allowed to react with artemisinin acid 50 (Scheme 4) using HBTU as the coupling agent, in order to afford the benzylated derivatives 58 and 59 in a 50% yield. The latter were then hydrogenated, leading to the final FSM-ART hybrids 22 and 23 in 60% and 80% yields, respectively, after flash column chromatography (FCC) purification.

Synthesis of FSM Derivatives 26-35
The third family of FSM derivatives synthesized is based on the functionalization of the acid 42. This compound can be an interesting platform, where the main functions of FSM are present, and a propyl carboxylic acid is attached as a linker. In this respect, the two first compounds of this family (56 and 57, Scheme 4) have already been synthesized as valuable intermediates in the synthesis of FSM-ART conjugates with spermidine and homospermidine linkages. By using analogous peptide coupling conditions, eight new compounds were synthesized possessing various amino and/or amido frameworks connected to the hydroxamic function (Scheme 5). Among the different amine reagents, Boc-monoprotected dianiline (used for derivative 29) and Trt-piperazine (used for derivative 66) were synthesized according to known procedures [39,40], while all other amines were commercially available.
Finally, two more compounds were synthesized, where the hydroxamic part was replaced by a fragment that potentially can interact with metal ions as is the case and the role of the hydroxamic function of FSM. In this respect, o-phenylenediamine (a known pharmacophore) was chosen due to its chelating capacity [41]. Synthesis of the two final compounds 34 and 35 was straightforward. Alkylation of o-phenylenediamine under sonicating conditions using the already available bromophosphonate 37 afforded in fair yield compound 34, which upon ethyl ester deprotection, followed by basic work-up, led to compound 35 (Scheme 6). The synthesized compounds were evaluated for their antiplasmodial activity against the CQ-resistant P. falciparum FcB1/Colombia strain [42,43], using as control drugs FSM, FR900098, ART, and CQ (see Figure 3). Moreover, their cytotoxicity was measured, as previously reported, upon the primary human fibroblast cell line AB943, which allowed the calculation of their selectivity index (SI) [35]. Specifically, IC 50 (µM) values for the antiplasmodial activities were determined from four independent experiments as the mean values ± standard deviations. IC 50 (µM) values for the cytotoxicity activities were determined from two independent experiments as the mean values ± standard deviations. IC 50, SIs (ratio between IC 50 (µM) of cytotoxicity/IC 50 (µM) of the antiplasmodial activity), and XLogGP3 values for the FSM derivatives, hybrid, and conjugates are reported in Table 1. The IC 50 values of ART, FSM, FR900098, and CQ were also measured and are provided as controls. Concerning the first family of compounds (FSM, 17, FR900098, 18), it was noticed that phosphonate esters are inactive. For example, FR900098 s diethyl phosphonate analogue 17 and the dimer 18, which possesses two diethylphosphonate ester FSM frames linked together with a succinic acid spacer, have IC 50 values >100 µM. FSM appears 2-fold less active than FR900098, with IC 50 values of 15.0 and 7.0 µM respectively, while their selectivity indexes also follow the same trends (>6.6 vs. >14.2 µM respectively).
The second family, constructed by introducing next to the carbonyl group of the FSM hydroxamate group active antimalarial pharmacophores like aminochloroquinoline or artemisinin, presents very interesting results. The hybrid compounds with aminochloroquinoline 19 and 20 showed strong and comparable activities of 2.7 and 4.2 µM, respectively. Compound 19 possesses the structure of piperazine linked through an amide chain to the FSM diester fragment. The 19 and 20 compounds are 3.5-5.5 times more active than FSM itself and more than 25-40-fold more active in comparison to the diester compound 17. Their activities are lower but closer to the CQ, indicating a major influence of the ACQ fragment, largely compensating the absence of acid functionality on the FSM. In addition, they showed higher selectivity indexes in comparison to FSM but much lower in comparison to CQ.
Concerning the hybrid compounds containing artemisinin, we observed a weaker activity for compound 21, where one artemisinin moiety is linked directly to the FSM diester fragment (IC 50 = 28.0 µM). On the contrary, the conjugation of the two ART frames with one FSM diester fragment through a spermidine and to a lesser extent to an homospermidine linker (22,23) afforded the most active molecules of our series with IC 50 values of 0.36 and 0.65 µM; and the best selectivity among all new compounds, with SI values of 94 and 34, respectively. Here, again, it is observed that the IC 50 values are 6.5-12 times higher than ART itself but much lower than FSM's (23-41 fold). These values indicate a stronger influence of the ART fragment to the conjugates. It is noteworthy that all molecules of this family, which exhibit strong antimalarial activities, possess the diethyl phosphonate group.
According to our recently reported work on other antimalarial hybrid compounds [35], the presence of two artemisinin moieties linked through spermidine or homospermidine to an amide chain bearing a third endoperoxide (GMeP) gave IC 50 values of the order of 10 nM. In the case of compounds 22 and 23, the presence of FSM diester (in the place of GMeP) was observed again, even if the activities were lower in this case, probably confirming that hybrid compounds with two different potential targets of Plasmodium can be an interesting option in the search for new antimalarial compounds.
Compounds possessing only the spermidine or homospermidine fragments attached through an amido linker to FSM diester (24, 25) are much less active; however, a better IC 50 value was observed for compound 25 bearing the homospermidine frame. Among the other compounds of this third family, those bearing two aromatic rings attached to the amido function (28, 29, 31) present activities in the range between 22 and 44 µM, while compounds 26, 27, and 30 are inactive. Nevertheless, the second-best compound of this family series (29) is cytotoxic since it has an SI value of only 1.7. Finally, the two compounds possessing a piperazine or N-methyl piperazine fragment (32,33) are inactive, indicating that piperazine alone (as spermidine and homospermidine alone) lacks activity like the diester 17. For the last two compounds of the third family (34,35), where the hydroxamic fragment was replaced by an ortho-benzenediamine fragment, we observed good activity for the diester 34 (IC 50 = 8.2 µM), while the mono sodium salt of the corresponding diacid (35) is inactive. However, despite the good antimalarial activity of 34, it seems to be cytotoxic, with an SI value of only 3.7.
In Table 1, the MWs, IC 50 values, and the evaluation of the XLogGP3 SwissADME values according to the online calculation [44] are also reported. The software can combine physicochemical properties along with pharmacokinetics predictions for the drug likeness of the examined compounds. Since the families of the synthesized and tested compounds are quite different, we first examined and compared the values for each family. The active and known compounds FSM and FR900098 show the most negative values (−2.22 and −2.24, respectively) in comparison to values of −0.43 and −1.22 for diester 17 and the dimer 18. For the second and most promising family of compounds, values vary between +1.42 and +5.33, with the two most active compounds having the highest ones. Concerning the third family of compounds, the most active show values between +1.92 and +2.80, while the inactive ones have negative values. In summary, apart from the first family, where all compounds possess negative XLOGP3 ADME values, the others show positive values for the active ones, the highest being for the most actives. Even if the calculation of the XLOGP3 values takes into consideration a number of different parameters, including the molecular weight, we feel that XLOGP3 could be one of many valuable tools to include when searching to optimize structures of the same family.
Results concerning comparative activities and the XLogGP3 SwissADME for all compounds tested indicated that different factors intervene for the activities of the compounds. Most important of them are related to the fragment-attached FSM derivative and to the linker next to that. The influence of the acid group of the FSM has to be more extensively studied, as its rol is not apparent in regard to some of the results obtained. Moreover, it is noteworthy to observe that compounds with a potential protonation site on the fragment adjacent to the FSM core (compounds 19, 20, 34) can be very active, along with those possessing two highly active pharmacophores (compounds 22, 23) connected through a polyamino linker to the FSM core.
In conclusion, a series of FSM analogs, derivatives, and conjugates with ART and ACQ were synthesized and evaluated against P. falciparum. The most active compounds, which in addition to possessing a good SI, are those that combine two ART frames with the one of FSM diester, through a spermidine or homospermidine linker and exhibit IC 50 values of 0.36 and 0.65 µM, respectively (compounds 22 and 23). Interestingly, the hybrid compounds with one ACQ frame (compounds 19 and 20) are 3.5-5.5 times more active than FSM itself with fairly good SIs. While for FSM and FR900098 the monosodium salt 17 is the most active form, for the hybrid compounds mentioned above, the diethyl phosphonates 19 and 20 show the best activity.
Further work is in progress to examine whether for the most active compounds, (i) the diester forms are better than the acidic ones and (ii) if they are active against strains of P. falciparum showing a different resistance status. Finally, according to our recent findings on hybrid compounds possessing the endoperoxide GMeP [35], we are now engaged in incorporating this frame in a series of FSM analogues and evaluating them.

General Methods
Melting points were determined with a Buchi SMP-20 apparatus and are uncorrected. 1 H NMR spectra were obtained at 600.13 MHz and 13 C NMR spectra at 150.90 MHz on a Bruker AVANCEIII HD spectrometer. Chemical shifts (δ) are indicated in parts per million (ppm) downfield from TMS and coupling constants (J) are reported in hertz (Copies of NMR spectra are available in the Supplementary Materials. ESI-HRMS spectra were recorded on a Waters Q-TOF UPLC Xevo G2 and ESI mass spectra were recorded at 30 V, on a Micromass-Platform LC spectrometer using MeOH as solvent. All solvents were dried and/or purified according to standard procedures prior to use. Anhydrous Na 2 SO 4 was used for drying solutions, and the solvents were then routinely removed at ca. 40 • C under reduced pressure using a rotary vacuum evaporator. All reagents employed in the present work were commercially available and used without further purification. When required, reactions were carried out under dry argon atmosphere in preflamed glassware. Flash column chromatography (FCC) was performed on Merck silica gel 60 (230-400 mesh) and analytical thin layer chromatography (TLC) was performed on Merck silica gel 60F 254 (0.2 mm) precoated on aluminum foil. Spots on the TLC plates were visualized with UV light at 254 nm and ninhydrine solution or charring agents. Then, the addition of succinic anhydride (1.1 mmol) was followed in small portions over 30 min and the reaction mixture was stirred at ambient temperature for 3h. Upon completion of the reaction, THF was removed under reduced pressure and the residue thus obtained was diluted with CH 2 Cl 2 and washed with 5% aqueous citric acid, water, and brine. The organic layer was dried over Na 2 SO 4 and evaporated to dryness under vacuum. The residue was subjected to FCC to give acid 42 as yellow oil (283 mg, 88%); R f (CHCl 3  , amine 45 (7 mg, 0.03 mmol), and Et 3 N (7 µL, 0.045 mmol) were added. After 4h, the reaction mixture was diluted with DCM, washed with pre-cooled 5% aqueous citric acid, water, 5% aqueous NaHCO 3 , water, and brine. The organic layer was dried over Na 2 SO 4 , filtered, and evaporated to dryness under reduced pressure. The residue thus obtained was subjected to FCC, affording the corresponding pure conjugate 49 as yellow oil (9. and Et 3 N (15 µL, 0.105 mmol) were added. The reaction mixture was stirred overnight at ambient temperature. Subsequently, it was diluted with CH 2 Cl 2 , washed with pre-cooled 5% aqueous citric acid, water, 5% aqueous NaHCO 3 , water, and brine. The organic layer was dried over Na 2 SO 4 , filtered, and evaporated to dryness under reduced pressure. The residue thus obtained was subjected to FCC, affording conjugate 51 as yellow oil (  Diethyl (3- (N-hydroxy-3-((3S,5aS,6R,8aS,9R,10R,12R) added. After 7-12 h, the mixture was diluted with CHCl 3 and washed with pre-cooled 5% aqueous citric acid, water, 5% aqueous NaHCO 3 , water, and brine. The organic layer was dried over Na 2 SO 4 , filtered and evaporated to dryness under reduced pressure. The residues thus obtained were subjected to FCC, affording the corresponding conjugates 54 and 55.      or 57 (21 mg, 0.03 mmol), and Et 3 N (20 µL, 0.135 mmol) were added. After 12 h, the mixture was diluted with CHCl 3 and washed with pre-cooled 5% aqueous citric acid, water, 5% aqueous NaHCO 3 , water, and brine. The organic layer was dried over Na 2 SO 4 , filtered, and evaporated to dryness under reduced pressure. The residues thus obtained were subjected to FCC, affording the corresponding conjugates 58 and 59. General procedure for the hydrogenolysis of 56-59. A solution of 56 or 57 or 58 or 59 (0.01 mmol) in methanol (3 mL) was subjected to hydrogenolysis over 10% Pd/C (5 mg) at ambient temperature and pressure for 4 h-6.5 h. Thus, the reaction mixture was filtered through Celite and the filter cake was washed several times with methanol. After evaporation of the solvent to dryness under reduced pressure, the residues were subjected to FCC, affording the corresponding pure deprotected molecules 24, 25, 22, and 23, respectively.  96 (m, 5H), 1.95-1.90 (m, 4H), 1.88-1.83 (m, 5H), 1.78-1.72 (m, 8H), 1.55 (s, 5H), 1.49 (t, J = 7.4 Hz, 3H), 1.40 (t, J = 6.9 Hz, 8H), 1.34 (s, 4H), 1.58-1.13 (m, 2H), 1.06-1.02 (m, 3H), 0.99-0.94 (m, 9H); 13 C NMR (CDCl 3 ) δ 213.4, 209.3, 173.7, 160.5, 107.3, 97.4, 82.7, 68.3, 62.2, 54.6, 47.9, 46.3

Synthesis of FSM-Amino and/or Amido Derivatives 26-33
General procedure for the synthesis of Fosmidomycin conjugates with amines. To a solution of 42 (1 mmol) in CH 2 Cl 2 (1.4 mL), the corresponding amines (1 mmol), HBTU (1.1 mmol), and DIPEA (1.5 mmol) were added. The reaction mixture was stirred at ambient temperature to complete the reaction (monitored by TLC) and then diluted with CH 2 Cl 2 , washed with 5% aqueous NaHCO 3 , pre-cooled 5% aqueous citric acid, water, and brine. The organic layer was dried over Na 2 SO 4 , filtered, and evaporated to dryness under reduced pressure. The residues thus obtained were subjected to FCC, affording the corresponding pure conjugates 60-67.    60−65, 67, and 68. A solution of 60−65, 67, and 68 (0.1 mmol) in methanol (3 mL) was subjected to hydrogenolysis over 10% Pd/C at ambient temperature and pressure until completion of the reaction. Thus, the reaction mixture was filtered through Celite and the filter cake was washed several times with methanol. After evaporation of the solvent to dryness under reduced pressure, the residues were subjected to FCC, affording the corresponding pure deprotected molecules 26-33.

In Vitro Growth Inhibition of P. falciparum
The chloroquine-resistant FcB1/Colombia strain of Plasmodium falciparum was maintained in vitro on human erythrocytes in RPMI 1640 medium supplemented by 8% (v/v) heat-inactivated human serum, at 37 • C, under an atmosphere of 3% CO 2 , 6% O 2 , and 91% N 2 [45]. In vitro drug susceptibility assays were measured by [ 3 H]-hypoxanthine incorporation. Drug stock solutions were prepared in DMSO. Compounds were serially diluted two-fold with 100 µL of culture medium in 96-well plates. Asynchronous parasite cultures (100 µL, 1% parasitaemia and 1% final hematocrite) were then added to each well and incubated for 24 h at 37 • C prior to the addition of 0.5µCi of [ 3 H]-hypoxanthine (GE Healthcare, France, 1 to 5Ci·mmol/mL) per well [42]. After a further incubation of 24 h, plates were frozen and thawed. Cell lysates were then collected onto fiberglass filters and counted in a liquid scintillation spectrometer. The growth inhibition for each drug concentration was determined by comparison of the radioactivity incorporated in the treated culture with that in the control culture maintained on the same plate. The IC 50 value was obtained from the drug concentration-response curve and the results were expressed as the mean values ± standard deviations determined from several independent experiments. Chloroquine and artemisinin were used as antimalarial drug controls.