Synthesis and Antimalarial Activities of New Hybrid Atokel Molecules

Abstract The currently spreading resistance of the malaria parasite Plasmodium falciparum to artemisinin‐based combination therapies makes an urgent need for new efficient drugs. Aiming to kill artemisinin‐resistant Plasmodium, a series of novel hybrid drugs named Atokels were synthesized and characterized. Atokels are based on an 8‐amino‐ or 8‐hydroxyquinoline entity covalently bound to a 1,4‐naphthoquinone through a polyamine linker. These drugs have been designed to target the parasite mitochondrion by their naphthoquinone moiety reminiscent of the antimalarial drug atovaquone, and to trigger a damaging oxidative stress due to their ability to chelate metal ions in order to generate redox active complexes in situ. The most effective Atokel drug shown a promising antimalarial activity (IC50=622 nm on an artemisinin‐resistant P. falciparum strain) and no cytotoxicity at 50 μm indicating a specific antiplasmodial mode of action.


Introduction
Malaria is a major public health issue for more than three billion of people living in endemic areas, and also a significant threat for millions of travelers. Five different Plasmodium species are responsible for infection of 230 million people and death of more than 400,000 people each year, making malaria one of the three main infectious causes of death with tuberculosis and AIDS. [1] Currently, treatment of malaria relies on chemotherapy by using medicines acting on the blood stage of this parasitic disease. Artemisinin-based combination therapies (ACTs) that associates a derivative of the naturally occurring endoperoxide artemisinin (ART) to an antimalarial drug belonging to another therapeutic class, are the current recommended drug regimens. Then, most commonly used ACTs consist of an artemisinin derivative (dihydroartemisinin, or artemether, or artesunate) associated to mefloquine, lumefantrine, amodiaquine, sulfadoxine-pyrimethamine, piperaquine, or pyronaridine. [2] Unfortunately, the emergence of Plasmodium falciparum resistance to artemisinin derivatives and ACTs in southeast Asia threatens world's malaria control and may reduce in the near future the efficacy of the first-in-class antimalarial drugs. [3,4] Therefore, it is urgent to develop new drugs efficient against currently resistant malaria parasites. [5] The antimalarial activity of ART is based on intraparasitic reductive activation of the peroxidic bond of the drug by iron(II)-heme generated by the parasite induced hemoglobin catabolism, and subsequent alkylation of heme by the drug derived C-centered radical. [6] Understanding of the main feature of the mechanism of action of artemisinin prompted us and later several other research groups to design synthetic peroxide hybrid molecules such as trioxaquines [5b,7-9] as new antimalarial drugs. Unfortunately, P. falciparum parasites resistant to both artemisinin derivatives and a partner drug such as mefloquine or piperaquine recently emerged. [10][11][12][13] Moreover, it was demonstrated that trioxaquines exhibited cross-resistance with artemisinin, in both the experimental artemisinin-resistant parasite line (F32-ART) and in Cambodian field isolates. Trioxaquine drug pressure in vitro selected trioxaquine-resistant parasites with the same genetic trait (K13 mutation) than artemisinin-resistant parasites, thus raising concerns about the potential place of endoperoxide-based antiplasmodials in the future therapeutic arsenal. [14] Then, with the aim to kill multiresistant strains, we designed new hybrid antimalarial drugs with a dual mode of action, due to the presence within a single molecule of two pharmacophores that are not necessarily expected to act on the same biological target. [9] As pharmacophores, we used the naphthoquinone ring reminiscent of atovaquone and different 2substituted-8-aminoquinolines related to the TDMQ series (Figure 1). Atovaquone has a potent antimalarial activity, [15] and is until to now the only antimalarial drug active on artemisininresistant Plasmodium strains in vitro. [13,16] This drug behaves as a competitive inhibitor of ubiquinone and is able to block the parasite respiratory chain, a mechanism that remains active in resistant and quiescent Plasmodium parasites. [16] On the other hand, TDMQ drug-candidates ( Figure 1) have been designed as copper chelators, in order to target the copper dyshomeostasis in the brain of patients with Alzheimer's disease (AD). [17][18][19] The ability of several TDMQ chelators to limit the oxidative stress created by storage of copper in amyloids is due to their specific affinity for copper(II). In fact, TDMQ20 or TDMQ22 providing a perfect square-planar N 4 -coordination sphere around copper(II) have very strong affinities for copper(II) (log K app = 15-17), and are consequently able to fully inhibit the oxidative stress induced by CuÀ Aβ [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] . In contrast, TDMQ5 or TDMQ19 providing a distorted N 3 X-coordination to copper, exhibit moderate affinities for copper(II) (log K app = 10). Consequently, TDMQ5 and TDMQ19 are able to provide a Cu II /Cu I redox cycle and, consequently, trigger ROS (reactive oxygen species) production.
In order to disrupt the mitochondrial activity of artemisininresistant parasites (and, consequently, kill them), our idea was to associate the naphthoquinone moiety of atovaquone to a chelator based on a TDMQ skeleton able to recruit in situ redoxactive metal ions and, consequently, able to create an oxidative stress within mitochondria that are rich in oxygen and electrons.
Here, we report synthesis and physico-chemical properties of such naphthoquinone-chelator hybrid molecules, named Atokel, and also their biological activities on Plasmodium parasites.

Synthesis and Characterization
Structural modulations of the Atokel series were carried out by preparing compounds with hydrogen and/or chlorine substituents at C5 and C7 of the 8-aminoquinoline nucleus (R 5 and R 7 , Table 1a). In fact, other factors being equal, these substituents allowed to modulate affinity of TDMQ ligands for copper(II) by 2 orders of magnitude. [18] The role of i) substituent at position 3' of the naphthoquinone moiety (R 3 ', Table 1a), ii) substituents at the R 3 '-R 8 ' positions of the naphthoquinone (Table 1b), and iii) a 8-hydroxyquinoline moiety to be compared to the drugs based on an 8-aminoquinoline (compounds 3, 4, and 14, Table 1c, to compare to 1, 2, and 13, respectively) were also investigated. This series includes 5',8'-dihydroxy-1',4'-naphthoquinone derivatives (Atokel-19 to -24), a scaffold known to have η 2 metal chelating properties. [20] We also tried to synthesize Atokel derivatives with a hydroxyl group at C3', like in atovaquone itself, by deprotection of the C3'-tosylate derivatives. Unfortunately, all attempts to get 3'-OH-Atokel derivatives failed until to now.
Atokel-3 and -4, which are the 8-hydroxyquinoline analogues of the 8-aminoquinoline derivatives Atokel-1 and -2, respectively, were obtained starting from the 8-hydroxy-2methyl quinoline (Scheme 3, L). The functionalization of the C2methyl group was achieved by using n-butyllithium at À 78°C, followed by addition of paraformaldehyde, to yield the 2hydroxyethyl derivative M. Then, reaction with methanesulfonyl chloride and triethylamine, followed by sodium ethylate afforded the C2-vinyl derivative (N, Scheme 3). Then, the α-NH 2ω-Boc-diamine chain reated with the C2-vinyl group. Subsequent acidic deprotection of the distal primary amine gave rise to intermediate P (Scheme 3). This primary amine derivative reacted with 2,3-dichloro-1,4-naphthoquinone derivative in the presence of K 2 CO 3 in acetonitrile, to afford the derivative Q. Protonation of Q yielded Atokel-4. Dechlorination of Q in the presence of Pd/C 5 wt %, followed by protonation, afforded Atokel-3 (Scheme 3).

Stoichiometry of the Cu II -Atokel Complexes. Titration of all
Atokel ligands was carried out by addition of aliquots of a CuCl 2 aqueous solution in an Atokel ligand solution (15 μm in Tris buffer 200 μm, pH 7.4), except Atokel-15 and -16 that could not be extensively studied due to their poor aqueous solubility. Chelation of copper was monitored by UV-Vis spectroscopy. The results allow to investigate (i) the behavior of ligands based on an 8-aminoquinoline moiety compared to ligands based on a 8-hydroxyquinoline (Atokel-1 or -2 compared to Atokel-3 or -4, respectively), (ii) the role of non-coordinating Cl and NO 2 substituents either on the quinoline or on the naphthoquinone moieties, (iii) the evidence of the role of potentially coordinating hydroxyl or tosyl (OTs) substituents on the naphthoquinone moiety (Atokel-24 compared to Atokel-2, or Atokel-14 compared to Atokel-3).
First of all, the titrations by Cu 2 + of all Atokel ligands without 3'-OTs or 5',8'-OH naphthoquinone, whether they contained an 8-NH 2 or an 8-OH quinoline, resulted in immediate and complete copper coordination in a complex having 1/1 Cu II /L stoichiometry. As examples, the titrations of Atokel-2 and -4 are depicted in Figures 2 and 3, respectively. Upon metalation by copper(II), the main absorption band of Atokel-2 (253 nm) decreased, and was splitted into two absorption bands (235 and 291 nm) assigned to the Cu II -Atokel-2 complex ( Figure 2). In addition, the small band of the ligand at 470 nm underwent a bathochromic shift to 555 nm. Well defined isosbestic points detected at 243 nm, 267 nm and 493 nm, and the plot of absorbance at 291 nm with respect to the Cu/ Atokel-2 molar ratio in the mixture (Figure 2a, Insert) unambiguously confirmed the metalation of Atokel-2 as a 1/1 complex.    [20] (for Atokel-24) or three monodentate tosylates (for Atokel-14) at low Cu/L molar ratios, followed by rearrangement to more stable 1/1 complexes while the available Cu is no more a limiting factor.

Titration of Atokels by ZnCl 2 and FeCl 3
Titration by Zinc. Titration of all Atokel ligands was carried out using ZnCl 2 as metal source, and monitored by UV-Vis spectroscopy. All the derivatives based on an 8-aminoquinoline moiety and bearing no hydroxy substituent on the naphthoquinone ring, exhibited unchanged spectra up to 20 mole equivalents of metal with respect to Atokel, indicating that they were not significantly able to chelate zinc(II).
For Atokels bearing a C5',C8'-naphthoquine moiety (= naphthazarin, Atokel-19, -20, -21, and -24), half-metallation was obtained after addition of 30 to 150 mol equivalents of metal salt and 300 to 1000 mol equivalent of zinc were required to achieve full metallation. These data indicated that these ligands have a very low affinity for zinc(II), with calculated log K app (pH 7.4) values below 3.5-3.0. As a matter of example, titration of Atokel-4 by ZnCl 2 is provided as Supporting Information ( Figure S1). This low affinity chelation of zinc(II) by the naphthazarin moiety is consistent with metal complexes already reported for this motif. [20] So, the selectivity of Atokel ligands for the redox active copper(II) with respect to zinc(II) was at least 7 orders of magnitude.
On the other hand, all Atokels based on a 8-hydroxyquinoline moiety (Atokel-2, -4, -14) were found to exhibit some significant affinity for zinc, with log K app (pH 7.4) values below 4 (half metallation at 7-9 mol equivalents of metal salt). This result is consistent with the lack of metal specificity of 8hydroxyquinolines, compared to 8-aminoquinolines as previously observed. [25,26] Titration by Iron. Titration of Atokel-2, -3, -4, -14, and -24 was carried out with FeCl 3 , and monitored by UV-Vis spectroscopy. No significant change occurred in the spectra upon addition of 50 mol equivalents of iron(III), indicating that Atokel have no significant affinity for iron(III) in these conditions, regardless of their substitution pattern was (8-amino-or 8hydroxyquinoline, hydrogen, chlorine, or tosylate at C3', or dihydroxy substitution at C5' and C8' of the naphthoquinone moiety).

Ability of Cu-Atokel Complexes to Induce the Reduction of Dioxygen
The aerobic oxidation of ascorbate induced by the 1/1 Cu-Atokels complexes was measured by kinetic UV-visible, as an indirect evaluation of their ability to reduce dioxygen and, consequently, to produce the damaging hydroxyl radicals. [18,27] Each Atokel ligand was first incubated with CuCl 2 and the chelation of copper(II) was confirmed by UV-Vis spectroscopy {[Atokel] = 11 μm in Hepes buffer pH 7.4, Cu/L molar ratio = 1/ 1.1}. An aliquot of aqueous ascorbate was then added in the UV-Vis cuvette, and the absorbance of ascorbate was immediately monitored at 265 nm. A selection of results is reported in Figure 6 and Table 3. First of all, all Atokel ligands, except for Atokel-3, in presence of one mol equivalent of copper(II), induced rapid oxidation of ascorbate, with conversion values higher than 60 % after 10 minutes and full ascorbate conversion before 30 minutes of reaction time ( Figure 6). The ascorbate oxidation was not correlated to the presence of a 8-amino-or a 8-hydroxyquinoline in the Atokel ligand (85 % and 61 % for 8amino Atokel-2 and -13, respectively, compared to 78 % and 91 % for the 8-hydroxy derivatives Atokel-4 and -14, respectively, Table 3). Similarly, it was not correlated with the 3'substituent (91 %, 85 % and 61 % for Atokel-1, -2, and -13, respectively), or with the 5',8'-dihydroxy pattern of the naphthoquinone moiety (85 % and 93 % for Atokel-2, and -24, respectively). Only Atokel-3 in the presence of copper(II) exhibited a low redox activity, with 5 % of ascorbate oxidation at 10 min (16 % at 30 min), while its 3'-chlorinated and 3'tosylated analogues Atokel-4 and -14 induced 78 % and 91 % of ascorbate oxidation, respectively. At this point, there is no clear correlation between the substitution pattern of Atokel ligands and the redox activity of their copper complexes.
The comparison of ascorbate oxidation by several Atokel ligands in presence of 1 or 0.5 mol equiv. of copper(II) is reported in Figure 7 and Table 3. At 10 min of reaction time, Atokel-22 and -24 induced 29 % and 66 % of ascorbate oxidation in presence of 0.5 equiv. of copper, respectively, while ascorbate conversion in presence of 1 mol equiv. of copper was determined to 91 % and 93 %, respectively. Atokel-14 induced only 5 % of ascorbate oxidation at 10 min in presence of 0.5 mol equiv. of copper, while oxidation was higher than 90 % in presence of 1 mol equiv. of copper. These results confirm that these ligands generate different copper complexes having different redox behaviors, according to the available quantity of copper.   Table 3 is redundant with footnotes of the Table. Atokel-n n = Cu/L molar ratio Ascorbate oxidation at 10 min (%) [

Antimalarial Activity
The antiplasmodial activities of these hybrid molecules have been evaluated in vitro on the strain of P. falciparum F32-ART. [28] On this strain, the IC 50 value of atovaquone, the reference antimalarial drug routinely tested in our laboratory, was 0.8 � 0.2 nm. Consequently, the Atokel derivatives having a IC 50 value higher than 1 μM, 1000-fold higher than that of atovaquone were considered inefficient. The percentage of F32-ART Plasmodium growth inhibition by Atokel drugs at 1 μM was found in the range 0-40 % (Table 4), except for the more active Atokel-13 (72 %). The IC 50 value of Atokel-13 on the F32-ART strain was 622 � 9 nm. Atovaquone acts by inhibiting the mitochondrial cytochrome bc 1 complex (cyt bc 1 ) of Plasmodium. [29] The X-ray structure of mitochondrial cyt bc 1 from Saccharomyces cerevisiae with atovaquone bound indicates that a polarized H-bond to His181 of the Rieske protein in cyt bc 1 interacts at 2.8 Å with the deprotonated hydroxyl group at C3' of the drug (pK a = 6.9). [29] So, the lack of this C3'À OH group in the Atokel series might be, at least in part, responsible for the low antimalarial activity of these compounds. However, beside this OH group, interaction of atovaquone with the Qo site of the protein involves the protons and carbonyl functions of the naphthalene-1,4-dione moiety (with the highly conserved residues Val-136 and Pro-271 on one hand, and Met-139, Gly-143 and Ile-269, on the other hand). [29] Consequently, any derivative containing the naphthalene-1,4dione residue (even without hydroxyl at C2 or the specific sidechain of atovaquone) may retain some affinity for the target protein.

Cytotoxicity
The IC 50 value of Atokel-13 against Vero cells was largely up to 50 μm (corresponding to the maximum concentration tested, with no cytotoxicity at this concentration). Then, the selectivity index of Atokel-13 was > 80 (> 50/0.6). In the same conditions, the IC 50 of atovaquone against Vero cells was 8 μm, indicating a selectivity index of this drug around 1 × 10 4 .

Conclusion
A series of ligands based on an 8-amino-or 8-hydroxyquinoline covalently bound to a 1,4-naphthoquinone through a polyamine linker, were synthesized and fully characterized. These ligands exhibited an affinity for copper(II) with log K app values in the range 7-11, and a high selectivity for copper(II) with respect to zinc(II) and iron(III). Despite a rational design that aimed to target the mitochondrion and to trigger a damaging oxidative stress, the synthesized Atokel ligands exhibited a deceiving antimalarial activity, that might due in part to the lack of hydroxyl substituent at C3'. The obtained results do not allow to deduce a possible correlation between the drug structures, their ability to induce ROS production in presence of a redox active metal ion like copper, and their biological activity. The determination of the structures of metalated Atokel complexes might provide useful information for pharmacomodulation in order to improve the antiplasmodial activity of these hybrid molecules.

Reagents and Methods
All solvents and commercially available reagents were purchased from Sigma-Aldrich, Fluka, Acros, TCI, or BLD Pharmatech, and were used without further purification. copper(II)-zinc(II) and iron(III) cloride were used as source of metal ions. 1 H-and 13 C NMR spectra were recorded on a Brucker Avance Neo 600 spectrometer et 298 K except otherwise stated, or on Bruker Avance 400 or Avance 500 spectrometers. Chemical shift values are given in ppm, using tetramethylsilane (TMS) as the external standard. All spectra have been recorded in the ranges À 1 to 13 ppm for 1 H, and À 10 to 210 ppm for 13 C. Full characterization of compounds by high resolution NMR (600 MHz in 1 H, 150 MHz in 13 C) is routinely based on COSY, ROESY, HSQC and HMBC analyses, at variable temperature when necessary. For all final Atokel ligands, the NMR signals have been listed along with assignment in the logic order of Atokel ligands building: first, quinoline positions from 1 to 8, then the substituent at C2, and finally the naphthoquinone ring. HPLC analyses were carried out using a GL Sciences Inc. equipment with InertSustain C18 column (5 μm, 4.6 mm x 150 mm), flow rate was 1.0 mL min À 1 , UV detection at λ = 254 nm. ESI + -Mass spectra were obtained on ThermoFisher or Xevo G2 QTOF (Waters). UV-visible spectra were recorded from 200 to 700 nm, at 25°C, on a Evolution 300 (ThermoFisher) or Cary 3500 spectrophotometers equipped with magnetic stirring.

Synthesis and Characterization of Atokels
The Atokel ligands and their synthetic intermediates have been prepared according to general protocols depicted in Schemes 1, 3, and 4. Each compound was numbered as "X-Atokel-n", where X stands for the letter of the given intermediate in Schemes 1, 3, and 4, followed by the target Atokel, with n = 1-24.

Atokel-2.
Protonation of J-Atokel-2 afforded Atokel-2, in the same experimental conditions as described above for the preparation of Atokel-1 from K-Atokel-1. Atokel-2 was isolated in form of a yellow

2-Vinylquinolin-8-ol (N-Atokel-3).
Triethylamine (184 μL, 13.75 mmol) and methanesulfonyl chloride (91 mg, 8.25 mmol) were added to a solution of M-Atokel-3 (50 mg, 2.75 mmol) in dichloromethane (2.5 mL) at 0°C, and the mixture was stirred at room temperature for 3 h. The solvent was evaporated, the resulting solid was solubilized in the minimum of EtOH (0.9 mL), and an aqueous solution of sodium hydroxide (1 m, 3 mL) was added. The mixture was stirred for 5 h at reflux, and then neutralized with HCl 1 m to pH = 7. The solution was extracted with dichloromethane, dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. The residue was purified by chromatography on silica gel with CH 2 Cl 2 as eluent, to give N-Atokel-3 as a white solid (36 mg, 80 %). 1 Atokel-3). To a mixture of N-Atokel-3 (1.3 g, 7.6 mmol) was added to a stirring solution of tert-butyl (2-(methylamino)ethyl)carbamate (3.06 g, 15.2 mmol) in methanol (20 mL), and then 2 mL of acetic acid were added and the mixture was stirred at 70°C for 24 h. It was then concentrated under reduced pressure. Dichloromethane was added, and the mixture was washed with saturated aqueous NaHCO 3 and water. The organic extracts were dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure, and the residue was purified by chromatography on silica gel (CH 2 Cl 2 /MeOH, 20/1, v/v) to give O-Atokel-3 as a yellow oil (2.3 g, 87 %). 1

Atokel-5 5,7-Dichloro-2-methylquinoline (B-Atokel-5).
To solution of 3,5dichloroaniline (2.0 g, 12.3 mM) in HCl (12 m, 8 mL) at 0°C, acetaldehyde was added dropwise under stirring. The reaction mixture was kept at 0°C for 15 min, and the temperature was gradually raised to 80°C and stirred at 80°C for 4 h. The resulting mixture was poured into ice-cold water and neutralized with aqueous ammonium hydroxide. After extraction with CH 2 Cl 2 , the organic phase was dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. The crude mixture was purified by silica gel flash column chromatography (ethyl acetate/petroleum ether, 1/20, v/v). After, evaporation of the eluent, B-Atokel-5 was obtained as a yellow solid (1.80 g, 69 %). 1

5,7-Dichloro-2-methyl-8-nitroquinoline (C-Atokel-5).
To a stirred solution of B-Atokel-5 (2.00 g, 9.5 mM) in neat sulfuric acid (10 mL), was added dropwise fuming nitric acid (2 mL) over a 1 h period, at room temperature. The resulting mixture was stirred for an additional hour, and was then poured into ice. The mixture was allowed to warm to ambient temperature, and neutralized with aqueous ammonium hydroxide. After extraction with CH 2 Cl 2 , the organic phase was dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. The crude mixture was purified by silica gel flash column chromatography (ethyl acetate/petroleum ether, 1/10, v/v). By evaporation of the solvent, C-Atokel-5 was isolated as a yellow solid (1.82 g, 91 %). 1

Evaluation of Cu II or Zn II to Ligand Affinity Constants
The affinity constants of Atokel ligands for copper(II) and zinc(II) were evaluated as previously reported, either directly from UVvisible titrations (for Zn) or using a competitive chelator, namely ethylenediamine (EDA) or sodium nitriloacetate (NTA). [18,23,30]

Oxidation of Ascorbic Acid by Cu-Atokel Complexes
The oxidation of ascorbate in presence of Atokel ligands and copper was carried out as reported in Ref. [18]. The final concentrations in the cuvette were as follows: Atokel, 11 μm; CuCl 2 , 10 or 5 μm, and sodium ascorbate, 100 μm in Hepes buffer, 50 mm, pH 7.4, containing less than 2 vol% of DMSO. For each kinetic experiment, the absorbance at 265 nm was corrected by subtracting the own absorbance or each copper complex at 265 nm at t 0 from the raw data.

Evaluation of Antiplasmodial Activity of Atokels and Cytotoxicity
The Plasmodium falciparum strain F32-ART, an artemisinin-resistant line obtained by artemisinin drug pressures, [28] was cultivated in vitro as previously described [31] with some modifications. [7] The antiplasmodial activity of the molecules was performed in 2 independant experiments by SYBR Green I method [32] on synchronized ring-stage parasites at 1 % parasitemia treated with different concentrations of each drug for 48 h, in 96-well culture plates. [16] The drug concentrations tested were in the ranges 0.1 nm-1 μm for Atokel-1-7, and 0.1 μm -50 μm for Atokel-8-24. All the compounds were dissolved in DMSO and serial dilutions were carried out in RPMI 1640 to yield a final DMSO concentration of 0.5 % in the well, a concentration that was verified as not affecting parasite growth. Each concentration tested was done in triplicate. The molecules were then washed off three times in PBS. The parasitized red blood cells were lysed by a cycle of freezing/thawing and subsequently probed with SYBR Green for fluorescence reading on Microplate Fluorescence Reader (excitation: 485 nm, emission: 528 nm). The control parasite culture (i. e., RPMI with 0.5 % DMSO) was referred to as 100 % growth. The IC 50 values (50 % Inhibitory Concentration of parasite growth) were calculated using GraphPad Prism software 7 (GraphPad Software, San Diego, CA, USA) by drawing the curve: % inhibition versus log of drug concentration. Because for all Atokels tested, except Atokel-13, their activity against P. falciparum in vitro was at least 1000-fold lower than that of atovaquone, we decided to present, for each molecule, the percentage of inhibition of P. falciparum obtained at 1 μm (Table 4). For Atokel-13 and atovaquone (tested in parallel as drug control), 3 and 6 independent experiments were respectively conducted and their IC 50 values were reported as a mean of the values � standard error of the mean (SEM).
The cytotoxicity of Atokel-13 and of atovaquone as antiplasmodial drug control (with phenol 0.5 % as control of cytotoxicity for each experiment), was carried out 3 times, on Vero cells. The drug concentrations tested ranged from 5 nm to 50 μm. The security index was calculated as the ratio of cytotoxicity/antiplasmodial activity.