Thomas Cheviet

Sharon Wein

Gabriel Bourchenin

Manon Lagacherie

Christian Périgaud

Rachel Cerdan

Suzanne Peyrottes

β-Hydroxy-and β-Aminophosphonate Acyclonucleosides as Potent Inhibitors of Plasmodium falciparum Growth

published or not. The documents may come

1 Beta-Hydroxy-and beta-aminophosphonate acyclonucleosides as potent inhibitors of

Plasmodium falciparum growth

Thomas Cheviet, [a] Sharon Wein, [b] Gabriel Bourchenin, [a] Manon Lagacherie, [a] Christian Périgaud, [a] Rachel Cerdan,* [b] and Suzanne Peyrottes* [a] [a] Institute for Biomolecules Max Mousseron (IBMM), UMR 5247 UM-CNRS-ENSCM, Univ.

INTRODUCTION

Drug discovery is mainly based on the complementarity of target-based (protein screening) and phenotypic (whole cell screening) approaches and both strategies have been used to identify novel antimalarials. 1 Cell-based phenotypic screening has proven to be a valuable approach for drug candidate identification and it is used since decades to identify new chemotypes. One of its limitation is that it fails to provide information on the molecular target and/or the mechanism of action. On the contrary, target-based approaches involve the screening of a library of compounds against a defined protein followed by structural optimization of a hit-compound for potency and selectivity.

In the case of parasitic diseases, there is a rather limited number of validated molecular targets if we consider the strict definition that a validated target is associated to a registered drug, for which the corresponding mode of action has been proven. As example, several proteins from the purine and pyrimidine synthesis pathways have been proposed as chemotherapeutic targets for various parasites. 2 Special attention was given to the purine salvage pathway as many parasitic protozoa lack the de novo pathway for the biosynthesis of nucleoside 5'-monophosphates. Indeed, they rely exclusively on the enzymes from the purine salvage pathway and related transporters for the supply of purines and purine-containing nucleotides. In the case of malaria, one of the most important human parasitic diseases and a major cause of mortality worldwide, parasite resistance to traditional antimalarial drugs is now a serious concern. Thus, increasing efforts have been made to develop new drugs with novel mechanisms of action. 1c, 3 We have recently reviewed the literature on inhibitors of the purine salvage pathway and highlighted two main families of potential drugs (Figure 1), 4 namely, immucillin derivatives and acyclonucleoside phosphonates (ANPs), which target the purine nucleoside phosphorylase (PNP) 5 , and the hypoxanthine-guaninexanthine phosphoryl transferase (HGXPRT), 6 respectively. Several generations of immucillin have been studied as transition state inhibitors of PNP and have exhibited potent antimalarial activities in vitro and in vivo. 7 Some have already been the subject of preclinical studies 7b but no further development has been reported to date. In addition, ANPs are also promising as a class of antimalarial agents because they inhibit HGXPRT. 8 This enzyme is crucial for the synthesis of inosine 5 '-monophosphate (IMP) from hypoxanthine, the central and unique precursor for all purine-containing nucleotides required during plasmodial nucleic acid biosynthesis. These compounds were designed on the basis of the crystal structure of 2-(phosphonoethoxy)ethylguanine (PEEG, Figure 1, X=O) in complex with HsHGPRT 9 and incorporate a purine base (preferably guanine or hypoxanthine), a five atoms linker (preferably including an heteroatom) and the phosphonate group. The selectivity of these series of ANPs is usually modest with the exception of the Aza- C-ANPs. In addition, due to the anionic nature of ANPs at physiological pH, these compounds generally require conversion to neutral prodrugs to observe effective biological activity in cell culture experiments. 8b, 10 This observation points to the fact that drugs must be able to enter the targeted cell, in this case the infected red blood cell, in order to block parasite processes.

Figure 1.

Examples of Immucilin 7b , ANP (X=O, 9 NH, 11 S, 12 CH2 13 ) and aza-C-ANP 14 derivatives described in the literature 4 as potent inhibitors of PNP and HG(X)PRT.

Herein, we describe the synthesis of a new series of ANPs (Figure 2). Their antimalarial potency was evaluated by a phenotypic approach against P. falciparum (in vitro) and P. berghei (in vivo) and revealed different properties from those of immucilins and aza-C-ANP. Chemistry. We focused specifically on the synthesis of α/β-hydroxyphosphonate derivatives as structural analogues of hydroxycarboxylic acids 15 (the latter are involved in various biological processes). Thus, we describe a limited number of regio-and stereo-selective synthetic approaches to obtain these derivatives as pure enantiomers under mild conditions. 16 We previously reported on the synthesis of -hydroxyphosphonate ribonucleoside derivatives using the efficient ringopening reaction of epoxides with phosphorus nucleophile, catalyzed by BF3.etherate. 17 In this case, chirality at the beta-position (relative to the phosphorus atom) is controlled and originated from the naturally occurring chiral pool. Similarly, our general approach to synthesis (Scheme 1) involved the use of D-or L-aspartic acid or (R)-or (S)-glycidol, as cheap and/or naturally occurring chiral starting materials. Thus, propyl derivatives (compounds 1-3, Figure 1, n=zero, namely C3series) may be obtained by the direct coupling of glycidol with the appropriate nucleobase precursors, followed by a ring-opening reaction. For butyl derivatives (compounds 4-10 and 12-13, Figure 1, n=1, namely C4-series), the required epoxide or aziridine should be obtained in a few steps, through intramolecular nucleophilic substitution from aspartic acid, in accordance with previously described procedures. 18 To avoid side reactions during the coupling steps, exocyclic functions of the purines were protected either by a tert-butyloxycarbonyl (Boc) group or a methoxy group (for 6-oxopurines).

Scheme 1. Proposed retrosynthesis

Thus, Boc protected adenine, 2-amino-6-O-methylpurine and 2,6-diaminopurine (obtained beforehand using previously reported protocols 19 ) were coupled either with (R)-or (S)-glycidol under Mitsunobu conditions 20 and yielded the enantiomeric pairs of epoxides 14-16 (Scheme 2).

Then, the ring-opening step was carried out in the presence of an excess of silylated diethylphosphite and BF3.Et2O and generated fully protected -hydroxyphosphonate derivatives 17-19. Removal of the various protecting groups was performed using a standard protocol with trifluoroacetic acid and then bromotrimethylsilane. The resulting phosphonic acids were eventually converted into their sodium salts, compounds 1-3, by percolation onto ion-exchange resin (Dowex Na+ form). We then envisaged obtaining the corresponding -hydroxyphosphonate derivatives, including a butyl chain, compounds 4-6 (Figure 2). The synthesis required the (R)-and (S)-2hydroxyethyloxirane 20 (Scheme 3), which were prepared in three steps starting from D-or Laspartic acid according to previously reported procedures. 18a Isolation of epoxide 20 was problematic (due to its volatility and solubility in water). Therefore, we decided to use it as a crude after filtration of the reaction media. The solution of 20 in dichloromethane was added directly in the Mitsunobu coupling step with the required nucleobases, providing the enantiomeric pairs of epoxides 21-23. The above-mentioned steps (ring-opening reaction and removal of protecting groups) were performed on these substrates and yielded isolated intermediates 24 to 26 and, then, the desired final compounds 4-6, as sodium salts. As xanthine and hypoxanthine derivatives are important substrates during the biosynthesis of P. falciparum nucleic acids (through the salvage pathway), we chose to synthesize these analogues in the C4-series (compounds 7-8, Figure 2) from derivatives 24 and 25 (Scheme 4). Briefly, acidic treatment allowed the removal of Boc protecting groups and yielded intermediates 27 and 28 quantitatively, which were then subjected to a diazotization reaction. 13 Thus, hydroxyphosphonate derivatives 29-30, including hypoxanthine and 2-oxo-6-O-methylpurine, were converted into their phosphonic acids (7 and 8 isolated as sodium salts) with trimethylsilyl bromide. We showed that compounds with (R)-stereochemistry and four carbons linker had the best activities (see in vitro data section, Table 1). We thus extended our SAR study to non-natural purines (Figure 2, compounds 9-10), incorporating 2-amino-and 6-amino-2-fluoropurines. Compounds 9-10 were prepared following the same synthetic approach as described for derivatives 4-6 (Scheme 5). Compound 20(R) was coupled under Mitsunobu conditions to protected N-(Boc) 2-6-amino-2- We then probed the essentiality of the hydroxyl group at the -position relative to the phosphorus atom by either removing it (Figure 2, compounds 11a and 11b including a butyl or butenyl linker) or replacing it by an amino group (Figure 2, compounds 12 and 13, corresponding to aminophosphonate derivatives).

The guanine derivatives 11a and 11b were obtained by adapting the existing procedure for 11a 13, 21 and using an alternative route for 11b 22 (cf. SI for details).

To access the -aminophosphonate in the C4-series (Scheme 6), we developed an original synthetic route. The preparation of enantiomerically pure or 35(R) was previously reported in a few steps from L-or D-aspartic acid, respectively. 18d Briefly, quantitative synthesis of aspartic acid dimethyl esters was carried out using SOCl2 in methanol, followed by the introduction of a tert-butyloxycarbonyl protecting group on the amine. The subsequent reduction of the diesters in the presence of NaBH4 and CaCl2 yielded the corresponding (S)-or (R)- N-Boc-amino diols. 18b The latter were converted to the 1,4-ditosylated intermediates. In situ this led to the intramolecular cyclization in the presence of potassium hydroxide and provided the enantiomeric pair of N-Boc-aziridine 35 in 45% overall yields (over five steps). 18d Then, nucleophilic displacement of the remaining tosyl group by the appropriate purine precursors was accomplished in the presence of potassium carbonate and led to the isolation of the N-9-regioisomers 36 and 37 (sole products). The ring-opening of activated aziridines was somewhat puzzling. It is highly dependent on both the nature of the protecting groups on the aziridine ring and the phosphorus nucleophilic species (data not shown). Best conditions involved the use of a Boc group and the addition of the lithium phosphite generated in situ from diethylphosphite and LiHMDS at low temperature. After overnight stirring, satisfactory yields of the desired -aminophosphonate derivatives 38-39 were isolated and we observed concomitant and partial removal of the Boc group on the exocyclic amino group of the nucleobases. Final removal of the remaining Boc groups was performed by treating derivatives 38-39 with trifluoroacetic acid in dichloromethane yielding compounds 40-41. On hydrolysis of the diethyl phosphonate moiety (using TMSBr in DMF), this led to the expected derivatives 12 and 13. Enantiomeric excess characterization. 31 P NMR spectroscopy was used in combination with a chiral solvating agent (CSA) to stereo-discriminate a pair of isomers and check the enantio-purity of our compounds. This simple rapid approach requires a small amount of compound. It has already been reported for chiral -amino and -hydroxy-phosphonic acids using -cyclodextrin as CSA. 23 Thus, several factors (temperature, pD, etc...) were tested to determine the best conditions for our derivatives based on -hydroxyphosphonates (SI, Figures S1-S3). The best separation was observed at T= 15°C, the lowest tested temperature. This may be because it slows the conformational exchange rate of both enantiomers inside the cyclodextrin cavity and, thus, enhances their differentiation. The optimal pD was slightly different (always under basic conditions), depending on the studied compounds (β-amino and β-hydroxyphosphonates). This suggests that the ionization of the hydroxyl and amino group plays an important role in the complexation of the compound inside the cavity. The two enantiomers of each compound were tested separately and showed only one peak at a specific δ, at the same pD (Figure 3). The procedure made it possible to differentiate the two peaks from a racemic mixture. Thus, we demonstrate the enantiopurity of our final compounds and the reliability of our synthesis. In vitro antimalarial activities. The ability of our novel derivatives to interfere with parasite growth in cell culture was monitored by adding the compounds to a P. falciparum-infected erythrocyte suspension for a complete 48 h parasite cycle before assessing parasite viability (Table 1). The C3-series analogues 1-3 showed low antimalarial activities with IC50 higher than 300 µM for both R and S enantiomers, irrespective of the nature of the nucleobase. In this series, the most potent analogue is the 2,6-diaminopurine. In the C4-series with an hydroxyl group in the -position relative to the phosphorus atom, we tested both enantiomers (compounds 4 to 8) and different nucleobases (compounds 4 to 10). The best activity was always observed for the R-enantiomer (Table 1) when the analogue was active (compounds 5, 6 and 8). Concerning the nature of the nucleobase, the guanine derivatives 5(R) and 5(S) were the most interesting compounds with an IC50 value of 74 nM and 6.1 µM, respectively. Compounds 6(R), 9(R), 10(R) exhibited modest antiplasmodial activities with IC50 values between 26 to 67 µM. The use of hypoxanthine (compounds 7, both R and S-isomers) and xanthine (compounds 8, both R and S-isomers) resulted in total loss of the activity indicating the important role of the amino substitution at position 2.

Finally, concerning the nature of the polar group in the -position relative to the phosphorus atom, the hydroxyl group remained the best option (Table 1). Its replacement by an NH2 group (compounds 12 and 13, with IC50 values >100 µM) or removal (compounds 11a-b) led to a significant decrease of the antiplasmodial activity, at least 1000-fold (in the µM range) compared to compound 5(R) (in the nM range).

Table 1. Comparative effects of ANP derivatives on the growth of P. falciparum (3D7 strain) and erythroblast (K562) human cell line. Values for X, Y and n are related to formulas shown in fig. For other values, the means of two independent experiments (each performed in duplicate) and differing by <30% are given. [b] SI (selectivity index) is the ratio of IC50 value for K562 cells to the IC50 value for P. falciparum. NA: not applicable. Parasite growth was assessed after 48 h of contact with the compound by measuring the incorporation of [ 3 H] hypoxanthine into nucleic acids from 48 to 66 h. 25 In order to assess the effect of the analogues on the development of P. falciparum parasites, an infected-erythrocyte culture treated with 5(R) was followed over 69 h. Thin blood smears showed that treated parasites developed normally for 48 h until the end of the cycle at the schizont stage (Figure 4A). However, only few new parasites were detected at the following cycle when treated with 2 µM of compound 5(R) and they died before 69 h. Immunofluorescence assays on mature parasites at late schizont stage showed that the daughter cells developed normally in presence of compound 5(R) and that the DNA content of the newly formed merozoites seemed unaffected by the treatment (Figure 4B). This suggests that the mode of action of compound 5(R) could be different from other classes of ANPs that have been described to target DNA synthesis (Keough et al. 9, 26 ). This hypothesis is supported by the comparison our data with the literature. It has been reported that both guanine and hypoxanthine ANP derivatives equally inhibited recombinant

PfHGXPRT. 12-13 In contrast, our guanine derivatives (compounds 5) have an antimalarial activity between nM and low µM range whereas our hypoxanthine derivatives (compounds 7) were not active at 1 mM. Additionally, Cesnek et al. 13 showed that the guanine derivative corresponding to our compound 11a has no affinity for PfHGXPRT with Ki>100µM. In our experiments, this compound 11a showed a modest inhibition of P. falciparum growth in vitro with an IC50 value of 74 µM. Altogether, these data suggest that our series of analogues have a different mode of action from other classes of ANPs and a target that is unlikely to be the PfHGXPRT. We then evaluated the compounds for toxicity on human blood cell lines (K562 cells) and calculated selectivity indexes (SI) (Table 1). None of the derivatives inhibited human blood cell growth below a concentration of 1 mM indicating a high selectivity for infected red blood cells.

All ANPs tested are of anionic character which generally limits their passive diffusion across biological membranes, explaining their low toxicity towards human cell lines. Our data showed that the ANPs developed in this study affected parasite growth and were therefore likely able to specifically enter into infected red blood cells. This could be due to the presence of channels transporting negatively-charged molecules on the infected erythrocyte membrane that are induced by the parasite. 27 Altogether, these data revealed a unique antimalarial pharmacophore and the compound 5(R)

shows considerable potential with an antimalarial activity of 74 nM and a SI higher than 1350.

Evaluation of compounds 5(R) and 5(S) on the drug-resistant strains of P. falciparum

Compounds 5(R) and 5(S) were selected for further studies with the multidrug-resistant W2 strain and the chloroquine-resistant FcM29 strain of P. falciparum (Table 2). Both derivatives showed equivalent potency in the three strains, 3D7, W2 and FcM29. Antimalarial activity in P. berghei-infected mice. According to the results obtained in vitro, compound 5(R) was evaluated in an in vivo assay using P. berghei-infected mice (Figure 5). After four days of treatment by intraperitoneal (i.p.) route, the parasitemia was quantified at day 5 and the dose required to decrease the parasitemia by 50% (ED50) was determined. In these conditions, the derivative 5(R) showed a potent efficacy in vivo with an ED50 value of 0.56 mg/kg (Figure 5A).

Parasites were not detected after a treatment with 30 mg/kg at day 5 but we observed a parasite recrudescence for two mice after day 11 (6.9 and 16.7% of parasitemia) and for one mouse after day 14 (1.7% of parasitemia). In parallel, we tested the effect of a single dose of 30 mg/kg administered at day 1. This treatment showed a decrease of the parasitemia by 95% as compared to the controls. The efficacy of the derivative may enable the dosage regimens to be modified for further development. The activity was also evaluated by oral administration after four days of treatment. In these conditions, the ED50 was found to be 30 mg/kg indicating a low bioavailability likely due to the polarity and anionic character of the compound (Figure 5B).

In comparison, artesunate, an artemisinin derivative, showed an ED50 value of ∼ 0.1 mg/kg by i.p.

route (Figure 5C) and a treatment of 27 mg/kg did not allow a complete cure. This suggests that the derivative 5(R) showed a potent activity comparable to this widely used antimalarial drug.

EXPERIMENTAL SECTION

Chemistry. General Information

Solvents were dried by standard procedures. Tetrahydrofuran (THF) was freshly distilled from Na under argon and in presence of benzophenone. All reagents and chemicals were obtained from commercial sources. Thin layer chromatography (TLC) was performed on pre-coated aluminum sheets of Silica Gel 60 F254 (Merck, Art. 5554). Product spots on TLC plate were visualized with a UV lamp (254 nm). Flash chromatography on normal phase (silica gel) and on reversed phase (C18AQ) was performed on Biotage or Interchim Flash Chromatography Systems, respectively.

NMR spectra were recorded on Bruker Avance 500 ( 1 H at 500 MHz, 13 C at 125.8 MHz) or 300

( 1 H at 300 MHz, 13 C at 125.8 MHz) spectrometer with TMS as internal standard or referenced to the residual solvent signal (CDCl3, DMSO-d6 or D2O) for 1 H and 13 C. H3PO4 was used as external standard for 31 P NMR and corresponding spectra were recorded with proton decoupling at ambient mL/mmol of nucleobase), was added under argon atmosphere triphenylphosphine (1.2 eq.) and the nucleobase (1 eq.). The reaction mixture was cooled to 0°C and diethylazodicarboxylate (1.2 eq.) was added dropwise. After 10 min, the reaction mixture was allowed to warm to room temperature and stirred overnight. The reaction mixture was concentrated under vacuum, dissolved in a small amount of diethyl ether, and stored at 4°C overnight. The triphenylphosphine oxide precipitate was filtrated off and washed with cold diethyl ether. The filtrate was then concentrated under vacuum and the crude purified by flash chromatography on silica gel, using either an isocratic of AcOEt or CH2Cl2/MeOH (0 to 5%) to isolate the desired product.

N, )- N-9-(2R-(oxiran-2-yl) 28 (2 eq.) in freshly distilled dichloromethane (6.5 mL/mmol of nucleobase), was added under argon atmosphere anhydrous cesium carbonate (Cs2CO3, 3.6 eq.). The reaction mixture was stirred at room temperature and followed by TLC (CH2Cl2/MeOH 95/5 v/v, with sulfuric acid revelation) until completion. After 24 h, the suspension was filtered and the precipitate washed with 3 mL of anhydrous dichloromethane. The filtrate was then transferred in a round-bottom flask flushed with argon and anhydrous tetrahydrofuran (3.8 mL/mmol of nucleobase), triphenylphosphine (1.2 eq.) and the nucleobase (1 eq.) were successively added. The reaction mixture was cooled to 0°C and diethylazodicarboxylate (1.2 eq.) was added dropwise. After 10 min, the reaction mixture was allowed to warm to room temperature and stirred for 24 h with TLC monitoring (CH2Cl2/MeOH 95/5 v/v). The reaction mixture was concentrated under vacuum, dissolved in a small amount of diethyl ether, and stored at 4°C overnight. The triphenylphosphine oxide precipitate was removed by filtration and washed with cold diethyl ether. The filtrate was then concentrated under vacuum and the crude purified by flash chromatography on silica gel, using CHCl3/AcOEt (0 to 100%) to remove the remaining triphenylphosphine oxide, and then CH2Cl2/MeOH (0 to 10%) to isolate the desired product, which was obtained as a pale yellow oil.

1H, CH2CH2N), 1.98 -1.95 (m, 1H, CH2CH2N), 1.43 (s, 18H, CH3). 13 N, amino-6-methoxy- N-9-(2S-(oxiran-2-yl) 13 C NMR (DMSO-d6) δ 153.9 (C-4), 150.7 (C-6), 148.0, 149.4, 149.5, 150.3 (C=O), 126.3 (C-5), 82.8, 83.4 N, - N-9-(2S-(oxiran-2-yl)ethyl) -2,6-diaminopurine (23(S))

Compound 23(S) (3.01 g) was obtained in 89% yield, according to procedure B, from 2-(S)-bromo-1, 4-butanediol (1.84 g). Rf = 0.23 in CH2Cl2/MeOH (95/5, v/v).

[α]D 20 -19.8° (c 0.85, CH2Cl2). N, )- N-9-(2R-(oxiran-2-yl) )- N-9-(2R-(oxiran-2-yl) was added under argon N, . The reaction mixture was stirred and refluxed for 4h.

Reaction progress was followed by 31 P NMR, to check the disappearance of diethylphosphite signal (δ~7 ppm) while silylated diethylphosphite appeared (δ ~ 127 ppm). The reaction mixture was allowed to reach to room temperature, and then cooled to -60°C. A solution of the epoxide (1 eq.) in freshly distilled dichloromethane (6.6 mL/mmol of epoxide) was added dropwise. Then, boron trifluoride diethyl etherate (6 eq.) was added dropwise, and the reaction mixture was stirred for 3h and then allowed to warm to room temperature overnight. Volatiles were removed under reduced pressure; the residue was dissolved in ethyl acetate and washed with water. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure. Purification by flash chromatography on silica gel using CH2Cl2/MeOH (0 to 10%) gave rise to the desired product as a white solid.

Diethyl(R)- (3)(4)(5)(6) J = 6.9 Hz, 3H, OCH2CH3). 13 C NMR (CDCl3) δ 153.7 (C-4), 152.0 (C-2), 150.6 (C-6), 150.4 (C=O), 146.4 (C-8), 128.7 (C-5), 84.0 (C(CH3)3), 65.5 (CHOH),62.5,62.4 (OCH2CH3),49.9,49.7 (CH2N),31.6,30.2 (CH2P), 27.9 (CH3), 16.5,16.4 (OCH2CH3). 31 Diethyl(S)- (3)(4)(5)(6) Diethyl(R)- Diethyl(S)- amino)- 6-methoxy-9H-purin-9-yl)-2-hydroxy-propyl) phosphonate (18(S))

Compound 18(S) (1.01 g) was obtained in 43% yield, according to procedure C, from 15(S) (1.75 g). Rf = 0.16 in CH2Cl2/MeOH (95/5, v/v).

[α]D 20 -6.8° (c 0.95, MeOH). 1 H NMR (CDCl3): δ = 8.08 (s, 1H, H-8), 5.23 [START_REF]MS (ESI) m/z 303.12 ]+; HRMS: calc. C9H16N6O4P[END_REF]1H,OH),4.72 (m,1H,CHOH),2H,CH2N), 4. 10-3.95 (m, 7H, OCH3, OCH2CH3), 2.00-1.67 (2m, 2H, CH2P), 1.37 (2s, 18H, CH3), 1.23 (m, 6H, OCH2CH3). 13 C NMR (CDCl3): δ 161.4 (C-6), 152.9 (C-4), 151.7 (C-2), 150.9 (C=O), 144.5 (CH- Diethyl(R)- Diethyl(S)- Diethyl (R)- (4)(5)(6) Diethyl (S)- (4)(5)(6)amino)-9H-purin-9-yl)-2-hydroxybutyl) phosphonate (24(S))

Compound 24(S) (746 mg) was obtained in 54% yield, according to procedure C, from 21(S) (1 g). Rf = 0.5 in CH2Cl2/MeOH (9/1, v/v).

[α]D 20 -19.3° (c 0.3, CH2Cl2). 1 Hz, 6H, OCH2CH3). 13 C NMR (CDCl3) δ 161.6 (C-6), 153.0 (C-4) Diethyl (S)- amino)- 6-methoxy-9H-purin-9-yl)-2-hydroxy-butyl) phosphonate (25(S))

Compound 25(S) (742 mg) was obtained in 45% yield, according to procedure C, from 22(S) (1.25 g). Rf = 0.53 in CH2Cl2/MeOH (9/1, v/v).

[α]D 20 -18.2° (c 0.1, CH2Cl2). 1 H NMR (CDCl3) δ 7.97 (s,1H,2H,CH2N),4.15 (s,1H,OCH3),4H,OCH2CH3),1H,CHOH), 2. 4H,CH2CH2N,CH2P), 1.46 (s, 18H, CH3), 1.29 (t, 6H, OCH2CH3). 13 C NMR (CDCl3) δ 161.6 (C-6), 153.0 (C-4) Diethyl(R)- Diethyl (S)- (CH2N), 37. 34.5,33.1 (CH2P),28.2 (CH3),16.7,16.6 (OCH2CH3). Diethyl(R)- (4)(5)(6) 4H,OCH2CH3),3.95 (m,1H,CHOH), 2. 4H,CH2CH2N,3.46 (m,1H,CHOH), 2. 3H,CH2CH2N,CH2P),1H,CH2CH2N), 1. 6H,CH3CH2). 13 C NMR (D2O) δ 166.9 (C-6), 162.0 63.2,63.1 (CH2CH3,CHOH),53.8 (OCH3), 39.7 (CH2N), 37.0, 36.9 (CH2CH2N), 32.9,31.8 (CH2P),15.4,15.3 (s,CH3CH2). 31 water was added to the reaction mixture and stirring was pursued 1 h at rt. Then, triethylammonium bicarbonate solution (TEAB 1M) was added to the reaction mixture until pH = 7, the volatiles were evaporated under high vacuum and the aqueous residue was freeze-dried. The resulting lyophilizate was purified by flash chromatography silica gel on C18 reverse phase using water/MeOH gradient (0 to 100%) and freeze-dried, giving rise to the desired phosphonic acid.

Eventually, the latter was dissolved in a small amount of water and percolated through a Dowex resin column (Na + form) to afford after freeze-drying the corresponding compound as sodium salt.

(R)- -8), 117.9 (C-5), 65.5 (CHOH), 40.9 (CH2N), 37.0, 36.9

(1.1 eq.) in anhydrous DMF (1.5 mL/mmol) was added dropwise over a 30 min period. The reaction mixture was kept under stirring for three days at room temperature with TLC monitoring (hexane/ethyl acetate, 5/5, v/v). Then, the volatiles were evaporated under high vacuum, and the resulting residue was dissolved in ethyl acetate, sonicated and the undissolved salts were filtered on a sintered glass (porosity 4). The filtrate was then evaporated and purified on silica gel by flash chromatography (petroleum ether/ethyl acetate gradient) to afford the N-Boc purinyl aziridine as a white foam.

(R)- )-6-methoxy- N-9-purine)ethyl) aziridine- N-carboxylate (36(R))

Compound 36(R) (983 mg) was obtained in 69% yield, according to procedure G, from 35(R) (1 g). Rf = 0.3 in hexane/AcOEt (5/5, v/v).

[α]D 20 -38.2 (c 1, CH2Cl2). 1 H NMR (CDCl3) δ 8.14 (s, 1H, H8), 4. 2H,CH2N) (S)- )-6-methoxy- N-9-purine)ethyl) aziridine- N-carboxylate (36(S))

Compound 36(S) (893 mg) was obtained in 44% yield, according to procedure G, from 35(S) (1.3 g). Rf = 0.3 in hexane/AcOEt (5/5, v/v) 6.11 (ddt,J = 45.8,13.0,7.4 Hz,1H,CHCH2),5.83 (ddt,J = 17.7,13.1,1.6 Hz, 1H, CHP), 4.15 (t, J = 7.1 Hz, 2H, CH2N), 2.93 (qdd, J = 7.0, 2.8, 1.5 Hz, 2H, CH2CH). 13

Biological experiments

In vitro antimalarial activity. Drug effects on in vitro P. falciparum growth (3D7, W2 and FcM29 strains) were measured in microtiter plates according to a modified Desjardins test. 25, 31 P.

  Montpellier, Team Nucleosides & Phosphorylated Effectors, Place E. Bataillon, cc 1704, 34095 Montpellier, France. [b] Laboratory of Pathogen Host Interactions (LPHI), UMR 5235 UM-CNRS, Univ. Montpellier, Place E. Bataillon, 34095 Montpellier, France. KEYWORDS. Malaria, Nucleotide analogue, Drug discovery.

Figure 2 .

 2 Figure 2. Structures of the studied compounds

Scheme 2 .

 2 Scheme 2. Synthetic route to C3--hydroxy-ANPs containing adenine, guanine and 2,6-

Scheme 3 .

 3 Scheme 3. Synthetic route to C4--hydroxy-ANPs containing adenine, guanine and 2,6-

Scheme 4 .

 4 Scheme 4. Synthetic route to C4--hydroxy-ANPs containing xanthine and hypoxanthine as

Scheme 5 .

 5 Scheme 5. Synthetic route to C4--hydroxy-ANPs containing 2-amino-and 2-fluoro-6-

Scheme 6 .

 6 Scheme 6. Synthetic route to C4--amino-ANPs containing guanine and 2,6-diaminopurine as

Figure 3 .

 3 Figure 3. Separation of pair of enantiomers of two families of ANPs by 31 P NMR. A, comparison of 31 P NMR of enantiomers of β-hydroxyphosphonates; B, comparison of 31 P NMR of enantiomers

  Values are the means of at least three independent experiments performed in duplicate (±SEM).

Figure 4 .

 4 Figure 4. Effect of compound 5(R) on the development of P. falciparum. A tightly synchronized

Figure 5 .

 5 Figure 5. In vivo antimalarial activity of 5(R) and artesunate in P. berghei-infected mice. 5(R)

  photodiode array detector 2996). The column was a Waters Symmetry Shield (50 mm × 4.6 mm,

  amino)-6-methoxy-9H-purin-9-yl)-2-hydroxy-propyl) phosphonate (18(R))Compound 18(R) (107 mg) was obtained in 41% yield, according to procedure C, from 15(R) (200 mg). Rf = 0.16 in CH2Cl2/MeOH (95/5, v/v). [α]D 20 + 8° (c 1.0, MeOH).1 H NMR (DMSO-d6)d6): 28.1; MS (ESI) m/z 560.3[M+H]+; HRMS: calc. C23H39N5O9P[M+H]+ 560.2485, obs. 560.2481.

7 (

 7 diamino)-9H-purin-9-yl)-2-hydroxypropyl) phosphonate (19(R)) Compound 19(R) (482 mg) was obtained in 36% yield, according to procedure C, from 16(R) (1.08 g). Rf = 0.18 in CH2Cl2/MeOH (95/5, v/v). [α]D 20 + 11.8° (c 1.2, MeOH). 1 H NMR (DMSO-d6): δ = 8.56 (s, 1H, H-8), 5.51 (1H, OH), 4.47 (m, 1H, CHOH), 4.21 (m, 2H, CH2N), 3.99 (m, 4H, OCH2CH3), 2.00 (m, 2H, CH2P),1.36 (s, 36H, CH3), 1.22 (pt, 6H, OCH2CH3).13 C NMR (DMSO-d6): δ 154.0(C-4), 150.7(C-2), 150.2, 149.6, 149.3, 148.5 (C-6, C=O), 126.1(C-5,), 83.4, 82.).31 P NMR (DMSO-d6): 27.9; MS (ESI) m/z 745.3[M+H]+; HRMS: calc. C32H54N6O12P[M+H]+ 745.3537, obs. 745.3543.

5 (

 5 amino)-9H-purin-9-yl)-2-hydroxybutyl) phosphonate (24(R)) Compound 24(R) (886 mg) was obtained in 52% yield, according to procedure C, from 21(R) (1.28 g). Rf = 0.5 in CH2Cl2/MeOH (9/1, v/v). [α]D 20 + 17.05° (c 0.09, CH2Cl2). 1 H NMR (CDCl3) δ 8.85 (s, 1H, H-2), 8.13 (s, 1H, H-8), 4.54-4.44 (m, 2H, CH2N), 4.09 (m, 4H, OCH2CH3), 3.95-3.84 (m,1H, CHOH),2.18, 2.01 (2m, 2H, CH2CH2N), 1.93 (m, 2H, CH2P), 1.46 (s, 18H, CH3), 1. 6H, OCH2CH3).13 C NMR (CDCl3) δ 153.6(C-4), 152.1(C-2), 150.7(C-6), 150.5 (C=O), 145.s). HRMS (TOF ESI>0) Found: 544.2534, Calculated for C23H39N5O8P: 544.2536 (M+H) + .

  31 P-NMR (CDCl3) δ 29.0 (s). MS (ESI) m/z 566.24 [M+Na]+.

  ), 31.9 (CHCH2N), 28.1 (C(CH3)3). HRMS (TOF ESI>0) Found: 535.2880, Calculated for C25H39N6O7: 535.2880 (M+H) +

  42.8 (CH2N), 30.2 (d, J = 21.8 Hz, 1C, CH2CH). 31 P NMR (202 MHz, D2O) δ 10.5. HMRS TOF ESI+ Found : 286.0708; Calculated for C9H13N5O4P : 286.0705 (M+H) + .

  falciparum infected erythrocyte suspension (1.5% final hematocrit and 0.6% parasitemia) were grown in complete medium (Hepes-buffered RPMI 1640 + 10% AB human serum) with or without ANP analogs. The compounds were dissolved in RPMI 1640 and then further diluted in culture medium. Parasites growth was assessed by measuring the incorporation of [ 3 H] hypoxanthine into nucleic acids as previously described.25 Suspensions of P. falciparum -infected erythrocytes were incubated with various concentrations of analogs during 48 h. Then 30 µl of [ 3 H]hypoxanthine (0.5 µCi/well) was added for an additional 18h-period. The reactions were stopped by freezing at -80°C. Cells were lysed by thawing and the parasite macromolecules including nucleic acids were recovered by harvesting the lysate on glass-fiber filter plates (Unifilter 96 GF/C, Perkin Elmer) using a FilterMate cell harvester (Packard Instruments). The radioactivity was counted on a TopCount microplate scintillation counter (Packard Instruments). Radioactivity background was obtained from incubation of non-infected erythrocytes under the same conditions and the value primary antibodies (-mTIP(myosin Tail Domain Interacting Protein)32 or mouse -MSP1(Merozoite Surface Protein 1)33 diluted in PBS containing 0.15 % BSA for 1h. After three washes, appropriate secondary antibodies were added for 1h before staining of nuclei with Hoechst (5 min). Observations were performed with a Zeiss Axioimager epifluorescence microscope equipped with a Zeiss Axiocam MRmCCD camera at Montpellier RIO imaging facility. Images were processed using Zen Blue 2.3 (Zeiss) for optical sectioning, luminosity and contrast adjustment.In vivo antimalarial activity. Female Swiss NMRI mice (Charles River Laboratories, France) were infected on day 0 by intravenous injection into the caudal vein of 10 7 P. berghei ANKAinfected erythrocytes (clone 2.34). Mice were treated once a day for four consecutive days from day 1 by i.p or oral route. Compound 5(R) was dissolved in 0.9% NaCl or water (100 µl per mouse and 3 mice per group) for i.p. or oral administration, respectively. Artesunate was dissolved in DMSO (100 µl per mouse and 3 mice per group). Doses were 0, 0.1, 0.3, 1, 3, 10 and 30 mg/kg for compound 5(R) and 0, 0.3, 1, 3 9 and 27 mg/kg for artesunate by i.p. By oral route, doses were 0, 1, 3, 10 and 30 mg/kg for compound 5(R). Control (untreated) group received only 0.9% NaCl or water. On day 5, parasitemia was counted on Giemsa-stained thin blood smears and the dose leading to the reduction of 50% of the parasitemia (ED50) was determined. Parasitemia was followed for 30 days.The experiments done here were conform to the national and European regulations and to French laws (EU directive no. 86/609 modified by the directive 2010/63 regarding the Protection of Animals used for Experimental and Other Scientific Purposes). The animal studies were performed at the "Centre d'Elevage et de Conditionnement Experimental des Modèles Animaux",

Table 2 :

 2 Comparative effects of derivatives 5(R) and 5(S) on the growth of P. falciparum strains,

	sensitive (3D7) or resistant (FcM29 and W2) to chloroquine.
	Compounds		IC50 (µM)
		3D7	FcM29	W2
	5(R) UA2239	0.074 ± 0.007	0.03 ± 0.003	0.045 ± 0.006
	5(S) UA2238	6.1 ± 1.1	4.5 ± 1.3	5.6 ± 1.4
	Chloroquine	0.016 ± 0.001	0.117 ± 0.010	0.131 ± 0.022

Values (±SEM) are the means of at least three independent experiments performed in duplicate.

  methyl)-adenine (14(R))Compound 14(R) (1.86 g) was obtained in 87% yield as an oil, according to procedure A, from (R)-glycidol (362 µL). Rf = 0.41 in AcOEt.[α]D 20 = + 22.5° (c 1.02, MeOH).1 H NMR (DMSO-

	154.0 (C-4), 151.0, 150.2 (C-6, C-2), 148.0, 149.5, 149.6, 150.2 (C=O), 125.9 (CH-8), 83.4, 82.8
	(C(CH3)3), 49.2 (CHOH), 44.7 (OCH2, CH2N), 27.2, 27.0 (CH3); MS (ESI) m/z 607.3 [M+H] + ;
	HRMS: calc. C28H43N6O9 [M+H] + 607.3092, obs. 607.3082.
	d6): δ = 8.86 (s, 1H, H-2), 8.60 (s, 1H, H-8), 4.67 (dd, 1H, 2 J = 15.0 Hz, 3 J = 2.7 Hz, CH2N), 4.40
	(dd, 1H, 2 J = 15.0 Hz, 3 J = 6.0 Hz, CH2N), 3.47 (m, 1H, CHO), 2.83 (m, 1H), 1.37 (s, 18H, CH3); General Procedure B: Preparation of epoxide derivatives in the C4-series. To a solution of
	13 C NMR (CDCl3): δ = 152.4 (CH-2), 145.1 (CH-8), 128.6 (C-5), 83.9 (C(CH3)3), 49.9 (CHO), 45.5 enantiomerically pure 2-bromo-1,4-butanediol
	(OCH2), 45.4 (CH2N), 27.9 (CH3); MS (ESI) m/z 392.2 [M+H] + ; HRMS: calc. C18H26N5O5
	[M+H] + 392.1934, obs. 392.1921.
	0 (CH-8), 127.5
	(C-5), 83.3 (C(CH3)3), 49.4 (CHO), 45.1 (OCH2), 45.0 (CH2N), 27.3 (CH3); MS (ESI) m/z 392.3
	[M+H] Compound 15(R) (1.72 g) was obtained in 74% yield as a solid, according to procedure A, from
	(R)-glycidol (336 µL). Rf = 0.2 in AcOEt. [α]D 20 = + 20.5° (c 1.05, MeOH). 1 H NMR (DMSO-
	d6): δ = 8.39 (s, 1H, H-8), 4.54-4.34 (m, 2H, CH2N), 4.07 (s, 3H, OCH3), 3.40 (m, 1H, CHO), 2.81
	(m, 1H, OCH2), 2.50 (m, 1H partially covered by DMSO, OCH2), 1.39 (s, 18H, CH3). 13 C NMR
	(DMSO-d6): δ = 160.6 (C-6), 152.7 (C-4), 151.0 (C-2), 150.3 (C=O), 144.9 (CH-8), 118.7 (C-5), 82.6

N,

-

N-9-(2S-(oxiran-2-yl)

methyl)-adenine (14(S)) Compound 14(S) (1.01 g) was obtained in 86% yield as a solid, according to procedure A, from (S)-glycidol (181 µL). Rf = 0.41 in AcOEt. [α]D 20 = -16.8° (c 1.01, MeOH). 1 H NMR (DMSO-d6): δ = 8.86 (s, 1H, H-2), 8.60 (s, 1H, H-8), 4.65 (dd, 1H, 2 J = 15.0 Hz, 3 J = 2.7 Hz, CH2N), 4.40 (dd, 1H, 2 J = 15.0 Hz, 3 J = 6.0 Hz, CH2N), 3.47 (m, 1H, CHO), 2.83 (m, 1H), 1.38 (s, 18H, CH3); 13 C NMR (DMSO-d6): δ = 153.3 (C-4), 151.6 (CH-2), 150.0, 149.1

(C-6, C=O)

, 147.+ ; HRMS: calc. C18H26N5O5

[M+H] + 392.1934, obs. 392.1941

. N,

methyl)-

2-aminopurine (15(R))

  P NMR (DMSO-d6): δ = 28.1; MS (ESI) m/z 530.3 [M+H] + ; HRMS: calc. C22H37N5O8P [M+H] + 530.2380, obs. 530.2391.

  . [α]D20 + 38.8°(c 1, CH2Cl2).1 H NMR (CDCl3) δ 8.13 (s, 1H, H8), 4. 2H, CH2N), 4.14 (s, 3H, CH3O), 4H, CHN, CHCH2N, CH2CH2N), 1.88 (d, J = 3.5 Hz, 1H, CHCH2N), 1. 1H, CH2CH2N), 1.46 (s, 9H, CH3), a white lyophilizate.The desired compound (130 mg) was obtained in 54% yield. Rf = 0.18 (iPrOH/H2O/NH4OH 7/2/1). Purity (HPLC) > 96%.1 H NMR (500 MHz, D2O) δ 7.82 (s, 1H, H-

8),

Funding Sources

This work is part of the research project " NAN-Pal" supported by the Région Occitanie and SATT AxLR. ACKNOWLEDGMENT This work is part of the research project "NAN-Pal" supported by the Région Occitanie and SATT AxLR. We thank the Centre d'Elevage et de Conditionnement Experimental des Modèles

CH2P), 1.49 (s,18H,CH3), 1.30 (m,6H,OCH2CH3). 13 C NMR (CDCl3) δ 155.8 (C-6), 154.1 (C-4), 150.9 (C-2), 149.0 (C=O), 144.8 (C-8), 83.4 (C(CH3)3), 62.4 (CHOH), 61.2 (OCH2CH3), 40.1 (CH2N),36.4,36.3 (CH2CH2N),33.2,31.8 (CH2P), 26.8 (CH3),15.4,15.3 (OCH2CH3). 31 δ 29.1 (s). MS (ESI) m/z 562.25 [M+H]+.

General Procedure D:

Removal of Boc protecting groups. To a stirred solution of βhydroxyphosphonate (1 eq.) in anhydrous dichloromethane (14 mL/mmol) under argon, and at room temperature, was added dropwise a solution of trifluoroacetic acid in anhydrous dichloromethane (19 mL/mmol, 3/7, v/v). The reaction mixture was stirred at room temperature for 5 h. Volatiles were removed under reduced pressure and the crude was purified by flash chromatography on silica gel using CH2Cl2/MeOH (0 to 10%). The desired compound was obtained as a white foam.

Diethyl (R)- (4-(6-amino-9H-purin-9-yl)- 2-hydroxybutyl) phosphonate (27(R))

The compound 27(R) (530 mg) was obtained in quantitative yield, according to procedure D, from 24(R) (840 mg). Rf = 0.42 in CH2Cl2/MeOH (90/10, v/v). [α]D 20 + 15° (c 0.2,CH2Cl2). 1 H NMR (CDCl3) δ 8.31 (s, 1H, H-2), 7.91 (s, 1H, H-8), 6.87 (s,2H,NH2),2H,CH2N), 4. 13-4.03 [START_REF]MS (ESI) m/z 303.12 ]+; HRMS: calc. C9H16N6O4P[END_REF]4H,CH2CH3),1H,CHOH), 2. 1H,CH2CH2N), 2. 02-1.88 [START_REF]MS (ESI) m/z 303.12 ]+; HRMS: calc. C9H16N6O4P[END_REF]3H,CH2CH2N,CH2P), 1. 6H,CH3CH2). 13 C NMR (CDCl3) δ 154.5 (C-6), 150.3 (C-2), 149.9 (C-4), 142.0 (CH-8), 119.5 (C-5), 63.2 (CHOH), 62. 40.9 (CH2N),38.2,38.1 (CH2CH2N),34.3,33.0 (CH2P), 16.5 (CH3CH2). 31 P-NMR (CDCl3) δ 28.7 (s). HRMS (TOF ESI>0) Found: 344.1487, Calculated for C13H23N5O4P: 344.1488 (M+H) + .

Diethyl (S)- (4-(6-amino-9H-purin-9-yl)- 2-hydroxybutyl) phosphonate (27(S))

The compound 27(S) (127 mg) was obtained in quantitative yield, according to procedure D, from 24(S) (200 mg). Rf = 0.39 in CH2Cl2/MeOH (90/10, v/v). [α]D 20 -16.2° (c 0.1, CH2Cl2). 1 H NMR (CDCl3) δ 8.31 (s, 1H, H-2), 7.91 (s, 1H, H-8), 6.87 (s,2H,NH2),2H,CH2N), 4. .00 [START_REF]MS (ESI) m/z 303.12 ]+; HRMS: calc. C9H16N6O4P[END_REF]4H,CH2CH3),1H,CHOH), 2. 1H,CH2CH2N), 1.96-1.89 [START_REF]MS (ESI) m/z 303.12 ]+; HRMS: calc. C9H16N6O4P[END_REF]2H,CH2P), 1.29 (m,CH3CH2). 13 C NMR (CDCl3) δ 154.5 (C-6), 150.3 (C-2), 149.9 (C-4), 142.0 (CH-8), 119.5 (C-5) Diethyl (R)- (4-(2-amino-6-methoxy-9H-purin-9-yl)-2-hydroxybutyl)phosphonate (28(R))

Compound 28(R) (470 mg) was obtained in quantitative yield, according to procedure D, from 25(R) (720 mg). Rf = 0.50 in CH2Cl2/MeOH (90/10, v/v). [α]D 20 + 20.95° (c 0.5, CH2Cl2). 1 H NMR (CDCl3) δ 7.81 (s,1H,2H,CH2N),4.13 (s,3H,OCH3),4H,CH2CH3), 4. 1H,CHOH), 2. 4H,CH2CH2N,CH2P), 1.30 (m,6H,CH3CH2). 13 , 157.8 (C-2), 149.3 , 114.7 63.4 (CHOH),62.4,62.3 (CH2CH3),55.1 (OCH3),41.4 (CH2N),38.1,38.0 (CH2CH2N),33.9,32.8 (CH2P), 16.5 (CH3CH2). 31 Diethyl (S)- (4-(2-amino-6-methoxy-9H-purin-9-yl)-2-hydroxybutyl)phosphonate (28(S))

Compound 28(S) (280 mg) was obtained in quantitative yield, according to procedure D, from 25(R) (430 mg). Rf = 0.50 in CH2Cl2/MeOH (90/10, v/v). [α]D 20 -17.62° (c 1.0, CH2Cl2). 1 H NMR (CDCl3) δ 7.81 (s,1H,2H,CH2N),4.13 (s,3H,OCH3),4H,CH2CH3),1H,CHOH), 2. 4H,CH2CH2N,CH2P), 1.31 (m,6H,40 CH3CH2). 13 C NMR (CDCl3) δ 162.6 63.4 (CHOH),62.4,62.3 (CH2CH3),55.1 (OCH3),41.4 (CH2N),38.1,38.0 (CH2CH2N),33.9,32.8 (CH2P),16.5,16.4 (CH3CH2). 31 General Procedure E: Preparation of oxo-purines β-hydroxyphosphonate derivatives. A solution of β-hydroxyphosphonate (1 eq.) in acetic acid 1M (15 mL/mmol) was heated at 65°C. Sodium nitrite (15 eq.) dissolved in water (2.22 mL/mmol of NaNO2) was added portion wise over a 30 min-period. The reaction mixture was stirred for 2 h at 65°C, then cooled to 0°C and quenched by addition of aqueous sodium 1 M until pH = 7. Volatiles were removed under reduced pressure and the crude purified by flash chromatography silica gel on C18 reverse phase using water/MeOH gradient (0 to 100% MeOH) to afford the product as a white solid after freeze-drying.

Diethyl (R)- (2-hydroxy-4-(6-oxo-1,6-dihydro-9H-purin-9-yl) butyl)phosphonate (29(R))

Compound 29(R) (156 mg) was obtained in 66% yield, according to procedure E, from 27(R) (236 mg). Rf = 0.30 in CH2Cl2/MeOH (90/10, v/v). [α]D 20 + 14.77° (c 1.2, CH2Cl2). 1 H NMR (CDCl3) δ 8.14 (s, 1H, H-2), 7.91 (s,1H,4.36 (m,2H,CH2N),4H,CH2CH3),3.94 (m,1H,CHOH), 2. 4H,CH2CH2N,CH2P), 1.30 (m,6H,CH3CH2). 13 C NMR (CDCl3) δ 159.2 (C-6), 149.2 145.4, 140.9 (CH-8), 124.7 (C-5), 63.2 (CHOH), 62. 40.9 (CH2N),38.4,38.3 (CH2CH2N),34.3,33.2 (CH2P), 16.5 (CH3CH2). 31 (S)- phosphonic acid (4(S))

Compound 4(R) (167 mg) was obtained in 69% yield, according to procedure F, from 24(S) (398 mg). Rf = 0.26 in iPrOH/H2O/NH4OHc (7/2/1, v/v/v). Purity (HPLC) > 95%. 1 H NMR (D2O): δ = 7.98 (s, 1H, H-2), 7.96 (s, 1H, H-8), 4. 2H,CH2N),1H,CHOH), 2. 1H,CH2CH2N),3H,CH2CH2N,CH2P). 13 C-NMR (D2O) δ 154.9 , 117.9 65.5 (CHOH),40.9 (CH2N),37.0,36.9 (CH2CH2N),36.7,35.5 (CH2P); 31 (R)- phosphonic acid (5(R))

Compound 5(R) (165.6 mg) was obtained in 68% yield, according to procedure F, from 25(R) (404 mg). Rf = 0.60 in iPrOH/H2O/NH4OHc (7/4/2, v/v/v). Purity (HPLC) > 95%. [α]D 20 + 7° (c 0.0097, H2O). 1 H NMR (D2O): δ = 7.75 (s, 1H, H-8), 4. 2H,CH2N),1H,CHOH), 2. 1H,CH2CH2N),3H,CH2CH2N,CH2P). 13 C-NMR (D2O) δ 158.7 (C-6), 153.5 65.4 (CHOH),40.6 (CH2N),37.1,37.0 (CH2CH2N),36.7,35.4 (CH2P); 31 (S)- phosphonic acid (5(S))

Compound 5(S) (171 mg) was obtained in 51% yield, according to procedure F, from 25(S) (558 mg). Rf = 0.10 in iPrOH/H2O/NH4OHc (7/2/1, v/v/v). Purity (HPLC) > 99%. 1 H NMR (D2O): δ = 7.77 (s, 1H, H-8), 4. 2H,CH2N),1H,CHOH), 2. 1H,1.43 (s,18H,CH3). 13 C NMR (CDCl3) δ 162.2 (C=O), 161.6 (C-6), 152.8 (C-4), 152.0 (C-2), 151.1 (C=O), 143.7 (C-8), 120.3 (C-5), 83.1, 81.9 (C(CH3)3), 54.6 (OCH3), 42.4 (CH2N), 35.0 (CHN), 32.8 (CH2CH2N), 31.9 (CHCH2N), 28.0 (C(CH3)3). HRMS (TOF ESI>0) Found: 535.2882, Calculated for C25H39N6O7: 535.2880 (M+H) + (R)- )- N-9-purine)ethyl)aziridine- Ncarboxylate (37(R))

Compound 37(R) (1 g) was obtained in 87% yield, according to procedure G, from 35(R) (600 mg). Rf = 0.3 in hexane/AcOEt (5/5, v/v). [α]D 20 -41.5° (c 1, CH2Cl2). 1 H NMR (CDCl3) δ 8.33 (s, 1H, H8), 4. 2H,CH2N),3H,CH2CH2N,CHN),1.91 (d,J = 3.3 Hz,1H,CHCH2N),1H,CH2CH2N), 1.47 (s, 9H, CH3), 1.42 (s, 36H, CH3). 13 C NMR(CDCl3) δ 162.3 (C=O), 154.3 (C-4), 151.1 (C-2), 151.0 (C=O), 150.3 (C-6), 146.3 (C-8), 127.6 83.8,83.3,81.9 (C(CH3)3), 42.53 (CH2N), 34.9 (CHN), 32.7 (CH2CH2N), 31.9 (CHCH2N), 28.1,28.0,27.8 (C(CH3)3). HRMS (TOF ESI>0) Found: 720.3937, Calculated for C34H54N7O10: 720.3932 (M+H) + .

(S)-Tert- )- N-9-purine)ethyl)aziridine- 1carboxylate (37(S)) Compound 37(S) (1.07 g) was obtained in 93% yield, according to procedure G, from 35(S) (600 1H,CH2CH2N), 1.47 (s, 9H, CH3), 1.42 (s, 36H, CH3). 13 (R) and 36(S). Diethylphosphite (5 eq.) was dissolved in anhydrous THF (1 mL/mmol DEP) under argon atmosphere and cooled at -78°C. LiHMDS (5 eq.) was added dropwise to the mixture. The reaction mixture was kept under stirring at -78°c for 15 min. Then, the solution of aziridine 36 (R) or 36(S) (1 eq.) in anhydrous THF (2.8 mL/mmol aziridine) was added dropwise. After 5 min, the cooled bath was removed and the mixture slowly warmed to room temperature. The reaction was stirred overnight and followed by TLC monitoring (CH2Cl2/MeOH 95/5 v/v). After 24 h, the mixture was quenched by addition of saturated aqueous solution of NH4Cl, until dissolution of the previously formed salts. The resulting solution was extracted twice by ethyl acetate and the organic layers were gathered, dried over MgSO4, concentrated under vacuum and finally purified on silica gel by flash chromatography (dichloromethane/methanol gradient up to 10% methanol) to afford the expected N-Boc βaminophosphonate 38 (R) or 38(S) as a white foam.

(R)-2-Amino- (N-tert-butoxycarbonyl)-6-methoxy- N-9-(3-amino-4-diethylphosphonobutyl) purine

Compound 38(R) (915 mg) was obtained in 62% yield, according to procedure H, from 36(R) (1.37 g). Rf = 0.4 in CH2Cl2/MeOH 95/5 (v/v). [α]D 20 -20.8° (c 1, CH2Cl2). 1 H NMR (CDCl3) δ 7.82 (s, 1H, H8), 7.33 (s, 1H, NH), 6.26 (d, J = 7.4 Hz, 1H, CHNH), 4. 1H,CH2N), 4. .17 (R) )- N-9-(3-amino-4-diethylphosphonobutyl)purine (39(R))

Compound 39(R) (1.47 g) was obtained in 76% yield, according to procedure I, from 37(R) (2.13 g). Rf = 0.4 in CH2Cl2/MeOH 95/5 (v/v)). [α]D 20 -20.9° (c 1, CH2Cl2). 1 CH3), 1. 6H,CH2CH3). 13 (S) )- N-9-(3-amino-4-diethylphosphonobutyl)purine (39(S))

Compound 39(S) (1.58 g) was obtained in 78% yield, according to procedure I, from 37(S) (2.22 g). Rf = 0.4 in CH2Cl2/MeOH 95/5 (v/v). [α]D 20 + 20.1° (c 1, CH2Cl2). 1 H NMR (CDCl3) δ 8.04 (s, 1H, H8), 7.87 (s, 1H, NH), 7.81 (s, 1H, NH), 6.32 (m, 1H, CHNH), 4. 32-4.18 (2 m, 2H, CH2N), 55 4. 4H,CH2CH3),1H,CHNH),4H,CH2CH2N,CH2P), 1.52 (s, 9H, CH3), 1.50 (s, 9H, CH3), 1.43 (s, 9H, CH3), 1. 6H,CH2CH3). 13 aminophosphonate (1 eq.) in anhydrous dichloromethane (14 mL/mmol) under argon, and at room temperature, was added dropwise a solution of trifluoroacetic acid in anhydrous dichloromethane (19 mL/mmol, 3/7, v/v). The reaction mixture was stirred at room temperature for 5 h. Volatiles were removed under reduced pressure and the crude was purified by flash chromatography on silica gel using CH2Cl2/MeOH (0 to 10%). The desired compound was obtained as a white foam.

Diethyl (R)-(4-(2-amino-6-methoxy-9H-purin-9-yl)-2-aminobutyl)phosphonate (40(R))

Compound 40(R) (594 mg) was obtained in quantitative yield, according to procedure D', from 38(R) (915 mg). Rf = 0.25 in CH2Cl2/MeOH (90/10, v/v). [α]D 20 -12.9° (c 1, MeOH). 1 H NMR (MeOD) δ 7.91 (s, 1H, H8), 4. 2H,CH2N), 4. 4H,CH2CH3)

Diethyl (S)-(4-(2-amino-6-methoxy-9H-purin-9-yl)-2-aminobutyl)phosphonate (40(S))

Compound 40(S) (290 mg) was obtained in quantitative yield, according to procedure D', from 38(S) (447 mg). Rf = 0.25 in CH2Cl2/MeOH (90/10, v/v). [α]D 20 + 13.5° (c 1, MeOH). 1 H NMR (MeOD) δ 7.92 (s, 1H, H8), 4.39-4.23 (2m, 2H, CH2N), 4. 4H,CH2CH3) 4- (2,6-diamino-9H-purin-9-yl)-2-aminobutyl) phosphonate (41(R))

Compound 41(R) (663 mg) was obtained in quantitative yield, according to procedure D', from 39(R) (1.47 g). Rf = 0.2 in CH2Cl2/MeOH (90/10, v/v). [α]D 20 -12.8° (c 1, MeOH). 1 H NMR (MeOD) δ 7.90 (s, 1H, H8), 4. 2H,CH2N), 4. 4H,CH2CH3),1H,CHNH2),4H,CH2CH2N,CH2P), 1.30 (m,6H,CH2CH3). 13

Diethyl (S)-(4-(2,6-diamino-9H-purin-9-yl)-2-aminobutyl) phosphonate (41(S))

Compound 41(S) (685 mg) was obtained in quantitative yield, according to procedure D', from 39(S) (1.58 g). Rf = 0.2 in CH2Cl2/MeOH (90/10, v/v). [α]D 20 + 12.4° (c 1, MeOH). 1 H NMR (MeOD) δ 7.86 (s, 1H, H8), 4. 2H,CH2N), 4. 4H,CH2CH3),1H,CHNH2),4H,CH2CH2N,CH2P),1.29 (m,6H,CH2CH3). 13 C NMR (MeOD) δ 155.9 (C-2), 153.1 , 117.2 64.3,64.1 (CH2CH3) General Procedure F': Removal of the diethyl phosphonate groups. To a stirred solution of diethyl-β-aminophosphonate (1 eq.) in anhydrous dimethylformamide (20 mL/mmol), cooled at 0°C, was added dropwise bromotrimethylsilane (6.6 eq.). The reaction mixture was stirred at room temperature until completion of the reaction was indicated by TLC monitoring (isopropanol/water/ammoniac 7/2/1 v/v/v). When in situ removal of Boc groups was required, water was added to the reaction mixture and stirring was pursued 1 h at rt. Then, triethylammonium bicarbonate solution (TEAB 1M) was added to the reaction mixture until pH = 7, the volatiles were evaporated under high vacuum and the aqueous residue was freeze-dried. The resulting lyophilizate was purified by flash chromatography silica gel on C18 reverse phase using water/MeOH gradient (0 to 100%) and freeze-dried, giving rise to the desired phosphonic acid.

Eventually, the latter was dissolved in a small amount of water and percolated through a Dowex resin column (Na + form) to afford after freeze-drying the corresponding compound as sodium salt.

(R)- phosphonic acid (12(R))

Compound 12(R) (520 mg) was obtained in 82% yield, according to procedure F, from 40(R) 20 -8.8 ° (c 0.1, H2O). 1 H NMR (D2O) δ 7.82 (s, 1H, H8), 4.21 (t,J = 7.4 Hz,2H,CH2N),1H,CHNH2), 2. 2H,CH2CH2N),1H,CH2P),1H,CH2P). 13 C-NMR (D2O) δ 158.9 (C-6), 153.8 (C-2), 151.5 46.6 (CHNH2),58 39.6 (CH2N),34.7,34.6 (CH2CH2N),31.5,30.7 (CH2P). 31 (S)- ) phosphonic acid (12(S))

Compound 12(S) (162 mg) was obtained in 64% yield, according to procedure F, from 40(S) (270 mg). Rf = 0.15 in iPrOH/H2O/ NH4OHc (7/2/1, v/v/v). Purity (HPLC) > 98%. [α]D 20 + 9.7 ° (c 0.1, H2O). 1 H NMR (D2O) δ 7.77 (s, 1H, H8), 4. 22-4.09 (m, 2H, CH2N), 3.49-3.36 (m, 1H, CHNH2), 2. 2H,CH2CH2N),1H,CH2P), 1. 1H,CH2P). 13 C-NMR (D2O) δ 159.0 (C-6), 153.9 (C-2), 151.6 (C-4), 139.8 46.6 (CHNH2),39.7 (CH2N),34.7,34.6 (CH2CH2N),31.5,30.7 (CH2P). 31 (R)- )-2-aminobutyl) phosphonic acid (13(R))

Compound 13(R) (471 mg) was obtained in 73% yield, according to procedure F, from 41(R) (663 mg). Rf = 0.15 in iPrOH/H2O/NH4OHc (7/2/1, v/v/v). Purity (HPLC) > 97%.

[α]D 20 -8.0 ° (c 0.1, H2O). 1 H-NMR (D2O) δ 7.87 (s, 1H, H8), 4. 2H,CH2N),1H,CHNH2), 2.17-2.04 (m, 2H, CH2CH2N), 1. 1H,CH2P), 1H,CH2P). 13 C-NMR (D2O) δ 156.1 (C-2), 151.0 , 113.0 45.3 (CHNH2),40.8 (CH2N),38.1,38.0 (CH2CH2N),37.0,36.1 (CH2P). 31 (S)- ) phosphonic acid (13(S))

Compound 13(S) (435 mg) was obtained in 71% yield, according to procedure F, from 41(S) (630 mg). Rf = 0.15 in iPrOH/H2O/ NH4OHc (7/2/1, v/v/v). Purity (HPLC) > 96%. [α]D 20 + 8.9 ° (c 0.1, H2O). 1 H-NMR (D2O) δ 7.89 (s, 1H, H8), 4. 2H,CH2N),1H,CHNH2),1H,CH2CH2N),1H,CH2CH2N),1H,CH2P), 1.48-1.41 59 [START_REF]MS (ESI) m/z 303.12 ]+; HRMS: calc. C9H16N6O4P[END_REF]1H,CH2P). 13 C-NMR (D2O) δ 156.1 (C-2), 151.0 (C-4), 140.1 (C-8), 117.3 (C-6), 113.0 45.3 (CHNH2),40.8 (CH2N),38.1,38.0 (CH2CH2N),37.0,36.1 (CH2P). 31 P-NMR (D2O) δ 17.2. HRMS (TOF ESI>0) Found: 346.0769, Calculated for C9H15N7O3PNa2: 346.0769 (M+H) + . guanine (11a) 13,21,30 Compound 11a was obtained in 58% yield according to literature. Rf = 0.18 (iPrOH/H2O/NH4OH 7/2/1). Purity (HPLC) > 98%. 1 H NMR (D2O) : δ = 1.3 (t, 9H), 1. 55-1.66 (m, 4H), 1.84-1.95 (m, 2H), 3.20 (m, 6H), 4.07 (m, 2H), 7.84 (s, 1H) ; 13 C NMR (D2O) : δ = 20.22, 20.25, 26.7, 27.7 (CH2), 30.2, 30.3, (CH2P), 43.2, 46.6 (CH2N), 115.8 (C-5), 140.1 (C-8), 151.4 (C-4), 153.6 (C-2), 158.8(C-6). 31 (Z)- (4-(2-amino-6-hydroxy-9H-purin-9-yl)-but- 1-en-1-yl)phosphonic acid disodic salt (11b)

The diethyl-(Z)- (4-(2-amino-6-methoxy-9H-purin-9-yl)-but-1-en-1-yl)phosphonate (261 mg, 1 eq.) was dissolved under argon atmosphere in anhydrous DMF (20 mL/mmol) at 0°C. TMSBr (6.6 eq.) was added dropwise to the solution, and the mixture was kept at 0°C for 5 min, then allowed to slowly warm to room temperature and stirred for 3 days. The reaction progress was followed by TLC monitoring (isopropanol/water/ammoniac 7/2/1 v/v/v). The reaction was quenched by addition of a triethylbutylammonium solution (1M, pH 7). The volatiles were removed under vacuum and the resulting aqueous solution was freeze-dried. The crude was purified on reverse phase flash chromatography (water/methanol gradient, 0 to 100% methanol), leading to the phosphonate triethylammonium salts as a white powder. The compound was percolated through a Na + Dowex resin and after freeze-dried of the require fractions the sodium salts were obtained as obtained was deduced. Results were expressed as IC50, which is the drug concentration leading to 50% inhibition of parasite growth. A nonlinear regression model (sigmoidal doseresponse/variable slope; GraphPad Prism6) was used to calculate the IC50s.

In vitro toxicity against erythroblast K562 cell line. K-562 cells were cultured in complete medium (RPMI 1640 complemented with 10% fetal calf serum in 96-well microplates (8000 cells per well) and incubated 24 h at 37°C and under 5% CO2 in presence of various concentrations of tested analogues as above. After 24 h incubation at 37 °C, 30 μL of complete medium containing 0.5 μCi [ 3 H]-thymidine were added to each well for an additional 6h-period. The reactions were stopped by freezing at -80°C. Cell macromolecules including nucleic acids were recovered as above. Radioactivity background was obtained from the incubation of culture medium alone. Animaux (University of Montpellier, France) for providing access to their facilities for animal studies. We wish to thank the technical excellence of Mathilde Gens and Natacha Venturini.

Effect of compounds

ABBREVIATIONS

Table of Contents graphic


Dionissia A Pepe

Dimitra Toumpa

Christiane André-Barrès

Christophe Me- Nendez

Elisabeth Mouray

Michel Baltas

Philippe Grellier

Dionissios Papaioannou

Constantinos M Athanassopoulos

Synthesis of novel G factor-or chloroquine-artemisinin hybrids and conjugates with polyamines with potent antiplasmodial activity

Keywords: artemisinin, phytormones G-factors, chloroquine, hybrids, conjugates, polyamines, antimalarials

A series of novel hybrids of artemisinin (ART) with either a phytormone endoperoxide G factor analogue (GMeP) or chloroquine (CQ) and conjugates of the same compounds with the polyamines (PAs) spermidine (Spd) and homospermidine (Hsd) were synthesized and their antimalarial activity were evaluated using the CQ-resistant P. falciparum FcB1/Colombia strain. The ART-GMeP hybrid 5 and compounds 9 and 10 which are conjugates of Spd and Hsd with two molecules of ART, and one molecule of GMeP, were the most potent with IC 50 values of 2.6, 8.4 and 10.6 nM, respectively.

The same compounds also presented the highest selectivity indexes against the primary human fibroblast cell line AB943 ranging from 16,372 for the hybrid 5 to 983 for the conjugate 10 of Hsd.

antimalarials (e.g. mefloquine or piperaquine), that have been developed thereafter. [START_REF] Çapci | Artemisinin-(Iso)quinoline Hybrids by C-H Activation and Click Chemistry: Combating Multidrug-Resistant Malaria[END_REF][START_REF] Rudrapal | Endoperoxide antimalarials: development, structural diversity and pharmacodynamic aspects with reference to 1,2,4-trioxane-based structural scaffold[END_REF] (Figure 1). Unfortunately, P. falciparum has already developed resistance for CQ and quinoline-based antimalarials. The second family is based on the natural product Artemisin (ART), that was first isolated from the Chinese medicinal plant Artemisia annua L. in 1972 by Tu Youyou. [START_REF] Tu | Artemisinin-A Gift from Traditional Chinese Medicine to the World (Nobel Lecture[END_REF] ART is a sesquiterpene lactone which incorporates an endoperoxide functionality demonstrated to be important for its antimalarial activity. [START_REF] Li | Biological Actions of Artemisinins: Insights from Medicinal Chemistry Studies[END_REF] Because of its low bioavailability and an unfavorable pharmacokinetic profile in vivo, intensive research efforts have been since concentrated to the development of improved hemisynthetic and fully synthetic ART analogs and derivatives. These efforts have been culminated to the development of the C-10 oxa-substituted ART-based drugs, such as the dihydroartemisinin (DHA), the lipophilic artemether, the hydrophilic sodium artesunate (Figure 1) but more importantly to the corresponding C-10carba-substituted ART with improved ADMET properties. In that respect we can mention as attractive intermediate the acid (1) 8,9 or ART hybrids (see below), such as the anilide 2. 9 In addition, C-10-carba-substituted ART dimers or trimers, molecules with two or even three ART moieties connected through symmetric or asymmetric linkers have shown more potent biological (antimalarial or anticancer) activity, which depended from the nature of the linker. [START_REF] Posner | Antimalarial, Antiproliferative, and Antitumor Activities of Artemisinin-Derived, Chemically Robust, Trioxan Dimers[END_REF][START_REF] Lombard | Antimalarial and anticancer activities of artemisinin-quinoline hybrid-dimers and pharmacokinetic properties in mice[END_REF] Unfortunately, P. falciparum resistance to artemisinin derivatives in the South East Asia and particularly in Cambodia has been observed. N12 In a seminal work, 5 molecular marker of artemisinin resistance that is the kelch propeller protein (K13-propeller) has been identified recently and should be important in the continuing efforts to restrain artemisinin resistance. [START_REF] Sraimer | K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates[END_REF][START_REF] Ouji | Malaria-Infected Mice Live until at Least Day 30 after New Monomeric Trioxane Combined with Mefloquine Are Administered Together in a Single Low Oral Dose[END_REF] Inspired by artemisinin, many groups developed methods for constructing trioxanes, entities known to exhibit antimalarial activity in a similar manner to artemisinin. Synthetic molecules of this type with strong activities have been reported by the groups of Posner, Kepler, Singh, Griesbeck. N13 Of high importance are also the efforts of Meunier's group towards the development of trioxaquines that are molecules based on the «covalent bitherapy» and are obtained by covalent attachement of a trioxane entity to an aminoquinoline entity. N14 Since many years now, we have been interested in the naturally occurring phytormones known as G-factors (G1, G2, G3) synthesized from syncarpic acid and incorporating an endocyclic endoperoxide frame. [START_REF] Benbakkar | Synthesis of Syncarpic Acid and Related β-Oxo δ-Enol Lactone via Selective O-or C-Acylation of Preformed Enolates[END_REF] The natural compounds and many synthetic analogs have been synthesized. [START_REF] Najjar | Crucial role of peroxyketal function for antimalarial activity in the G-factors series[END_REF][START_REF] Ruiz | Antimalarial bicyclic peroxides belonging to the G-factor family: mechanistic aspects of their formation and iron (II) induced reduction[END_REF] Among them, the methylated compound G3Me (Figure 1), and its derivatives have been shown to be potent antimalarial agents.

In particular, the G3Me-derived amino-endoperoxide spiro compound (endoperoxide GMeP, 3), suitable for the synthesis of G factor hybrids through a succinic acid type linker (compound 4) has been developed and two compounds incorporating also CQ proved to be very active. 17 Due to the emerging resistance of P .falciparum to artemisinins, WHO urgently recommends the use of Artemisinin-based Combination Therapies (ACTs), which involve the combination of a potent but short-lived artemisinin derivative, e.g. artemether or sodium artesunate, with one or even two longer-lasting partner drugs with a different mechanism of action, e.g. a quinoline-based drug. Such combinations are for example ''artesunate-MQ'' or ''artesunate-MQ-PQ''. Recently, the combination of two antimalarial drugs in one single molecular entity (coined as hybrid or conjugate) has been described as being more efficient against diseases, such as malaria and cancer. Hybrid molecules have probably less chance to develop drug resistance. [START_REF] Çapci | Artemisinin-(Iso)quinoline Hybrids by C-H Activation and Click Chemistry: Combating Multidrug-Resistant Malaria[END_REF] Examples of ARTCQ (S1-S2) [START_REF] Çapci | Artemisinin-(Iso)quinoline Hybrids by C-H Activation and Click Chemistry: Combating Multidrug-Resistant Malaria[END_REF][START_REF] Lombard | Antimalarial and anticancer activities of artemisinin-quinoline hybrid-dimers and pharmacokinetic properties in mice[END_REF] or a G-factorCQ (S3) hybrids, [START_REF] Lombard | Antimalarial and anticancer activities of artemisinin-quinoline hybrid-dimers and pharmacokinetic properties in mice[END_REF] and of conjugates (S4-S7) of ART with the naturally occurring polyamines (PAs), e.g. spermidine (Spd) or spermine (Spm), [START_REF] Pearce | Synthesis and antimalarial evaluation of artesunate-polyamine and trioxolane-polyamine conjugates[END_REF][START_REF] Magoulas | Synthesis of novel artemisinin dimers with polyamine linkers and evaluation of their potential as anticancer agents[END_REF][START_REF] Chadwick | synthesis and antimalarial/anticancer evaluation of spermidine linked artemisin conjugates designed to exploit polyamine transporters in Plasmodium falciparum and HL-60 cancer cell lines[END_REF] of relevance to the present work are provided in Figure S1 (Supporting Information). It should be noted that naturally occurring and synthetic PAs, their analogs and conjugates with other biologically active molecules are compounds with a variety of interesting biological properties. [START_REF] Karigiannis | Structure, Biological Activity and Synthesis of Polyamine Analogues and Conjugates[END_REF][START_REF] Hahn | Solid phase chemistry for the directed synthesis of biologically active polyamine analogs, derivatives, and conjugates[END_REF] Conjugation of organic or bioorganic molecules with PAs has been used as a mean to improve or combine the biological properties of the constituent molecules. PAs can form the basis for the development of novel pharmaceuticals. [START_REF] Drug Discovery; Woster | [END_REF][START_REF] Melchiorre | Polyamines in drug discovery: From the universal template approach to the multitargeted ligand design strategy[END_REF] Along this line of research, we now wish to report our first results on the synthesis of two types of artemisinin hybrids/conjugates with the endoperoxide GMeP (3) or the 4-amino-7-chloroquinoline (ACQ) core of CQ, and the biological evaluation of their antimalarial potential. Hybrids of the first type are the compounds 5, 6 and 7 (Figure 2) in which the two pharmacophores are joined with an amide bond using suitable linkers. For the sake of comparison, the hybrid 8 [START_REF] Benbakkar | Synthesis of Syncarpic Acid and Related β-Oxo δ-Enol Lactone via Selective O-or C-Acylation of Preformed Enolates[END_REF] of endoperoxide-GMeP (3) with ACQ was also synthesized. Conjugates of the second type (Figure 2 With these new hybrids/conjugates, we wished to determine the various factors affecting their potential antimalarial activity, such as (a) the influence of the other antimalarial agent linked with ART (compare for example conjugates 5 and 6 or 9 and 11), (b) the structure (conformationally restricted or not) or the length (Spd against Hsd chain) of the linker (compare for example conjugates 6 and 7 or 9 and 10, respectively) and (c) the number (one against two) of ART or endoperoxide-GMeP residues in the conjugate (compare for example conjugates 5 and 9 or 8 and 13, respectively).

The synthesis of ART conjugates 5, 6 and 7 requires as key-intermediate the ART-derived carboxylic acid 1. This compound was prepared (Scheme S1, Supporting Information) in 58% overall yield from ART, according to a five-steps published procedure. 8,9 On the other hand, endoperoxide GMeP (3) was synthesized from syncarpic acid in a sequence of three in situ reactions (Mannich, dienol formation, oxygen uptake) followed by tert-butyloxycarbonyl (Boc) deprotection in an overall yield of 50%, according to our published procedure (Supporting Information). [START_REF] Ruiz | Antimalarial bicyclic peroxides belonging to the G-factor family: mechanistic aspects of their formation and iron (II) induced reduction[END_REF] The required syncarpic acid was synthesized (Supporting Information), for the needs of the present work, essentially through a reported procedure starting from 2',4',6'trihydroxyacetophenone. 25 An alternative synthesis of this key-intermediate has been described. [START_REF] Benbakkar | Synthesis of Syncarpic Acid and Related β-Oxo δ-Enol Lactone via Selective O-or C-Acylation of Preformed Enolates[END_REF] Finally for the needs of the present work, the N 1 -(7-chloroquinolin-4-yl)ethane-1,2-diamine (15) and the 7-chloro-4-(piperazin-1-yl)quinoline (16) were readily obtained in 56% and 76% yield, respectively, from the commercially available 4,7dichloroquinoline through the nucleophilic aromatic substitution of the latter with 1,2diaminoethane and piperazine, respectively [START_REF] Ruiz | Antimalarial bicyclic peroxides belonging to the G-factor family: mechanistic aspects of their formation and iron (II) induced reduction[END_REF] (Scheme S1, Supporting Information).

Condensation of acid 1 with amines 3, 15 and 16, in the presence of the coupling agent 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-phosphate (HBTU) and diisopropylethylamine (DIPEA), produced unexceptionally the anticipated conjugates 5, 6 and 7 in 95%, 80% and 72% yields, respectively (Scheme 1). On the other hand, the synthesis of the conjugate 8 involved the initial reaction of amine 15 with succinic anhydride to give the corresponding carboxylic acid 17 in 100% yield. This acid was then coupled with endoperoxide GMeP (3), also in the presence of HBTU and DIPEA, to give conjugate in 72% yield.

The synthesis of ART-Spd and Art-Hsd mixed conjugates 9, 10, 11 and 12 with endoperoxide GMeP (3) or ACQ, as well as the endoperoxide GMeP-Spd and endoperoxide GMeP-Hsd mixed conjugate 13 and 14 with ACQ, required the availability of suitably protected triamines, such as the N 1 ,N 8 -ditritylspermidine (18) [START_REF] Magoulas | Synthetic studies toward the development of novel minoxidil analogs and conjugates with polyamines[END_REF] and N 1 ,N 9 -ditritylhomospermidine (19). These compounds are readily available, in 48% overall yield, from butane-1,4-diamine (putrescine, Put) through a three-steps sequence depicted in Scheme S1 (Supporting Information).

Reaction of PA derivatives 18 and 19 with succinic anhydride gave the anticipated monocarboxylic acid derivatives 20 and 21, respectively, in quantitative yield.

These compounds were then coupled with endoperoxide GMeP (3) in the presence of HBTU and DIPEA to give the protected PA conjugates 22 and 23 in 40% and 67% yield, respectively. From these intermediates the final products 9 and 10 were obtained in 64% and 40% yield, respectively, upon N-deprotection followed by coupling with ART-derived acid 1, in the presence of HBTU and DIPEA (Scheme 1).

On the other hand, coupling of partially protected PAs 18 and 19 with the ACQderived acid 17, in the presence of HBTU and DIPEA, produced PA conjugates 24 and 25 in 86% yield. From these conjugates, the final products 11 and 12 were unexceptionally obtained in 61% and 32% yields upon detritylation and coupling with ARTderived acid 1. Similarly, the conjugates 13 and 14 were readily obtained (Scheme 1) from the same polyamine conjugates 24 and 25, in 40% and 34% yield, respectively, upon deprotection of the latter followed by coupling with endoperoxide GMeP-derived acid 4. The latter was readily obtained in 98% from the reaction of endoperoxide GMeP and succinic anhydride (Scheme S1). The experimental details for the synthesis of (a) all above described fragments, which are necessary for the assembly of the proposed hybrids/conjugates, and (b) the final products, that is hybrids/conjugates 5-14 are provided in the Supporting Information section. The synthesized conjugates were evaluated for their antiplasmodial activity against the CQ-resistant P.falciparum FcB1/Colombia strain, according to published procedure. [START_REF] Grayfer | Total Synthesis, Antiparasitic Evaluation, and Preliminary SAR Studies[END_REF][START_REF] Bosc | Highly improved antiparasitic activity after introduction of an N-benzylimidazole moiety on protein farnesyltransferase inhibitors[END_REF] The cytotoxicity of the compounds was evaluated using the primary human fibroblast cell line AB943, also according to published procedure, [START_REF] Kryshchyshyn | Thiazolidinone/thiazole based hybrids -new class of antitrypanosomal agents[END_REF] which allowed the calculation of their selectivity index (SI). In Table 1 (see also (IC 50 >167), incorporating two molecules of endoperoxide GMeP per molecule of conjugate, showed that the former was slightly more active than the latter conjugates.

In conclusion, a series of novel hybrids of ART with either endoperoxide GMeP or the ACQ core of CQ and conjugates of the same compounds with the PAs Spd (an asymmetric PA) and Hsd (a symmetric, with longer chain, PA), bearing either two molecules of ART or of endoperoxide GMeP, were synthesized and their antiplasmodial activities and selectivity indexes (SI) were evaluated using the CQresistant P.falciparum FcB1/Colombia strain and the primary human fibroblasts cell line AB943 (for SI calculations), respectively. These studies showed that the most potent combination with the highest SI value was that between ART and the endoperoxide GMeP, in particular when the two molecules participate in the construct (hybrid/conjugate) in the ratio 1:1, that is without the intervention of a PA chain.

Combinations between endoperoxide GMeP and ACQ seem not to be particularly ef-fective, whereas the length of the PA chain or the geometry (symmetric or asymmetric) of the construct, defined by the PA used, does not seem to play any significant role. It seems that combinations of two endoperoxide-type compounds can form the basis for the development of more potent and safer antimalarial and therefore we are currently engaged in this line of research. Moreover, it should be important to study in a further work, if the three hybrid compounds possessing two different trioxane frames and different linkers between them can affect the spread of ART resistance and mitigate its impact on malaria treatment.

Supporting Information. Synthetic and Bioassay protocols, as well as, copies of 1 H and [START_REF] Kepler | 1,2,4-Trioxanes as potential antimalarial agents[END_REF] C NMR spectra for all the synthesized compounds.

Figure 1 .

 1 Figure 1. Structures of antimalarial agents encountered in the present work.

  ) are conjugates bearing two molecules of ART per molecule of the PAs Spd and Hsd and, in addition, one molecule of endoperoxide-GMeP (compounds 9 and 10) or ACQ (compounds 11 and 12). For the sake of comparison, conjugates bearing two molecules of endoperoxide GMeP (3) per molecule of the PAs Spd and Hsd and, in addition, one molecule of ACQ (conjugates 13 and 14) were also synthesized.

Figure 2 .

 2 Figure 2. Structures of synthesized ARTendoperoxide GMeP (5), ARTACQ (6 and 7) and ACQendoperoxide GMeP (8) hybrids, and ARTSpd and Hsd mixed conjugates with endoperoxide GMeP and ACQ (9, 10 and 11, 12, respectively) and endoperoxide GMePSpd and Hsd mixed conjugates with ACQ (13 and 14).

Scheme 1 .

 1 Scheme 1. Synthesis of ARTendoperoxide GMeP (5), ARTACQ (6 and 7) and ACQendoperoxide GMeP (8) hybrids, as well as ARTSpd and Hsd mixed conjugates with endoperoxide GMeP or ACQ (9-12) and endoperoxide GMePSpd and Hsd mixed conjugates with ACQ (13 and 14). Reagents and reaction conditions: (i)

Table S1 ,

 S1 Supporting Information), the measured IC 50 values or the % inhibition at 10 nΜ, in cases IC 50 values could not be determined, and SIs for the synthesized hybrids/conjugates are tabulated. IC 50 values for ART, CQ and endoperoxide GMeP(3) are also provided for the sake of comparison.

Table 1 .

 1 Evaluation of antiplasmodial activities of synthesized compounds with CQresistant P. Falciparum FcB1/Colombia strain and selectivity indexes (SI). In particular the hybrid 5 of ART with endoperoxide GMeP is the most potent in this series of compounds with an IC 50 value 21 times more potent than that of ART. Interestingly, this compound appears to be the safest compound for use as antimalarial (SI = 16,372) as its SI is significantly higher than that of ART and ca. 8-17 times higher than the other two best synthesized conjugates (9 and 10). A comparison of hybrid 5 (IC 50 =2.6, SI=16,372), bearing one molecule of ART, with the conjugates 9 (IC 50 =8.4, SI=2,108) and 10 (IC 50 =10.6, SI=983), bearing two molecules of ART held together through a PA chain, showed that a second ART molecule does not offer any particular advantage but also leads to significant lowering of the SI value. On the other hand, comparison of the two conjugates 9 and 10, differing only in the length of the PA chain, shows that the two compounds have comparable IC 50 but the one with the Hsd longer chain is ca. 2.1 times less safe than the one with the Spd shorter chain. On the other hand, the hybrids 6 and 7 of ART with the CQ core showed identically low inhibition values (11% inhibition. at 10 nM), indicating that (a) the CQ core does not offer any advantage to ART and (b) the structure of the diamine linker seems not to be important for the antiplasmodial activity.Comparison of the couples of conjugates 9 (IC 50 =8.4, SI=2,108) and 11 (33% inh.

	Compd	IC 50 (nΜ) or	SI	Compd	IC 50 (nΜ) or	SI
		% inhibition			% inhibition
		at 10 nM			at 10 nM
	5	2.61.0	16,372	12	52.315.9	328
	6	11%		13	>167	
	7	11%		14	>167	
	8	124.340.4	144	ART	5513.6	>4,545
	9	8.43.7	2,108	CQ	727.4	347
	10	10.62.3	983	3	5.7 μM 14	
	11	33%	
	From Table 1 it is apparent that three out of seven of the synthesized hy-
	brids/conjugates of ART, incorporating one or two molecules of ART per hy-
	brid/conjugate, are significantly more potent (IC 50 values ranging from 2.6 to 10.6
	nM) than the parent compound, one conjugate (12) was almost equipotent to ART but
	with at least 13 times lower SI value and the rest showed low inhibition values (11-
	33%) at 10 nM.

at 10 nM) and 10 (IC 50 =10.6, SI=983) and 12 (IC 50 =52.3, SI=460), bearing the Spd and the Hsd PA core linkers respectively, two ART molecules and either a molecule of the endoperoxide GMeP or the CQ core, showed that the endoperoxide GMeP substructure is more important for biological activity that the CQ core and provides safer conjugates. Finally, the hybrid 8 of endoperoxide GMeP with the CQ core was almost 2.5 times less active than ART but had almost 10 times higher SI value. Also, comparison of conjugate 8 (IC 50 =124.3, SI=144) with the conjugates 13 (IC 50 >167) and 14

  resistance to artemisinin-based combination therapies: A sword of Damocles in the Ma, J.; Katz, E.; Kyle, D. E.; Ziffer, H. Syntheses and Antimalarial Activities of 10-Substituted Deoxoartemisinins. J. Med. Chem. 2000, 43, 4228─4232.

	path	toward	malaria	elimination.	Parasite	2018,	25,	24;
	doi.org/10.1051/parasite/2018021.
	8.

Acknowledgments: We thank the CNRS, the University Paul Sabatier and the University of Patras for financial support. We thank the Erasmus+ Program for financing D. Pepe's traineeship and the University Paul Sabatier for financing the visiting professorship of Assoc. Prof. C.M. Athanassopoulos, 2017. The authors would like to acknowledge networking contribution by the COST Action CM1407 "Challenging organic syntheses inspired by nature -from natural products chemistry to drug discovery". The NMR Facility (ICT, Toulouse, France, http://ict.ups-tlse.fr/?RMN-103) and the Instrumental Analysis Lab (School of Natural Sciences, University of Patras) are gratefully acknowledged for carrying out the NMR analyses.

AUTHOR INFORMATION

Conflicts of Interest:

The authors declare no conflict of interest.