Nicolas Primas

Peggy Suzanne

Pierre Verhaeghe

Sébastien Hutter

Charline Kieffer

Michèle Laget

Anita Cohen

Julie Broggi

Jean-Charles Lancelot

Aurélien Lesnard

Patrick Dallemagne

Pascal Rathelot

Sylvain Rault

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

Nadine Azas
email: nadine.azas@univ-amu.fr.

Synthesis and in vitro evaluation of 4-trichloromethylpyrrolo[1,2-a]quinoxalines as new antiplasmodial agents

Keywords: Plasmodium falciparum, Pyrrolo[1,2-a]quinoxaline, Trichloromethyl goup, In vitro antiplasmodial activity, In vitro HepG2 cytotoxicity, SARs

Introduction

Nowadays, malaria still remains one of the most prevailing and lethal infectious disease worldwide. The protozoa parasite responsible for this infection belongs to the Plasmodiumgenus and is transmitted to humans by the bite of the infectedAnopheles mosquito female. Among the fivePlasmodium species responsible for malaria, the most lethal is P. falciparum, causing cerebral malaria. According to the WHO, in 2012, malaria affected about 207 million people in the inter-tropical area and caused an estimated 627,000 deaths [1].

Artemisinin-based combination therapies (ACTs) are recommended by the WHO as the firstline treatment for treating malaria caused by P. falciparum. Unfortunately, parasite resistance to artemisinin derivatives has now been detected in 4 countries of the Greater Mekong subregion: Cambodia, Myanmar, Thailand and Vietnam [2][START_REF] Fidock | Drug discovery: Priming the antimalarial pipeline[END_REF][4][START_REF] Müller | Artemisinin resistance on the Thai-Cambodian border[END_REF][START_REF] Sibley | Monitoring antimalarial drug resistance: Applying lessons learned from the past in a fast-moving present[END_REF]. This emerging resistance of P. falciparum strains is a serious problem for the containment and the treatment of this disease as it could lead to a resurgence of more virulent levels of malaria, unless new chemical entities,displaying novel mechanisms of action, be rapidly found [START_REF] Anthony | The global pipeline of new medicines for the control and elimination of malaria[END_REF][8][START_REF] Zhang | Targeting protein kinases in the malaria parasite: update of an antimalarial drug target[END_REF][START_REF] Schrader | The antimalarial pipeline -An update[END_REF].

Aiming at synthesizing original molecules displaying antiparasitic properties [START_REF] Kabri | Original quinazoline derivatives displaying antiplasmodial properties[END_REF][START_REF] Crozet | Lowering of 5-nitroimidazole's mutagenicity: towards optimal antiparasitic pharmacophore[END_REF][START_REF] Cohen | Tandem synthesis and in vitro antiplasmodial evaluation of new naphtho[2,1-d]thiazole derivatives[END_REF][START_REF] Gellis | Preparation and antiprotozoal evaluation of promising beta-carboline alkaloids[END_REF][START_REF] Kieffer | Sonogashira cross-coupling reaction in 4-chloro-2trichloromethylquinazoline series is possible despite a side dimerization reaction[END_REF], our research group previously reported promising results in 4-substituted-2trichloromethylquinazoline series, showing that the trichloromethyl group was crucial for the in vitro activity against Plasmodium falciparum [START_REF] Verhaeghe | Synthesis and antiplasmodial activity of new 4-aryl-2trichloromethylquinazolines[END_REF][START_REF] Verhaeghe | Synthesis and in vitro antiplasmodial evaluation of 4-anilino-2-trichloromethylquinazolines[END_REF][START_REF] Castera-Ducros | Targeting the human malaria parasite Plasmodium falciparum: In vitro identification of a new antiplasmodial hit in 4-phenoxy-2-trichloromethylquinazoline series[END_REF][START_REF] Verhaeghe | 4-Thiophenoxy-2trichloromethyquinazolines display in vitro selective antiplasmodial activity against the human malaria parasite Plasmodium falciparum[END_REF][START_REF] Primas | A New Synthetic Route to Original Sulfonamide Derivatives in 2-Trichloromethylquinazoline Series: A Structure-Activity Relationship Study of Antiplasmodial Activity[END_REF]. These results prompted us to investigate wider the anti-infectious potential of various aromatic nitrogen-containing heterocyclic scaffolds bearing a trichloromethyl group, in a view to identify new antiplasmodial candidates.

Results and discussion

Preliminary antiplasmodial screening of various trichloromethylated heterocyclic scaffolds

An antiplasmodial screening was first made from a series of closely related compounds corresponding to aromatic 6-membered bicyclic nitrogen-containing heterocycles bearing a CCl 3 group. These compounds were selected in our internal chemical library, part of the French National Chemical Library(Chimiothèque Nationale). Thus, quinoline (series 1), isoquinoline (series 2), quinazoline (series 3 and 4) and quinoxaline (series 5)derivatives (Figure 1) were evaluated in vitro toward both their antiplasmodial activity against the K1 multi-resistant P. falciparum strain (determination of the IC 50 =inhibitory concentration 50%)

and their cytotoxicity (determination of the CC 50 = cytotoxic concentration 50%) on the HepG2 human cell line.

<Insert Figure 1.> Molecules 1a, 2a, 4a and 5a were prepared from their corresponding methylated precursors (1b, 2b and 5b, purchased from Sigma-Aldrich) by a microwave-assisted chlorination reaction using PCl 5 and POCl 3 [START_REF] Verhaeghe | Highly efficient microwave assisted α-trichlorination reaction of α-methylated nitrogen containing heterocycles[END_REF][START_REF] Verhaeghe | Convenient preparation of original vinylic chlorides with antiparasitic potential in quinoline series[END_REF]. Molecule 4b was prepared as described by Liu and co-workers by reacting acetamidine hydrochloride with 2-bromobenzylamine in the presence of CuBr as catalyst [START_REF] Liu | Copper-Catalyzed Sequential N-Arylation and Aerobic Oxidation: Synthesis of Quinazoline Derivatives[END_REF].Compound 3awas obtained by using an alternative route which was previously developed in our lab [START_REF] Since | First SNAr reaction using TDAE-initiated carbanions in quinazoline series[END_REF][START_REF] Broggi | Organic Electron Donors as Powerful Single-Electron Reducing Agents in Organic Synthesis[END_REF]. Thus, 4-chloroquinazoline was reacted with bromotrichloromethane in the presence of tetrakis(dimethylamino)ethylene (TDAE) instead of using hazardous chlorine gas, as described in the literature [START_REF] Mencarelli | Formation of a neutral covalent adduct in the nucleophilic aromatic substitution reaction involving a carbon leaving group[END_REF]. Then, all these molecules were tested in vitro and compared with two commercial reference-drugs: chloroquine and doxycycline. For all tested compounds, the corresponding selectivity indexes (SI) were calculated. The results are presented in Table 1.

<Insert Table 1.> Among all tested compounds, only the quinoline, isoquinoline or quinoxaline derivatives bearing a CCl 3 group showed some potential in vitro antiplasmodial activity (respectively compounds 1a, 2a and 5a), in comparison with chloroquine or doxycycline. The quinazoline derivatives including a CCl 3 group at position 2 or 4 (compounds 4a and 3a) did not display significant activity. These last results indicated that the CCl 3 group was not sufficient alone for providing antiplasmodial activity in quinazoline series and suggested that the position of the CCl 3 group on the quinazoline ring could also play a key-role toward biological activity.

Moreover, when comparing the activities of the CCl 3 containing compounds to their CH 3 analogs, in all tested series, it clearly appeared that the CCl 3 group was involved in the antiplasmodial activity, in accordance with the experimental results which we previously obtained in 4-substituted quinazoline series [START_REF] Verhaeghe | Synthesis and antiplasmodial activity of new 4-aryl-2trichloromethylquinazolines[END_REF][START_REF] Verhaeghe | Synthesis and in vitro antiplasmodial evaluation of 4-anilino-2-trichloromethylquinazolines[END_REF][START_REF] Castera-Ducros | Targeting the human malaria parasite Plasmodium falciparum: In vitro identification of a new antiplasmodial hit in 4-phenoxy-2-trichloromethylquinazoline series[END_REF][START_REF] Verhaeghe | 4-Thiophenoxy-2trichloromethyquinazolines display in vitro selective antiplasmodial activity against the human malaria parasite Plasmodium falciparum[END_REF].

Although displaying antiplasmodial activity, quinoline 1a and isoquinoline 2a did not appear to be the most potent compounds because of their rather high IC 50 values (respectively 8.5 and 8 M), in comparison with quinoxaline 5a (IC 50 = 1.5 M)and reference-drugs chloroquine (IC 50 = 0.6 µM) and doxycycline (IC 50 = 6.0 µM). Nevertheless, 5a showed a significant in vitro cytotoxicity (CC 50 = 3.1 M) in comparison with doxorubicine (CC 50 = 0.2 µM), making this compound poorly selective and unsuitable for further development. In order to validate the SARs of 5a, in addition to its methylated precursor 5b, three closely related analogs (5c, 5d and 5e) were also tested (Figure 2). Compound 5c was prepared by a chlorination reaction of 5b using PCl 5 and POCl 3 ,under milder conditions than those used to prepare 5a, while compounds 5d and 5e were purchased from Aldrich.

<Insert Figure 2.>

The comparison of the results noted for the tested quinoxaline derivatives 5a-e indicated that the quinoxaline ring, itself, did not show any antiplasmodial potential (IC 50 of 5e> 50 M) and that the CCl 3 group was the only one that could confer antiplasmodial potential to the studied series, when located at position 2 of the quinoxaline ring (Table 1).

Then, trying to reach a new 2-trichloromethylquinoxaline derivative presenting an improved antiplasmodial profile, we synthesized compound 5f in a view to compare its biological profile to the one of its antiplasmodial quinazoline position isomer which we previously reported [START_REF] Verhaeghe | Synthesis and antiplasmodial activity of new 4-aryl-2trichloromethylquinazolines[END_REF](Figure 3). 5f was prepared from its corresponding methylated precursor which was synthesized according to the literature [START_REF] Delpivo | A Green Synthesis of Quinoxalines and 2,3-Dihydropyrazines[END_REF] by a microwave-assisted chlorination reaction using PCl 5 and POCl 3 . Concerning the influence of the nature of the substituents at position 7 of the pyrrolo[1,2a]quinoxaline scaffold, it appeared that halogen substituents have a negative influence on the cytotoxicity profile without improving the antiplasmodial activity. Moreover, the carboxylic acid derivative 10j was not active. Globally, no substituent at position 7 of the chemical scaffold was required for providing antiplasmodial activity; some substituents (bromine, iodine, methyl carboxylate or hydroxyamidine) could even play a negative role toward either cytotoxicity or solubility in the biological media.

In order to study the influence of the trichloromethyl group at position 4 of the pyrroloquinoxaline scaffold, 4-methylsubstituted analogs 11a, 11f and 11h of respective hit compounds 10a, 10f and 10h were prepared and evaluated. The results clearly showed that these 4-methylpyrrolo[1,2-a]quinoxaline analogs do not show any activity against P.

falciparum. Moreover, the 4-CF 3 substituted analog 12a of hit compound 10a was not active neither, demonstrating that the CCl 3 group is very specifically required for providing antiplasmodial activity in 4-trichloromethylpyrrolo[1,2-a]quinoxaline series.

Conclusion

From a preliminary screening, highlighting that the 2-trichloromethylquinoxaline scaffold displayed antiplasmodial potential, a new series of 7-substituted-4-trichloromethylpyrrolo- HRMS spectra were recorded on QStar Elite (Applied Biosystems SCIEX) spectrometer. PEG was the matrix for HRMS. The experimental exact mass was given for the ion which has the maximum isotopic abundance. Purity of synthetized compounds was checked with LC-MS analyses which were realized at the Faculté de Pharmacie de Marseille with a Thermo Scientific Accela High Speed LC System ® coupled with a single quadrupole mass spectrometer Thermo MSQ Plus ® . The RP-HPLC column used is a Thermo Hypersil Gold ® 50 × 2.1 mm (C18 bounded), with particles of 1.9 µm diameter. The volume of sample injected on the column was 1 µL. The chromatographic analysis, total duration of 8 min, is made with the gradient of following solvents: t = 0 min, water/methanol 50/50; 0 < t < 4 min, linear increase in the proportion of water to a ratio water/methanol 95/5; 4 < t < 6 min, water/methanol 95/5; 6 < t < 7 min, linear decrease in the proportion of water to return to a ratio 50/50 water/methanol; 6 < t < 7 min, water/methanol 50/50. The water used was buffered with 5 mM ammonium acetate. The retention times (t R ) of the molecules analyzed are indicated in min. The preparation of compounds1a [START_REF] Verhaeghe | Highly efficient microwave assisted α-trichlorination reaction of α-methylated nitrogen containing heterocycles[END_REF][START_REF] Kato | Trichloromethylquinolines : Synthesis and Reaction with Trimethyl Phosphite[END_REF], 3a [START_REF] Since | First SNAr reaction using TDAE-initiated carbanions in quinazoline series[END_REF][START_REF] Mencarelli | Formation of a neutral covalent adduct in the nucleophilic aromatic substitution reaction involving a carbon leaving group[END_REF], 4b [START_REF] Verhaeghe | Convenient preparation of original vinylic chlorides with antiparasitic potential in quinoline series[END_REF], 5a [START_REF] Verhaeghe | Highly efficient microwave assisted α-trichlorination reaction of α-methylated nitrogen containing heterocycles[END_REF],

11a [START_REF] Cheeseman | The synthesis of pyrrolo[1,2-a]quinoxalines from N-(2acylaminophenyl)pyrroles[END_REF][START_REF] Trager | Human malaria parasites in continuous culture[END_REF] and 12a [START_REF] Zhu | Synthesis of 6-trifluoromethylindolo[1,2c]quinazolines and related heterocycles using N-(2-iodophenyl)trifluoroacetimidoyl chlorides as starting material via C-H bond functionalization[END_REF] was achieved as described in the literature. Compounds 1b, 2b, 5b, 5d and 5e were purchased from Sigma-Aldrich.

1-(Trichloromethyl)isoquinoline (2a)

A 100-mL round-bottomed flask equipped with a drying tube-condenser and a magnetic stir bar was charged with 1-methylisoquinoline (500 mg, 3.49 mmol), PCl 5 (

2-(Dichloromethyl)quinoxaline (5c)

A 100-mL round-bottomed flask equipped with a drying tube-condenser and a magnetic stir bar was charged with 2-methylquinoxaline (2.0 g, 13.87 mmol), POCl 3 (2.59 mL, 27.74 mmol) and PCl 5 (5.78 g, 27.74 mmol). The mixture was refluxed for 12 h. After the reaction mixture was cooled with an ice bath, the mixture was carefully poured into 200 mL of ice.

The mixture was made alkaline by addition of NaHCO

2-Phenyl-3-(trichloromethyl)quinoxaline (5f)

A 50-mL round-bottomed flask equipped with a drying tube-condenser and a magnetic stir bar was charged with 2-methyl-3-phenylquinoxaline [START_REF] Delpivo | A Green Synthesis of Quinoxalines and 2,3-Dihydropyrazines[END_REF]

N-[2-(1H-Pyrrol-1-yl)phenyl]-2,2,2-trichloroacetamide (9a)

Yield 65%. Off-white powder. Mp 79°C. 1 A 100-mL round-bottomed flask equipped with a condenser and a magnetic stir bar was charged with methyl 4-(trichloromethyl)pyrrolo[1,2-a]quinoxaline-7-carboxylate (500 mg, 1.45 mmol) and 15 mL of conc. HCl. The mixture was refluxed until disappearance of starting material (ca. 5 h). After the reaction mixture was cooled to rt, the mixture was extracted five times with CH 2 Cl 2 . The combined organic layers were washed twice with water. Then the organic layer was dried with anhydrous sodium sulphate and was concentrated under vacuum. After cooling at rt, the mixture was filtered through a pad of Celite ® and thoroughly washed with CHCl 3 . Volatiles were removed under vaccum then the mixture was made alkaline with a saturated solution of Na 2 CO 3 . The aqueous layer was extracted with CHCl 3 , and then the organic layer was dried with anhydrous sodium sulphate and was concentrated under vacuum.

Purification by chromatography on silica gel (CHCl 3 /acetone) gave the expected product.

In vitroCytotoxicity evaluation

HepG2 cell line was maintained at 37 °C, 6% CO 2 , 14% O 2 , 80% N 2 , with 90% humidity in RPMI supplemented with 10% foetal bovine serum, 1% L-glutamine (200 mM) and penicillin (100 U/mL) / streptomycin (100 µg/mL) (complete RPMI medium). The evaluation of the tested molecules cytotoxicity on the HepG2 (hepatocarcinoma cell line purchased from ATCC, ref HB-8065) cell line was performed according to the method of Mosmann [START_REF] Mosmann | Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays[END_REF] with slight modifications. Briefly, 5.10 3 cells in 100 µL of complete medium were inoculated into each well of 96-well plates and incubated at 37 °C in a humidified 6% CO 2 . After 24 h incubation, 100 μL of medium with various product concentrations dissolved in DMSO (final concentration less than 0.5% v/v) were added and the plates were incubated for 72 h at 37 °C.

Triplicate assays were performed for each sample. Each plate-well was then microscopeexamined for detecting possible precipitate formation before the medium was aspirated from the wells. 100 μL of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) solution (0.5 mg/mL in medium without FCS) were then added to each well. Cells were incubated for 2 h at 37 °C. After this time, the MTT solution was removed and DMSO (100 μL) was added to dissolve the resulting blue formazan crystals. Plates were shaken vigorously (700 rpm) for 10 min. The absorbance was measured at 570 nm with 630 nm as reference wavelength using a BIO-TEK ELx808 Absorbance Microplate Reader. DMSO was used as blank and doxorubicin (purchased from Sigma Aldrich) as positive control. Cell viability was calculated as percentage of control (cells incubated without compound). The 50% cytotoxic concentration (CC 50 ) was determined from the dose-response curve by using the TableCurve software 2D v.5.0. CC 50 values represent the mean value calculated from three independent experiments.

Figures captions:

Figure 1.Structures of the heterocyclic compounds which were selected from our chemical library for the preliminary in vitro antiplasmodial screening Tables:

Table 1.In vitro antiplasmodial and cytotoxicity evaluation of compounds 1-5.

Entry

<Insert Figure 3 <Insert Figure 5 .> 2 . 1 .

 3521 .> Indeed, quinoxaline 5f appeared to be much more selective than 5a(SI = 17.5 versus 2.1, respectively), presenting a quite low antiplasmodial IC 50 value (0.2 M), better than the one of its quinazoline position isomer (IC 50 = 8 M). However, quinoxaline 5f remained too cytotoxic (CC 50 = 3.5 M) for allowing further investigations. Such interesting result noted with compound 5f prompted us to carry on the investigation of the antiplasmodial potential of the 2-trichloromethylquinoxaline scaffold, especially when considering that various pyrrolo[1,2-a]quinoxaline derivatives (Figure 4) had been reported in the literature as quite promising antimalarial compounds [28-31]. <Insert Figure 4.> Taking advantage of the synthetic know-how of our research teams in the fields of both trichloromethylated heterocycles [15-21] and pyrrolo[1,2-a]quinoxalines [32] preparation, we studied the synthesis and antiplasmodial properties of a series of original molecules, based on the quinoxaline ring. These molecules, corresponding to 4-(trichloromethyl)pyrrolo[1,2a]quinoxaline derivatives, were a combination between the pyrrolo[1,2-a]quinoxaline scaffold and the trichloromethyl group (Figure 5), aiming at identifying new antiplasmodial quinoxalines with improved biological profile, being less cytotoxic than 5f. Synthesis of the 4-(trichloromethyl)pyrrolo[1,2-a]quinoxaline series The 7-substituted-4-(trichloromethyl)pyrrolo[1,2-a]quinoxalines derivatives were synthesized from various commercially available 4-substituted-2-nitroanilines 6a-i (Scheme

[ 1 ,

 1 2-a]quinoxaline derivatives was synthesized in four to five reaction steps. Thus, three hit compounds were identified, presenting in vitro IC 50 values in the micromolar range (1.5 ≤ K1 P. falciparumIC 50 ≤ 2.4 µM), good cytotoxity profiles (17 ≤ HepG2CC 50 ≤ 53 µM) and promising selectivity indexes (11.3 ≤ SI ≤ 27.5), in comparison with the antimalarial drug references chloroquine and doxycycline. Structure-activity relationships showed that the CCl 3 group was mandatory for providing antiplasmodial activity to the pyrrolo[1,2-a]quinoxaline scaffold of the studied series. They also pointed out that the attachment of a pyrrole moiety to the quinoxaline ring improved the cytotoxicity profile without disturbing the anti-infectious activity. It finally showed that the substitution of the 4-trichloromethylpyrrolo[1,2a]quinoxaline scaffold at position 7 was neither necessary nor beneficial, as regards of both cytotoxicity or solubility in the biological media. Complementary pharmacomodulation works will be carried out to study the influence of the substitution of the same scaffold at other positions toward antiplasmodial activity. Commercial reagents were used as received without additional purification. Melting points were determined on a Kofler bench and are uncorrected. Elemental analysis and HRMS were carried out at the Spectropole, Faculté des Sciences et Techniques de Saint-Jérôme, Marseille, France. NMR spectra were recorded on a Bruker ARX 200 spectrometer at the Faculté de Pharmacie de Marseille ( 1 H-NMR: 200 MHz, 13 C-NMR: 50 MHz) or on aJEOL Lambda 400 Spectrometer at the Centre d'Etudes et de Recherche sur le Médicament de Normandie( 1 H-NMR: 400 MHz, 13 C-NMR: 100 MHz). NMR references were the following: 1 H: CHCl 3 δ = 7.26, DMSO-d 6 δ = 2.50 and 13 C: CHCl 3 δ = 76.9, DMSO-d 6 δ = 39.5. Solvents were dried by conventional methods. The following adsorbent was used for column chromatography: silica gel 60 (Merck, particle size 0.063-0.200 mm, 70-230 mesh ASTM). TLC was performed on 5 cm × 10 cm aluminium plates coated with silica gel 60F-254 (Merck) in an appropriate eluent. Visualization was made with ultraviolet light (234 nm).

3. 1 . 5 .

 15 Representative procedure for the preparation of 1-(4-substituted-2nitrophenyl)-1H-pyrrole (7a-i)A 100-mL round-bottomed single-neck flask equipped with a condenser and a magnetic stir bar was charged with appropriated 4-substituted-2-nitroaniline (25.0 mmol), 2,5dimethoxytetrahydofurane (25.0 mmol) and 40 mL of glacial acetic acid. The flask was refluxed until disappearance of starting material (ca. 2 h). After the reaction mixture was cooled to rt, removal of the volatiles under reduced pressure gave the crude product which was purified by column-chromatography (Petroleum Ether/EtOAc = 9:1 then 7:3) to give the desired 1-(4-substituted-2-nitrophenyl)-1H-pyrrole.3.1.5.

3. 1 . 6 . 3 . 1 . 6 . 1 . 5 -

 1631615 Representative procedure for the preparation of 5-substituted-2-(1Hpyrrol-1-yl)anilineA 250-mL round-bottomed single-neck flask equipped with a condenser and a magnetic stir bar was charged with appropriated 1-(4-substituted-2-nitrophenyl)-1H-pyrrole (15.0 mmol), tin (II) chloride dihydrate (16.92 g, 75.0 mmol) and 60 mL of ethanol. The flask was refluxed until disappearance of starting material (ca. 3 h). After the reaction mixture was cooled to rt, the mixture was quenched with a saturated solution of sodium carbonate. The white precipitate was filtered through a pad of Celite ® and was thoroughly washed with ethanol. Then, removal of the volatiles under reduced pressure gave the crude 5-substituted-2-(1Hpyrrol-1-yl)aniline which was engaged in the next reaction step without any further purification Iodo-2-(1H-pyrrol-1-yl)aniline (8e) Yield 71%. Orange powder. Mp 80°C. 1 H NMR (400 MHz, CDCl 3 ) δ = 7.15 (d, J = 1.7 Hz, 1H, H-6), 7.09 (dd, J = 2.0; 8.1 Hz, 1H, H-4), 6.85 (d, J = 8.0 Hz, 1H, H-3), 6.80 (t, J = 1.7

3. 1 . 7 .

 17 Representative procedure for the preparation of 2,2,2-trichloro-N-[5substituted-2-(1H-pyrrol-1-yl)phenyl]acetamide (9a-i) A 100-mL round-bottomed flask equipped with a condenser, an Ar inlet and a magnetic stir bar was charged with appropriated 5-substituted-2-(1H-pyrrol-1-yl)aniline (15.0 mmol), 25 mL of anhydrous dioxane, triethylamine (3.14 mL, 22.5 mmol). The mixture was cooled with a ice bath and trichloroacetyl chloride was added dropwise (1.67 mL, 15.0 mmol). The flask was refluxed until disappearance of starting material (ca. 4 h). After the reaction mixture was cooled to rt, removal of the volatiles under reduced pressure gave the crude product which was dissolved in EtOAc. The organic layer was washed with a aqueous solution of sodium bicarbonate and brine, then the organic layer was dried with anhydrous sodium sulphate and was concentrated under vacuum. Purification by chromatography on silica gel (Petroleum Ether/EtOAc) gave the expected 2,2,2-trichloro-N-[5-substituted-2-(1H-pyrrol-1yl)phenyl]acetamide.

3. 1 . 8 .

 18 Representative procedure for the preparation of 7-substituted-4-(trichloromethyl)pyrrolo[1,2-a]quinoxaline (10a-i)A 100-mL round-bottomed flask equipped with a condenser, an Ar inlet and a magnetic stir bar was charged with appropriated 2,2,2-trichloro-N-[5-substituted-2-(1H-pyrrol-1yl)phenyl]acetamide (5.0 mmol), 15 mL of freshly distilled POCl 3 and anhydrous pyridine (1 mL). The mixture was refluxed until disappearance of starting material (ca. 4 h). After the reaction mixture was cooled with an ice bath, it was carefully poured over 200 mL of ice. The mixture was made alkaline with concentrated ammonia to pH 10. The alkaline aqueous phase was extracted three times with CH 2 Cl 2 . The combined organic layers were washed with 10% hydrochloric acid and then by water. Then the organic layer was dried with anhydrous sodium sulphate and was concentrated under vacuum. Purification by chromatography on silica gel (Petroleum Ether/EtOAc) gave the expected 7-substituted-4-(trichloromethyl)pyrrolo[1,2-Yield 57%. Beige powder. Mp 202°C.1 H NMR (400 MHz, CDCl 3 ) δ = 8.10 (dd, J = 1.4; 8.3

Figure 2 .Figure 3 .Figure 4 .Figure 5 .Figure 1 .

 23451 Figure 2.Structures of 5a and its 2-substituted analogs in quinoxaline series

Figure 2 .

 2 Figure 2.

Figure 3 .

 3 Figure 3.

Figure 4 .

 4 Figure 4.

Figure 5 1 .

 51 Figure 5.

  Doxorubicine was used as a cytotoxic reference-drug; b Chloroquinine and doxycycline were used as antimalarial reference-drugs, c Selectivity indexes were calculated according to the formula : SI = HepG2 CC 50 / K1 IC 50 . d No activity was observed at the highest concentration tested.

►

  Doxorubicine was used as a cytotoxic reference-drug; b Chloroquinine and doxycycline were used as antimalarial reference-drugs; c Selectivity index (SI) was calculated according to the formula: SI = CC 50 /IC 50 ; d No activity was observed at the highest concentration tested; *Not determined (nd) because of lack of solubility in the culture media. Antiplasmodial screening of CCl 3 -substituted-nitrogen containing heterocycles was made. ► 2-Trichloromethylquinoxaline scaffold was the most interesting but was too cytotoxic. ► Original 4-trichloromethylpyrrolo[1,2-a]quinoxalines analogs were prepared. ►3 Compounds displayed an IC 50 <2.4 µM in vitro on K1 P. falciparum. ► Cytotoxicity was assessed in parallel on the human HepG2 cell line showing good selectivity.

2.2. Biological evaluation of the 7-substituted-4-(trichloromethyl)pyrrolo[1,2- a]quinoxaline derivatives

	carboxylic acid derivative 10j was prepared in 44% yield from methylic ester 10i by an acidic
	hydrolysis in concentrated HCl. Amidoxime10k was obtained in 74% yield by reacting nitrile
	derivative 10h with hydroxylamine hydrochloride in a refluxing EtOH/H 2 O mixture.
	Compounds 11a, 11f and 11h were obtained, respectively, by reducing the trichloromethyl
	group of 10a, 10f and 10h with iron in refluxing glacial AcOH. Compound 12a [33] was
	prepared by trifluoroacetylation of aniline 8a in the presence of trifluoroacetic acid,
	trichloroacetonitrile and triphenylphosphine [34] followed by a Bischler-Napieralski
	cyclisation.
	<Insert Scheme1.>
	Thus, a series of fifteen 4-trichloromethylpyrrolo[1,2-a]quinoxaline derivatives was then
	tested in vitro for both its in vitro cytotoxicity (human HepG2 cell line) and its antiplasmodial
	activity (K1 P. falciparummulti-resistant strain). The results (Table 2) were compared to the
	ones obtained with chloroquine and doxycycline, chosen as antimalarial reference-drugs and
	doxorubicin, chosen as a reference cytotoxic agent.
	<Insert Table2.>
	Among the eleven tested compounds bearing a trichloromethyl group, seven exerted a
	significant antiplasmodial activity in comparison with the 2 antimalarial drug references,
	presenting IC 50 values in the micromolar range (1.2 to 2.4 M). Out of these seven hit
	compounds, four molecules displayed a limited cytotoxicity toward the HepG2 human cell
	line, with CC 50 values ranging from >15 to 53 M, in comparison with reference-drug
	1). The Clauson-Kaas reaction between anilines and 2,5-dimethoxytetrahydrofuran (2,5-doxorubucine (CC 50 = 0.2 µM). Thus, three of these molecules reached a SI superior to 10
	DMTHF), in refluxing glacial AcOH, provided the pyrrolic derivatives7a-i in 63-86% yields. µM: 10a, 10f and 10h. Indeed, hit compound 10a demonstrated the same antiplasmodial
	The nitro group was then reduced, either by BiCl 3 /NaBH 4 or tin (II) chloride in refluxing activity as starting compound 5a (IC 50 = 1.5 M) but showed a greatly improved cytotoxicity
	ethanol, affording the expected 1-(2-amino-4-substituted-phenyl)pyrroles8a-i in moderate to (CC 50 = 41.2 M), making its selectivity (SI = 27.5) intermediate between the one of
	good yields(47-90%). Subsequent acylation of the aniline moiety with trichloroacetyl chloride chloroquine (SI = 50) and the one of doxycycline (SI = 3.3). It could then be concluded that
	furnished the corresponding trichloroacetamides 9a-i in 46-88% yields. These final the attachment of a pyrrole moiety to the quinoxaline ring clearly improved the antiplasmodial
	intermediates were cyclized under Bischler-Napieralski conditions, leading to the expected 7-profile of the 2-trichloromethylquinoxaline scaffold. Due to a lack of solubility in the
	substituted-4-(trichloromethyl)pyrrolo[1,2-a]quinoxalines 10a-i in 20-69% yields. The biological media, compounds 10d and 10i could not be tested.

  ice bath, it was carefully poured into 50 mL of ice and was made alkaline (pH 8) by addition of NaHCO 3 . Aqueous layer was extracted three times with CH 2 Cl 2 , and then dried with anhydrous sodium sulphate and finally concentrated under vacuum. Purification by chromatography on silica gel (Petroleum Ether/ CH 2 Cl 2 ) gave the expected 1-

	(trichloromethyl)isoquinoline.
	Yield 24%. Yellow powder. Mp 66°C. 1 H NMR (200 MHz, CDCl 3 ) δ = 8.88 (dd, J = 1.8; 7.4
	Hz, 1H, H-3), 8.53 (d, J = 5.4 Hz, 1H, H-8), 7.94 (dd, J = 2.2; 7.2 Hz, 1H, H-5), 7.67-7.80 (m,
	3H, H-4, H-6 and H-7). 13 C NMR (50 MHz, CDCl 3 ) δ = 153.9, 139.0, 138.1, 130.3, 127.9,
	127.4, 127.1, 124.3, 123.3, 98.2. Anal. Calcd for C 10 H 6 Cl 3 N: C, 48.72; H, 2.45; N, 5.68.
	Found: C, 48.32; H, 2.41; N, 5.64.

4.40 g, 20.

94 mmol) and a minimal amount of POCl 3 to allow the stirring of the mixture. The reaction mixture was heated during 1 h under microwave heating (800 W, 110 °C). After the reaction mixture was cooled with an

3.1.2. 2-(Trichloromethyl)quinazoline (4a)

A 100-mL round-bottomed flask equipped with a drying tube-condenser and a magnetic stir bar was charged with 2-methylquinazoline

[START_REF] Liu | Copper-Catalyzed Sequential N-Arylation and Aerobic Oxidation: Synthesis of Quinazoline Derivatives[END_REF]

(200 mg, 1.4 mmol), PCl 5 (870 mg, 4.2 mmol) and a minimal amount of POCl 3 to allow the stirring of the mixture. The reaction mixture was heated during 15 min under microwave irradiation (800 W, 150 °C). After the reaction mixture was cooled with an ice bath, it was carefully poured into 50 mL of ice and was made alkaline (pH 8) by addition of NaOH. Aqueous layer was extracted twice with CH 2 Cl 2 , the organic layers were washed twice with brine and then dried with anhydrous sodium sulphate and finally concentrated under vacuum. Purification by chromatography on silica gel (Petroleum Ether/ CH 2 Cl 2 ) gave the expected 2-(trichloromethyl)quinazoline. Yield 30%. Off-white powder. Mp 116°C.

1

H NMR (200 MHz, CDCl 3 ) δ = 9.57 (s, 1H, H-4), 8.22 (d, J = 9.1 Hz, 1H, H-8), 8.01-8.08 (m, 2H, H-5 and H-7), 7.81 (dt, J = 1.2; 6.7 Hz, 1H, H-6).

13

C NMR (50 MHz, CDCl 3 ) δ = 161.8, 161.3, 149.3, 135.3, 129.7, 129.3, 127.2, 123.8, 97.0. HRMS (ESI): m/z [M + H] + calcd for [C 9 H 6 Cl 3 N 2 ] + : 246.9596; found: 246.9599.

  was concentrated under vacuum. Purification by chromatography on silica gel (CHCl 3 ) gave the expected 2-(dichloromethyl)quinazoline. Yield 13%. Off-white powder. Mp 117°C.1 H NMR (200 MHz, CDCl 3 ) δ = 9.33 (s, 1H, H-4),

	8.18-8.05 (m, 2H, H-5 and H-8), 7.87-7.78 (m, 2H, H-6 and H-7), 6.88 (s, 1H, CHCl 2 ). 13 C
	NMR (50 MHz, CD 2 Cl 2 ) δ = 151.9, 143.3, 142.5, 140.1, 131.2, 131.0, 129.5, 129.3, 70.2.
	Anal. Calcd for C 9 H 6 Cl 2 N 2 : C, 50.73; H, 2.84; N, 13.15. Found: C, 50.66; H, 2.99; N, 12.92.

[START_REF] Fidock | Drug discovery: Priming the antimalarial pipeline[END_REF]

.The resulting aqueous solution was extracted three times with CHCl 3 , and then the organic layer was dried with anhydrous sodium sulphate and

  (441 mg, 2.0 mmol), POCl 3 (1.86 mL, 20.0 mmol) and PCl 5 (2.08 g, 10.0 mmol). The mixture was refluxed for 1h30. After the reaction mixture was cooled with an ice bath, the mixture was carefully poured into 200 mL of ice. The mixture was extracted three times with EtOAc. Then the organic layer was dried with anhydrous sodium sulphate and was concentrated under vacuum. Purification by

chromatography on silica gel (Petroleum Ether/EtOAc) gave the expected 2-phenyl-3-(trichloromethyl)quinoxaline. Yield 26%. White powder. Mp 125°C. 1 H NMR (200 MHz, CDCl 3 ) δ = 8.31-8.15 (m, 2H, H-2'), 7.95-7.83 (m, 2H, H-5 and H-8), 7.64 (dd, J = 2.9; 6.8 Hz, 2H, H-6 and H-7), 7.54-7.44 (m, 3H, H-3' and H-4'). 13 C NMR (50 MHz, CDCl 3 ) δ = 152.6, 149.7, 141.6, 139.6, 138.6, 132.3, 131.3, 129.8, 129.8, 129.0, 129.0, 127.9, 97.1. HRMS (ESI): m/z [M + Na] + calcd for [C 15 H 9 Cl 3 N 2 Na] + : 344.9723; found: 344.9723.

  Yield 82%. Brown powder. Mp 70°C.1 H NMR (400 MHz, CDCl 3 ) δ = 8.15 (d, J = 1.7 Hz, 1H, H-3'), 7.96 (ddd, J = 0.7; 2.0; 8.6 Hz, 1H, H-5'), 7.21 (d, J = 8.2 Hz, 1H, H-6'), 6.76 (t, J = 1.7 Hz, 2H, H-2), 6.37 (t, J = 2.0 Hz, 2H, H-3).13 C NMR (100 MHz, CDCl 3 ) δ =145.5, 142.2, 134.0, 133.6, 129.3, 121.3, 111.7, 90.6. HRMS (ESI): m/z [M + H] + calcd for [C 10 H 8 N 2 O 2 I] + : 314.9625; found: 314.9626.

	3.1.5.2. Methyl 3-nitro-4-(1H-pyrrol-1-yl)benzoate (7i)
	Yield 79%. Yellow powder. Mp 118°C. 1 H NMR (200 MHz, CDCl 3 ) δ = 8.47 (d, J = 1.5 Hz,
	1H, H-2), 8.28 (dd, J = 1.7; 8.4 Hz, 1H, H-6), 7.54 (d, J = 8.4 Hz, 1H, H-5), 6.81 (t, J = 2.1
	Hz, 2H, H-2'), 6.39 (t, J = 2.1 Hz, 2H, H-3'), 3.98 (s, 3H, CH 3 ). 13 C NMR (50 MHz, CDCl 3 )
	δ =164.5, 144.4, 137.4, 134.0, 129.2, 127.3, 126.5, 121.0, 112.2, 53.0. HRMS (ESI): m/z [M +
	H] + calcd for [C 12 H 11 N 2 O 4 ]

1. 1-(4-Iodo-2-nitrophenyl)-1H-pyrrole (7e) + : 247.0713; found: 247.0714.

  110.8, 105.1, 92.4, 55.8. HRMS (ESI): m/z [M + H] + calcd for [C 13 H 12 N 2 O 2 Cl 3 ] + : 332.9959;

	Yield 63%. Yellow powder. Mp 98°C. 1 H NMR (400 MHz, CDCl 3 ) δ = 8.43 (d, J = 2.2 Hz,
	1H, H-6), 8.29 (bs, NH), 7.26 (d, J = 8.6 Hz, 1H, H-3), 7.20 (dd, J = 2.2; 8.6 Hz, 1H, H-4), found: 332.9958.
	6.73 (t, J = 1.7 Hz, 2H,H-2 pyrrole), 6.38 (t, J = 1.7 Hz, 2H,H-3 pyrrole). 13 C NMR (100 3.1.7.8. 2,2,2-Trichloro-N-(5-cyano-2-(1H-pyrrol-1-yl)phenyl)acetamide MHz, CDCl 3 ) δ =159.3, 135.1, 133.4, 130.0, 128.2, 125.9, 122.1, 120.8, 111.6, 92.3. HRMS (9h)
	(ESI): m/z [M -H] -calcd for [C 12 H 7 N 2 OCl 4 ] -: 336.9289; found: 336.9288. Yield 77%. Yellow powder. Mp 129°C. 1 H NMR (200 MHz, CDCl 3 ) δ = 8.75 (d, J = 1.6 Hz,
	3.1.7.4. N-[5-Bromo-2-(1H-pyrrol-1-yl)phenyl]-2,2,2-trichloroacetamide 1H, H-6), 8.54 (bs, NH), 7.59 (dd, J = 1.7; 8.2 Hz, 1H, H-4), 7.48 (d, J = 8.1 Hz, 1H, H-3),
	(9d) 6.84 (t, J = 2.1 Hz, 2H, H-2 pyrrole), 6.48 (t, J = 2.1 Hz, 2H, H-3 pyrrole). 13 C NMR (50
	Yield 63%. Beige powder. Mp 90°C. 1 H NMR (400 MHz, CDCl 3 ) δ = 8.57 (d, J = 2.0 Hz, MHz, CDCl 3 ) δ =159.5, 135.1, 132.8, 129.6, 127.9, 124.5, 121.7, 117.6, 112.8, 112.4, 92.1.
	1H, H-6), 8.29 (bs, NH), 7.37 (dd, J = 2.2; 8.6 Hz, 1H, H-3), 7.20 (d, J = 8.6 Hz, 1H, H-4), HRMS (ESI): m/z [M + H] + calcd for [C 13 H 9 N 3 OCl 3 ] + : 327.9806; found: 327.9802.
	6.73 (t, J = 1.7 Hz, 2H, H-2 pyrrole), 6.38 (t, J = 1.7 Hz, 2H, H-3 pyrrole). 13 C NMR (100 MHz, CDCl 3 ) δ =159.3, 133.5, 130.6, 128.9, 128.5, 123.7, 122.8, 122.0, 111.7, 92.3. HRMS 3.1.7.9. Methyl 4-(1H-pyrrol-1-yl)-3-(2,2,2-trichloroacetamido)benzoate (9i)
	(ESI): m/z [M + H] + calcd for [C 12 H 9 N 2 OBrCl 3 ] MHz, CDCl 3 ) δ =159.3, 135.1, 133.3, 131.3, 129.5, 128.6, 122.0, 111.6, 93.8, 82.3. HRMS Yield 77%. Amber oil.
	(ESI): m/z [M + H] + calcd for [C 12 H 9 N 2 OICl 3 ] + : 428.8820; found: 428.8818.
	3.1.7.6. 2,2,2-Trichloro-N-(5-methyl-2-(1H-pyrrol-1-yl)phenyl)acetamide
	(9f)
	Yield 46%. Amber oil. 1 H NMR (200 MHz, CDCl 3 ) δ = 8.31 (bs, NH), 8.23 (s, 1H, H-6), 7.26
	(d, J = 8.0 Hz, 1H, H-3), 7.09 (d, J = 8.0 Hz, 1H, H-4), 6.79 (t, J = 2.1 Hz, 2H, H-2 pyrrole),
	3.1.7.2. 2,2,2-Trichloro-N-[5-fluoro-2-(1H-pyrrol-1-yl)phenyl]acetamide 6.41 (t, J = 2.1 Hz, 2H, H-3 pyrrole), 2.46 (s, 3H, CH 3 ). 13 C NMR (50 MHz, CDCl 3 ) δ
	(9b) =159.2, 139.5, 132.0, 129.3, 126.9, 126.5, 122.2, 121.2, 110.9, 92.6, 21.6. HRMS (ESI): m/z
	Yield 65%. Brown powder. Mp 64°C. 1 H NMR (400 MHz, CDCl 3 ) δ = 8.34 (bs, NH), 6.85 [M + H] + calcd for [C 13 H 11 N 2 OCl 3 ] + : 317.0010; found: 317.0013.
	(dd, J = 2.7; 10.2 Hz, 1H, H-6), 7.36 (dd, J = 5.6; 8.8 Hz, 1H, H-3), 6.98 (td, J = 2.9; 7.8 Hz, 3.1.7.7. 2,2,2-Trichloro-N-(5-methoxy-2-(1H-pyrrol-1-yl)phenyl)acetamide 1H, H-4), 6.78 (t, J = 2.0 Hz, 2H, H-2 pyrrole), 6.43 (t, J = 2.0 Hz, 2H, H-3 pyrrole). 13 C (9g)
	NMR (100 MHz, CDCl 3 ) δ =162.4 (d, J = 266.8 Hz), 159.3, 134.1 (d, J = 12.3 Hz), 128.6 (d, Yield 88%. Yellow powder. Mp 67°C. 1 H NMR (200 MHz, CDCl 3 ) δ = 8.29 (bs, NH), 8.04 J = 9.9 Hz), 127.5, 122.3, 112.5 (d, J = 23.0 Hz), 111.5, 108. 1 (d, J = 29.6 Hz), 92.3. HRMS (d, J = 2.8 Hz, 1H, H-6), 7.28 (d, J = 9.3 Hz, 1H, H-3), 6.79 (dd, J = 2.2; 8.2 Hz, 1H, H-4),

H NMR (200 MHz, CDCl 3 ) δ = 8.41 (m, 1H, H-6), 8.37 (bs, NH), 7.48 (dt, J = 1.8; 8.

4

Hz, 1H, H-5), 7.37 (dt, J = 1.6; 7.8 Hz, 1H, H-3), 7.28-7.24 (m, 1H, H-4), 6.82 (t, J = 2.2 Hz, 2H, H-2 pyrrole), 6.43 (t, J = 2.2 Hz, 2H, H-3 pyrrole). 13 C NMR (50 MHz, CDCl 3 ) δ =159.3, 132.4, 131.8, 129.2, 127.3, 125.9, 122.1, 120.8, 111.2, 92.6. HRMS (ESI): m/z [M + H] + calcd for [C 12 H 10 N 2 OCl 3 ] + : 302.9853; found: 302.9852. (ESI): m/z [M + H] + calcd for [C 12 H 9 N 2 OFCl 3 ] + : 320.9759; found: 320.9762. 3.1.7.3. 2,2,2-Trichloro-N-[5-chloro-2-(1H-pyrrol-1-yl)phenyl]acetamide (9c) + : 382.8934; found: 382.8933. 3.1.7.5. 2,2,2-Trichloro-N-[5-iodo-2-(1H-pyrrol-1-yl)phenyl]acetamide (9e) Yield 69%. Brown powder. Mp 90°C. 1 H NMR (400 MHz, CDCl 3 ) δ = 8.79 (d, J = 1.7 Hz, 1H, H-6), 8.32 (bs, NH), 7.63 (dd, J = 1.9; 8.3 Hz, 1H, H-4), 7.10 (d, J = 8.3 Hz, 1H, H-3), 6.79 (t, J = 1.9 Hz, 2H, H-2 pyrrole), 6.43 (t, J = 1.9 Hz, 2H, H-3 pyrrole). 13 C NMR (100 6.76 (t, J = 2.1 Hz, 2H, H-2 pyrrole), 6.40 (t, J = 2.1 Hz, 2H, H-3 pyrrole), 3.88 (s, 3H, OCH 3 ). 13 C NMR (50 MHz, CDCl 3 ) δ =159.9, 159.2, 133.5, 128.0, 124.3, 122.4, 111.6, 1 H NMR (200 MHz, CDCl 3 ) δ = 9.00 (d, J = 1.8 Hz, 1H, H-6), 8.46 (bs, NH), 7.98 (dd, J = 1.8; 8.2 Hz, 1H, H-4), 7.44 (d, J = 8.2 Hz, 1H, H-3), 6.85 (t, J = 2.1 Hz, 2H, H-2 pyrrole), 6.45 (t, J = 2.1 Hz, 2H, H-3 pyrrole), 3.96 (s, 3H, CH 3 ). 13 C NMR (50 MHz, CDCl 3 ) δ =165.9, 159.4, 135.5, 131.8, 130.7, 127.5, 127.0, 112.5, 121.8, 111.8, 92.4, 52.7. HRMS (ESI): m/z [M + H] + calcd for [C 14 H 12 N 2 O 3 Cl 3 ] + : 360.9908; found: 360.9904.

  J = 1.2; 8.[START_REF] Fidock | Drug discovery: Priming the antimalarial pipeline[END_REF] Hz, 1H, H-8), 7.50 (dt, J = 0.7; 7.3 Hz, 1H, H-7), 7.40 (dd, J = 0.7; 4.2 Hz, 1H, H-3), 6.98 (dd, J = 2.7; 4.2 Hz, 1H, H-2). 13 C NMR (100 MHz, CDCl 3 ) δ =150.1, 133.5, 131.5, 130.0, 127.8, 125.8, 120.6, 115.2, 114.2, 113.8, 110.4, 96.3. Anal. Calcd for C 12 H 7 Cl 3 N 2 : C, 50.47; H, 2.47; N, 9.81. Found: C, 50.35; H, 2.37; N, 9.67.

	3.1.8.2. 7-Fluoro-4-(trichloromethyl)pyrrolo[1,2-a]quinoxaline (10b)
	Yield 56%. Yellow powder. Mp 192°C. 1 H NMR (400 MHz, CDCl 3 ) δ = 7.97 (dd, J = 1.2;
	2.7 Hz, 1H, H-1), 7.85 (dd, J = 4.9; 9.0 Hz, 1H, H-6), 7.77 (dd, J = 2.9; 9.0 Hz, 1H, H-9),
	7.41-7.34 (m, 2H, H-3 and H-8), 6.97 (dd, J = 2.7; 4.1 Hz, 1H, H-2). 13 C NMR (100 MHz,
	CDCl 3 ) δ =160.0 (d, J = 244.3 Hz), 151.1, 134.6 (d, J = 11.5 Hz), 124.4, 120.4, 117.9 (d, J =
	23.9 Hz), 116.6 (d, J = 23.0 Hz), 115.4, 115.0 (d, J = 9.1 Hz), 114.4, 110.7, 96.2. Anal. Calcd
	for C 12 H 6 Cl 3 FN 2 : C, 47.48; H, 1.99; N, 9.23. Found: C, 47.81; H, 1.76; N, 9.10.
	5,
	115.0, 114.6, 110.9, 95.9. Anal. Calcd for C 12 H 6 Cl 4 N 2 : C, 45.04; H, 1.89; N, 8.75. Found: C,
	45.44; H, 1.89; N, 8.75.
	3.1.8.4. 7-Bromo-4-(trichloromethyl)pyrrolo[1,2-a]quinoxaline (10d)
	Yield 59%. Yellow-green powder. Mp 156°C. 1 H NMR (400 MHz, CDCl 3 ) δ = 8.27 (d, J =
	2.2 Hz, 1H, H-6), 7.99 (dd, J = 1.0; 2.7 Hz, 1H, H-1), 7.77 (d, J = 8.8 Hz, 1H, H-9), 7.72 (dd,
	J = 2.2; 8.8 Hz, 1H, H-8), 7.41 (dd, J = 1.0; 4.2 Hz, 1H, H-3), 6.99 (dd, J = 2.7; 4.2 Hz, 1H,
	H-2). 13 C NMR (100 MHz, CDCl 3 ) δ =151.0, 134.7, 133.9, 132.9, 126.8, 120.6, 118.3, 115.5,
	115.2, 114.7, 111.0, 95.9. Anal. Calcd for C 12 H 6 BrCl 3 N 2 : C, 39.55; H, 1.66; N, 7.69. Found:
	C, 39.39; H, 1.74; N, 7.61.
	3.1.8.5. 7-Iodo-4-(trichloromethyl)pyrrolo[1,2-a]quinoxaline (10e)
	Yield 56%. Brown powder. Mp 170°C. 1 H NMR (400 MHz, CDCl 3 ) δ = 8.45 (d, J = 2.0 Hz,
	1H, H-6), 7.97-7.95 (m, 1H, H-1), 7.87 (dd, J = 1.7; 8.6 Hz, 1H, H-8), 7.62 (d, J = 8.8 Hz, 1H,

Hz, 1H, H-6), 8.02 (dd, J = 1.0; 2.7 Hz, 1H, H-1), 7.90 (dd, J = 1.2; 8.

[START_REF] Fidock | Drug discovery: Priming the antimalarial pipeline[END_REF]

Hz, 1H, H-9), 7.63 (dt, 3.1.8.3. 7-Chloro-4-(trichloromethyl)pyrrolo[1,2-a]quinoxaline (10c) Yield 52%. Yellow-green powder. Mp 132°C. 1 H NMR (400 MHz, CDCl 3 ) δ = 8.09 (d, J = 2.2 Hz, 1H, H-6), 7.97 (dd, J = 1.2; 2.9 Hz, 1H, H-1), 7.81 (d, J = 8.8 Hz, 1H, H-9), 7.57 (dd, J = 2.4; 8.8 Hz, 1H, H-8), 7.41 (dd, J = 1.0; 4.2 Hz, 1H, H-3), 6.98 (dd, J = 2.7; 4.2 Hz, 1H, H-2). 13 C NMR (100 MHz, CDCl 3 ) δ =151.1, 134.4, 131.0, 130.8, 130.0, 126.4, 120.5, 115.

H-9), 7.40 (d, J = 4.2 Hz, 1H, H-3), 6.98 (dd, J = 2.7; 4.0 Hz, 1H, H-2).

13

C NMR (100 MHz, CDCl 3 ) δ =150.8, 139.9, 138.

3, 134.6, 127.4, 120.5, 115.4, 115.3, 114.5, 110

.9, 95.9, 88.6. Anal. Calcd for C 12 H 6 ICl 3 N 2 : C, 35.03; H, 1.47; N, 6.81. Found: C, 34.69; H, 1.49; N, 6.69.

  Trituration of the residue in Et 2 O afforded the expected compound after filtration.A small reactor equipped with a magnetic stir bar was charged with the appropriate 7substituted 4-(trichloromethyl)pyrrolo[1,2-a]quinoxaline (1.1 mmol) and iron fine powder (880 mg, 15.8 mmol) in 15 mL of glacial AcOH. The mixture was heated at reflux for 1 h.

	Yield 44%. Yellow powder. Mp >260°C. 1 H NMR (200 MHz, DMSO-d 6 ) δ = 8.70 (d, J = 1.8
	Hz, 1H, H-6), 8.50-8.45 (m, 2H, H-1 and H-9), 8.22 (dd, J = 1.6; 8.7 Hz, 1H, H-8), 7.36 (d, J
	= 3.8 Hz, 1H, H-3), 7.18-7.05 (m, 1H, H-2). 13 C NMR (50 MHz, DMSO-d 6 ) δ =166.3, 149.8,
	132.0, 131.7, 130.6, 130.1, 128.3, 119.4, 118.3, 115.5, 115.2, 110.8, 95.8. Anal. Calcd for
	C 13 H 7 Cl 3 N 2 O 2 : C, 47.38; H, 2.14; N, 8.50. Found: C, 47.52; H, 2.21; N, 8.47.
	3.1.10. N'-Hydroxy-4-(trichloromethyl)pyrrolo[1,2-a]quinoxaline-7-
	carboximidamide (10k)
	A 50-mL round-bottomed flask equipped with a condenser and a magnetic stir bar was
	charged with 4-(trichloromethyl)pyrrolo[1,2-a]quinoxaline-7-carbonitrile (311 mg, 1.0
	mmol), hydroxylamine hydrochloride (70 mg, 1.0 mmol) and 5 mL of a mixture of EtOH/H 2 O
	7/3. The mixture was refluxed until disappearance of starting material (ca. 24 h). After the
	reaction mixture was cooled to rt and the volatiles were removed, the mixture was extracted
	five times with CH 2 Cl 2 . The combined organic layers were washed twice with water. Then,
	the organic layer was dried with anhydrous sodium sulphate and was concentrated under
	vacuum. Purification by chromatography on silica gel (Petroleum Ether/EtOAc 7/3 then 5/5)
	gave the expected product.
	Yield 74%. Yellow powder. Mp 114°C. 1 H NMR (200 MHz, DMSO-d 6 ) δ = 9.92 (s, 1H,
	OH), 8.68 (m, 1 H, H-6), 8.42 (d, J = 8.8 Hz, 1H, H-9), 8.35 (d, J = 1.6 Hz, 1H, H-1), 8.10
	(dd, J = 1.6; 8.8 Hz, 1H, H-8), 7.36 (dd, J = 1.0; 4.2 Hz, 1H, H-3), 7.12 (dd, J = 2.7; 4.2 Hz,
	1H, H-2), 6.23 (sl, 2H). 13 C NMR (50 MHz, DMSO-d 6 ) δ =151.3, 149.6, 132.2, 129.4, 128.0,
	127.8, 127.6, 119.3, 117.9, 115.1, 114.9, 110.4, 96.0. HRMS (ESI): m/z [M + H] + calcd for
	[C 13 H 10 N 4 OCl 3 ] + : 342.9915; found: 342.9920.
	3.1.11. Representative procedure for the preparation of 4-methyl-7-substituted-
	pyrrolo[1,2-a]quinoxaline (11f,h)

2 Biology 3.2.1 In vitro Antiplasmodial evaluation

	erythrocytes according to a SYBR Green I fluorescence-based method [39] using a 96-well
	fluorescence plate reader. Compounds, previously dissolved in DMSO (final concentration
	less than 0.5% v/v) were incubated in a total assay volume of 200 µL (RPMI, 4% hematocrit
	and 1% parasitaemia) for 72 h in a humidified atmosphere (14% O 2 and 6% CO 2 ) at 37 °C, in
	96-well flat bottom plates. Duplicate assays were performed for each sample. After
	incubation, 125 µL supernatant was discarded and cells were washed twice with 125 µL 1X
	PBS. 15 µL re-suspended cells were transferred to 96-well flat bottom nonsterile black plates
	(Greiner Bio-one) already containing 15 µL of the SYBR Green I lysis buffer (2X SYBR
	Green I, 20 mM Tris base pH 7.5, 20 mM EDTA, 0.008% w/v saponin, 0.08% w/v Triton X-3.1.11.1. 4,7-Dimethylpyrrolo[1,2-a]quinoxaline (11f) 100). Negative control, treated by solvents (DMSO or H 2 O) and positive controls
	Yield 56%. Beige powder. Mp 140°C. 1 H NMR (200 MHz, CDCl 3 ) δ = 7.85 (dd, J = 1.3, 2.6 (chloroquine and doxycycline) were added to each set of experiments. Plates were incubated
	Hz, 1H, H-1), 7.72 (s, 1H, H-6), 7.69 (d, J = 8.4 Hz, 1H, H-9), 7.27 (dd, J = 2.2, 7.6 Hz, 1H, for 15 min at 37 °C and then read on a TECAN Infinite F-200 spectrophotometer with
	H-8), 6.90-6.80 (m, 2H, H-2 and H-3), 2.73 (s, 3H, CH 3 ), 2.47 (s, 3H, CH 3 ). 13 C NMR (50 excitation and emission wavelengths at 485 and 535 nm, respectively. The concentrations of
	MHz, CDCl 3 ) δ = 153.5, 135.3, 135.2, 128.8, 128.3, 126.1, 125.2, 114.5, 113.6, 113.5, 106.9, compounds required to induce a 50% decrease of parasite growth (IC 50 K1) were calculated
	21.8, 21.2. Anal. Calcd for C 13 H 12 N 2 : C, 79.56; H, 6.16; N, 14.27. Found: C, 79.89; H, 2.18; from three independent experiments.
	N, 13.99.
	3.1.11.2. 4-Methylpyrrolo[1,2-a]quinoxaline-7-carbonitrile (11h)
	Yield 57%. White powder. Mp 140°C. 1 H NMR (200 MHz, CDCl 3 ) δ = 8.17 (d, J = 1.6 Hz,
	1H, H-1), 7.97-7.77 (m, 2H, H-6 and H-9), 7.68 (dd, J = 1.7; 8.5 Hz, 1H, H-8), 7.01-6.87 (m,
	2H, H-2 and H-3), 2.73 (s, 3H, CH 3 ). 13 C NMR (50 MHz, CDCl 3 ) δ = 155.9, 135.9, 133.8,
	130.3, 129.8, 126.4, 118.6, 115.4, 115.2, 114.9, 108.6, 108.4, 22.1. HRMS (ESI): m/z [M +
	H] + calcd for [C 13 H 10 N 3 ] + : 208.0869; found: 208.0871.
	3.In this study, a K1 culture-adapted P. falciparum strain resistant to chloroquine,
	pyrimethamine and proguanil was used in an in vitro culture. Maintenance in continuous
	culture was done as described previously by Trager and Jensen [38]. Cultures were
	maintained in fresh A+ human erythrocytes at 2.5% hematocrit in complete medium (RPMI
	1640 with 25 mM HEPES, 25 mM NaHCO 3 , 10% of A+ human serum) at 37 °C under
	reduced O 2 atmosphere (gas mixture 14% O 2 , 6% CO 2 , and 80% N 2 ). Parasitaemia was
	maintained daily between 1% and 6%. The P. falciparum drug susceptibility test was carried
	out by comparing quantities of DNA in treated and control cultures of parasite in human

Table 2 .

 2 In vitro antiplasmodial and cytotoxicity evaluation of the 4-trichloromethylpyrrolo-[1,2-a]quinoxaline series

	Entry Molecule	R 1 -	R 2 -	HepG2CC 5 0 (µM)	PfK1 IC 50 (µM)	SI c
	1	10a	-CCl 3	-H	41.2	1.5	27.5
	2	10b	-CCl 3	-F	>15 d *	1.7	>8.8
	3	10c	-CCl 3	-Cl	9.8	1.6	6.1
	4	10d	-CCl 3	-Br	nd*	nd*	-
	5	10e	-CCl 3	-I	6.6	1.2	5.5
	6	10f	-CCl 3	-CH 3	53	2.4	22.1
	7	10g	-CCl 3	-OCH 3	35	4.2	8.3
	8	10h	-CCl 3	-CN	17	1.5	11.3
	9	10i	-CCl 3	-COOCH 3	nd*	nd*	-
	10	10j	-CCl 3	-COOH	106	22	<10.6
	11	10k	-CCl 3 -C(NH 2 )=NOH	>15 d *	1.75	>8.6
	12	11a	-CH 3	-H	120	>50 d	<2.4
	13	11f	-CH 3	-CH 3	57	>50 d	<4.7
	14	11h	-CH 3	-CN	>31.5 d	>50 d	-
	15	12a	-CF 3	-H	160	>50 d	<3.2
		Doxorubicine a		0.2	-	-
		Chloroquine b		30	0.6	50
		Doxycycline b

Acknowledgement

This work was supported by the CNRS and Aix-Marseille Université. The authors warmly thank Dr Vincent Remusat and Dr Rémi Legay for the 1 H-and 13 C-NMR spectra recording.


Recurrent Neural Network for Syntax Learning with Flexible Representations

Xavier Hinaut 1Abstract-We present a Recurrent Neural Network (RNN), namely an Echo State Network (ESN), that performs sentence comprehension and can be used for Human-Robot Interaction (HRI). The RNN is trained to map sentence structures to meanings (e.g. predicates). We have previously shown that this ESN is able to generalize to unknown sentence structures in English and French. The meaning representations it can learn to produce are flexible: it enables one to use any kind of "series of slots" (or more generally a vector representation) and are not limited to predicates. Moreover, preliminary work has shown that the model could be trained fully incrementally. Thus, it enables the exploration of language acquisition in a developmental approach. Furthermore, an "inverse" version of the model has been also studied, which enables to produce sentence structure from meaning representations. Therefore, if these two models are combined in a same agent, one can investigate language (and in particular syntax) emergence through agent-based simulations. This model has been encapsulated in a ROS module which enables one to use it in a cognitive robotic architecture, or in a distributed agent simulation.

I. INTRODUCTION

How do children learn language? In particular, how do they associate the structure of a sentence to its meaning? This question is linked to the more general issue: how does the brain associate sequences of symbols to internal symbolic or sub-symbolic representations? We propose a framework to understand how language is acquired based on a simple and generic neural architecture (Echo State Networks) [START_REF] Jaeger | The "echo state" approach to analysing and training recurrent neural networks[END_REF] which is not hand-crafted for a particular task.

First, we present the general ESN architecture. Then, we detail the reservoir sentence processing model. Finally we give some perspectives.

II. METHODS & RESULTS

A. Echo State Networks

The language module is based on an ESN [START_REF] Jaeger | The "echo state" approach to analysing and training recurrent neural networks[END_REF] with leaky neurons: inputs are projected to a random recurrent layer and a linear output layer (called "read-out") is modified by learning (which can also be done in an online fashion). The units of the recurrent neural network have a leak rate (α) hyper-parameter which corresponds to the inverse of a time constant. These equations define the update of the ESN:

x(t + 1) = (1 -α)x(t) + αf (W in u(t + 1) + W x(t)) (1)
y(t) = W out x(t) (2)
sité de Bordeaux, Bordeaux, France xavier.hinaut@inria.fr with x(t), u(t) and y(t) the reservoir (i.e. hidden) state, the input and the read-out (i.e. output) states respectively at time t, α the leak rate, W , W in and W out the reservoir, the input and the output matrices respectively and f the tanh activation function. After the collection of all reservoir states the following equation defines how the read-out (i.e. output) weights are trained:

W out = Y d [1; X] + (3)
with Y d the concatenation of the desired outputs, X the concatenation of the reservoir states (over all time steps for all train sentences) and M + the Moore-Penrose pseudoinverse of matrix M . Hyper-parameters that can be used for this task are the following: spectral radius: 1, input scaling: 0.75, leak rate: 0.17, number of reservoir units: 100.

B. Reservoir Sentence Processing Model

The reservoir sentence processing model has been adapted from previous experiments on a neuro-inspired model for sentence comprehension using ESN [START_REF] Hinaut | Real-time parallel processing of grammatical structure in the fronto-striatal system: a recurrent network simulation study using reservoir computing[END_REF] and its application to HRI [START_REF] Hinaut | Exploring the acquisition and production of grammatical constructions through human-robot interaction with echo state networks[END_REF]. The model learns the mapping of the semantic words (SW; e.g. nouns, verbs) of a sentence onto the different slots (the thematic roles: e.g. action, location) of a basic event structure (e.g. action(object, location)). As depicted in Fig. 1, the system processes a sentence as input and generates corresponding predicates. Before being fed to the ESN, sentences are transformed into a sentence structure (or grammatical construction) semantic words (SW), i.e. nouns, verbs and adjectives that have to be assigned a thematic role, are replaced by the SW item. The processing of the grammatical construction is sequential (one word at a time) and the final estimation of the thematic roles for each SW is read-out at the end of the sentence.

By processing constructions [START_REF] Goldberg | Constructions: A construction grammar approach to argument structure[END_REF] and not sentences per se, the model is able to bind a virtually unlimited number of sentences to these sentence structures. Based only on a small training corpus (a few tens of sentences), this enables the model to process future sentences with currently unknown semantic words if the sentence structures are similar. One major advantage of this neural network language module is that no parsing grammar has to be defined a priori: the system learns only from the examples given in the training corpus.

Here are some input/output transformations that the language model performs:

• "Please give me the mug" → give(mug, me)

• "Could you clean the table with the sponge?"

→ clean(table, sponge) • "Find the chocolate and bring it to me" → find(chocolate), bring(chocolate, me) As shown, the system can robustly transform different types of sentences. Recently, we have explored how flexible our system is: we found that it can handle various kinds of representations at the same time. For instance, it allows to use nouns as main elements of a predicate, and use its arguments to fill in adjectives:

• "Search for the small pink object" → search(object), object(small, pink) It also allows to process various kinds of complex sentences:

• "Bring me the newspaper which is on the table in the kitchen" → bring(newspaper, me), newspaper(

C. Perspectives

In Hinaut et al. [START_REF] Hinaut | A recurrent neural network for multiple language acquisition: Starting with english and french[END_REF], it has been shown that the model can learn to process sentences with out-of-vocabulary words. Moreover, we demonstrated that it can generalize to unknown constructions in both French and English at the same time. To illustrate how the robot interaction works, a video can be seen youtu.be/FpYDco3ZgkU [6] [START_REF] Twiefel | Using Natural Language Feedback in a Neuro-inspired Integrated Multimodal Robotic Architecture[END_REF]. The source code, implemented as a ROS module, is available at github. com/neuronalX/EchoRob. With an ESN of 100 units, the training of 200 sentences takes about one second on a laptop computer. Testing a sentence is of the order of 10 ms.

This ROS module could be employed to process various hypotheses generated by a speech recognition system (like in [START_REF] Twiefel | Using Natural Language Feedback in a Neuro-inspired Integrated Multimodal Robotic Architecture[END_REF]), then returning the retrieved predicates for each hypothesis, thus, enabling a semantic analyser or world simulator to choose the predicates with the highest likelihood. Preliminary work has shown that the model could be trained fully incrementally (i.e. changing weights at each time step) [START_REF] Hinaut | An incremental approach to language acquisition: Thematic role assignment with echo state networks[END_REF].Moreover, an "inverse" version of the model has been also studied, which enables to learn mapping from meaning representations to sentence structure [START_REF] Hinaut | Exploring the acquisition and production of grammatical constructions through human-robot interaction with echo state networks[END_REF]. The representations may not be limited to "series of slots": neural networks are able to deal with hierarchical structures when they are embedded in a vector representation (e.g. Fluid Construction Grammar using Holographic Reduced Representations like in [START_REF] Knight | A vector representation of Fluid Construction Grammar using Holographic Reduced Representations[END_REF]. With these properties, one can investigate language syntax emergence through agent-based simulations.

In a nutshell, the objectives of this model are to improve HRI and provide models of language acquisition. From the HRI point of view, the aim of using this neural networkbased model is (1) to gain adaptability because the system is trained on corpus examples (no need to predefine a parser for each language), ( 2) to be able to process natural language sentences instead of stereotypical sentences (i.e. "put cup left"), and (3) to be able to generalize to unknown sentence structures (not in the training data set). Moreover, this model is quite flexible when changing the output predicate representations, as we have shown here. From the computational neuroscience and developmental robotics point of view, the aim of this architecture is to model and test hypotheses about child learning processes of language acquisition [START_REF] Tomasello | Constructing a language: A usage based approach to language acquisition[END_REF].

Fig. 1 .

 1 Fig. 1.Sentences are converted to a sentence structure by replacing semantic words by a SW marker. The ESN is given the sentence structure word by word. Each word activates a different input unit. During training, the connections to the readout layer are modified to learn the mapping between the sentence structure and the arguments of the predicates. When a sentence is tested, the most active units are bound with the SW kept in the SWs memory to form the resulting predicate. (Adapt. from[START_REF] Hinaut | Exploring the acquisition and production of grammatical constructions through human-robot interaction with echo state networks[END_REF].)

X. Hinaut is with Inria Bordeaux Sud-Ouest, Talence, France; LaBRI, UMR 5800, CNRS, Bordeaux INP, Université de Bordeaux, Talence, France; and Institut des Maladies Neurodégénératives, UMR 5293, CNRS, Univer-

ACKNOWLEDGMENT

I thank William Schueller for interesting discussions and ideas.