Dionissia A Pepe

Dimitra Toumpa

Christiane André-Barrès

Christophe Menendez

Elisabeth Mouray

Michel Baltas

Philippe Grellier

Dionissios Papaioannou

Constantinos M Athanassopoulos

Synthesis of Novel G Factor or Chloroquine-Artemisinin Hybrids and Conjugates with Potent Antiplasmodial Activity

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

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

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-10-carba-substituted ART with improved ADMET properties. In that respect we can mention as attractive intermediate the acid (1) [START_REF] Ma | Syntheses and Antimalarial Activities of 10-Substituted Deoxoartemisinins[END_REF][START_REF] Woodward | 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] or ART hybrids (see below), such as the anilide 2. [START_REF] Woodward | 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] 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 | 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, [START_REF] Ariey | A molecular marker of artemisinin-resistant Plasmodium falciparum malaria[END_REF] 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 | Plasmodium falciparum resistance to artemisinin-based combination therapies: A sword of Damocles in the path toward malaria elimination[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. [START_REF] Kepler | 1,2,4-Trioxanes as potential antimalarial agents[END_REF] 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. [START_REF] Dechy-Cabaret | Synthesis and antimalarial activity of trioxaquine derivatives[END_REF] 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 multi-targeted 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] The synthesis of ART conjugates 5, 6 and 7 requires as key-intermediate the ARTderived carboxylic acid 1. This compound was prepared (Scheme S1, Supporting Information) in 58% overall yield from ART, according to a five-steps published procedure. [START_REF] Ma | Syntheses and Antimalarial Activities of 10-Substituted Deoxoartemisinins[END_REF][START_REF] Woodward | 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] 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 4,7-dichloroquinoline through the nucleophilic aromatic substitution of the latter with 1,2-diaminoethane 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 [START_REF] Woodward | 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] 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 ART-derived 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 Table S1, Supporting Information), the measured IC50 values or the % inhibition at 10 nΜ, in cases IC50 values could not be determined, and SIs for the synthesized hybrids/conjugates are tabulated.

IC50 values for ART, CQ and endoperoxide GMeP (3) are also provided for the sake of comparison. 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 (IC50=124.3, SI=144) with the conjugates 13 (IC50>167) and 14 (IC50>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 CQ-resistant 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 effective, 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.

  of endoperoxide-GMeP (3) with ACQ was also synthesized. Conjugates of the second type (Figure 2) 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).

  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).

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) HBTU,

Table 1 .

 1 Evaluation of antiplasmodial activities of synthesized compounds with CQresistant P. Falciparum FcB1/Colombia strain and selectivity indexes (SI). of ART with endoperoxide GMeP is the most potent in this series of compounds with an IC50 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 (IC50=2.6, SI=16,372), bearing one molecule of ART, with the conjugates 9 (IC50=8.4, SI=2,108) and 10 (IC50=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 IC50 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.

	Compd	IC50 (nΜ) or	SI	Compd	IC50 (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 hybrids/con-
	jugates of ART, incorporating one or two molecules of ART per hybrid/conjugate, are
	significantly more potent (IC50 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. In partic-
	ular the hybrid 5

at 10 nM) and 10 (IC50=10.6, SI=983) and 12 (IC50=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

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. Dedication: This article is dedicated to the memory of our good friend, Professor Maurizio Botta.

AUTHOR INFORMATION

Corresponding Authors *C.M. Athanassopoulos; Email: kath@chemistry.upatras.gr ; P. Grellier; Email: philippe.grellier@mnhn.fr M. Baltas; Email: baltas@chimie.ups-tlse.fr

Conflicts of Interest:

The authors declare no conflict of interest.