Anurag Kumar

Abderrahmane Hamdi

Yannick Coffinier

Ahmed Addad

Pascal Roussel

Rabah Boukherroub
email: rabah.boukherroub@iri.univ-lille1.fr

Suman L Jain

Visible light assisted oxidative coupling of benzylamines using heterostructured nanocomposite photocatalyst

Keywords: Photocatalysis, oxidative coupling, quantum dots, hybrid catalyst, benzylamine

Highlights of the research

 Facile synthesis of hybrid nanocomposite consisting of copper (I) oxide and carbon quantum dots.  Heterogeneous photocatalyst for organic transformation  Visible light assisted oxidative coupling of benzyl amine  A green, economical and efficient photochemical approach  Higher activity and robustness of hybrid photocatalyst than neat Cu2O and CQDs.  Facile recovery and efficient recycling of the photocatalyst.

Sunlight is a unique natural energy source which is inexpensive, nonpolluting and abundantly available. Hence, the replacement of high-energy synthetic processes with clean, cost effective and green photochemical approaches has become an area of tremendous importance in recent decades [START_REF] Yoon | Visible light photocatalysis as a greener approach to photochemical synthesis[END_REF]. So far, many semiconductor photocatalysts have been reported for organic transformations [START_REF] Lang | Heterogeneous visible light photocatalysis for selective organic transformations[END_REF]; however, less efficiency, limited visible light absorbance and poor conversion rates are certain drawbacks associated with these methodologies [START_REF] Zhang | Metal-organic frameworks for artificial photosynthesis and photocatalysis[END_REF]. Copper(I) oxide (Cu2O) is a promising visible light active semiconductor which possesses some unique properties such as narrow band gap (2.2 eV), low cost, non-toxicity, better environmental acceptability and good adsorption of molecular oxygen [START_REF] Huang | Preparation of cuprous oxides with different sizes and their behaviours of adsorption, visible-light driven photocatalysis and photocorrosion[END_REF]. Owing to these physical and chemical properties, Cu2O has extensively been investigated in solar conversion processes and catalysis [START_REF] Wang | Enhanced Photoreduction CO2 Activity over Direct Z-Scheme α-Fe2O3/Cu2O Heterostructures under Visible Light Irradiation[END_REF].

Furthermore, the coupling of Cu2O with other semiconductors to form heterostructured composites is a promising approach for enhancing the charge separation as well as photo-activity [START_REF] Zhang | TiO2 Film/Cu2O microgrid heterojunction with photocatalytic activity under solar light irradiation[END_REF]. In this regard, a number of heterostructured composites consisting of Cu2O and semiconductors like TiO2, CdS, ZnO and CuO have been reported for numerous photocatalytic applications [START_REF] Wang | Design of a Novel Cu2O/TiO2/Carbon Aerogel Electrode and its Efficient Electrosorption-assisted visible light photocatalytic degradation of 2,4,6-trichlorophenol[END_REF].

In the recent years, carbon quantum dots (CQD), novel nanosized carbon materials with less than 10 nm, have gained significant interest in the area of catalytic and photocatalytic applications. Owing to their excellent optical and electronic properties including higher absorbance and superior charge transfer, these materials have widely used in photocatalytic applications [START_REF] Li | Carbon nanodots: synthesis, properties and applications[END_REF]. In addition, low cost, non-toxicity, facile functionalization, good biocompatibility and chemical inertness make CQD as the material of choice in place of commonly known inorganic semiconductor and organic dye photocatalysts [START_REF] Maaoui | A Prussian blue/carbon dot nanocomposite as an efficient visible light active photocatalyst for C-H activation of amines[END_REF]. In recent years, photocatalytic oxidation processes using visible light irradiation are gaining considerable interest for the maximum utilization of solar energy as it consists of nearly 45% of the solar spectrum [START_REF] Dong | Recent developments in heterogeneous photocatalytic water treatment using visible lightresponsive photocatalysts: a review[END_REF]. In addition, most of the photo-catalytic transformations occur under ambient conditions which are advantageous over thermocatalytic processes, which require higher reaction temperatures [START_REF] Zhang | Transforming CdS into an efficient visible light photocatalyst for selective oxidation of saturated primary C-H bonds under ambient conditions[END_REF]. Among the various oxidation processes, oxidative coupling of amines to corresponding imines is an important chemical transformation in organic synthesis. These compounds have found wide applications as synthetic intermediates in the preparation of bioactive molecules of therapeutic interest [START_REF] Zhang | Iron-catalyzed direct synthesis of imines from amines or alcohol and amines via aerobic oxidative reactions under air[END_REF]. Conventional oxidation methods involve the use of stoichiometric oxidants such as 2-iodoxybenzoic acid, N-test-butylphenylsulfinimidoyl chloride, which produce copious amounts of undesirable waste and are deterimental to the environment. Subsequently, catalytic methods using ruthenium based catalysts such as RuCl3, [RuCl2(RCH2NH2)2(PPh3)2], ruthenium (II) porphyrin, ruthenium-hydroxyapatite, Ru2(OAc)4Cl and Ru/Al2O3 in conjunction with molecular oxygen as oxidant have been reported for this transformation [START_REF] Gomez | Diastereospecific and Diastereoselective synthesis of Ruthenium(II) complexes using N, N'-bidentate ligands aryl-pyridin-2-ylmethyl-amine ArNH-CH2-2-C5H4N and their oxidation to imine ligands[END_REF]. However, very few reports are known on the photocatalytic oxidation of benzylic amines to the corresponding imines using O2 as an oxidant. In this context, Su and coworkers reported mesoporous graphite carbon nitride (mpg-C3N4) for the oxidation of benzylic alcohols and amines with O2 under visible light [START_REF] Su | Aerobic Oxidative coupling of amines by carbon nitride photocatalysis with visible light[END_REF]. Higher pressure (0.5 MPa) and need of halogenated solvent (trifluorotoluene) make this methodology of limited applicability. Furukawa et al. [START_REF] Furukawa | Selective amine oxidation using Nb2O5 photocatalyst and O2[END_REF] described niobium oxide/O2 system for the photo-oxidation of amines. Apart from these semiconductor photocatalysts, recently, Berlicka et al. [START_REF] Berlicka | Porphycene-mediated photooxidation of benzylamines by visible light[END_REF] investigated porphyrin or porphycene-mediated photo-oxidation of primary amines to N-benzylidene benzylamines in excellent conversion yields. Despite the excellent activity of these dyes under visible light, homogeneous nature and non-recyclability are the obvious drawbacks associated with this system. Hence, the development of heterogeneous photocatalyst along with the use of low cost

A C C E P T E D M A N U S C R I P T
metals under visible light irradiation is highly desired from both environmental and economic viewpoints. Raza et al. [START_REF] Raza | Visible-light-driven oxidative coupling reactions of amines by photoactive WS2 nanosheets[END_REF] studied oxidative coupling reactions of amines by photoactive WS2 nanosheets. More recently, Kumar et al. [START_REF] Kumar | A [Fe(bpy)3] 2+ grafted graphitic carbon nitride hybrid for visible light assisted oxidative coupling of benzylamines under mild reaction conditions[END_REF] reported an efficient semiconductor-metal complex hybrid photocatalyst, namely, graphitic carbon nitride (g-C3N4) and iron (III) bipyridine complex for the oxidation of the benzylic amines with molecular oxygen under visible light irradiation.

However, the multi-step synthesis of the hybrid photocatalyst makes the developed methodology of limited practical applicability.

In this paper, we report an efficient and simple visible light assisted heterostructured nanocomposite consisting of cuprous oxide (Cu2O), carbon quantum dots (CQD) i.e. Cu2O/CQD catalyzed photo-oxidation of benzylamines using molecular oxygen as an oxidant to the corresponding imines under ambient conditions (Scheme 1). Scheme 1: Photo-oxidation of benzylamines.

Experimental Section

Materials

Fructose, sodium hydroxide (NaOH), and copper perchlorate (Cu(ClO4)2) were obtained from Sigma Aldrich (France) and used as received. PHl 5000 VersaProbe -Scanning ESCA Microprobe (ULVAC-PHI, Japan/USA) instrument at a base pressure below 5×10 -9 mbar. Monochromatic AlKα radiation was used and the X-ray beam, focused to a diameter of 100 µm, was scanned on a 250×250 µm surface, at an operating power of 25 W (15 kV). Photoelectron survey spectra were acquired using a hemispherical analyzer at pass energy of 117.4 eV with 0.4 eV energy step. Core-level spectra were acquired at pass energy of 23.5 eV with 0.1 eV energy step. All spectra were acquired at 90° between X-ray source and analyzer and with the use of low energy electrons and low energy argon ions for charge neutralization. After subtraction of the Shirley-type background, the core-level spectra were decomposed into their components with mixed Gaussian-Lorentzian (30:70) shape lines using the CasaXPS software. Quantification calculations were performed using sensitivity factors supplied by PHI. The sample was prepared by casting a concentrated aqueous solution of the material onto silicon substrate followed by drying at 100 °C for 1 h.

A C C E P T E D

M A N U S C R I P T

Synthesis of carbon dots

Carbon quantum dots (CQDs) were synthesized by a one-pot method at a relatively low reaction temperature [START_REF] Maaoui | Non-Enzymatic Glucose Sensing Using Carbon Quantum Dots Decorated with Copper Oxide Nanoparticles[END_REF]. In a typical procedure, fructose (500 mM) and sodium hydroxide (500 mM) were added to 20 mL water. The resulting solution was placed into a Teflon-lined stainless-steel autoclave and heated at 50 °C for 1 h. The solution turned from a colorless into brown.

Synthesis of Cu2O/CQDs

Copper perchlorate (1.35 g) was dissolved in 10 mL of previously prepared CQDs solution and heated at 80 °C for 3 h. The formed precipitate was separated by centrifugation, washed with water three times and annealed at 120 °C overnight [START_REF] Maaoui | Non-Enzymatic Glucose Sensing Using Carbon Quantum Dots Decorated with Copper Oxide Nanoparticles[END_REF]. For comparison, Cu2O was prepared by the same procedure using copper chloride and sodium hydroxide without adding fructose as mentioned above. The Cu content in the Cu2O/CQDs was determined by ICP-AES which was found to be 38.04 wt% (2.9 mmol Cu/g catalyst).

Photocatalytic oxidative coupling of benzylamines

All the photocatalytic experiments were carried out by using a 20 watt white cold LED light (Model No. HP-FL-20W-F-Hope LED Opto-Electric Co., Ltd, λ >400 nm) as a light source. In a typical experiment, benzylamine (1 mmol) and photocatalyst (25 mg) were mixed in acetonitrile (10 mL). The resulting mixture was irradiated with visible light with stirring for 8 h in the presence of molecular oxygen. The progress of the reaction was monitored by TLC. After completion of the reaction, the photocatalyst was separated by filtration and the obtained residue was concentrated under reduced pressure to obtain a crude product. The pure product was obtained via purification using column chromatography on silica gel by using ethyl acetate: hexane (9:1) as eluent.

Synthesis and characterization of photocatalyst

The Cu2O/CQDs investigated in this work was prepared according to a previous protocol published by some of us [START_REF] Maaoui | Non-Enzymatic Glucose Sensing Using Carbon Quantum Dots Decorated with Copper Oxide Nanoparticles[END_REF]. The morphology of the synthesized Cu2O/CQD nanocomposite was investigated by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM image of the synthesized nanocomposite as shown in Figure 1a revealed nanoparticles in the aggregated form. The TEM image of the prepared nanocomposite indicated Cu2O NPs of 20-30 nm in size (Fig. 1b,c). In addition, in the HR-TEM image of a single Cu2O NP an interplanar distance of 0.21 nm between adjacent planes, related to the interplanar spacing of the (002) plane of Cu2O was observed (Fig. 1d). The appearance of five diffraction rings in the selected-area electron diffraction (SAED) pattern corresponding to the (022), ( 112), (002), (111), and (011) planes of Cu2O suggested the poly crystalline nature of the sample (Fig. 1e).

X-ray diffractogram of Cu2O/CQD composite as shown in Figure 2 (311) crystalline planes, respectively. Indeed, very weak peaks of Cu appeared in the XRD pattern of Cu2O/CQD. However, the material is mostly Cu2O and also the chemical transformation is a photochemical process, the contribution of Cu is believed to be negligible.

Hence we have denoted active photocatalyst as Cu2O/CQD. Additionally, a small peak at ~22.8° confirmed the presence of carbon from the CQD. These characterization results were found to be in good agreement with our previous reports on reduced graphene oxide modified with Cu2O [START_REF] An | Cu2O/Reduced graphene oxide composites for the photocatalytic conversion of CO2[END_REF], and Cu2O modified multi-walled carbon nanotube nanostructures [START_REF] Zhou | Facile synthesis of nanospindlelike Cu2O/straight multi-walled carbon nanotube hybrid nanostructures and their application in enzyme free glucose sensing[END_REF] and CQD [START_REF] Li | Carbon quantum dots/octahedral Cu2O nanocomposites for non-enzymatic glucose and hydrogen peroxide amperometric sensor[END_REF]. In t e n s ity (a .u .) The UV-Vis spectra of nanocomposite Cu2O/CQD and its components are shown in Figure . 3.

2 t h e t a ( °) C Q D s Cu 2 O (1 1 0 ) Cu 2 O (1 1
The UV-vis absorption spectrum of CQDs in water revealed a main absorption peak at 293 and a shoulder at 340 nm attributed to the π-π* transition of the conjugated C=C and the n-π* transition of the C=O, respectively [START_REF] Maaoui | Non-Enzymatic Glucose Sensing Using Carbon Quantum Dots Decorated with Copper Oxide Nanoparticles[END_REF]. Interestingly, both Cu2O and Cu2O/CQD absorb remarkably in the visible region, making these materials suitable for photocatalytic processes under visible light irradiation. The band gap of Cu2O was determined with the help of Tauc plot which was found to be 2.2 eV (Fig. S3). According to the band gap, the absorption edge wavelength for Cu2O was estimated to be 563 nm. The chemical composition of the synthesized nanocomposite was examined by X-ray photoelectron spectroscopy (XPS). In survey scan as shown in Figure S1, the appearance the corresponding peaks of all the desired elements such as Cu3s (75.0 eV), Cu3p (122.5 eV), C1s

(285.0 eV), O1s (531.5 eV) and Cu2p (532 eV) confirmed the successful synthesis of the material.

The high-resolution XPS spectrum of C1s (Fig. 3) revealed the main peak at ~285 eV due to C-H, C-C and C=C bonds from the CQD along with two components at 286.2 and 288.6 eV assigned to C-O and C=O bonds, respectively.

The XPS high-resolution spectrum of Cu2p displayed two peaks at 932.5 and 952.4 eV attributed to Cu2p3/2 and Cu2p1/2, respectively from Cu2O (Fig. 4). The XPS high-resolution spectrum of O1s exhibited peaks at 530.7 and 531.8 eV respectively, due to the oxygen bonded to the carbon skeleton of the CQD and Cu-O in Cu2O (Fig. S2).

Catalytic Activity

The photocatalytic activity of the synthesized nanocomposite Cu2O/CQD and its components were checked for the oxidation of benzylamine with O2 in acetonitrile solvent under visible light irradiation at ambient temperature and pressure conditions (Scheme 1). At first, optimization experiments using variable reaction parameters were performed and the results of these experiments are summarized in Table 1. In a blank reaction, no product was formed even in the prolonged time (24h) in the absence of photocatalyst under otherwise identical conditions. (Table 1, entry 1). Similarly, in the absence of a visible light source, no reaction was occurred (Table 1, entry 2), suggesting that the reaction was truly photocatalytic in nature. However, in the presence of visible light irradiation, the oxidation of benzylamine (1 mmol) using 25 mg Cu2O/CQD as a photocatalyst in CH3CN under an oxygen atmosphere at an ambient temperature (25 o C) and pressure (1 atm) afforded an almost quantitative yield of the corresponding imine (Table 1, entry 3). Furthermore, the use of neat Cu2O or CQDs as photocatalysts under identical conditions

A C C E P T E D M A N U S C R I P T
afforded only 12 and 30% conversion, respectively (Table 1, entry 4-5). Furthermore, the effect of various solvents was studied to select the optimum reaction medium. Among the different solvents such as methanol, dimethylformamide, tetrahydrofuran, acetonitrile, and ethanol (Table 1, entries 3, 6-9) studied, acetonitrile (CH3CN) was found to be the best solvent for the reaction (Table 1, entry 3). Molecular oxygen played an essential role and in its absence (irradiation under nitrogen atmosphere), a negligible conversion was achieved (Table 1, entry 10). Although the reaction occurred efficiently at room temperature (24 o C) at 1 atmospheric pressure, to check the effect of temperature, the reaction was performed at 50 o C under described conditions (Table 1, entry 3). A marginal enhancement in the conversion (97.5%) was observed at higher temperature; therefore we selected room temperature as the optimum temperature for the reaction. Reaction conditions: benzylamine (1 mmol), photocatalyst (25 mg), solvent (10 mL), light source: white cold LED λ > 400 nm, time: 8-24 h under oxygen balloon; Power at reaction vessel: 70 W/m 2 ;

[b] determined by GC-MS; [c] TON is defined as the amount of product formed to that of the active site Cu in the photocatalyst; [d] at 50 o C temeprature; [e] in the absence of molecular oxygen under N2 atmosphere.

A C C E P T E D

M A N U S C R I P T
Furthermore, the reaction was generalized to variously substituted benzylamines under optimized reaction conditions using Cu2O/CQD/O2 system under visible light at room temperature. The results of these reactions are summarized in Table 2. All the substrates afforded corresponding imine products selectively without observing any by-product. Moreover, marginal substituent effect was observed and all the substrates containing either electron-releasing or withdrawing groups were smoothly and efficiently converted to the resultant N-benzylidene benzylamines in good to excellent yields (75-95%). The slightly higher activity of the electron rich amines might be due to the easy formation of an intermediate imine. Furthermore, o-substituted benzylamine displayed slightly poor product yield as compared to the p-isomer, which is mainly due to the obvious reason of steric hindrance (Table 2, entry 4). Secondary benzylamine (dibenzylamine) also showed high reactivity and afforded moderate product yield (Table 2, entry 8). However, cyclohexylamine was not reacted under the described conditions (Table 2, entry 10). Further, to check the recyclability of the material, recycling experiments were performed by choosing benzylamine as a representative example. After completion of the reaction; the photocatalyst was separated by simple filtration, washed with acetonitrile, dried and reused in the subsequent experiments for six runs (Fig. 5). Almost similar conversion and product yield were obtained in all cases, suggesting that the synthesized photocatalyst was highly stable in nature.

Furthermore, to ascertain the leaching of metal, selected filtrate samples of recycling Although, the exact mechanism of the reaction is not clear at this stage, based on our experimental findings and existing literature reports [START_REF] Raza | Visible-light-driven oxidative coupling reactions of amines by photoactive WS2 nanosheets[END_REF] a plausible mechanism of the reaction is depicted in Scheme 2. As shown in a controlled experiment that oxygen played a pivotal role and in its absence the reaction did not occur (Table 1, entry 10). It is believed that after absorbing the light, charge separation takes place in photocatalyst Cu2O/CQD. The photogenerated electrons were excited from the VB to the CB of Cu2O. Subsequently the photo-generated electrons were transferred to CQD, which act as electron collector, transporter to provide better mobility and charge separation. Furthermore, molecular oxygen gets excited under light and converted to the singlet oxygen which can accept an electron from the conduction band of nanocomposite to give superoxide radical. To confirm the formation of superoxide radical, the reaction was performed by adding a common radical scavenger, i.e. p-benzoquinone (BQ). A significant decrease in the

Formation of singlet oxygen in Cu2O/CQD system under visible light irradiation

The formation of singlet oxygen ( 1 O2) during the photocatalytic oxidative coupling of benzylamine was investigated by adding sodium azide (NaN3), a quencher of 1 O2. For this study, a comparison was made between the original photocatalytic experiment and that obtained after the addition of sodium azide (NaN3) at different time intervals (Fig. 6). As shown in Fig. 6, the

Conclusions

We have demonstrated an efficient, readily synthesized and cost effective photocatalyst i.e.

heterostructured nanocomposite consisting of cuprous oxide and carbon quantum dots (Cu2O/CQD) for the oxidative coupling of benzylamines using molecular oxygen as oxidant under visible light at ambient conditions. The developed nanocomposite Cu2O/CQD exhibited superior activity as compared to bare Cu2O and CQD. In addition, the nanocomposite showed

  SEM images were obtained using an electron microscope Hitachi S4700 equipped with a cold field emission emitter and two different detectors (high efficiency In-lens SE detector, Everhart-Thornley Secondary Electron Detector). The samples were prepared by casting 50 μL aqueous of Cu2O-CQDs composite on a clean silicon wafer followed by drying in an oven at 80 °C to remove adsorbed water. Transmission electron microscopy (TEM) imaging was performed on a FEI Tecnai G2-F20 microscope operating at an accelerating voltage of 200 kV. Powder X-ray diffraction (XRD) patterns were collected on a Bruker D8 advance diffractometer (Cu-Kα radiation, 1.54056 Å) with an applied voltage of 40 kV and an anode current of 40 mA in the 2θ range of 10-80°. UV/Vis absorption spectra were recorded using a Perkin Elmer Lambda UV/Vis 950 spectrophotometer in quartz cuvettes with an optical path of 10 mm. The wavelength range was 200-800 nm. X-ray photoelectron spectroscopy (XPS) experiments were performed in a

Figure 1 .

 1 Figure 1. SEM (a), low (b) and high (c) resolution TEM images, (d) HR-TEM of a nanoparticle

  exhibited several reflections having at least two different crystalline phases of Cu (ICDD No. 65-9026) and Cu2O (ICDD No. 78-2076). The diffraction planes of the Cu phase observed at 43.2° and 50.3° were corresponded to the Cu(111) and Cu(200), respectively. The other diffraction peaks at 29.5°, 36.5°, 42.2°, 61.3°, and 73.7° were related to the Cu2O (110), Cu2O (111), Cu2O (200), Cu2O (220) and Cu2O

Figure 2 .

 2 Figure 2. Powder X-ray diffraction pattern of Cu2O/CQD.

Figure 3 .

 3 Figure 3. UV-Vis spectra of CQD, Cu2O/CQD and Cu2O

Figure 4 .

 4 Figure 4. High-resolution XPS spectra of C1s and Cu2p of Cu2O/CQD.

a

  Reaction conditions: benzylamine (1 mmol), Cu2O/CQD (25 mg), acetonitrile (10 mL) under visible light irradiation with white cold LED λ > 400 nm for 8 h; Power at reaction vessel: 70 W/m 2 ; b determined by GC-MS; c Isolated yield.

  experiments were subjected to ICP-AES analysis. No metal trace was detected in the solution phase, indicating no leaching had occurred and the reaction was truly heterogeneous in nature.

Figure 5 .

 5 Figure 5. Results of recycling experiments.

Scheme 2 :

 2 Scheme 2. In the final step, superoxide radical abstracts a hydrogen atom from the benzylic

  conversion of benzylamine using Cu2O/CQDs photocatalyst with oxygen under visible light irradiation was found to be 21, 58, 87.4 and 97.5% after 2, 4, 6 and 8h, respectively in the absence of NaN3. However, the conversion was reduced significantly, and maximum conversion reached only up to 12.5% after 8h when NaN3 was added into the system. These experimental results confirmed the generation of singlet oxygen ( 1 O2) in the photocatalytic system under visible light irradiation.

Figure 6 .

 6 Figure 6. Conversion of benzylamine using Cu2O/CQD photocatalyst with and without adding

  excellent stability and efficient recyclability without any detectable leaching during the reaction.The superior performance of Cu2O/CQD was mainly attributed to the enhanced charge transportation, effective separation of the photogenerated electron-hole pair and improved contact between photocatalyst and substrate molecules. Owing to the low cost and better efficiency, such Cu2O/CQDs nanocomposite can be used as a promising alternative to the noble metals (Au, Pd, Pt etc) in improving the photocatalytic ability of semiconductor photocatalysts.

Table 1 :

 1 Optimization of oxidative coupling of benzylamine with molecular oxygen under

	different reaction conditions [a]
	Entry	Visible	Solvent	Photocatalyst Time	Conv.	TON [c]	TOF
		light		(h)		(%) [b]		(h -1 )
	1	Yes	CH3CN	No	24	-	-	-
	2	No	CH3CN	Cu2O/CQDs	24	-	-
	3	Yes	CH3CN	Cu2O/CQDs	8	97, 97.5 [d]	13.8 13.9	1.7 1.7
	4	Yes	CH3CN	Cu2O	10	15	-	-
	5	Yes	CH3CN	CQDs	10	30	-	-
	6	Yes	MeOH	Cu2O/CQDs	10	74	10.6	1.1
	7	Yes	DMF	Cu2O/CQDs	10	92	13.1	1.3
	8	Yes	THF	Cu2O/CQDs	10	54	7.7	0.8
	9	Yes	EtOH	Cu2O/CQDs	10	75	10.7	1.1
	10 [d]	Yes	CH3CN	Cu2O/CQDs	8	trace	-	-
	[a]

Table 2 :

 2 Cu2O/CQD-catalyzed oxidative coupling of benzylamines[a]

	Entry	Substrate	Product	Conv. (%) [b]	Yield (%) [c]
	1		N	97	95
		NH 2
	2
		H 3 CO
		NH

Acknowledgments

Authors are thankful to Director IIP for granting permission to publish these results. AK is thankful to CSIR, New Delhi, for providing research fellowships. The CNRS, the University of Lille and the Hauts-de-France region are acknowledged for financial support.


Jérémy Ruiz

Joëlle Azéma

Corinne Payrastre

Michel Baltas

Béatrice Tuccio

Henri Vial

Christiane André-Barrès

Antimalarial bicyclic peroxides belonging to the G-factor family: mechanistic aspects of their formation and iron (II) induced reduction

Keywords: malaria, cyclic peroxide, C-centered radical, hybrid molecules

Artemisinin and its derivatives are peroxide-containing compounds targeting P. falciparum. We review here structural analogues of bicyclic peroxides belonging to the G factors family presenting antimalarial properties. They were synthesised under Mannich type conditions, followed by an autoxidation step resulting exclusively in the peroxide. As the electron transfer from haem or free iron to the peroxide is the first step in the mode of action of artemisinin-like compounds, the redox properties of some endoperoxides were studied by electrochemistry allowing the evaluation of the reduction standard potentials. The Fe(II) induced reduction was also investigated and the reactivity of the C-centered radical intermediate formed was linked to the antimalarial activity. These bicyclic peroxides both with various hybrid molecules containing the endoperoxide framework were evaluated in vitro against Plasmodium falciparum. They exhibited moderate to good activities.

1. Introduction 2. Synthesis of endoperoxides via spontaneous oxygen uptake 3. Crucial role of the peroxyhemiketal function 4. Dissociative electron transfer studies 5. Fe(II)-induced Reduction 6. Structural variations of the G-factors.

6.1 Modifications on the lateral chain 6.2 -Spiro-endoperoxides synthesis 6.3 Hybrid molecules 6.3.1 Streptocyanine-endoperoxide 6.3.2

Ciprofloxacin-endoperoxide 6.3. [START_REF] Wells | New medicines to improve control and contribute to the eradication of malaria[END_REF] 4-Aminoquinoline-endoperoxide Conclusion

Introduction

Malaria caused by protozoan parasites of the genus Plasmodium is the most prevalent parasitic disease with approximately 50% of the world's population at risk, mainly in many tropical and subtropical regions, with an estimate of 216 million cases of malaria and 655,000 deaths worldwide annually [1]. Four species of Plasmodium, P. falciparum, P. vivax, P. ovale and P. malariae commonly infect humans, and a fifth, P. knowlesi, has recently been identified as being responsible for a significant number of human cases in South-East Asia. [START_REF] Cox-Singh | Plasmodium knowlesi malaria in humans is widely distributed and potentially life threatening[END_REF] Malaria remains a major world health problem following the emergence and spread of P. falciparum, the most dangerous of the Plasmodium parasites that infect and is resistant to the majority of antimalarial drugs. [START_REF] Wells | New medicines to improve control and contribute to the eradication of malaria[END_REF] The discovery of the sesquiterpene lactone artemisinin (known as Qing hao su) in 1972 by Chinese scientists and the subsequent development of potent derivatives have been a key event in the malaria treatment because the artemisinins rapidly kill all the blood stages of the parasite, resulting in the shortest fever clearances times of all antimalarials. Artemisinin-based combination therapies (ACTs) which combine a potent but short-lived artemisinin derivative such as artesunate, artemether or dihydroartemisinin with a longer-lasting partner drug are now the first-line recommendation for P. falciparum malaria in most endemic areas and are increasingly used against the chloroquine-resistant P. vivax malaria problem [START_REF] Anthony | The global pipeline of new medicines for the control and elimination of malaria[END_REF].

Since the discovery of artemisinin, the search for new generation of artemisinin-based therapeutics has been intensively pursued resulting in new classes of peroxide-containing compounds such as 1,2,4-trioxane, [START_REF] Jefford | Synthesis of new 1,2,4-trioxanes and their antimalarial activity[END_REF][START_REF] Jefford | Synthetic peroxides as potent antimalarials. News and views[END_REF][START_REF] Dechy-Cabaret | Synthesis and antimalarial activity of trioxaquine derivatives[END_REF][START_REF] Singh | Orally active 1,2,4-trioxanes: Synthesis and antimalarial assessment of a new series of 9-functionalized 3-(1arylvinyl)-1,2,5-trioxaspiro[5.5undecanes against multi-drug-resistant Plasmodium yoelii nigeriensis in mice[END_REF][START_REF] O'neill | Enantiomeric 1,2,4trioxanes display equivalent in vitro antimalarial activity versus Plasmodium falciparum malaria parasites: implications for the molecular mechanism of action of the artemisinins[END_REF] 1,2,4-trioxolanes [START_REF] Vennerstrom | Identification of an antimalarial synthetic trioxolane drug development candidate[END_REF][START_REF] Charman | Synthetic ozonide drug candidate OZ439 offers new hope for a single-dose cure of uncomplicated malaria[END_REF], 1,2,4,5-teraoxanes [START_REF] Terent'ev | Synthesis of five and six-membered cyclic organic peroxides: Key transformations into peroxide ring-retaining products[END_REF], cyclic peroxyketal [START_REF] Posner | Antimalarial cyclic peroxy ketals[END_REF] and endoperoxides [START_REF] Hofheinz | RO-42-1611 (arteflene), a new effective antimalarial -chemical-structure and biological-activity[END_REF][START_REF] Bachi | A short synthesis and biological evaluation of potent and nontoxic antimalarial bridged bicyclic beta-sulfonyl-endoperoxides[END_REF][START_REF] O'neill | Design and synthesis of endoperoxide antimalarial prodrug models[END_REF] targeting P. falciparum.

For the chemical synthesis of these peroxide-containing compounds, several oxygen sources were used in order to introduce the O-O bond such as: hydrogen peroxide, ozone, singlet oxygen, and to a less extent molecular oxygen.

Hydrogen peroxide was thus added to carbonyl compounds or to epoxide leading respectively to gem-dihydroperoxides or to hydroperoxyhemiketals [START_REF] Zmitek | alpha-Substituted organic peroxides: synthetic strategies for a biologically important class of gemdihydroperoxide and perketal derivatives[END_REF] and to -hydroxy-hydroperoxides [START_REF] Subramanyam | Synthesis and reactions of beta-hydroxyhydroperoxides[END_REF] which were precursors of several cyclic peroxides such as 1,2,4,5-tetraoxanes, [START_REF] Sanderson | Macrocycles -synthesis and thermal-decomposition of some disubstituted dicyclohexylidene diperoxides[END_REF][START_REF] Vennerstrom | Dispiro-1,2,4,5-tetraoxanes -a new class of antimalarial peroxides[END_REF] 1,2-dioxolanes, [START_REF] Wang | Spiro-and dispiro-1,2-dioxolanes: contribution of iron(II)-mediated one-electron vs two-electron reduction to the activity of antimalarial peroxides[END_REF] 1,2,4-trioxanes [START_REF] Zhang | A facile access to bridged 1,2,4-trioxanes[END_REF] or 1,2-dioxanes. [START_REF] Murakami | Facile construction of 6-carbomethoxymethyl-3-methoxy-1,2-dioxane, a core structure of spongean anti-malarial peroxides[END_REF][START_REF] Murakami | New readily accessible peroxides with high anti-malarial potency[END_REF] In a same way, ozone was reacted with alkenes or more generally enol ethers and oxime ethers leading to peroxycarbenium ion species [START_REF] Tang | Dispiro-1,2,4-trioxane analogues of a prototype dispiro-1,2,4-trioxolane: Mechanistic comparators for artemisinin in the context of reaction pathways with iron(II)[END_REF] which in turn were the reactive intermediates in peroxide formation as described for the synthesis of the natural compound Yingzhaosu, [START_REF] O'neill | A carbonyl oxide route to antimalarial yingzhaosu A analogues: Synthesis and antimalarial activity[END_REF] plakinic acid A analogues [START_REF] Dai | Intramolecular reactions of hydroperoxides and oxetanes: Stereoselective synthesis of 1,2-dioxolanes and 1,2dioxanes[END_REF] or various 1,2,4-trioxanes. [START_REF] Ushigoe | Synthesis of 1,2,4-trioxanes and 1,2,4-trioxepanes by N-halogenosuccinimide-mediated cyclisations of unsaturated hydroperoxyacetals[END_REF][START_REF] Dong | Dispiro-1,2,4,5-tetraoxanes via ozonolysis of cycloalkanone O-methyl oximes: A comparison with the peroxidation of cycloalkanones in acetonitrile-sulfuric acid media[END_REF] In addition, Vennerstrom has shown that ozone treatment of oxime ethers and addition of the peroxycarbenium ion on ketones, led to 1,2,4-trioxolanes which revealed to be promising antimalarial candidates. [START_REF] Vennerstrom | Identification of an antimalarial synthetic trioxolane drug development candidate[END_REF] The fully synthetic 1,2,4-trioxolane peroxide OZ439, appears as an exciting prospect demonstrating high potency and longer plasma exposure compared to artemisinin [START_REF] Charman | Synthetic ozonide drug candidate OZ439 offers new hope for a single-dose cure of uncomplicated malaria[END_REF]. This compound offers new hope for a single-dose cure of uncomplicated malaria and is currently into phase 2 trials supported by medicine for Malaria Venture [32].

Singlet oxygen was also used as a source of oxygen to access peroxide derivatives via the Schenck reaction or [4+2] or [2+2] cycloadditions. Arteflene [START_REF] O'neill | Enantiomeric 1,2,4trioxanes display equivalent in vitro antimalarial activity versus Plasmodium falciparum malaria parasites: implications for the molecular mechanism of action of the artemisinins[END_REF] was synthesized using the Schenck reaction. Singh [START_REF] Singh | Preparation of beta-hydroxyhydroperoxides by photooxygenation of allylic alcohols and their elaboration into 1,2,4-trioxanes[END_REF] and Bloodworth [START_REF] Bloodworth | A halogenocyclization route to 1,2,4-trioxanes[END_REF][START_REF] Bloodworth | Synthesis of 1,2,4-trioxanes via intramolecular oxymercuriation[END_REF] independently synthesized trioxanes by photooxygenation of allylic alcohols or olefins followed by acid -catalyzed condensation of carbonyl compounds on hydroperoxides. Singlet oxygen [4+2] cycloaddition on cyclopentadiene was developed by Jefford [START_REF] Jefford | structure, and antimalarial activity of some enantiomerically pure, cis-fused cyclopenteno-1,2,4trioxanes[END_REF] during Fenozan (BO7) synthesis. This methodology was reinvestigated by Meunier in the synthesis of trioxaquines. [START_REF] Dechy-Cabaret | Preparation and antimalarial activities of "trioxaquines", new modular molecules with a trioxane skeleton linked to a 4-aminoquinoline[END_REF] Jefford et al. [START_REF] Jefford | structure, and antimalarial activity of tricyclic 1,2,4-trioxanes related to artemisinin[END_REF] have also shown that singlet oxygen reacts through a [2+2] cycloaddition on enol ethers yielding the 1,2-dioxetane intermediates, which, at low temperature and acid catalysis react on ketones to give the desired 1,2,4-trioxanes. Posner [START_REF] Posner | Antimalarial sulfone trioxanes[END_REF] modified this method, by using thio enol ethers instead of enol ethers to prepare sulfone trioxanes.

Molecular oxygen was scarcely used as its reactivity is low. Interestingly however, it could be used in the preparation of 3hydroxy-1,2-dioxanes. O'Neill et al. [START_REF] O'neill | thd)(2): a superior catalyst for aerobic epoxidation and hydroperoxysilylation of unactivated alkenes: application to the synthesis of spiro-1,2,4-trioxanes[END_REF] optimized a route to spirotrioxanes via the Co(II) catalyzed preparation of triethylsilyl peroxides. Posner and Bachi [START_REF] Bachi | Synthesis and in vitro antimalarial activity of sulfone endoperoxides[END_REF] perfected a radical process called TOCO (Thiol-Olefine Co-Oxygenation) giving access to dioxanes or trioxanes via formation of a peroxyl radical intermediate. O'Neill reinvestigated this process [START_REF] O'neill | Application of thiol-olefin co-oxygenation methodology to a new synthesis of the 1,2,4-trioxane pharmacophore[END_REF] by describing a 1,2,4-trioxane synthesis. Posner et al. [START_REF] Posner | Antimalarial cyclic peroxy ketals[END_REF] synthesized cyclic peroxyketals by using the Snider and Shi protocol, [START_REF] Snider | Total synthesis of (+/-)-Chondrillin, (+/-)-Plakorin, and related peroxy ketals -development of a general-route to 3,6dihydro-1,2-dioxin-3-ols[END_REF] e.g. photoenolisation followed by oxygenation. Blaumstark et al. [START_REF] Baumstark | Synthesis of pentasubstituted 3-hydroxy-1,2-dioxolanes[END_REF] described the synthesis of pentasubstituted 3-hydroxy-1,2-dioxolanes via O 2 trapping of intermediates generated during the thermolysis of cyclic azo hydroperoxides.

The mode of action of those antimalarial peroxides prodrugs is based on Fe(II) activation causing oxygen-oxygen bond breaking; the oxy radical intermediate undergoes rearrangement furnishing primary or secondary carbon-centered radical. These highly reactive intermediates are thought to cause irreversible damage to certain parasite's biomolecules. [START_REF] Slack | Antimalarial peroxides: advances in drug discovery and design[END_REF] They trigger damage to cellular lipids and digestive vacuole membranes, inactivation of Plasmodium proteins, alkylation of haem and interference with haem sequestration into crystals and finally parasite death. The effect on intraerythrocytic stages is broad, including strong action on rings stages as well as trophozoits and early schizonts.

Our lab is interested in new types of antimalarial agents potentially acting as artemisinin. We focused our efforts on the syntheses of new endoperoxides, related to the phytohormones known as G factors (G1, G2, G3) which are natural compounds extracted from leaves of Eucalyptus grandis (Figure 1). These factors act as growth regulators and are abundant in leaves after a frost period or hydric stress. They cannot be present in their physiological forms, but rather in some inactive form, and are readily released in response to damage to the plant or to a biological stimulus. [START_REF] Ghisalberti | Bioactive acylphloroglucinol derivatives from Eucalyptus species[END_REF] They constitute a family of six-membered unsaturated cyclic endoperoxides fused to another six-carbon ring derived from syncarpic acid.

The present work reviews our synthetic program on the construction of bicyclic endoperoxides belonging to the G-factor family and our mechanistic studies which aimed at elucidating their mode of action. On the basis of this analysis, new structures and hybrid molecules are presented in order to pave the way for new type of antimalarial endoperoxides.

Synthesis of endoperoxides via spontaneous oxygen uptake

First, we developed a straightforward synthesis of endoperoxides belonging to the G-factor series. This synthesis is based on two key-steps: formation of a Mannich base as described by Bolte [START_REF] Bolte | Plant-growth regulators in Eucalyptus-grandis .4. Synthetic approaches to G-regulator analogs[END_REF] followed by autoxidation (Scheme 1). Mannich bases were formed from reaction between syncarpic acid and an iminium issued from the reaction of different aldehydes with piperidine. Preparation of Mannich bases intermediates allows minimizing or avoiding the formation of Michael bis-adduct. Ethylenic ketones were then easily released under acidic conditions. Depending on the substituents, equilibrium between enone and dienol can take place. [START_REF] Gavrilan | Bicyclic peroxydes in the G factors series: synthesis and electrochemical studies[END_REF] The addition of the molecular dioxygen occured more or less rapidly, depending on the substituents of the dienols, affording the corresponding endoperoxides. This quite surprising autoxidation operates in absence of any sensitizer, even in the dark and without solvent or any solvent excepted hydroxylated ones. Molecular dioxygen, in its fundamental triplet state has been shown to be trapped by the dienol system, singlet dioxygen being not implied. In fact, the presence of DABCO, a well-known singlet quencher, didn't inhibit the autoxidation. On the contrary, as tert-butyl mercaptan in catalytic amount was found to inhibit the reaction, we investigated this autoxidation by spin trapping / EPR technique. The spontaneous addition of dioxygen on 1a was followed by spin trapping (ST) combined with EPR spectroscopy and mass spectrometry (MS). [START_REF] Triquigneaux | A combined spin trapping/EPR/mass spectrometry approach to study the formation of a cyclic peroxide by dienolic precursor autoxidation[END_REF] Two different nitrones, i.e. the commercially available 4-{(E)-[tert-butyl(oxido)imino]methyl}pyridine 1oxide (POBN), and the 31 P-labelled trinitrone diethyl {1-[(Z)- (3,5-bis{(E)-[1 (diethoxyphosphoryl)-1-methylethyl] (oxido) imino] methyl}-benzylidene)(oxido)amino]-1-methylethyl}phosphonate (TN), were used to trap the radical intermediates formed during the autoxidation. The EPR spectra of TN-spin adducts provide more information about the addend structure because the strong hyperfine coupling constant with the phosphorus nucleus (a P ) is usually very sensitive to the nature of the radical trapped. Whatever the nitrone, a single mono-radical nitroxide, i.e. a carbon-centered radical spin adduct, was EPR detected. Similar experiments were performed with two 13 C-labelled precursors 1b and 1c, obtained from labelled syncarpic acid on two different and crucial positions, which allowed us to identify precisely the radical center in the addend. Actually, identical EPR spectra were obtained for 1a and 1b, while an extra coupling with the 13 C nucleus was EPR-observed in the case of 1c (a 13C = 1.14 mT), as shown in Scheme 2. The hyperfine coupling constants obtained for the POBN-C spin adduct with 1a and 1b were a N = 1.64 mT, a H = 0.16 mT and with 1c a N = 1.64 mT, a H = 0.16 mT and a 13C = 1.14 mT. Similarly, the hyperfine coupling constants obtained for the TN-C spin adduct with 1a and 1b were a N = 1.55 mT, a H = 0.19 mT and with 1c a N = 1.55 mT, a H = 0.19 mT and a 13C = 1.14 mT. In order to define more precisely the spin adduct structures, the ST/MS technique was then employed without preliminary chromatographic separation. The diamagnetic hydroxylamine derivatives of the spin adduct were detected by electrospray ionization mass spectrometry (ESI-MS), in both positive and negative modes, and their structures were elucidated by tandem mass spectrometry (MS/MS).

Following this ST/EPR/MS study, the mechanism pathway presented in Scheme 3 was proposed for this oxidation. The results obtained are consistent with the pathway B but this does not mean that the pathway A could not occur simultaneously. Under the experimental conditions considered here, the biradical formed from 1a in pathway B was unambiguously identified. However, the ring closure into the biradical formed in pathway A could be much faster than the trapping reaction. Thus, pathway A could not be dismissed as an alternative or even as a main process. Whatever it may be, this ST/EPR/MS study clearly demonstrates that the autoxidation corresponds to a radical process.

Crucial role of the peroxyhemiketal function

Based on the synthetic methodology described above, several compounds were prepared by varying the aldehyde used in the Mannich based preparation. After elimination of the piperidine and oxygen uptake, several endoperoxides were alkylated or benzylated on the peroxyhemiketal position in order to mimic trioxane. The compounds were evaluated against both chloroquine susceptible (Nigerian and 3D7) and resistant (FcM29 and W2) P. falciparum strains, the results are presented in Table 1. [START_REF] Najjar | Synthesis and electrochemical studies of new antimalarial endoperoxides[END_REF][START_REF] Najjar | Crucial role of the peroxyketal function for antimalarial activity in the G-factor series[END_REF][START_REF] Najjar | Alkylation of natural endoperoxide G3-factor. Synthesis and antimalarial activity studies[END_REF] The natural compound G3 (R=OH) (2) possess a mild activity (36 μM) on Nigerian P. falciparum strain and appears similarly active against resistant strains. Remarkably, methylation of the peroxyhemiketal function yielded G3Me (R=OMe) (3) derivative which appears 100 fold more active on the same strain with IC 50 of 0.28 μM. Fluorination of this position gave an inactive compound (4). The extension of the alkyl chain did not change the activity as compounds 5, 6 and 7, showed similar activities to G3Me. Finally, the benzylation yielded endoperoxide 8 which exhibited among the highest activities (IC 50 = 0.37 and 0.10 M on chloroquine sensitive and resistant strains of Plasmodium falciparum, respectively). The enantiomers were separated by chiral column chromatography, both enantiomers displayed similar activity. This last result is a clue to rule out the hypothesis of an enzymatic target in the mode of action. This work demonstrates that alkylation of the peroxyhemiketal function is crucial for antiplasmodial activity and that antimalarial activity of this type of endoperoxide is quite substantial with IC 50 below 1 M. Activities of endoperoxides are always related to O-O bond cleavage induced by iron(II) activation. In this context we decided to study and compare the reduction of the G3 and G3Me both by electrochemistry and by iron(II) induced degradation.

Dissociative electron transfer studies

As the first step in the mode of action of artemisinin-like compounds has been shown to be a dissociative electron transfer from the iron(II) of the haem to the peroxidic bond, [START_REF] Meshnick | Artemisinin (qinghaosu): The role of intracellular hemin in its mechanism of antimalarial action[END_REF][START_REF] Zhang | Hemin-catalyzed decomposition of artemisinin (qinghaosu)[END_REF][START_REF] Meshnick | Iron-dependent free radical generation from the antimalarial agent artemisinin (qinghaosu)[END_REF] we focused our efforts in determining the reductions potentials of our endoperoxides G3 (2) and G3Me (3) in the aim to compare them to each other and to artemisinin. The reduction was thus studied by cyclic voltammetry and convolution analysis. [START_REF] Najjar | Electrochemical reduction of G3-factor endoperoxide and its methyl ether: Evidence for a competition between concerted and stepwise dissociative electron transfer[END_REF] One electron was detected during the reduction of both compounds and a slow and irreversible system was observed. Electron transfer led to irreversible cleavage of the O-O bond. Detailed analysis of the voltammetry curves using Marcus theory, revealed a non-linear dependence on the transfer coefficient indicating a change from a stepwise mechanism to one with more concerted character with increasing potential (Figure 2). In the concerted mechanism, the electron is accepted into the * orbital, largely associated with the O-O bond, resulting in simultaneous cleavage to generate a distonic radical anion . ORRO -. There is also the possibility of a stepwise dissociative mechanism when there is an energetically accessible * orbital within the molecule. In the stepwise mechanism, the initial electron transfer results in the formation of an intermediate radical anion, followed in a second step, by cleavage of the O-O bond (k frag ), giving then the radical anion.

The O-O bond dissociation energies, estimated by quantum calculations using density functional theory (DFT), from the difference between the total energy of the most stable form for each molecule in its singlet and triplet state at the B3LYP 6-31G* level, were taken into account in the experimental data when evaluating the standard reduction potentials and other pertinent thermochemical informations such as reorganization energy and activation energy. The values of the standard reduction potentials of the stepwise mechanism (E°s t ) for both G3 and G3Me were estimated at respectively -1.70 and -1.64 V, very close to the values of the peak potentials (-1.67 and -1.61 V at the rate of 2 V/s ). The values of the standard reduction potentials of the concerted mechanism (E°c) were about -0.82 and -0.84 V close to the value of artemisinin standard reduction potential (-0.89 V) for which only a purely concerted mechanism is present. Using density functional theory (DFT) with B3LYP hybrid functional and 6-311+G(d,p) basis set, [START_REF] Lacaze-Dufaure | First computational evidence of a competitive stepwise and concerted mechanism for the reduction of antimalarial endoperoxides[END_REF] the reaction pathway of the electron transfer was also carried out. The stationary points and related energy differences were determined. Both for G3 and G3Me, the formation of two intermediate radical anions (A and B, Figure 2 The occurrence of the stepwise mechanism for the electron transfer, can be explained by the presence of the more accessible LUMO of the G3 with a π* character (Figure 2b). Then the electron is transferred into the LUMO+1 which is situated on the O-O bond and has a * character, leading to the breaking O-O bond and furnishing the second stable radical anion. The reorganization energy upon electron transfer in relation to the Marcus theory and the intrinsic activation energy were also calculated using the DFT method. The calculated values are in good agreement with their available experimental counterparts obtained from analysis of the voltammetry curves.

This interesting result does not explain the differences encountered in the biological activity of the two compounds. Indeed, they present quite similar standard potentials as artemisinin and their O-O bond dissociation energies (BDE) are in the same range. Nevertheless, this mechanism allows understanding the reason why these endoperoxides (especially G3Me) can exhibit antimalarial properties even if they present a steric hindrance of the O-O bond, hampering the approach of the haem, as the electron will first go into the π* orbital of the conjugated double bond.

Fe(II)-induced Reduction

A chemical approach to the reduction process was then undertaken. [START_REF] André-Barrès | Fe(II)-induced reduction of labelled endoperoxides. NMR degradation studies on G3 factor and its methyl ether[END_REF] We studied the Fe(II)-induced reduction of G3 (R=OH), G3Me (R=OMe) and G3F (R=F) and then characterized the degradation products. The conditions (FeSO 4 ; CH 3 CN / H 2 O : 1/1) were chosen to be closer to biological conditions than the THF-based system. In that case pH medium is acidic (pH 5) mimicking the pH of the parasite food vacuole (pH 4.5-5.0). [START_REF] Spiller | The pH of the Plasmodium falciparum digestive vacuole: holy grail or dead-end trail?[END_REF] A major product 10 (Scheme 4) was obtained in 82% yield from G3, whereas under the same conditions, three major compounds, 11, 12, and 13 (respectively, 23%, 24%, and 24%), were isolated from G3Me. In both cases starting material was still present (about 5%). One major compound 14 (60%) was generated by reduction of the fluoroendoperoxide G3F.

In order to get insight in the mechanisms involved in each reduction, 13 C-labelled derivatives for G3 and G3Me were synthesized from 13 C-labelled syncarpic acid on two crucial positions 13 C 1 and 13 C 2 . In this way, two series of endoperoxides were obtained. The first one labelled on 13 C 6 was issued from 13 C 1 syncarpic acid whereas the second one was issued from 13 C 2 syncarpic acid and consisted in mixture 1/1 of compounds labelled on 13 C 1 or 13 C 7 . The analysis of the 13 C-labelled distribution and studies of multiplicity obtained with coupling of 1 H-and 13 C allowed elucidation of the mechanisms involved (Schemes 5 and 6).

Scheme 4 : End-products issued from the Fe(II) induced reduction of G3, G3Me and G3F.

In the case of the G3, transfer of a single electron from Fe(II) to the O-O bond is followed by the homolytic cleavage of the peroxidic linkage affording only products issued from the O 1 -centered radical. Thus, the O 1 centered radical quickly evolves to tertiary gem dimethyl C-centered radical (Scheme 5). This tertiary radical rearranged within 5-exo-trig cyclisation, and after ring expansion and loss of acetone and iron(II), acid 10 is obtained.

In the case of G3Me, the tertiary radical evolves following two different pathways (Scheme 6). In pathway (a) a mechanism analogue to the one involved in G3 reduction was observed leading to the methyl ester 11 whereas in pathway (b) disproportionation and lactonisation with concomitant elimination of Fe(III), occurred affording compounds 12 and 13.

In the case of G3F, only the lactone 14 was obtained (Scheme 7); its formation is likely due to the formation of the tertiary gem dimethyl C-centered radical which quickly rearranges (5-exo-trig cyclisation, addition on the carbonyl and ring expansion) and then to the loss of Fe(II) and HF. In comparing the reduction of the three endoperoxides, we suggest a preponderant role of the tertiary C-centred radicals. The 5 exo-trig cyclisation will be more or less rapid depending on the substituent of the double bond (COOMe, COOH or COF). In the case of the endoperoxide G3Me (3), the C-centered radical could last long enough to combine with haem (or to act on proteins), a reaction which is impeded when the intramolecular 5-exo-trig cyclisation occurs rapidly. This could explain why G3F (4) does not possess significant antiplasmodial activity and G3Me (3) is far more potent than G3 [START_REF] Cox-Singh | Plasmodium knowlesi malaria in humans is widely distributed and potentially life threatening[END_REF].

A relationship between antiplasmodial activity and a good balance between reactivity and stability of the C-centered radical is thus suggested. This C-centered radical could then alkylate haem or parasitic vital proteins. On this basis we could explain why alkylation of the peroxyhemiketal function is crucial for antimalarial activity and how to design new compounds.

Structural variations of the G-factors.

Our synthetic strategy allows easy modifications of the endoperoxides by variation of the aldehydes (R 1 R 2 CHCHO) involved in the Mannich-type reaction. The use of various aldehydes permits the introduction of : i) a lateral chain comprising a hydroxyl group for further functionalization ii)

-spiro-alkyl moieties.

Modifications on the lateral chain

The use of aldehydes (R 1 R 2 CHCHO) with R 1 or R 2 bearing a hydroxyl group (R 1 or R 2 equals to CH 2 OH or CH 2 CH 2 OH, and CH 3 ) allows the obtention of endoperoxides with a functionalized lateral chain which are presented in Table 2 together with their antiplasmodial activities. In all cases, this hydroxyl group was protected as a tert-butyldiphenyl silyl (TBDPS) [START_REF] Najjar | Synthesis and electrochemical studies of new antimalarial endoperoxides[END_REF][START_REF] Givelet | New amino endoperoxides belonging to the antimalarial G-factors series[END_REF] or a paramethoxybenzyl (PMB) group. [START_REF] Bernat | Synthesis of antimalarial G-factors endoperoxides: relevant evidence of the formation of a biradical during the autoxidation step[END_REF] Methylation of the peroxyhemiketal moiety, cleavage of the protective group, and terminal hydroxyl transformation afforded mono and difluoro-endoperoxides 25, 26 with interesting antiplasmodial activities (lower than 1 . From this study, we can notice that methylation of the hydroxyl of the peroxo-hemiketal led to compounds with significant more powerful antiplasmodial activities in most cases excepted when TBDPS was introduced (19, 21 versus 20, 22 and 27, 29 versus 28, 30). Lipophilicity was also of great importance as the deprotection of the hydroxyl group on the lateral chain led to less active compounds. Endoperoxides 28 and 32 are 115-and 52-times more potent than 35. This is even more striking with the endoperoxide 19 (0.62 M) which can be compared to the bis hydroxylated endoperoxide 23 which does not present antimalarial activity anymore (>100 M). Hydroxylated compounds 19, 21 and 27, 29 are the most potent of this series with R=OH. The lipophilicity afforded by the presence of the TBDPS group could explain the gain in activity. No increase in activity was observed on their methylated analogues 20, 22 and 28, 30. This observation let us think that the mechanism of the Fe(II)-induced reduction may be different than the one observed in the case of the G3 and G3Me.

Scheme 7: Proposal of mechanism of Fe(II) induced reduction of G3F. endoperoxide. a IC50 values are duplicated and were considered acceptable when values did not vary by more than a factor of 2.5 to 3.

Introduction of mono or diamine frames, known to improve antiplasmodial activities by accumulation in the digestive vacuole of the parasite [START_REF] Hindley | Mechanism-based design of parasitetargeted artemisinin derivatives: Synthesis and antimalarial activity of new diamine containing analogues[END_REF] and thereby increasing its acidic pH, was also accomplished (Scheme 8). Substitution of the chloromethyl sulfonyl group by substituted piperazines or mopholine allowed obtention of amino-endoperoxides 36a-36d in moderate to good yields (58-70%). However, these endoperoxides (36a-d) have significant lower antiplasmodial activities with values ranging from 7.7 to 54 M. [START_REF] Bernat | Synthesis of antimalarial G-factors endoperoxides: relevant evidence of the formation of a biradical during the autoxidation step[END_REF] Presumably, these compounds did not reach the digestive vacuole of the parasite as they were too polar.

Scheme 8: Synthesis and in vitro antiplasmodial activities on Nigerian P. falciparum strains of amino-endoperoxides 36a-36d.

-Spiro-endoperoxides synthesis

The preparation of -spiro-endoperoxides was carried out in order to explore the possibility that the Fe(II)-induced reduction leads to homolytic cleavage of the peroxo linkage following the competitive route b) in Scheme 9. The O 2 -centered radical may then be formed and rearranged to the primary C-centered radical, either via -scission or via a secondary C-centered radical followed by a 1,5-H shift. Primary and secondary C-centered radical are indeed produced after Fe(II) activation of artemisinin or artemisinin-like analogues with antiplasmodial activities.

A recent rational approach involving "covalent bitherapy" is based on the use of hybrid molecules with a dual mode of action. [START_REF] Dechy-Cabaret | Synthesis and antimalarial activity of trioxaquine derivatives[END_REF] The two active entities are covalently linked. These drugs allow bypassing the development of resistance, enhancing patient compliance and reducing drug-drug interactions. The synthesis of hybrid molecules containing an endoperoxide moiety linked to a second pharmacophore, e.g. a streptocyanine, a fluoroquinolone or a 4-amino-7-chloroquinoline, mimicking chloroquine was thus undertaken. The reaction sequence to the amino-endoperoxide 47 capable of tighting a second pharmacophore is described in Scheme 10. The piperidine moiety in spiro position of the cyclic peroxide was chosen in order to avoid the introduction of a second asymmetric center and obtention of diastereoisomeric mixture. Following the methodology previously described, methylation of the peroxyhemiketalic hydroxyl group, and subsequent tert-butyl-oxy-carbonyl (BOC) cleavage, amino-endoperoxide 47 was obtained. As expected, as 47 is more polar than G3Me, its antimalarial activity against Nigerian P. falciparum strain (5.7 M) was less potent than the G3Me (0.28 M).

Scheme 10 : Synthesis of the amino-endoperoxide 47. a) piperidine, dichloromethane, RT, 95%. b) NH4Cl/HCl 1N, 95%. c) O2, EtOAc, 68%. d) BuLi/MeOTf, THF, -78°C, 62%. e) TFA, CH2Cl2, 95%.

Streptocyanine-endoperoxide

We previously described the synthesis of streptocyanine dyes as potential new antimalarial drugs. [START_REF] Maether | Synthesis and Antimalarial Properties of Streptocyanine Dyes[END_REF] SAR studies have shown the influence of the polymethine chain length (5C-, 7C-or 9C-) and also the importance of the structural modifications at the nitrogen end groups. The most active compounds displayed sub-micromolar in vitro activities against P. falciparum and the best selectivity between activity and cytotoxicity toward mammalian cells was obtained for 5C-streptocyanines with morpholino end groups. Streptocyanines are obtained by reaction of carboxonium salts with amines. This methodology was used to design peroxo-streptocyanines containing both the endoperoxide moiety related to the G-factor and the streptocyanine. The target dual molecules synthesis was carried out either via the hemicarboxonium, or the more reactive but less stable carboxonium (Scheme 11). Symmetrical [START_REF] Bolte | Plant-growth regulators in Eucalyptus-grandis .4. Synthetic approaches to G-regulator analogs[END_REF] and unsymmetrical compounds [START_REF] Gavrilan | Bicyclic peroxydes in the G factors series: synthesis and electrochemical studies[END_REF][START_REF] Triquigneaux | A combined spin trapping/EPR/mass spectrometry approach to study the formation of a cyclic peroxide by dienolic precursor autoxidation[END_REF][START_REF] Najjar | Synthesis and electrochemical studies of new antimalarial endoperoxides[END_REF] were obtained using this strategy. [START_REF] Maether | Synthesis and antiplasmodial activity of streptocyanine/peroxide and streptocyanine/4-aminoquinoline hybrid dyes[END_REF] Their antiplasmodial activities, against chloroquine sensitive (Nigerian) and resistant (FcB1-Columbia and FcM29-Cameroon) strains of P. falciparum are presented in Table 3 . All hybrid unsymmetrical molecules 49, 50 and 51 exhibited potent in vitro antiplasmodial activities (0.039 to 0.436 M) similar or better than those of their corresponding streptocyanines 52, 53, 54 (0.085 to 5.47 M). [START_REF] Maether | Synthesis and antiplasmodial activity of streptocyanine/peroxide and streptocyanine/4-aminoquinoline hybrid dyes[END_REF] Antiplasmodial activity is not improved by the introduction of two endoperoxide moieties as the hybrid symmetrical compound 48 did not display more potent IC 50 values than its corresponding hybrid unsymmetrical compound 49 or streptocyanine 52. Hybrid peroxo-streptocyanines are thus ten-to one hundred-fold more active than the cyclic peroxide 47 (IC 50 5.7 M) which is likely too hydrophilic due to the NH group. Interestingly, hybrid unsymmetrical compound 51 showed increased activities with regard to the corresponding symmetrical streptocyanine 53, with an IC 50 very low: 0.057 and 0.039M against respectively FcB1 and FcM29 P. falciparum strains and nearly 10-fold better than our lead actual lead compounds G3Me.

Ciprofloxacin-endoperoxide

The ciprofloxacin is a second generation of fluoroquinolone antibiotic. Fluoroquinolones affect bacterial DNA by targeting two essential enzymes, DNA gyrase and topoisomerase IV that belong to the type II topoisomerase family. Ciprofloxacin acts by forming a quaternary drug-topoisomerase-DNA complex involving Mg 2+ that causes DNA to rewind after being copied, stopping thus DNA and protein synthesis resulting in cell death. This antibiotic was also reported to be active against P. falciparum [67] with an IC 50 of 50 M against chloroquine sensitive 3D7 strains. [START_REF] Dubar | Enhancement of the Antimalarial Activity of Ciprofloxacin using a Double Prodrug/Bioorganometallic Approach[END_REF] Gyrase has moreover been localized in the apicoplast of the malarial parasite Plasmodium falciparum. [START_REF] Mcfadden | Plastid in human parasites[END_REF] To bring together the mode of action of endoperoxide, potentially capable to alkylate haem and the fluoroquinolone capable to inhibit DNA replication, the two pharmacophores were covalently linked to furnish a compound acting or in the digestive vacuole or in the apicoplast of the Plasmodium. Ciprofloxacine was first added to succinic anhydride leading to the corresponding acid in 76% yield (Scheme 12). A peptidic coupling using EDCI/HOBt (N-ethyl-N'-dimethylaminopropylcarbodiimide, /1hydroxybenzotriazole) was then used involving both entities providing after purification by column chromatography the hybrid molecule 55 in 44% yield. [70] Scheme 12: Synthesis of ciprofloxacine-endoperoxide hybrid molecule 55.

The activity of the hybrid molecule 55 was of 1.2 M against the chloroquine susceptible Nigerian P. falciparum strain. Although this compound is slightly more potent than the amino-endoperoxide 47 (IC 50 5.7 M), it is 50-fold more active than ciprofloxacin against chloroquine susceptible strain. It can also be compared with the new promising prodrugs of ciprofloxacin developed by Biot et al. which present activities ten to one hundred better than ciprofloxacin [START_REF] Dubar | Enhancement of the Antimalarial Activity of Ciprofloxacin using a Double Prodrug/Bioorganometallic Approach[END_REF]. Their hydrophobic properties lead indeed, to a better penetration capacity through the multiple membranes of the apicoplast before targeting DNA gyrase. [START_REF] Dubar | Ester prodrugs of ciprofloxacin as DNA-gyrase inhibitors: synthesis, antiparasitic evaluation and docking studies[END_REF] Ciprofloxacin derivatives or hybrid molecules based on fluoroquinolone pharmacophore could represent a new promising class of antimalarial drugs, and in a larger context, quinolone family could represent a new antimalarial series as Riscoe's team identified potent and selective quinolone-3-diarylether with good activity against P. falciparum both in vitro and in vivo.

[72]

4-Aminoquinoline-endoperoxide

Several strategies were investigated to design hybrids of 4-amino-7-chloroquinoline-endoperoxide, with various cyclic and acyclic links. The first one was a peptidic coupling between the amino-endoperoxide 47 and the 4-amino-7-chloroquinoline intermediate acids [START_REF] Meshnick | Iron-dependent free radical generation from the antimalarial agent artemisinin (qinghaosu)[END_REF][START_REF] Najjar | Electrochemical reduction of G3-factor endoperoxide and its methyl ether: Evidence for a competition between concerted and stepwise dissociative electron transfer[END_REF]. The synthesis of both hybrid compounds is described in Scheme 13. The intermediate acids [START_REF] Meshnick | Iron-dependent free radical generation from the antimalarial agent artemisinin (qinghaosu)[END_REF][START_REF] Najjar | Electrochemical reduction of G3-factor endoperoxide and its methyl ether: Evidence for a competition between concerted and stepwise dissociative electron transfer[END_REF] were obtained quantitatively by reaction of the succinic anhydride with the two amino derivatives of the 7-chloroquinoline. [START_REF] Maether | Synthesis and antiplasmodial activity of streptocyanine/peroxide and streptocyanine/4-aminoquinoline hybrid dyes[END_REF] Then, the coupling was carried out between the amino-endoperoxide 47 and the intermediate acids (56, 57) using the reagent HBTU (O-Benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluoro-phosphate) and di-isopropylethylamine.

Scheme 13: Synthesis of hybrid 4-amino-7-chloroquinoline-endoperoxides 58, 59 related to G-factors: a) succinic anhydride, DMAP, CH2Cl2, 72% (56) and 86% (57) b) 47, HBTU, (i-Pr)2EtN, CH2Cl2, 0 to 25°C, 77% (58) and 70% [START_REF] André-Barrès | Fe(II)-induced reduction of labelled endoperoxides. NMR degradation studies on G3 factor and its methyl ether[END_REF].

The second strategy involved linking the 7-chloroquinoline directly or via an amino ethylene coupling arm to the piperidineendoperoxide moiety. As the direct coupling was not successful, the 7-chloroquinoline was attached before the autoxidation step, with or without the linker as described in respectively Schemes 14 and 15. Starting from the 4,7-dichloroquinoline, aldehyde 60 was obtained after substitution of one chloride by the 4-piperidinylmethanol followed by oxidation with 2-iodoxybenzoic acid (IBX) in acetone. The so-obtained aldehyde could react with syncarpic acid in previous modified Knoevenagel conditions giving the precursor 61 which after slow oxygen uptake provide the expected endoperoxide 62 in 54% on the two last steps. It was then methylated using BuLi/MeOTf, at low temperature giving 63 in 50% yield. The same strategy was used to prepare endoperoxides 67 and 68. 4-aminoquinoline 64 was firstly prepared by reaction of the 4,7-dichloroquinoline with ethanolamine at reflux, followed by tosylation of the hydroxyl in a 90% yield for the two steps. After substitution of the tosyl group by 4-piperidinyl-methanol, followed by oxidation with IBX in DMSO, the aldehyde 65 was obtained in 45% yield. As previously described, dienol 66 could be obtained within Knoevenagel modified conditions between syncarpic acid and aldehyde 65. After a slow oxygen uptake (15 days) in dichloromethane, the endoperoxide 67 was obtained in 61% yield as the salt of the trifloroacetic acid. Finally, methylation with BuLi/MeOTf, at low temperature furnished the target endoperoxide 68 with 27% yield. These hybrid endoperoxides were evaluated in vitro, on both chloroquine susceptible and resistant strains (3D7 and W2 respectively). Values of the antiplasmodial activities are presented in Table 4. From these values, it seems that a link is necessary between the 4-amino-7-chloroquinoline part and the endoperoxide moiety as hybrid compounds 62 and 63 are the less potent of the series. Introduction of the ethylene link within compounds 67, 68 affords an increase in activity with values around 100 nM, these compounds being slightly more potent than G3Me. But the best improvements were obtained using a peptidic coupling between the amino-endoperoxide and the intermediate acids obtained by reaction of the succinic anhydride with the two cyclic and acyclic amino derivatives of the 7-chloroquinoline furnishing the hybrids 58 and 59. The compound 59 is the most potent with an IC 50 of 31nM on 3D7 and of 13 nM on W2. It is noteworthy that IC 50 of endoperoxides are generally lower on chloroquine resistant strain than on sensitive strain as it is the case for this compound. It seems that for this compound both the peroxide and the 4-amino-7-chloroquinoline moieties afford activity. This compound is indeed more potent than chloroquine alone on W2 and in the same range on 3D7. Moreover, it is 10-fold better than G3Me on both strains.

Conclusion

Synthesis of bicyclic peroxides related to the G-factor family was developed based on an autoxidation step to introduce the O-O bond. This methodology allows preparation of a large family of endoperoxides. An extensive electrochemical study of the dissociative electron transfer allows us to understand the mechanism implied during the electron transfer and then a chemical study, the characterization of the radical species obtained after iron(II) induced reduction. The endoperoxide moiety has been linked to other active compounds furnishing hybrid molecules. An increase in antiplasmodial activity was observed for some of them, especially for two kinds of hybrids: endoperoxide-streptocyanine 53 and endoperoxide-7-chloro-4-aminoquinoline 59 for which activities are in the low nanomolar range (10 to 50 nM).

Figure 1

 1 Figure 1 Natural G-Factors : G1, G2, G3.

Scheme 2 :

 2 Scheme 2: Experimental EPR signals (black full lines) and superimposed simulations (red dotted lines) obtained in deoxygenated benzene after 1 h of reaction between molecular oxygen and (a) 1a or 1b (0.2 mol dm -3 ) in the presence of POBN (0.2 mol dm -3 ), and (b) 1c (0.2 mol dm -3 ) in the presence of TN (0.1 mol dm -3 ). The simulations led to the following hyperfine coupling constant values: a) aN = 1.64 mT, aH = 0.16 mT ; b) aN = 1.55 mT, aH = 0.19 mT, aP = 4.26 mT and a13C = 1.14 mT).

Scheme 3 :

 3 Scheme 3: Proposal for triplet biradical intermediates formation during the autoxidation step.

Figure 2 :

 2 Figure 2: a) Competition between concerted and stepwise mechanism of the dissociative electron transfer, and b) LUMO of the G3.

Scheme 6 :Scheme 3 :

 63 Scheme 6: Proposal of mechanism of Fe(II) induced reduction of13 C labelled G3Me.

Scheme 11 :

 11 Scheme 11: Synthesis of symmetrical and unsymmetrical hybrid streptocyanines.

Scheme 14 :

 14 Scheme 14: Synthesis of hybrid 4-amino 7-chloroquinoline-endoperoxides 62, 63 related to G-factors: a) 4-piperidinyl-methanol, K2CO3, DMF, 170°C, 91% b) IBX, acetone, 60°C, 99% c) syncarpic acid, piperidine then NH4Cl/HCl 1N d) O2, EtOAc, 54% e) BuLi, MeOTf, -78°C, 50%.

Table 1

 1 In vitro antimalarial activity on different strains. Antiplasmodial activity was evaluated after contact with the different strains of P. falciparum for 48 h (duration of the erythrocyte cycle), and is expressed as IC50 which is the concentration that inhibit by 50 % the growth of the parasite.

			IC50(μM) a for the various strains
	R		Nigerian	3D7	FcM29	W2
	OH	2	36	62	41	38.5
	OCH3	3	0.28	0.40	0.23	0.14
	F	4	>100	-	-	-
	O(CH2)2CH3	5	0.16	-	-	-
	O(CH2)3CH3	6	0.76	1.04	0.19	0.44
	O(CH2)7CH3	7	0.18	-	-	-
	OCH2C6H5	8	0.21	0.37	0.10	0.20
	(+) OCH2C6H5 (+) 8	0.36	-	-	-
	(-) OCH2C6H5 (-) 8	0.34	-	-	-
	O-CH2C5H4N	9	0.28	0.82	0.12	0.33
	Artemisinin		0.008	0.019	0.004	0.004
	Chloroquin		0.03	0.019	0.155	0.125
	a IC50 values are duplicated and were considered acceptable when values
	did not vary by more than a factor of 2.5 to3.

Table 2

 2 In vitro antimalarial activity on Nigerian P. falciparum strains.

	compound	R	R 1	R 2	IC50(μM) a	compound	R	R 1	R 2	IC50(μM) a
	2	OH	CH3	CH3	36	3	OCH3	CH3	CH3	0.28
	15	OH	CH3	Ph	25	16	OCH3	CH3	Ph	2.00
	17	OH	Ph	CH3	27	18	OCH3	Ph	CH3	0.70
	19	OH	CH3	CH2OTBDPS	0.62	20	OCH3	CH3	CH2OTBDPS	1.45
	21	OH	CH2OTBDPS	CH3	0.45	22	OCH3	CH2OTBDPS	CH3	1.00
	23	OH	CH3	CH2OH	>100	24	OCH3	CH3	CH2OH	1.4
						25	OCH3	CH3	CH2F	0.69
						26	OCH3	CH3	CHF2	0.74
	27	OH	CH3	(CH2)2OTBDPS	0.74	28	OCH3	CH3	(CH2)2OTBDPS	1.4
	29	OH (CH2)2OTBDPS	CH3	0.73	30	OCH3 (CH2)2OTBDPS	CH3	1.6
	31	OH	CH3	(CH2)2OPMB	10	32	OCH3	CH3	(CH2)2OPMB	0.67
	33	OH	(CH2)2OPMB	CH3	9.9	34	OCH3	(CH2)2OPMB	CH3	0.47
						35	OCH3	CH3	(CH2)2OH	73

Table 3 .

 3 In vitro activity of the synthesized compounds against chloroquine susceptible (Nigerian) and chloroquine resistant (FcB1, FcM29) strains of Plasmodium falciparum. Values reflect the mean of two experiments carried out in duplicate. b Values reflect the mean of three independent experiments, as SD was constantly lower than 15 % they were not indicated in the table.

	IC50 P. falciparum (M)

a

Table 4 :

 4 In vitro activity of the synthesized compounds against chloroquine susceptible (3D7) and chloroquine resistant (W2) strains of P. falciparum.

	IC50 (nM)

Scheme 15 : Synthesis of hybrid 4-ethylamino-7-chloroquinoline-endoperoxides 67, 68 related to G-factors: a) ethanolamine, reflux 95% b) TsCl, pyridine, 95% c) 4-piperidinyl-methanol, K2CO3, DMF, 160°C, 85%, d) IBX, TFA, DMSO , 45%, e) syncarpic acid, piperidine then NH4Cl/HCl 1N, f) O2, CH2Cl2, TFA (1eq), 61%, g) BuLi, MeOTf, -78°C, 27%.

ACKNOWLEDGMENT

The authors thank the "Ministère de l'Education Nationale" for the grant of J. Ruiz.

Scheme 9: Hypothesis of mechanism of Fe(II)-induced reduction of -spiro-endoperoxides.

In regard to that, the synthesis of an α-cyclopropyl endoperoxide derivative which should provide a primary C-centered radical after Fe(II) induced reduction was first considered. However, the endoperoxide could not be obtained since the precursor exists only in the enone form and could not be enolized, whatever photo-activation, acidic or basic treatment. Consequently, neither autoxidation nor photo-oxygenation (via singlet oxygen) could lead to the desired endoperoxide.

Variation of the ring size and use of more elaborated aldehydes, such as norbornene-carboxaldehyde [START_REF] O'neill | thd)(2): a superior catalyst for aerobic epoxidation and hydroperoxysilylation of unactivated alkenes: application to the synthesis of spiro-1,2,4-trioxanes[END_REF][START_REF] Baumstark | Synthesis of pentasubstituted 3-hydroxy-1,2-dioxolanes[END_REF], or protected glyceraldehyde [START_REF] Bachi | Synthesis and in vitro antimalarial activity of sulfone endoperoxides[END_REF][START_REF] Slack | Antimalarial peroxides: advances in drug discovery and design[END_REF], were then investigated. The latter might be precursor to epoxide.

The endoperoxides obtained along with their methylated analogs are presented in Figure 3.

They were prepared following the previously described methodology, and were evaluated for their antimalarial activities against a chloroquine sensitive Nigerian P. falciparum strains. [START_REF] Bernat | -Spiro endoperoxides: synthesis and evaluation of their antimalarial activities[END_REF] Methylation of the peroxyhemiketal of 37, 38, 39 very substantially increases their antimalarial activity from ten to thirty fold. In order to understand their mode of action, Fe(II) induced reduction was performed on cyclohexyl and cyclopentyl endoperoxides 37 and 38. The main products of the reaction were cyclopentyl (or cyclohexyl) ketone and the acid previously obtained during Fe(II) reduction of G3 (Scheme 5). As in the case of G3 reduction, only route a) is involved and the same Fe(II) induced degradation mechanism can be invoked. Norbornene [START_REF] O'neill | thd)(2): a superior catalyst for aerobic epoxidation and hydroperoxysilylation of unactivated alkenes: application to the synthesis of spiro-1,2,4-trioxanes[END_REF] and acetonide (41) endoperoxides along with their methylated analogues [START_REF] Baumstark | Synthesis of pentasubstituted 3-hydroxy-1,2-dioxolanes[END_REF][START_REF] Slack | Antimalarial peroxides: advances in drug discovery and design[END_REF] exhibited very low antimalarial activities with values of the same range for both hydroxylated and methylated compounds which is a clue for probably another type of Fe(II) degradation mechanism.

In conclusion, considering all endoperoxidic analogues of the G-factor family studied, G3Me (3) remains the lead compound with its potent in vitro antimalarial activity in the submicromolar range.

Hybrid molecules