Dr Pierre Thuéry
email: pierre.thuery@cea.fr

Dr Jack Harrowfield
email: harrowfield@unistra.fr

Chain, Network and Framework Formation in Uranyl Ion Complexes with 1,1 -Biphenyl-3,3 ,4,4 -tetracarboxylate

Keywords: Carboxylic acids, Metal-organic frameworks, Structure elucidation, Tetracarboxylate ligands, Uranyl cation

Chain, network and framework formation in uranyl

ion complexes with 1,1-Biphenyl-3,3,4,4-tetracarboxylate Pierre Thuéry, Jack Harrowfield

Introduction

When using polycarboxylates for synthesizing uranyl-containing networks or polymers, [1][2][3][4][5] one means of obtaining large rings, cavities or channels relies on ligands with rigid and elongated carbon skeletons, among which the biphenyl motif is most obvious. The simplest biphenyl-based, divergent ligand is 1,1′-biphenyl-4,4′-dicarboxylate, which has given several original diperiodic networks, [6][7][8][9] some of which display polycatenation, [8] or guestdependent single-crystal-to-single-crystal phase transitions. [9] A larger, curved dicarboxylate ligand including three aromatic rings, 1,1′,3′,1″-terphenyl-4,4″-dicarboxylate, has been shown to give ribbon-like chains, [10] and a linear dicarboxylate ligand including four aromatic rings (two phenyl and two pyridinium) has also been investigated, [11] as well as a species involving three biphenylcarboxylate units. [12] The biphenyldicarboxylate motif itself can be modulated through modification of the positions of the carboxylate groups, and/or through addition of other groups, coordinating or not. In the first category, 1,1′-biphenyl-2,4′-dicarboxylate, which is a distinctly curved and not a divergently linear ligand, yields diperiodic networks effective in photocatalytic degradation of rhodamine B, [13] and 1,1′-biphenyl-2,2′-dicarboxylate gives mainly monoperiodic polymers with different chelating modes of the two carboxylate groups. [14] In the second category, the ligand with one additional methyl group on each ring has been used to synthesize a mixed uranyl-thorium(IV) framework, [15] while 1,1'-biphenyl-3,3′disulfonyl-4,4′-dicarboxylate gives mixed uranyl-potassium frameworks. [16] Several biphenylbased ligands with four carboxylate groups, and thus a potentially increased assembling power, are known. 1,1'-Biphenyl-4-fluoro-3,3′,5,5′-tetracarboxylate only forms a diperiodic network with uranyl ions, [17] whereas 1,1'-biphenyl-2,2′,6,6′-tetracarboxylate gives mono-, di-and triperiodic assemblies depending on the choice of additional metal cations present. [18] We have previously used the anions derived from the less symmetrical 1,1'-biphenyl-3,3′,4,4′-tetracarboxylic acid (H4bptc) to synthesize two homometallic uranyl ion complexes, the monoperiodic [UO2(H2bptc)(H2O)2]2H2O, [19] and [(UO2)2(bptc)(NMP)1.5(H2O)1.5]1.5H2O (NMP = N-methyl-2-pyrrolidone), which crystallizes as a triperiodic framework, [20] and also two heterometallic complexes involving either Ag I or Pb II as additional cations, [UO2Ag(bptc)(4,4′-bipyH)] and [UO2Pb(bptc)(2,2′-bipy)2] (bipy = bipyridine), which are both diperiodic networks with the additional cations having a simple decorating role. [20] Notwithstanding the ability of bptc 4-to form a framework with uranyl as sole cation, which is unusual in this family of biphenyl-based ligands, few complexes have been synthesized and crystallographically characterized, possibly as a result of the difficulty in growing single crystals of sufficient quality. This ligand is nonetheless of some particular interest as a phthalate homologue and thus as a species favouring 7-membered chelate ring formation on uranyl ion, a relatively uncommon feature in uranyl carboxylate coordination chemistry. We have now obtained three novel anionic uranyl complexes with bptc 4-which illustrate all the range of periodicity attainable upon variation of the counterions, these being [Co(en)3] 3+ (en = ethylenediamine) and H2NMe2 + , which are both hydrogen bond donors, and PPh4 + , which influences the structure through its bulkiness and ability to be involved in aromaticaromatic interactions.

Results and Discussion

The complex [Co(en)3][UO2(bptc)(HCOO)]2.5H2O (1) involves a counterion known to be an efficient hydrogen bond donor [21] and which has proven to be an efficient structure-directing species in uranyl complexes. [22][23][24][25] As noted above, the bptc 4-ligand is effectively a bis(phthalate) species and thus would be expected to form a 7-membered chelate ring at each phthalate-like site. This, in fact, is what is observed but, unlike most other known phthalate complexes, there are no other interactions of the oxygen donors with the metal cation. The unique uranyl cation is chelated by two ligands and bound to one more oxygen donor from the formate moiety, the latter formed in situ from N,N-dimethylformamide and one in formate in coordination severely limits the periodicity of the coordination polymer, which is a zigzag chain parallel to [010] having a marked S-shape when viewed endon. This undulating form defines gaps which, somewhat like the cavities found in a Kemp's tricarboxylate complex where the same countercation is present, [25] are approached from both sides by cations forming NHO bonds. The cation is present in its lel2ob conformation, [26] again as found in the Kemp's tricarboxylate complex, and all 12 amino group protons are involved in 15 NHO bonding interactions, some of them bifurcated, with two pairs of protons being bridged by water molecules and the others interacting with both bptc 4-and formate oxygen atoms, either coordinated or not [NO distances, 2.846(3)-3.369(3) Å; N-HO angles, 112-169°]. In particular, two protons of type C3 (i.e. such that the C-H bond is nearly parallel to the pseudo-C3 axis of the anion) form three hydrogen bonds with the formate ligand, possibly explaining the rather unusual monodentate nature of the latter (less than one third of all uranyl formate species reported in the Cambridge Structural Database (CSD, version 5.42 [27] ) are neither chelating, nor bridging). The water molecules form hydrogen bonds between one another and with carboxylate groups, thus giving an intricate hydrogen bonding pattern. The packing does not contain significant free spaces and it has a Kitaigorodski packing index (KPI, calculated with PLATON [28] ) of 0.74. 2). The uranium atom is polymer formed is triperiodic and it has the point symbol {4.12 2 }2{4 2 .12 4 }{4 2 .6.12 3 }2{4 2 .6}2.

While

Two views of the nodal representation of the framework, drawn with ToposPro, [29] are shown in Figure 4. The framework contains near-orthogonal strands, or monoperiodic The two types of monoperiodic subunits run in alternate horizontal planes.

subunits, in which the ligand-uranium interactions are quite different. In strands running parallel to [100], dinuclear units containing U1 are linked by ligands forming 7-membered chelate rings and bridging so that the dinuclear units are 8-membered dimetallacycles. In contrast, the strands running along [010] and containing U2 consist of alternating 14-and 22-membered metallacyclic units which are clearly visible in Figure 3b (much larger rings can be identified in the complete framework). These two strands are connected to one another by the  2 O,O -chelating carboxylate group containing O5 and O6 and bound to U1, each strand being connected to its neighbours on both sides along the [001] axis, thus generating the triperiodic assembly. If only the arrangement and linking of strands is considered, it can be viewed as akin to the cds rod packing. [30] Large channels based on a section of 13  18 Å 2 (the available space being reduced by protruding oxygen atoms) run along [010], each of them containing two rows of PPh4 + counterions (Figure 5). In each of these rows, the counterions are associated through "phenyl embrace" interactions, [31] with an alternation of short and long P1P2 distances, 6.656(2) and 8.380(2) Å corresponding to interactions involving three or two aromatic rings from each cation, respectively. Short contacts analysis with PLATON [28] indicates the possible presence of two parallel-displaced -stacking interactions between bptc 4-and PPh4 + [centroidcentroid distances, 3.901(5) and 4.083(4) Å; dihedral angles, 10.9(4) and 17.2(4)°; slippage, 1.38 and 1.48 Å]. Examination of the Hirshfeld surface (HS) [32] calculated with CrystalExplorer (ver. 3.1) [START_REF] Wolff | CrystalExplorer[END_REF] indicates that, as usual, the PPh4 + cations are involved in several CHO hydrogen bonds, [34] while water solvent molecules occupy the remaining spaces (KPI, 0.68).

Whereas 4-membered chelate ring formation through  2 O,O'-chelation is expected to be a favoured mode for carboxylate binding to a large metal cation [35] like uranyl ion, in diand higher polycarboxylates, formation of larger chelate rings through binding to one oxygen donor in each of two carboxylate groups is possible. 5-Membered chelate rings are found with oxalate, [36] as well as with various 2-hydroxycarboxylates, [37] and 6-membered rings are found with malonate. [38] There are 90 cases of 7-membered rings reported in the CSD, which involve, for example, citrate, [37] phthalate, [39] 4,4′-(1,1,1,3,3,3hexafluoroisopropylidene)diphthalate [40] and 1,2-cyclohexanedicarboxylates. [41] Larger, 8membered rings are known with citrate [42] and Kemp's tricarboxylate, [25,43] and 9-membered rings are found with 2,2'-bipyridine-3,3'-dicarboxylate, [44] 1,1′-biphenyl-2,2′-dicarboxylate, [14] and 1,1′-biphenyl-2,2′,6,6′-tetracarboxylate. 7-Membered ring formation is present in all complexes 1-3, and it is the only coordination mode found in 1, in which two such rings are found in the equatorial plane of the uranyl ion, but this is at variance with previously reported uranyl ion complexes with this ligand, in which the only 7-membered ring involves Ag I and not U VI . The coordination modes observed in all known uranyl ion complexes with this ligand are shown in Scheme 1. The situation in which one 4-membered and one 7membered rings are found around the same uranyl ion is found in 2 and 3, but there is no example here of two 4-membered rings associated with a 7-membered one, although one such case is known in which the two 4-membered rings are formed by nitrate anions [45] (no example is known for larger rings, while such coexistence is possible, although not frequent, Scheme 1. Coordination modes of bptc 4-or H2bptc 2-in uranyl ion complexes (solvent molecules are omitted from formulas).

with a 6-membered ring [38a,46] ). In complex 3, one ligand is exclusively involved in 4membered ring formation, while the other is involved in 7-membered ring formation (with extra bridging) indicating that the difference between these two modes must be small.

Apart from the coordination mode of the ligand, rotation of the two aromatic rings around the central bond, related to the proximal or distal geometry of the two carboxylate groups in the 3-position, and of the carboxylate groups with respect to the aromatic rings introduces another source of structure variations. Table 1 gives the values of the dihedral angles 1 and 2 formed by the carboxylate groups in the 4-position with respect to the attached aromatic ring, 3 and 4 formed by the carboxylate groups in the 3-position with respect to the attached aromatic ring, 1 and 2 between the two adjacent carboxylate groups on each ring, and  between the two aromatic rings; the relative location of the two carboxylate groups in the 3-position, proximal or distal, is also indicated. As could be Table 1. Dihedral angles (°) in bptc 4-/H2bptc expected, the smaller rotations between the two aromatic rings are found in species with the distal geometry, and the two rings are coplanar in centrosymmetric, distal ligands, whereas the largest rotation is found in the proximal heterometallic complex with Pb II additional cations. The rotation angles of the carboxylate groups with respect to the aromatic rings vary widely, with no clearcut relation to the coordination mode, and the two adjacent carboxylate groups on each ring make dihedral angles greater than 48°, and most often in the 70-89° range. A similar situation has been found in a series of uranyl phthalate complexes, [47] and it suggests that easy rotation of the coordinating groups endows these ligands with sufficient flexibility to be very sensitive to the effect of structure-directing species, while the proximity of the carboxylate groups bound to the same aromatic ring ensures that these ligands are never perfectly planar.

Taking a broader viewpoint, an eclectic selection from the recent literature [13,[48][49][50][51][52][53] illustrates that the solid state coordination chemistry of uranyl carboxylates continues to be an active area of research concerned with a variety of possible applications. In our own work in this area, largely focussed on anionic coordination polymers, it has been apparent that hydrogen bonding interactions can have a major influence on the form of the complexes and even upon the coordination sphere of the uranyl ion. Our use of solvo-hydrothermal methods for the syntheses has also made it apparent that the solvents employed can not only influence the solubility of a product but that, through their own reactions under the conditions imposed, they can as well influence the composition of the product in various ways. The present work has provided further illustration of the operation of both these factors. Thus, complex 1 provides a unique example of a phthalate-like ligand being bound to uranyl ion exclusively through the formation of 7-membered chelate rings, apparently as a result of proton competition (hydrogen bonding) for the donor sites which in other complexes bind to uranyl ion and expand the periodicity of coordinative interactions.

Hydrogen bonding by the multivalent [Co(en)3] 3+ cation may also explain in part why the solvent-derived formate ligand is bound in a  1 O and not a  2 O,O' fashion, although the coordination of U VI in the complex can also be considered a consequence of steric crowding of the equatorial coordination plane due to the presence of two 7-membered chelate rings, such a restriction being long known with 6-membered chelates such as 1,3-diketonates. Where even just a single 7-membered ring is present, as in complex 2 and one component of complex 3, pentagonal-bipyramidal coordination of U VI applies, as is the case in various complexes of phthalate itself. Hexagonal-bipyramidal coordination can be attained, however, when three bptc 4-ligands act as  2 O,O'-chelates to one uranyl centre, as seen in one component of complex 3 and as is again known for phthalate itself. The somewhat unpredictable influence of the cosolvent in solvohydrothermal synthesis is well illustrated in the structures of complexes 1 and 2, where the two products of DMF hydrolysis, formate and dimethylammonium, respectively, are separately present. As an NH donor, dimethylammonium ion is sterically a rather different species to [Co(en)3] 3+ and the 2:1 cation/U stoichiometry in 2 does not provide even an equal number of interaction sites. These differences are reflected in the structures, though it may be noted that the Hirshfeld surfaces for both cations provide evidence of secondary CHO interactions that are more prominent for dimethylammonium and thus may also play a role in determining the structures. Substitution in complex 3 of a CH donor, tetraphenylphosphonium cation, for the NH donors of 1 and 2 results not only in the crystallisation of a species lacking any solvent-derived components but also in a triperiodic polymer in which all known modes of phthalate ligand coordination to uranyl ion are present. Aromaticaromatic interactions, both between cations and between cations and bptc 4-units, have an influence on the polymer structure but it is again evident from consideration of all three present structures that equatorial coordination of carboxylate to uranyl ion must be an interaction of energy comparable to that of an NHO bond. While this means that an effective mechanism of structure control exists, it is not yet obvious how this may be rationally exploited to generate triperiodic frameworks.

Conclusions

We have reported the synthesis and crystal structure of three uranyl ion complexes with 1,1'-biphenyl-3,3′,4,4′-tetracarboxylate, a ligand which can be viewed as a phthalate dimer.

These three complexes, which include [Co(en)3] 3+ , H2NMe2 + or PPh4 + as counterions, are mono-, di-and triperiodic coordination polymers, respectively. These structures provide further evidence that the appropriate juxtaposition of two carboxylate groups on a scaffold

Experimental Section

General: [UO2(NO3)2(H2O)2]•4H2O (RP Normapur, 99%) was purchased from Prolabo, 1,1'biphenyl-3,3′,4,4′-tetracarboxylic acid dianhydride was from Aldrich, and [Co(en)3]Cl33H2O was from Alfa-Aesar. The elemental analysis of 1 was performed by MEDAC Ltd. Except for 1, the low yields of the syntheses prevented further characterization. For all syntheses, the mixtures in demineralized water were placed in 10 mL tightly closed glass vessels and heated at 140 °C in a sand bath, under autogenous pressure. The crystals formed directly from the pressurized and heated reaction mixtures and not as a result of subsequent cooling.

Caution! Uranium is a radioactive and chemically toxic element, and uranium-containing samples must be handled with suitable care and protection. Small quantities of reagents and solvents were employed to minimize any potential hazards arising both from the presence of uranium and the use of pressurized vessels for the syntheses.

[Co(en)3][UO2(bptc)(HCOO)]2.5H2O (30 mg, 0.04 mmol) were dissolved in a mixture of water (0.9 mL) and DMF (0.3 mL), giving a few light yellow crystals of complex 2 within one month.

[PPh4]4[(UO2)4(bptc)3]6H2O (3): 1,1'-Biphenyl-3,3′,4,4′-tetracarboxylic acid dianhydride (30 mg, 0.10 mmol), [UO2(NO3)2(H2O)2]•4H2O (50 mg, 0.10 mmol), and PPh4Br (42 mg, 0.10 mmol) were dissolved in a mixture of water (0.9 mL) and DMF (0.3 mL), giving a few light yellow crystals of complex 3 within ten days.

Crystallography: The data were collected at 100(2) K either on a Bruker D8 Quest diffractometer equipped with an Incoatec Microfocus Source (IS 3.0 Mo) and a PHOTON III area detector, and operated through the APEX3 software [START_REF] Bruker | APEX3. Version[END_REF] (complexes 1 and 2), or on a Nonius Kappa-CCD detector diffractometer [START_REF] Hooft | [END_REF] using graphite-monochromated Mo K radiation (complex 3). The data were processed with SAINT [START_REF]SAINT. Version 8.40A[END_REF] or HKL2000, [START_REF] Otwinowski | [END_REF] and absorption effects were corrected for empirically with SADABS [58] or SCALEPACK. [START_REF] Otwinowski | [END_REF] The structures were by intrinsic phasing with SHELXT [59] and refined by full-matrix least-squares on F 2 with SHELXL, [60] using the ShelXle interface. [61] All non-hydrogen atoms were refined with anisotropic displacement parameters. In complex 1, the hydrogen atoms bound to water oxygen atoms were found on a residual electron density map and were refined with restraints, but those in 2 were neither found, nor introduced, and those in 3 only partially found. The hydrogen atoms bound to nitrogen atoms in 1 and 2 were introduced at calculated positions. In 2 and 3, some water molecules have been given occupancy factor of 0.5 in order to retain an acceptable displacement parameter. The carbon-bound hydrogen atoms were introduced at calculated positions and were treated as riding atoms with an isotropic displacement parameter equal to 1.2 times that of the parent atom (1.5 for CH3, with optimized geometry). The Flack parameter for complex 2 was 0.031 (9). Crystal data and structure refinement parameters are given in Table 2. The molecular plots were drawn with ORTEP-3 and the polyhedral representations with VESTA. [63] CCDC 2084850 (for 1), 2084851 (for 2), and 2084852 (for 3) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

(Figure 1 .

 1 Figure 1. (a) View of complex 1. Displacement ellipsoids are drawn at the 50% probability level. Carbon-bound

Figure 2 .

 2 Figure 2. (a) View of complex 2. Displacement ellipsoids are drawn at the 50% probability level. Carbon-bound

Figure 3 .

 3 Figure 3. (a) View of complex 3 with displacement ellipsoids drawn at the 50% probability level. Counterions,

Figure 4 .

 4 Figure 4. Two views of the nodal representation of the framework in complex 3 (uranium, yellow; bptc 4-, blue).

Figure 5 .

 5 Figure 5. Space filling view of the section of one channel in complex 3, with one of the two rows of counterions

  of limited flexibility can favour the formation of 7-membered chelate rings, which are present in all cases, either exclusively in the monoperiodic polymer, or mixed with other coordination modes in the other two cases. The relatively large bite angle of such a ring appears to limit the number of additional donor atoms in the equatorial region of uranyl ion complexes to three and to thus enforce pentagonal bipyramidal coordination geometry, although two of the three additional donor atoms may derive from another 7-membered chelate ring. The presence of hydrogen bond donor species is clearly an important influence on the structure in the present series, with  2 O,O' carboxylate chelation being favoured the weaker is this influence.

  2-ligands in uranyl ion complexes.

	Complex	1	2	3	4	1	2		Geometry
	1	31.9(2)	33.9(2)	71.50(9)	65.24(10)	86.1(3)	83.5(3)	12.12(11)	distal
	2	67.4(3)	11.9(10)	29.4(6)	53.1(5)	63.1(5)	52.7(7)	39.6(2)	distal
	3	78.6(3)	45.4(4)	0.9(4)	21.1(8)	77.8(5)	48.3(5)	28.0(2)	proximal
		15.4(6)		80.8(3)		88.6(6)		0	distal
	[UO2(H2bptc)(H2O)2] [19]	12.8(9)		74.1(2)		76.5(5)		0	distal
	[(UO2)2(bptc)(NMP)1.5(H2O)1.5] [20]	9.6(5)	20.9(5)	71.3(5)	67.5(6)	69.9(10)	72.0(10)	19.4(4)	proximal
	[UO2Ag(bptc)(4,4′-bipyH)] [20]	75.98(12)	18.22(11)	21.0(2)	72.68(13)	79.1(2)	70.1(3)	9.67(5)	distal
	[UO2Pb(bptc)(2,2′-bipy)2] [20]	9.4(8)		72.6(3)		75.4(5)		65.41(15)	proximal

Table 2 .

 2 Crystal data and refinement details.

		1	2	3
	Empirical formula	C23H36CoN6O14.5U C20H23N2O10.5U	C144H110O38P4U4
	M (g mol 1 )	925.54	697.43	3524.31
	Crystal system	monoclinic	orthorhombic	triclinic
	Space group	P21/c	Fdd2	Pī
	a (Å)	8.3035(3)	30.2257(8)	13.5485(8)
	b (Å)	15.7282(6)	43.1520(11)	13.7423(4)
	c (Å)	23.0166(9)	7.4235(2)	18.2212(10)
	(°)	90	90	93.975(3)
	 (°)	94.2492(17)	90
	(°)	90	90	99.613(3)
	V (Å 3 )	2997.7(2)	9682.5(4)	3270.4(3)
	Z	4	16	1
	collected	170406	162891	145767
	Independent reflections	7730	7401	12421
	Observed reflections [I > 2(I)] 7122	7324	9526
	Rint	0.057	0.063	0.055
	Parameters refined	433	317	850
	R1	0.019	0.026	0.038
	wR2	0.041	0.060	0.092
	S	1.085	1.313	1.056
	min (e Å 3 )	0.70	1.83	1.38
	max (e Å 3 )	1.26	1.54	2.09


Dyhia Amrane

Nicolas Primas
email: nicolas.primas@univ-amu.fr

Christophe-Sébastien Arnold

Sébastien Hutter

Béatrice Louis

Julen Sanz-Serrano

Amaya Azqueta

Nadia Amanzougaghene

Shahin Tajeri

Dominique Mazier

Pierre Verhaeghe

Nadine Azas

Cyrille Botté
email: cyrille.botte@univ-grenoble-alpes.fr

Patrice Vanelle
email: patrice.vanelle@univ-amu.fr

PharmD-PhD José Maldonado

Antiplasmodial 2-thiophenoxy-3-trichloromethyl quinoxalines target the apicoplast of Plasmodium falciparum

Keywords:

come

Introduction

Malaria remains the leading cause of death among parasitic infections worldwide, mainly affecting children under the age of 8, the elderlies and pregnant women. According to the World Malaria Report 2020 [1], malaria caused an estimated 229 million clinical cases leading to 409 000 deaths. Eighty nine countries from the sub-tropical zone are endemic, where the mosquito vectors are present. Most cases and deaths occur in sub-Saharan Africa and South-East Asia. Malaria is caused by a unicellular eukaryote, i.e. protist, of the Apicomplexa phylum and the Plasmodium genus, among which the species falciparum is responsible for most fatal malaria cases, including cerebral malaria. The parasite is transmitted to humans by a bite of an infected female Anopheles mosquito during a blood meal. Despite numerous and ambitious malaria control efforts, Plasmodium strains that resist commercial anti-malarial drugs such as chloroquine [2], quinine [3], atovaquone [4], mefloquine [5], piperaquine [6] and associations of pyrimethamine-cycloguanil [7] or sulfadoxine-pyrimethamine [8] are spreading worldwide. In addition, the emergence of resistances toward the first line treatments against severe P. falciparum infections, Artemisinin-based Combination Therapies (ACTs), is now threatening the efficacy of malaria treatment particularly in the Greater Mekong Subregion [START_REF] Menard | Antimalarial Drug Resistance: A Threat to Malaria Elimination[END_REF], Africa [START_REF] Lu | Emergence of Indigenous Artemisinin-Resistant Plasmodium Falciparum in Africa[END_REF][START_REF] Uwimana | Emergence and Clonal Expansion of in Vitro Artemisinin-Resistant Plasmodium Falciparum Kelch13 R561H Mutant Parasites in Rwanda[END_REF] and India [START_REF] Rosenthal | Artemisinin Resistance in Eastern India[END_REF]. Therefore, new chemotypes with original modes of action, for use in combination therapies, are urgently needed to overcome these resistances and move towards malaria eradication.

Working on the synthesis and reactivity of new heterocycles bearing a trichloromethyl group as potential antiparasitic agents [13][START_REF] Primas | A New Synthetic Route to Original Sulfonamide Derivatives in 2-Trichloromethylquinazoline Series: A Structure-Activity Relationship Study of Antiplasmodial Activity[END_REF][START_REF] Gellis | A New DMAP-Catalyzed and Microwave-Assisted Approach for Introducing Heteroarylamino Substituents at Position 4 of the Quinazoline Ring[END_REF], our research group has previously reported an antiplasmodial 2-trichloromethylquinazoline derivative (Hit A, Table 1) bearing a thiophenoxy group at position 4 [START_REF] Verhaeghe | 4-Thiophenoxy-2-Trichloromethyquinazolines Display in Vitro Selective Antiplasmodial Activity against the Human Malaria Parasite Plasmodium falciparum[END_REF]. From this initial compound, a quinoxaline position isomer (Hit B, Table 1) was prepared and evaluated, showing an improved antiplasmodial profile [START_REF] Desroches | Discovery of New Hit-Molecules Targeting Plasmodium Falciparum through a Global SAR Study of the 4-Substituted-2-Trichloromethylquinazoline Antiplasmodial Scaffold[END_REF]. Based on this encouraging result, we decided to synthesize a series of original quinoxaline derivatives bearing a thiophenoxy moiety at the 2-position of the quinoxaline ring, with the aim of identifying a new optimized hitcompound and investigating the antiplasmodial structure-activity relationships.

Table 1.

Comparison between antiplasmodial Hit A and position isomer Hit B

Hit A

Hit B EC50 PfK1 (µM) 0.9 0.5 CC50 HepG2 (µM) >25 38.6 Selectivity index (SI) >28 77.2 Indeed, several quinoxalines have already been identified as promising antiplasmodial molecules, including BQR695 (Figure 1), identified from a phenotypic screening of the Novartis chemical library, exerting antiplasmodial activity through inhibition of the plasmodial protein kinase PfPI(4)K [START_REF] Mcnamara | Targeting Plasmodium PI(4)K to Eliminate Malaria[END_REF]. Guillon et al. [START_REF] Guillon | Synthesis and Antimalarial Activity of Novel Bis N-[(Pyrrolo[1,2-a]Quinoxalin-4-Yl)Benzyl]-3-Aminopropylamine Derivatives[END_REF] previously described a series of bis-and tris-pyrrolo [1,2-a]quinoxaline derivatives (C, Figure 1) showing good antiplasmodial activity during the intraerythrocytic stage of P. falciparum W2 and 3D7 with EC50 in the micromolar range. In addition, (R)-enantiomers of pyrrolo [1,2-a]quinoxalines (D) showed good antiplasmodial activities [START_REF] Jonet | Synthesis and Antimalarial Activity of New Enantiopure Aminoalcoholpyrrolo[1,2-a]quinoxalines[END_REF]. In addition, our lab has previously described 2-trichloromethylpyrroloquinoxaline derivatives (E) which also show selective antiplasmodial activity [START_REF] Primas | Synthesis and in Vitro Evaluation of 4-Trichloromethylpyrrolo [1,2-a] Quinoxalines as New Antiplasmodial Agents[END_REF], illustrating the potential of quinoxaline ring in antiplasmodial drugdesign. Figure 1. Structures of some previously described antiplasmodial derivatives containing a quinoxaline moiety. With the aim of discovering and developing new antimalarial compounds, Medicines for Malaria Ventures (MMV) defined target candidates (TCPs) and target product profiles (TPPs) for the treatment and chemoprevention of malaria [22]. MMV currently lists four molecules in pre-clinical trials and 14 compounds in clinical trials [23]. Therefore, new compounds with novel mechanisms of action are urgently required to overcome the resistance of Plasmodium against commercial drugs. In this context, apicoplast, a plant plastid-like organelle that is essential for parasite survival provide potentially an interesting antimalarial target.

The discovery in Australian corals of a photosynthetic apicomplexan Chromera velia confirmed the common plant and photosynthetic evolutionary origin of P. falciparum and its peculiar organelle, the apicoplast [START_REF] Weatherby | The Missing Link in the Evolution of Parasitism[END_REF]. Indeed, the apicoplast is a non-photosynthetic plastid acquired by secondary endosymbiosis from a plastid-containing red algal ancestor [START_REF] Mcfadden | The Apicoplast: Now You See It, Now You Don[END_REF]. As a consequence of this secondary endosymbiotic origin, the apicoplast is delimited by four membranes [START_REF] Botté | Plasmodium falciparum Apicoplast Drugs: Targets or off-Targets?[END_REF]. This P. falciparum organelle possesses a housekeeping machinery maintained in its small 35 kb circular genome, which encodes for tRNAs, ribosomal proteins and a handful of proteins that are targeted to the apicoplast [START_REF] Mukherjee | Anti-Malarial Drug Design by Targeting Apicoplasts: New Perspectives[END_REF]. Despite being non photosynthetic, the apicoplast retains several anabolic pathways that are essential for parasite survival. These pathways include fatty acids biosynthesis, isoprenoid biosynthesis precursors, iron-sulfur cluster assembly and heme biosynthesis [START_REF] Mukherjee | Anti-Malarial Drug Design by Targeting Apicoplasts: New Perspectives[END_REF]. These metabolic pathways have no equivalent in the human host. Isoprenoid precursors such as isopentenyl pyrophosphate (IPP) have previously been described as the only required function of the apicoplast in the asexual blood stage of malaria parasite in vitro under rich culture conditions [START_REF] Uddin | Validation of Putative Apicoplast-Targeting Drugs Using a Chemical Supplementation Assay in Cultured Human Malaria Parasites[END_REF]. Thus, it was confirmed that IPP inhibitors, such as fosmidomycin, mainly target the apicoplast. On the other hand, the apicoplast fatty acid biosynthesis plays a vital role during the in vitro liver stage [START_REF] Vaughan | Type II Fatty Acid Synthesis Is Essential Only for Malaria Parasite Late Liver Stage Development[END_REF], and can be reactivated during the blood stages and become critical in a low host nutritional/lipid environment [START_REF] Botté | Atypical Lipid Composition in the Purified Relict Plastid (Apicoplast) of Malaria Parasites[END_REF][START_REF] Amiar | Division and Adaptation to Host Environment of Apicomplexan Parasites Depend on Apicoplast Lipid Metabolic Plasticity and Host Organelle Remodeling[END_REF]. Furthermore, even if disrupted and absent in blood stage parasites by antiplastidial treatment (azythromycin, doxycycline, fosmidomycin), the apicoplast remains metabolically active in the form of vesicles, showing once again that the IPP pathway is not the only essential pathway of the apicoplast [32]. Moreover, Prigge et al. [33] have recently demonstrated the importance of carbon metabolism in the apicoplast of the malaria parasite. Indeed, a loss of pyruvate kinase II activity which is an essential Plasmodium enzyme providing the pyruvate necessary for the generation of essential isoprenoid precursors, leads to a reduction of apicoplast RNA, following by apicoplast disruption and parasite death. Seven enzymes of the isoprenoid precursors pathway are drug-target in the apicoplast, which are DOXP synthase, IspC, IspD, IspE, IspF, IspG and IspH. Knowing that the isoprenoid precursors biosynthesis is localized only to apicoplast, chemical rescue by adding IPP to the growth medium identified MMV-08138 as a potent IspD inhibitor during the blood stage of P. falciparum with an EC50 of 110 nM (Figure 2) [START_REF] Ghavami | Biological Studies and Target Engagement of the 2-C-Methyl-d-Erythritol 4-Phosphate Cytidylyltransferase (IspD)-Targeting Antimalarial Agent (1R,3S)-MMV008138 and Analogs[END_REF].

Figure 2. MMV-08138 acting at the level of the Plasmodium apicoplast.

Currently very few molecules targeting the apicoplast have been reported, but some known antibiotics have been studied, such as ciprofloxacin and doxycycline belonging to the fluoroquinolone and tetracycline classes respectively, which target DNA replication and rRNA translation respectively in P. falciparum apicoplast, leading to a delayed death drug-response [START_REF] Botté | Plasmodium falciparum Apicoplast Drugs: Targets or off-Targets?[END_REF].

The delayed death phenomenon in the apicomplexan parasite is well studied and its molecular mechanism appears to be related to the inhibition of functions in the apicoplast that induce death of daughter cell after one life cycle. It is related to apicoplast housekeeping processes such as DNA replication, transcription and translation mechanisms [START_REF] Mukherjee | Anti-Malarial Drug Design by Targeting Apicoplasts: New Perspectives[END_REF].

In addition to these antibiotics, several biocides such as triclosan (Figure 3) are reported to be active against Plasmodium through the metabolic pathway of fatty acid synthesis located in the apicoplast [START_REF] Botté | Plasmodium falciparum Apicoplast Drugs: Targets or off-Targets?[END_REF] although it was further proved that the apicoplast is not the main target [START_REF] Baschong | Triclosan Is Minimally Effective in Rodent Malaria Models[END_REF]. As P. falciparum apicoplast is an essential chloroplast-like organelle, studies have demonstrated that herbicides active against plants are also active against P. falciparum and therefore could act as antimalarials [36,[START_REF] Corral | Herbicidal Properties of Antimalarial Drugs[END_REF]. Indeed, clomazone [START_REF] Clastre | The Methylerythritol Phosphate Pathway for Isoprenoid Biosynthesis in Coccidia: Presence and Sensitivity to Fosmidomycin[END_REF] and fluridone [START_REF] Nagamune | Abscisic Acid Controls Calcium-Dependent Egress and Development in Toxoplasma Gondii[END_REF] affect DOXP synthase and block plastidial abscisic acid synthesized by the plastid DOXP pathway respectively, localized in the apicoplast (Figure 3). After working for 15 years on the development of trichloromethylated heterocycles without identifying their mechanism of action, we decided herein to evaluate the effect of our molecules toward this vital organelle in P. falciparum in order to look for the mechanism of action of such trichloromethyl derivatives.

Results

Synthesis

As shown in Scheme 1, the key substrate (1) was easily obtained by reacting ophenylenediamine with ethyl pyruvate to provide the corresponding lactam which was then chlorinated using POCl3 (Scheme 1) [START_REF] Gellis | A New DMAP-Catalyzed and Microwave-Assisted Approach for Introducing Heteroarylamino Substituents at Position 4 of the Quinazoline Ring[END_REF]. Next, chlorimine (1) was reacted through a nucleophilic aromatic substitution reaction (SNAr) with various thiophenol derivatives in the presence of cesium carbonate in DMF at 70 °C. The desired intermediates (2a-2p) were obtained in low to quantitative yields (14 to 100%). Yield variation does not seem to be correlated with the electron-donating orwithdrawing behavior of the different thiophenol substituents. Finally, from the methylated precursors (2a-2p), a chlorination reaction using PCl5 and POCl3 was performed under microwave heating at 100 °C for 30 min, according to a previously reported method [START_REF] Verhaeghe | Highly Efficient Microwave Assisted a -Trichlorination Reaction of a -Methylated Nitrogen Containing Heterocycles[END_REF], leading to the target compounds (3a-3p) in low to very good yields (15-86%). A series of 16 original derivatives (3a-3p) was then prepared, bearing electron-donating or electron-withdrawing substituents in ortho, meta and para positions of the thiophenol moiety. It is to note that attempt to chlorinate the intermediate (2i), bearing a 3'-OMe substituent on the thiophenoxy group, led to the only the unexpected 2'chlorinated product. Scheme 1. Preparation of 2-thiophenoxy-3-trichloromethylquinoxaline derivatives (3a-3p). Reagents and conditions: (i) ethyl pyruvate 1 equiv, H2O, 50 °C, 15 min; (ii) POCl3, reflux, 2 h; (iii) appropriate thiophenol derivative 1 equiv, Cs2CO3 1 equiv, anh. DMF, 70 °C, 12 h, sealed vial, N2; (iv) PCl5 6 equiv, POCl3 as solvent, 100 °C, MW, 20 min, 800 W.

Biological results

Structure-Activity Relationship (SAR) study

These new derivatives were evaluated in vitro against the K1 multidrug-resistant P. falciparum strain, by determining their 50% efficacy concentration (EC50), and compared with three reference antimalarial drugs: chloroquine, atovaquone and doxycycline. The in vitro 50% cytotoxic concentrations (CC50) were assessed on the HepG2 human hepatocyte cell line and compared with cytotoxic reference drug: doxorubicin. Selectivity indexes were calculated: SI = CC50/EC50. The results are presented in Table 2.

All the 16 newly synthesized compounds showed good aqueous solubility except for compound (3j), which precipitated above 1.9 µM in the cell viability assay. The derivatives showed cytotoxicity values ranging from 19.9 to 69.7 µM, comparable or better than chloroquine (30 µM) or doxycyclin (20 µM). It appears that strong electron-withdrawing groups (EWG) (3m-3o) at 4-position of 2-thiophenoxy group led to slightly more cytotoxic compounds (19.9-29.9 µM), contrary to electron-donating groups (EDG) (3p) or the unsubstituted thiophenoxy moiety (3k) (69.7-56.0 µM, respectively).

With regard to the antiplasmodial activity, all derivatives showed submicromolar activity except for (3p) bearing a 4-Me group (1.5 µM). A strong EWG such as 4-NO2 group (3o) gave the best antiplasmodial activity with only 0.2 µM. The 2, 3 or 4 fluoro-substituted thiophenoxy moiety (3e-3g) led to quite similar activities (0.32, 0.31, 0.36 µM respectively). Other 4-EWG such as 4-CF3 (3m) and 4-OCF3 (3n) gave quite similar activities to the fluoro-substitued thiophenoxy group (0.40 and 0.30 µM respectively). Substitution by a methoxy group was slightly detrimental to the activity, especially for the 2-OMe compound (3h) (0.74 µM). The disubstituted 2-chloro-3-methoxy-thiophenoxy moiety (3i) led to a better antiplasmodial activity than only 2-or 4-methoxy substitution (3h, 3j). For the dichloro-containing molecules (3c, 3d), 2-3 substitutions were better than the 2-4 substitutions (0.31 vs 0.90 µM).

Finally, the best compound in this series was the unsubstituted thiophenoxy group (3k) which combined both good antiplasmodial activity and low cytotoxicity, leading to the highest selectivity index in the series (175) (Table 2). It is to note that there is no correlation between the calculated logP and activity. Table 2. Antiplasmodial activity, cLogP and human cell toxicity on HepG2 cell line of the 2-phenoxy-3trichloro-methylquinoxaline compounds (3a-3p). -0.20 -a Chloroquine, Atovaquone and doxycycline were used as antimalarial reference-drugs; b Doxorubicin was used as a cytotoxic reference-drug; c Highest concentration tested due to a lack of solubility; d Selectivity index (SI) was calculated according to the formula: SI = CC50/EC50, e Weighted clogP was computed by swissADME; f The best antiplasmodial molecule.

Toxicity data

In order to complete the cytotoxicity data of the best molecule (3k) in this series, we tested it on CHO Chinese Hamster Ovary cells and Vero cells (Table 3). After 72 h of treatment, low cytotoxicities was observed both on cell lines (58.5 µM and 63.5 µM respectively), similar to that measured on HepG2 cell line (56.0 µM).

To complete the in vitro toxicological evaluation of compound (3k), its genotoxic potential was studied, using the comet assay on the HepG2 cell line (Table 3). Compound (3k) did not induce DNA strand breaks or alkali labile sites after either short (2 h) or long (72 h) exposure at two concentrations (5.6 and 28 µM; CC50/2 and CC50/10 after 72 h of treament) ( see supplementary material). The positive control (i.e., cells treated with 1 mM Methyl MethaneSulphonate, MMS) showed the expected results.

Role of the 2-CCl3 group

We have previously shown that the -CCl3 group is mandatory to provide antiplasmodial activity [START_REF] Desroches | Discovery of New Hit-Molecules Targeting Plasmodium Falciparum through a Global SAR Study of the 4-Substituted-2-Trichloromethylquinazoline Antiplasmodial Scaffold[END_REF][START_REF] Primas | Synthesis and in Vitro Evaluation of 4-Trichloromethylpyrrolo [1,2-a] Quinoxalines as New Antiplasmodial Agents[END_REF][START_REF] Amrane | Synthesis and Antiplasmodial Evaluation of 4-Carboxamido-and 4-Alkoxy-2-Trichloromethyl Quinazolines[END_REF]. To confirm the key role of the CCl3 group in the activity of 3k, we compared the antiplasmodial activity of (3k) with that of three analogs bearing a proton (3r), a methyl group (2k) or a trifluoromethyl group (3s) at position 3 of the quinoxaline ring respectively. None of these analogues lacking a CCl3 group was active against P. falciparum (EC50 PfK1 > 50 µM). These activity cliffs revealed once again the key role of the CCl3 group in antiplasmodial activity in the studied series (Figure 4) [START_REF] Stumpfe | Exploring Activity Cliffs in Medicinal Chemistry[END_REF].

• Treatment with molecule 3k affects parasites growth and apicoplast development

Using an epifluorescence microscope, we monitored the effect of molecule (3k) on different intraerythrocytic stages of P. falciparum parasite in order to identify the mechanism by which the molecule could affect parasite survival and whether this was directed against the apicoplast. The loss of apicoplast by molecule (3k)-treatment can then be visualized via fluorescence microscopy. The EC50 treatment was performed on synchronized cultures of P. falciparum 3D7 strain during a 48-hour parasite life cycle (Figure 5A).

Parasites treated with 0.3 µM of molecule (3k) shows a severe defect in apicoplast biogenesis when compared to the DMSO control. The apicoplast development, elongation and branching typically happening during asexual blood stage intracellular development is highly affected by the treatment and strongly suggest a direct effect on Plasmodium apicoplast (Figure 5A). Furthermore, overall parasite development is also slightly reduced, as the nucleus staining shows a defect in replication and the culture is enriched in mid trophozoite parasites (Figure 5A), suggesting either that apicoplast disruption induces parasite death or that the drug could have some sort of off target mechanism. If the apicoplast is the primary target of the (3k) molecule, one could expect a so-called delayed death phenotype, which is characteristic of some apicoplast targeting drugs such as chloramphenicol, doxycycline or ciprofloxacin. To confirm this, we performed a growth assay on three life cycles (3x 48 h) by measuring the development of treated parasite with 0.3 µM of (3k) and 0.3 µM of (2k). The replacement of CCl3 by a methyl group in (2k) analog shows to have no effect on the apicoplast development (Figure 5B) compared to untreated parasites (DMSO control). Results show a slight reduction of the parasites development at the first cycle (48 h), and a drastic reduction of the growth at the second and third cycles, confirming the delayed effect of apicoplast targeting drugs, compared to inactive (2k) molecule, which has no significative difference with DMSO (Figure 5C). Nevertheless, the slight decrease of the growth at the first cycle confirms the potential off target of (3k) molecule, outside of the apicoplast, coherent with the reduction of nucleus replication shown by IFA at 48 h treatment (Figure 5A). It should be noted out that all derivatives (3a-p) including (3k) have a high lipophilicity (3.92 < clogP < 5.69). This may be necessary property to cross the four membranes that envelop the apicoplast.

Evaluation on human hepatocyte infection

In order to further explore the potency of molecule (3k), an evaluation was made on the hepatic stage of P. falciparum infection. Cryopreserved primary human hepatocyte cultures were infected with P. falciparum NF135 strain. Treatment with (3k) at various doses was applied simultaneously with the sporozoites addition to hepatocytes and contact was maintained until postinfection day 6 (pi). (3k) treatment significantly reduced parasite size without affecting parasite numbers in a dose-dependent manner. Atovaquone (ATQ), a potent hepatic schizonticide was used as positive control.

We then tried to understand if apicoplast of P. falciparum schizonts was targeted by (3k). In untreated control wells fixed at day 6 pi, the parasitic apicoplast has a uniform appearance. The apicoplast is in its terminal stage of development (considering the 5-6 days schizogony of P. falciparum) and the Apicoplast protein acyl Carrier Protein (ACP) signal looks like small dots densely decorating the periphery of the organelle. In (3k) treated cultures, the integrity of small dots shown in the control parasites appears to be affected and the ACP signal is condensed leading to the appearance of larger aggregates. At the high dose of 30 µM approximatively 93% of P. falciparum schizonts showed partial or complete alteration of the apicoplast signal (Figure 6). Interestingly, no cytotoxicity was observed on human hepatocytes up to 30 µM (Figure 7). Figure 6. The effect of (3k) on liver stage P. falciparum growth. a) D0 pi-D6 (3k) exposure of human hepatocytes infected with P. falciparum and its impact on parasite numbers expressed as mean +/-SD and parasite size expressed as surface area expressed as μm 2 mean +/-SD. Parasites were immunostained with anti-HSP70 antibody and plates were scanned. b) Quantification of different apicoplast morphologies (normal and affected) in 100 parasites with percentage distribution of each defined category. c) Confocal microscopic images of control and treated P. falciparum schizonts. The schizonts were immunolabelled with anti-HSP70 antibody. Host and parasite DNA were visualized by 4′,6-diamidino-2-phenylindole (DAPI) dye. The apicoplast was labelled by Anti-PyACP (Scale bar = 10 µm). Figure 7. Toxicity of (3k) on primary human hepatocytes. The number of hepatocyte nuclei quantified from a certain number of microscopic scanning fields from control and (3k) treated groups.

Conclusion

An antiplasmodial SAR study was conducted through the synthesis and evaluation of 16 original thiophenoxyquinoxaline derivatives. All of these molecules displayed good in vitro antiplasmodial activity. Among these compounds, (3k) showed the best antiplasmodial activity (EC50 = 0.32 µM) and low cytotoxicity towards three cell-lines and no cytotoxicity on human hepatocytes. Moreover, compound (3k) presented no genotoxic effect in the comet assay. The activity cliffs showed that the CCl3 group was essential to provide antiplasmodial activity. Although no effect was demonstrated on hepatic stage, at high concentration, the size of parasite was affected. Interestingly, the molecule (3k) shows a delayed death inhibition and apicoplast biogenesis branching defect, confirming that it affects growth of P. falciparum by targeting the apicoplast, and probably another target outside of the apicoplast which affects the cell division. Essential metabolic pathways reside within the apicoplast which provide promising new drug targets for the development of new antimalarials, with a novel mechanism of action. Further investigations will be necessary in order to decipher the action of trichloromethylquinoxaline derivatives on the apicoplast.

Acknowledgments

Experimental section 2.1. Chemistry 2.1.1. Generality

Melting points were determined on a Köfler melting point apparatus (Wagner & Munz GmbH, München, Germany) and are uncorrected. Elemental analyses were carried out at the Spectropole, Faculté des Sciences de Saint-Jêrome (Marseille) with a Thermo Finnigan EA1112 analyzer (Thermo Finnigan, San Jose, CA, USA). NMR spectra were recorded on a Bruker AV (Billerica, MA, USA) 200 or AV 250 spectrometers or on a Bruker Avance NEO 400MHz NanoBay spectrometer at the Faculté de Pharmacie of Marseille or on a Bruker Avance III nanobay 400 MHz spectrometer at the Spectropole, Faculté des Sciences de Saint-Jêrome (Marseille). ( 1 H NMR: reference CDCl3 δ = 7.26 ppm, reference DMSO-d6 δ = 2.50 ppm and 13 C NMR: reference CHCl3 δ = 76.9 ppm, reference DMSO-d6 δ = 39.52 ppm). The following adsorbent was used for column chromatography: silica gel 60 (Merck KGaA, Darmstadt, Germany, particle size 0.063-0.200 mm, 70-230 mesh ASTM). TLC was performed on 5 cm × 10 cm aluminum plates coated with silica gel 60F-254 (Merck) in an appropriate eluent. Visualization was performed with ultraviolet light (234 nm). The purity of synthesized compounds was checked by LC/MS analyses, which were performed at the Faculté de Pharmacie of Marseille with a Thermo Scientific Accela High Speed LC System® (Waltham, MA, USA) coupled to a single quadrupole mass spectrometer Thermo MSQ Plus®. The RP-HPLC column is a Thermo Hypersil Gold® 50 × 2.1 mm (C18 bounded), with particles of a diameter of 1.9 mm. The volume of sample injected on the column is 1 μL. The chromatographic analysis with a total duration of 8 min, is performed on the following solvents gradient: t = 0 min, methanol/water 50:50; 0 < t < 4 min, linear increase in the proportion of methanol to a methanol/water ratio of 95:5; 4 < t < 6 min, methanol/water 95:5; 6 < t < 7 min, linear decrease in the proportion of methanol to return to a methanol/water ratio of 50:50; 6 < t< 7 min, methanol/water 50:50. The water used was buffered with ammonium acetate 5 mM. The flow rate of the mobile phase was 0.3 mL/min. The retention times (tR) of the molecules analyzed are indicated in min. The microwave reactions were performed using multimode reactors: ETHOS Synth Lab station and MicroSYNTH® Lab terminal 1024 (Ethos start, MLS GmbH, Leutkirch, Germany.); or monomode reactors: Biotage Initiator® classic in sealed vials with a power output of 0 to 400 W. Reagents were purchased and used without further purifications from Sigma-Aldrich or Fluorochem.

2-Chloro-3-methylquinoxaline (1)

Step 1: To a solution of o-phenylenediamine (6 g, 1.0 equiv) in H2O was added ethyl pyruvate (1.0 equiv) dissolved in H2O (80 mL). The reaction mixture was heated at 50 °C for 15 to 30 min. After cooling, the precipitate product of 3-methylquinoxalin-2(1H)-one was recrystallized from ethanol as a white solid, 7.92 g, yield 89%. This was used directly in step 2.

Step 2: To a solution of 3-methyl-2(1H)-quinoxalinone (12.5 g, 121.8 mmol, 1.0 equiv) was added cold POCl3 (65 mL) in portions to get a slurry. The mixture was refluxed for 2 h. Then, the reaction was added to ice cold water (500 mL) and basified slowly under cooling with Na2CO3 to pH 8. The organic phase was separated and washed with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuum to afford the crude product, which was purified by silica gel flash chromatography (using dichloromethane) to afford (1) as an off-white solid, yield 80% (7g). Mp 84 °C.

General procedure for 3-methyl-2-substituted-quinoxaline (2a-2p)

To a solution of 2-chloro-3-methylquinoxaline (1) (500 mg, 2.8 mmol) and the appropriate thiophenol (2.8 mmol, 1.0 equiv) in anhydrous DMF (10 mL), Cs2CO3 (912 mg, 2.8 mmol, 1.0 equiv) was added under inert atmosphere. The mixture was stirred at 70 °C overnight. After completion of the reaction, water was added, leading to a precipitate which was separated by filtration. The resulting precipitate was then thoroughly washed with water. The precipitate was dissolved in CH2Cl2 and dried with Na2SO4. After filtration and evaporation, the resulting solid was purified by silica gel column chromatography (using the appropriate eluant) to afford the desired compound.

Step 1: To a solution of o-phenylenediamine (2g, 18.5 mmol, 1.0 equiv) in H2O was added ethyl trifluoropyruvate (3g 146, 18.5 mmol, 1.0 equiv) dissolved in H2O (30 mL). The reaction mixture was heated at 50 °C for 15 min. After cooling, the precipitate was filtered off and washed with H2O. 3-(Trifluoromethyl)quinoxalin-2(1H)-one was recrystallized from ethanol, precipitating as a white solid and engaged directly in step 2. Yield 93%.

Step 2: 3-(Trifluoromethyl)quinoxalin-2-ol (3.5 g, 16.8 mmol, 1.0 equiv) was heated to reflux in phosphorus oxychloride (30 mL) for 2 h. After the starting material was consumed, the reaction mixture was cooled to r.t. and then quenched with ice at 0 °C. The precipitate was purified by flash chromatography, to afford 2-chloro-3-(trifluoromethyl)quinoxaline (3q) as a white solid, yield 96%. Mp 140 °C. NMR was consistent with description [START_REF] Wei | Visible Light-Induced Photocatalytic C-H Perfluoroalkylation of Quinoxalinones under Aerobic Oxidation Condition[END_REF].

Preparation of compounds (3r, 3s)

To a solution of 2-chloroquinoxaline or 2-chloro-3-(trifluoromethyl)quinoxaline (300 mg, 1.3 mmol, 1.0 equiv.) and thiophenol reagent (143 mg, 1.3 mmol, 1.0 equiv.) in anhydrous DMF (5 mL), Cs2CO3 (1.0 equiv.) was added under inert atmosphere. The mixture was stirred at 70 °C overnight. After completion of the reaction, water was added, leading to a precipitate which was separated by filtration. The resulting precipitate was then thoroughly washed with water. The resulting solid was purified by silica gel column chromatography (eluent: Cyclohexane/CH2Cl2, 4:6) to afford the desired compound.

, J = 35.8 Hz), 137.9, 136.0 (2C), 132.5, 129.8, 129.8, 129.4 (2C), 129.2, 128.3, 127.9 (d, J = 1.9 Hz), 121.2 (q, J = 276.0 Hz). LC-MS (ESI+) tR 6.8 min, m/z [M+H] + 307.07. MW: 306.31 g.mol -1 , purity 99%. HRMS m/z [M+H] + calcd for C15H9F3N2S: 307.0511, Found: 307.0511.

Biology 3.2.1. In Vitro Cytotoxicity Evaluation HepG2

HepG2 cell line was maintained at 37 °C, 5% CO2, at 90% humidity in MEM supplemented with 10% fetal bovine serum, 1% L-glutamine (200 mM) and penicillin (100 U/mL) / streptomycin (100 μg/mL) (complete RPMI medium). The cytotoxicity of the tested molecules on the HepG2 (hepatocarcinoma cell line purchased from ATCC, ref HB-8065) cell line was assessed according to the method of Mosmann [START_REF] Mosmann | Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays[END_REF] with slight modifications. Briefly, 5.103 cells in 100 μL of complete medium were inoculated into each well of 96-well plates and incubated at 37 °C in humidified 5% CO2. After 24 h incubation, 100 μL of medium with various product concentrations dissolved in DMSO (final concentration less than 0.5% v/v) were added and the plates were incubated for 72 h at 37 °C. Triplicate assays were performed for each sample. Each plate-well was then microscope examined for possible precipitate formation before the medium was aspirated from the wells. 100 µl of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) solution (0.5 mg/mL in medium without FBS) were then added to each well. Cells were incubated for 2 h at 37 °C. After this time, the MTT solution was removed and DMSO (100 μL) was added to dissolve the resulting blue formazan crystals. Plates were shaken vigorously (700 rpm) for 10 min. The absorbance was measured at 570 nm with 630 nm as reference wavelength using a BIO-TEK ELx808 Absorbance Microplate Reader (LabX, Midland, ON, Canada). DMSO was used as blank and doxorubicin (purchased from Sigma Aldrich) as positive control. Cell viability was calculated as percentage of control (cells incubated without compound). The 50% cytotoxic concentration (CC50) was determined from the doseresponse curve, using TableCurve software 2D v.5.0. CC50 values represent the mean value calculated from three independent experiments.

In vitro Cytotoxicity evaluation CHO

CHO cell line was maintained at 37 °C, 5% CO2 with 90% humidity in RPMI supplemented with 10% foetal bovine serum, 1% L-glutamine (200 mM) and penicillin (100 U/mL) / streptomycin (100 μg/mL) (complete RPMI medium). The evaluation of the tested molecules cytotoxicity on the CHO cell line (purchased from ATCC, ref CCL-61) was performed according to the method of Mosmann with slight modifications. Briefly, 5.10 3 cells in 100 μL of complete medium were inoculated into each well of 96well plates and incubated at 37 °C in a humidified 5% CO2. After 24 h incubation, 100 μL of medium with various product concentrations dissolved in DMSO (final concentration less than 0.5% v/v) were added and the plates were incubated for 24 h at 37 °C. Triplicate assays were performed for each sample. Each plate-well was then microscope-examined for detecting possible precipitate formation before the medium was aspirated from the wells. 100 μL of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide) solution (0.5 mg/mL in medium without FCS) were then added to each well. Cells were incubated for 2 h at 37 °C. After this time, the MTT solution was removed and DMSO (100 μL) was added to dissolve the resulting blue formazan crystals. Plates were shaken vigorously (700 rpm) for 10 min. The absorbance was measured at 570 nm with 630 nm as reference wavelength using a BIO-TEK ELx808 Absorbance Microplate Reader. DMSO was used as blank and doxorubicin (purchased from Sigma Aldrich) as positive control. Cell viability was calculated as percentage of control (cells incubated without compound). The 50% cytotoxic concentration (CC50) was determined from the dose-response curve by using the TableCurve software 2D v.5.0. CC50 values represent the mean value calculated from three independent experiments.

In vitro Cytotoxicity evaluation VERO

VERO cell line was maintained at 37 °C, 5% CO2 with 90% humidity in MEM supplemented with 10% foetal bovine serum, 1% L-glutamine (200 mM) and penicillin (100 U/mL) / streptomycin (100 μg/mL) (complete MEM medium). The evaluation of the tested molecules cytotoxicity on the VERO cell line (purchased from ATCC, ref CRL-1586) was performed according to the method of Mosmann with slight modifications. Briefly, 5.10 3 cells in 100 μL of complete medium were inoculated into each well of 96-well plates and incubated at 37 °C in a humidified 5% CO2. After 24 h incubation, 100 μL of medium with various product concentrations dissolved in DMSO (final concentration less than 0.5% v/v) were added and the plates were incubated for 48 h at 37 °C. Triplicate assays were performed for each sample. Each plate-well was then microscope-examined for detecting possible precipitate formation before the medium was aspirated from the wells. 100 μL of MTT (3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) solution (0.5 mg/mL in medium without FCS) were then added to each well. Cells were incubated for 2 h at 37 °C. After this time, the MTT solution was removed and DMSO (100 μL) was added to dissolve the resulting blue formazan crystals. Plates were shaken vigorously (700 rpm) for 10 min. The absorbance was measured at 570 nm with 630 nm as reference wavelength using a BIO-TEK ELx808 Absorbance Microplate Reader. DMSO was used as blank and doxorubicin (purchased from Sigma Aldrich) as positive control. Cell viability was calculated as percentage of control (cells incubated without compound). The 50% cytotoxic concentration (CC50) was determined from the dose-response curve by using the TableCurve software 2D v.5.0. CC50 values represent the mean value calculated from three independent experiments.

In vitro Antiplasmodial Evaluation

In this study, a K1 culture-adapted P. falciparum strain resistant to chloroquine, pyrimethamine, and proguanil was used in an in vitro culture. It was maintained in continuous culture as described previously by Trager and Jensen [START_REF] Trager | Human Malaria Parasites in Continuous Culture[END_REF]. Cultures were maintained in fresh A+ human erythrocytes at 2.5% hematocrit in complete medium (RPMI 1640 with 25 mM HEPES, 25 mM NaHCO3, 10% of A+ human serum) at 37 °C under reduced O2 atmosphere (gas mixture 10% O2, 5% CO2, and 85% N2). Parasitemia was maintained daily at between 1 and 3%. The P. falciparum drug susceptibility test was carried out by comparing quantities of DNA in treated and control cultures of parasite in human erythrocytes according to an SYBR Green I fluorescence-based method [START_REF] Guiguemde | Chemical Genetics of Plasmodium falciparum[END_REF] using a 96-well fluorescence plate reader. Compounds, previously dissolved in DMSO (final concentration less than 0.5% v/v), were incubated in a total assay volume of 200 μL (RPMI, 2% hematocrit and 0.4% parasitemia) for 72 h in a humidified atmosphere (10% O2 and 5% CO2) at 37 °C, in 96-well flat bottom plates. Duplicate assays were performed for each sample. After incubation, plates were frozen at -20 °C for 24 h. Then, the frozen plates were thawed for 1 h at 37 °C. Fifteen µL of each sample were transferred to 96-well flat bottom non-sterile black plates (Greiner Bio-one) already containing 15 µL of the SYBR Green I lysis buffer (2X SYBR Green I, 20 mM Tris base pH 7.5, 20 mM EDTA, 0.008% w/v saponin, 0.08% w/v Triton X-100). Negative control treated by solvents (DMSO or H2O) and positive controls (chloroquine and doxycycline) were added to each set of experiments. Plates were incubated for 15 min at 37 °C and then read on a TECAN Infinite F-200 spectrophotometer with excitation and emission wavelengths at 485 and 535 nm, respectively. The concentrations of compounds required to induce a 50% decrease of parasite growth (EC50 K1) were calculated from three independent experiments.

Comet assay 3.2.5.1. Cell culture and treatment

The human hepatocarcinoma cell line HepG2 was obtained from the American Type Culture Collection (ATCC, ref. HB-8065). Cells were cultured in Eagle's Minimum Essential Medium (EMEM, ref. ATCC® 30-2003TM) supplemented with 10% heat-inactivated foetal bovine serum, 100 U/mL penicillin and 0.1 mg/mL streptomycin (all from Gibco). Cells were maintained at 37 °C in a humidified atmosphere with 5% CO2. Cells were used in passage number 11 to 15. Two concentrations of the compound 3k (5.6 and 28 µM) were tested for 2 different times of incubation (2 and 72 h). Briefly, HepG2 cells were seeded at 1.13 x 10 5 cells/mL in 6-well plates (3 mL of cell suspension per well) and incubated at 37 °C in a humidified atmosphere with 5% CO2. After 24 and 94 h of incubation, cells were treated with different concentrations of the compound or the vehicle (0.5% dimethylsulfoxide, DMSO) for 72 and 2 h, respectively. Additionally, in the 2 h treatment plate, cells in an additional well were treated with 1 mM MMS as positive control for the comet assay. After treatment, medium was removed from the wells and cells were washed with phosphate buffered saline (PBS). Finally, cells were trypsinized and trypsin was neutralized with fresh medium. From this point, cells were kept ice-cold to avoid DNA repair.

Comet assay

The standard alkaline comet assay was employed for the detection of DNA strand breaks (SBs) and alkali-labile sites (ALS) in cells treated with molecule 3k. Trypsinized HepG2 cells were centrifuged at 125 g for 5 min at 4 °C and resuspended in cold PBS at 1 x 10 6 cells/mL. For the preparation of the agarose gels, 30 µL of cell suspension were mixed with 140 µL of 1% low melting point agarose in PBS at 37 °C and 2 aliquots of 70 µL of cell/agarose mixture were placed on agarose-precoated microscope slides. Each droplet was covered with a 20 x 20 mm coverslip and after 2-3 min on a cold metal plate, the coverslips were removed. Then, slides were immersed in lysis solution (2.5 M NaCl, 0.1 M Na2EDTA, 0.01 M Tris base, pH 10, and 1% Triton X-100) at 4 °C for 1 h. After lysis, slides were transferred to the electrophoresis tank and incubated for 40 min at 4 °C in the electrophoresis solution (0.3 M NaOH, 1 mM Na2EDTA, pH > 13) to allow DNA unwinding. After that, electrophoresis was carried out at 1 V/cm for 20 min (4 °C). Then, gels were neutralized and washed by immersing the slides in PBS for 10 min and distilled water for another 10 min (both at 4 °C). Gels were then air dried at room temperature. Comets were stained by adding 30 µL of 1 mg/mL of 4,6-diamidino-2phenylindole (DAPI) on top of each gel and placing 22 x 22 mm coverslips on top. Slides were incubated with DAPI at room temperature for 30 min before the analysis. The semiautomated image analysis system Comet Assay IV (Instem) was used to evaluate 50 comets per gel (i.e., 100/condition). The percentage of DNA in tail was the descriptor used for each comet.

Statistics

The median percentage of DNA in tail for 50 comets was calculated for each of the duplicate gels in each experiment, and the mean of the two medians was then calculated. The mean percentage of DNA in tail of 3 independent experiments and the standard deviation (SD) were calculated. 3.2.6. APICOPLAST 3.2.6.1 Culturing Plasmodium-infected red blood cells Plasmodium falciparum blood stage parasites were maintained at 2% hematocrit in 1640 RPMI-HEPES supplemented with 5% AlbuMAX II (GIBCO) and 0.25% gentamycin. Parasites were grown sealed Perspex chambers gassed with beta mix gas (1% O2, 5% CO2 and 94% N2) at 37 °C and maintained on 48-hour cycles. Cultures were tightly synchronized at ring stage using sorbitol treatment (5% v/v) as previously described [START_REF] Amiar | Division and Adaptation to Host Environment of Apicomplexan Parasites Depend on Apicoplast Lipid Metabolic Plasticity and Host Organelle Remodeling[END_REF].

IFA on treated parasites

Prior to the treatment parasites are synchronized using 5% sorbitol. After 48h treatment (0.3 µM of (3k) molecule, 0.3 µM of (2a) molecule or DMSO) parasites are fixed using 4% paraformaldehyde (PFA) and 0.0075% glutaraldehyde for 30 minutes at room temperature. Fixing solution is washed 3x times with PBS and cells are permeabilized with 0.1% TX-100 for 10 minutes at room temperature. Permeabilization solution is washed 3x times with PBS and cells are blocked with 3% fetal bovine serum (FBS) for 1 h. Primary antibody (Rat IgG anti-HA, Roche, 1/500 in 3% FBS) is incubated 1h at room temperature. Primary antibody is washed out 3x times with PBS and cells are incubated with secondary antibody (Alexa Fluor 488 goat anti-mouse IgG, Invitrogen, 1/1000 in 3% FBS) for 1h at room temperature. Secondary antibody is washed out 3x times with PBS and cells are incubated with

Figure 3 .

 3 Figure 3. Chemical structures of antiapicomplexe biocids.

Figure 4 .

 4 Figure 4. Comparison of the in vitro antiplasmodial activities of hit molecule 3k with analogs 3r, 2k and 3s. Evaluation on P. falciparum apicoplast

Figure 5 .

 5 Figure 5. (3k) molecule treatment affects apicoplast biogenesis. (A, B) Triose Phosphate Transporter (TPT) fluorescent signal is shown in green (TPT labeled with an 3xHA tag). DAPI, as a marker for the nucleus, is shown in blue. Apicoplasts of treated parasites with 0.3 µM of (3k) molecule or 0.3 µM of (2k) molecule are compared to normal plastid visualized in DMSO-treated control. The microscopic images were obtained by epifluorescence microscope. DIC, differential interference contrast. (C) Plastid associated with the delayed death of P. falciparum trophozoite was observed after treatment with molecule (3k) on three life cycles (n = 3, error bar = standard deviation, SD).

1 H

 1 NMR (250 MHz, CDCl3) δ 8.08 -7.84 (m, 2H), 7.79 -7.62 (m, 2H), 2.82 (s, 3H). 13 C NMR (63 MHz, CDCl3) δ 152.9, 147.9, 141.0, 141.0, 130.2, 130.1, 128.5, 128.2, 23.4. Spectral data matched with literature [43].

1 H

 1 NMR (400 MHz, CDCl3) δ 8.25 -8.20 (m, 1H), 8.15 -8.09 (m, 1H), 7.99 -7.86 (m, 2H).

3. 1 . 5 . 2 . 1 . 2 - 3 . 1 . 5 . 2 . 2 . 2 -

 15212315222 (Phenylthio)quinoxaline (3r) Yield 90% (279 mg). orange solid. Mp 88 °C (isopropanol). 1 H NMR (400 MHz, CDCl3) δ 8.44 (s, 1H), 7.99 (dd, J = 8.2, 1.4 Hz, 1H), 7.90 (dd, J = 8.3, 1.3 Hz, 1H), 7.73 -7.60 (m, 4H), 7.50 -7.44 (m, 3H). 13 C NMR (101 MHz, CDCl3) δ = 157.3, 143.6, 142.3, 140.0, 135.1 (2C), 130.6, 129.9 (2C), 129.8, 129.3, 129.1, 128.9, 128.4. LC-MS (ESI+) tR 3.4 min, m/z [M+H] + 239.16. MW: 238.31 g.mol -1 , purity 99%. HRMS m/z [M+H] + calcd for C14H10N2S: 239.0637, Found: 239.0638. (Phenylthio)-3-(trifluoromethyl)quinoxaline (3s) Yield 89% (354 mg). white solid. Mp 93 °C (isopropanol). 1 H NMR (400 MHz, CDCl3) δ 8.14 -8.07 (m, 1H), 7.75 -7.67 (m, 3H), 7.66 -7.61 (m, 2H), 7.51 -7.45 (m, 3H). 13 C NMR (101 MHz, CDCl3) δ = 153.5, 143.0, 139.

Table 3 .

 3 Cytotoxic and genotoxic data for hit compound (3k).

	Entry	CC50 HepG2	CC50 CHO	CC50 Vero	Comet
		(µM)	(µM)	(µM)	assay a
		56.0 ± 11.0	58.5 ± 3.9	63.5 ± 8.6	Negative

a Comet assay was performed on HepG2 cell line at 5.6 and 28 µM for 2 or 72 h.

DAPI, 1/25000 in PBS. Samples are fixed between a slide and a coverslip with fluorogel and observed by epi-fluorescent microscopy.

Growth assay

To observe a potential effect of delayed death of the molecule on the parasite, Plasmodium is maintained on Three life cycles (48h). At 48 h, 96 h and 144 h, 100 µL of cultures are transferred into a 96 wells black wall flat bottom plate and mixed with 100 µL SYBR Green lysis buffer (20 mM Tris, pH 7.5; 5 mM EDTA; 0.008 % (w/v) saponin; 0.08% (v/v) Triton X-100) with freshly added SYBR Green I (10000X), and incubated 1h at room temperature protected from the light. Fluorescence from each well is measured with TECAN infinite M200 plate reader (excitation: 485 nm, emission: 538 nm and integration time: 1000 μs). The rest of the cultures are diluted 1/10 as the untreated cultures. Graph is obtain by doing the ration the treated culture fluorescence intensity on the untreated culture fluorescence intensity (n= 3). 3.2.7. Study on the liver stage 3.2.7.1. Parasite strain and sporozoites isolation P. falciparum (NF135 strain) sporozoites were isolated by dissection of the salivary glands of infected A. stephensi 14-21 days after an infective blood meal (Department of medical microbiology, University Medical Centre, St Radboud, Nijmegen Netherland). All infected salivary glands were removed by hand dissection and crushed in a potter for sporozoites isolation and filtrated through a 40 μm filter to remove mosquito debris (Cell Strainer, BD BioSciences, USA). The sporozoites were counted using a disposable Glasstic microscope slide (KOVA, USA).

Primary hepatocytes culture

Conserved primary human hepatocytes were purchased from Biopredic International (France). Cells were seeded in 96-well plates (Falcon by Becton Dickinson Labware Europe, France) coated with collagen I (BD Biosience, USA), at a density of 80 000 cells per well. They are cultured at 37 °C in 5% CO2 in William's E medium (Gibco, Life Technologies, Saint Aubin, France) supplemented with 10% of Foetal Bovine Serum FCIII, 5x10 -5 M hydrocortisone hemisuccinate (Upjohn Laboratoires SERB, France), 5 µg per ml Insulin (Sigma Aldrich, USA), 2 mM L-glutamine, and 0.02 U per ml -0.02 µg per ml penicillin -streptomycin (Life Technologies) until infection with sporozoites.

In vitro infection and drug assays

P. falciparum sporozoites were re-suspended in the above complete medium used for hepatocytes culture. Cultured human hepatocytes were inoculated with P. falciparum sporozoites (30,000 sporozoites/well of 96 well plates each) in 50 µl of complete media. The infected culture plate was centrifuged 10 min at 2000 rpm to allow fast parasite sedimentation onto the target cells and further incubated with serial dilution of drugs that were prepared in advance. After 3 hours incubation, cultures were washed and further incubated in fresh medium containing the appropriate drug concentration which were changed daily until cells fixation at day 6 post infection with 4%PFA for 20 min at room temperature. The exo-erythrocytic forms (EEFs) were stained using the anti-HSP70 serum for P. falciparum and revealed with an Alexa 488-conjugated goat anti-mouse immunoglobulin (Molecular Probes). Host cell and parasite nuclei were labelled with 4′,6-diamidino-2-phenylindole (DAPI). The EEFs were counted under a fluorescence microscope (Leica DMI4000B) or by using a CellInsight High Content Screening platform equipped with the Studio HCS software (Thermo Fisher, Scientific). To investigate the effect of drugs inhibition on the apicoplast maturation during liver-stage development, the EEFs were stained using Acyl carrier protein (ACP) antibody and revealed with an Alexa Fluor 680conjugated goat anti-rabbit immunoglobulin (Molecular Probes), and then imaged using a confocal microscope (Olympus FV1200). The images were analyzed using ImageJ software.

Data analysis

GraphPad Prism 7 statistical Software (GraphPad. Software, San Diego, CA, USA) were used for data analysis and graphing. All values were expressed as means and standard deviations (SD).