Guillaume Bentzinger

Etienne Pair

Jean Guillon

Mathieu Marchivie

Catherine Mullié

Patrice Agnamey

Alexandra Dassonville-Klimpt

Pascal Sonnet
email: pascal.sonnet@u-picardie.fr

Enantiopure substituted pyridines as promising antimalarial drug candidates

Keywords:

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Malaria is still today a major public health threat on a worldwide scale. In 2018, 228 million cases of malaria were reported worldwide and led to 405,000 deaths, 67% of them children aged under 5 years. 1 Since 2014, investments for malaria control and elimination were robust and resolute (2.7 billion dollars in 2018) but falling far the 5 billion dollars funding target of the World Health Organization (WHO) global strategy. Plasmodium falciparum is the most prevalent malaria parasite in Africa and is responsible for most malaria cases. It has shown great adaptive potential and the apparition of resistances towards antiplasmodial molecules always calls for new drug candidates. In the fight against malaria, the family of arylaminoalcohol (AAA) drugs including quinine (QN), mefloquine (MQ) and lumefantrine play an important role. 1 Not only do they have good activities against P. falciparum but their slow clearance makes them perfect partners for artemisinin-based combination therapy (ACT), widely recommended by the WHO since 2006. [START_REF] Who | Guidelines for the Treatment of Malaria[END_REF] MQartesunate and lumefantrine-artemether are among the six ACT commercially available. 1,[START_REF] Who | Guidelines for the treatment of malaria[END_REF] The first AAA used in antimalarial therapy was the enantiomerically pure quinine, one of the four cinchona alkaloids. Quinidine (QD), the dextrorotatory diastereomer of quinine, was discarded because it differs from QN in many pharmacological respects. Although, QD is about three to four times more active as an antimalarial drug, [START_REF] White | [END_REF] the QT (Q wave-T wave interval) prolongation observed with it is about four times more than with QN. 5 Accordingly, QN was used as antimalarial drug while QD was for years available as an anti-arrhythmic. For the MQ, another AAA, several studies highlighted that the (R,S) enantiomer of MQ is the least active (Fig. 1) and crosses more easily the blood-brain barrier. [6][7][8][9][10] This latter ability making it, in part, responsible for the important neurological side effects of MQ. [11][12][13] Despite important disparities in activities, toxicities and pharmacokinetic profiles of the AAA stereoisomers, current commercial drugs containing MQ or lumefantrine are still used as racemates.

For those reasons, our group works on the enantioselective synthesis of new AAA analogs. Previously, the synthesis of 4-(2amino-1-hydroxyethyl)quinolines (4-AHQs) as enantiopure open analogs of MQ was achieved (Fig. 1). Structure-activityrelationship (SAR) studies on the aminoalcohol moiety allowed us to identify a few molecules 1a-d with alkyl side chains, as active as MQ in vitro and in vivo on the selected P. falciparum strains 3D7 and W2. [14][15][16][17][18] These compounds 1a-d also showed a clearer (S)/(R) selectivity in vitro than MQ with IC 50 up to 15-fold higher for the more active enantiomer.

The importance of the aminoalcohol stereochemistry for the antimalarial activity was reinforced with our study on the pyrroloquinoxaline family. 18 Although their corresponding activity was low (micromolar range), strong (S)/(R) disparities have been observed (eudysmic ratio up to 15.5). As a continuation of our SAR studies, we decided now to focus our work on the aromatic ring role by restructuring the ring system. Dissociation of the quinoline core by splitting the benzo compound could lead to new antimalarial drugs such as 4-(2amino-1-hydroxyethyl)pyridines (4-AHPs) 2a-d. Furthermore, 4-AHPs could also be considered as enpiroline (ENP) analogs (Fig. 2). ENP is an AAA based on a pyridine core and was previously studied as a drug candidate. [19][20][21][22] Surprisingly, this compound was never fully exploited despite promising activities, good pharmacokinetic parameters, and low toxicity. Also, ENP enantiomers have similar activities with eudysmic ratios around 1.2. 6,23 We thought that 4-AHPs 2a-d could display improved antimalarial activities and enhanced eudysmic ratios compared to 4-AHQs 1a-d and lower long-term toxicity than MQ. 15 Herein, we describe the enantioselective synthesis and biological evaluation of 4-AHPs 2a-d as new antimalarial potential drugs. In order to take advantage of the strategy we had optimized for generating the library of enantiopure 4-AHQs 1 for making the enantiopure 4-AHPs 2, we needed to access to the epoxides 3 through an indirect asymmetric epoxidation of the 4-vinylpyridine 4 (scheme 1). 14 Two routes to obtain the keyintermediate 4-vinyl-pyridine 4 are studied based on a Kröhnketype cyclization 24 or metal-catalyzed reactions. The respective advantages and inconveniences of these ones are discussed below. Finally, the 4-AHPs 2a-d were prepared in three steps from the vinyl pyridine 4 and their in vitro activities were measured against P. falciparum 3D7 and W2 strains. Thus, novel SAR were highlighted with regard to their stereochemistry, alkyl substitution and in comparison with the corresponding quinolines 4-AHQs 1.

Results and discussion

Chemistry

Two ways of making the 4-vinylpyridine 4 were explored either via a Kröhnke-type cyclization (7 steps, 46% overall yield) 24 or metal-catalyzed reactions (9 steps, 37% overall yield). Scheme 1. Retrosynthesis strategies for the 4-AHPs 2a-d.

. 1 . 1 . S yn t h es i s o f 4 u s i n g a K r ö h n ke -t yp e cyc l i z a t i o n

Having a Wittig reaction in mind for the synthesis of the enone 6, the commercial 2-bromo-4'-(trifluoromethyl)acetophenone 8 was treated with PPh 3 (Scheme 2). The resulting phosphonium salt 9, obtained quantitatively, was then reacted with glyoxylic acid to obtain 6 as the Wittig product in quantitative yield. The 1 H NMR analysis and the coupling constant between the ethylenic protons ( 3 J H1/H2 = 15.6 Hz) confirmed the selective formation of the (E)-isomer. In parallel, the commercially available 3-bromo-1,1,1trifluoroacetone 10 was reacted with pyridine in EtOH at 70 °C. The corresponding pyridinium salt 5 was afforded in 72% yield with the ketone function completely hydrated due to its electrowithdrawing environment. 19,24,25 This structure was confirmed by 13 C NMR (δC(C(OH) 2 ): 148.2 ppm) and X-ray diffraction (Scheme 2, supplementary material). With the enone 6 and the pyridinium salt 5 in hands, a Kröhnke-type cyclization was performed in order to form the isonicotinic acid 11 in 94% yield. Thereafter, the acid function is converted into aldehyde in order to introduce the vinyl group with a Wittig reaction. Different reducing agents were tested to achieve the direct reduction of the acid moiety to the aldehyde 13, but mixtures of the aldehyde 13 and the alcohol 12 were afforded systematically. Thus, a two steps aldehyde synthesis was performed: i) borane (BH 3 ) was used as reducing agent to obtain the primary alcohol 12 in 96% yield and ii) Dess-Martin Periodinane as selective oxidant allowed to afford the aldehyde 13 in 81% yield. For the Wittig reaction, the methyltriphenylphosphonium bromide was deprotonated in situ with n-BuLi as the base at 0 °C in THF to generate the corresponding ylide. Due to the high reactivity of this ylide with aldehydes, the reaction required serious optimization (temperature, additives, reaction times). In our best conditions, the addition of aldehyde 13 was carried out dropwise at -78 °C. Then, the Wittig reaction itself was performed at -30 °C for six hours. Thus, the vinyl derivative 4 was obtained in 63% yield. When one gram or more of aldehyde were used, yields dropped between 15% and 30%. This reaction being very temperature sensitive, it proved to be hard to reproduce when scaled-up, probably due to a lack of temperature-control. With this synthetic pathway, the desired vinyl intermediate 4 is accessible in a 46% yield in an overall 7 steps sequence. Because of the scalability issues with the final Wittig step, we considered this method to be unsuitable for making a library of compounds and devised a new route to obtain the key derivative 4, based on robust and reliable metal-catalyzed reactions. BrPPh3CH3, n-BuLi, LiOH, THF, 0°C, 2) 13, -78 °C, 1 h then -30 °C, 6 h.

. 1 . . S yn t h es i s o f 4 u s i n g m et a l -ca t a l y z ed r ea ct i o n s .

The commercially available 2,6-dibromopyridine 7 was converted into 4-hydroxypyridine 14 via the formation of a boronate intermediate using the [Ir(cod)Cl] 2 catalyst in the presence of pinacolborane and 4,4′-di-tert-butyl-2,2′-dipyridyl (dtbpy). Then, a one-pot oxidative cleavage of the boronate was performed with Oxone ® to afford 14 in an 82% yield. 26 An Oprotecting reaction was carried out with 4-methoxybenzyl chloride to give 15. A mono-halogen exchange, using Grignard reagent in the presence of I 2 , afforded the mono-iodinated compound 16 in 85% yield. After optimization, the best conditions were found by using 1.6 equivalents of both Turbo Grignard and I 2 . 27 The mono-iodination allowed to perform a selective Suzuki coupling with 4-(trifluoromethyl)phenylboronic acid affording 17 in 95% yield. 28 At that point, direct conversion of 17 into 19 using reaction conditions described by Z. Gonda et al. 29 was unsuccessful. Consequently, a copper-catalyzed aromatic Finkelstein reaction was used in order to convert 17 into 18 in 88% yield. 30,31 The iodo derivative 18 proved to be more reactive and was converted into a -CF 3 affording 19 in a satisfactory 89% yield. 29 After removal of the -PMB group, the resulting alcohol 20 was reacted with POBr 3 to isolate the bromo compound 21 in 93% yield. This allowed a Suzuki coupling with potassium (vinyl)trifluoroborate affording the key intermediate 4 in 98% yield. The global yield for this new synthetic pathway is of 37% over 9 steps. The selected reactions proved to be very robust whatever the scale (up to 5 g of vinyl 4 per sequence).

. 1 . 3 . S yn t h es i s o f 4 -(2-a mi n o -1 -h yd r o x ye t h yl ) p yr i d i n es (4-AH Ps ).

With intermediate 4 in hands, the diols (S)-22 and (R)-22 were obtained enantioselectively using a Sharpless dihydroxylation in the presence of AD-mix-α or -β respectively in 99% yield and 99% ee. A ring-closure was carried out in order to form the epoxides (S)-3 and (R)-3 by means of a slightly modified -one-pot‖ reaction, previously described. 14 Those were obtained with complete retention of configuration in 95% and 85% yield from (S)-22 and (R)-22 respectively. X-ray diffraction of epoxide (S)-3 was performed and the (S)-geometry was ascribed unambiguously (Scheme 4, supplementary material). The last step of the synthesis consisted in a regioselective S N 2 ring-opening with diverse primary amines (Scheme 4, Table 1). Using microwave irradiations allowed for reduced reaction times compared to the classical heating that was used for making the corresponding quinoline derivatives. Eight (S)-or (R)-4-AHPs 2a-d were thus synthesized using four alkylamines of growing size in good to excellent yields (60-96%) and with enantiomeric excesses superior to 98%.

Biological evaluation.

The activities of all 4-AHPs 2a-d were evaluated against P. falciparum 3D7 and W2 using the SYBR Green I method. 32 PfW2 is a chloroquine (CQ) resistant strain and is MQ sensitive while Pf3D7 is chloroquine sensitive and displays a decreased susceptibility to MQ. The two antiplasmodial compounds CQ and MQ were used as references. The half maximal inhibitory concentration (IC 50 ) calculated are reported in Table 2. Pleasingly, all IC 50 are in the nanomolar range and all compounds are more active than the references whatever the strain. As previously observed for the AHQs 1a-d, 15 the 4-AHPs 2a-d are more active against PfW2 than Pf3D7, the CQ-resistant strain, with IC 50 ranging from 3.5 (entry 8) to 10.0 nM (entry 2). For the Pf3D7 strain, less sensitive to MQ, the IC 50 are between 17.7 (entry 7) and 56.7 nM (entry 2). In general, it appears that a longer side chain enhances the efficacy of the compound and the differences between (S)-and (R)-enantiomers. Interestingly, this trend correlates with a higher calculated logP of the corresponding molecules. Compound 2d, with a heptyl side chain, shows the best activities against both Pf3D7 and PfW2 and has a clear difference between (S)-and (R)-enantiomers against Pf3D7 with a eudysmic ratio of 3.1. The newly synthesized 4-AHPs 2a-d are as much active as their quinoline counterparts 4-AHQs 1, but clearly show less differences between (S)-and (R)-enantiomers which had a eudysmic ratio between 2.2 and 15.1. 15 Nonetheless, disparities could appear more clearly in pharmacokinetic properties like in the case of MQ. These pharmacokinetic studies are currently under progress.

Conclusion

The synthesis of eight enantiopure AHPs 2a-d with varying alkyl substituents was achieved from the 4-vinylpyridine 4. This key-intermediate was obtained by two routes using either a Kröhnke-type cyclization or metal-catalyzed reactions. The cyclisation route led to 4 in a 46% yield in an overall 7 steps sequence. However, this pathway was not suitable for making a library of compounds, as the scaling-up was limited by the last step of the sequence. The metal-catalyzed route proved to be more robust. However, 4 was obtained in a lower yield of 37% yield over 9 steps. Nonetheless, this new synthesis allowed us to obtain large quantities of the AHPs precursor 4 (up to 5 g per sequence). Consecutively, a regioselective S N 2-ring opening of enantiopure epoxides 3 with four alkyl primary amines gave 4-AHPs 2a-d with good yields and excellent ee. These pyridines 2a-d showed strong antimalarial activity with IC 50 ranging from 3.5 to 10.0 nM against Pf3D7 and 17.7 to 56.7 nM against PfW2. Compared with their quinoline counterparts AHQs 1, the differences between (S)-and (R)-enantiomers IC 50 are less marked with eudysmic ratio between 1.1 and 3.1. These novel promising antimalarial 4-AHPs 2a-d and derivatives were recently patented. 32 Pharmacokinetic studies and in vivo experiments in a mouse model are under progress to validate their potential as antimalarial drug candidates.

Experimental

Chemistry

Reactions were monitored by thin-layer chromatography with silica gel 60 F 254 pre-coated aluminium plates (0.25 mm). Visualization was performed under UV light and PMA oxidation. Filtrations were performed on Celite ® 545. Chromatographic purification of compounds was achieved with 60 silica gel (40-63 m). Unless otherwise noted, all reagent-grade chemicals and solvents were used as supplied (analytical or HPLC grade) without prior purification. Melting points were measured on a Stuart SMP3 apparatus with a precision of +/-1.5 °C and are uncorrected. Infrared spectra (IR) were recorded on a FT/IR-4200 Jasco with an ATR-Golden gate. Liquids and solids were applied on the Single Reflection Attenuated Total Reflectance (ATR) Accessories. Data are reported in cm -1 . Optical rotations were determined with a Jasco P1010 polarimeter with a 10 cm cell. Specific rotations are reported in 10 -1 deg.cm 2 .g -1 and concentrations in g per 100 mL. 1 H NMR Spectra (400 MHz) and 13 C NMR spectra (100 MHz) were recorded on a Bruker 400 MHz NMR. The field was locked by external referencing to the relevant deuteron resonance. Data appear in the following order: chemical shifts in ppm which were referenced to the internal solvent signal, number of protons, multiplicity (s, singlet; d, doublet; t, triplet; dd, doublet of doublet, ddd, doublet of doublet of doublet, ddt, doublet of doublet of triplet, m, multiplet) and coupling constant J in Hertz. The abbreviation Ar is used to denote aromatic, br. to denote broad and app. to denote apparent. Coupling constants, J, are measured to the nearest 0.1 Hz and are presented as observed. LC-HRMS analyses were performed on an ACQUITY UPLC H-Class system (Waters-Micromass, Manchester, UK) coupled with a SYNAPT G2-Si Q-TOF hybrid quadrupole time-of-flight instrument (Waters-Micromass, Manchester, UK), equipped with an electrospray (ESI) ionization source (Z-spray) and an additional sprayer for the reference compound (Lock Spray)., Torrance, CA, USA) heated at 50 °C. High-resolution mass spectra (HRMS) were obtained from a Micromass-Waters Q-TOF Ultima spectrometer, in electrospray ionization (ESI) mode (positive or negative). Enantiomeric excesses were measured with Schimadzu LC-20AD equipped with a Chiralpak column (IA, IB, IC, ID or IG).

. 1 . 1 . (2-Oxo-2 -(4-(t r i f l u o r o m e t h yl )p h en y l ) et h yl ) t r i p h en yl p h o s p h o n i u m b r o m i d e ( 9 )

To a solution of 2-bromo-1-(4-(trifluoromethyl)phenyl)ethanone 8 (1.00 g, 3.70 mmol, 1 eq.) in THF (20 mL) were added triphenylphosphine (1.08 g, 4.10 mmol, 1.1 eq.). The reactional mixture was heated to reflux for 15 h and concentrated under reduced pressure. The residue was washed with Et 2 O. The solid was filtered, washed with toluene and then with Et 2 O to afford 9 (2.00 g, quant.) as a white solid. m.p. 136 °C; NMR 1

. 1 . 2 . (E ) --o xo --(4-(t r i f l u o r o m et h yl )p h en y l )b u t -2 -en o ï c a c i d ( 6 )

To a solution of 6.00 g (11.4 mmol, 1 eq.) of 9 in 30 mL of CHCl 3 were added 2.00 mL (1.45 g, 17.0 mmol, 1.5 eq.) of NEt 3 . The solution was stirred at 25 °C for 5 min, before to add 1.57 g (17.0 mmol, 1.5 eq.) of glyoxylic acid in 30 mL of MeOH. The reactional mixture was stirred at 25 °C for 15 h and then concentrated in vacuo. AcOEt (30 mL) were added before to be treated with 4 x 30 mL sat. aq. NaHCO 3 . The aqueous layers were combined and acidified (pH ~ 1) with 1 M aq. HCl. The solid was filtered and dried to afford 6 (3.07 g, quant.) as a white solid. m.p. 154 °C; NMR 1 H (300 MHz, CD 3 OD): δH 6.80 (d, J = 15.6 Hz, 1H), 7.82 (d, J = 8.1 Hz, 2H), 7.90 (d, J = 15.6 Hz, 1H), 8.15 (d, J = 8.1 Hz, 2H) ppm; NMR 13 C (75 MHz, CD 3 OD): δC 129.4 (q, J = 272.0 Hz), 127.0 (q, J = 3.8 Hz), 130.5, 134.8, 135.7 (q, J = 32.5 Hz), 137.0, 140.9, 168.

. 1 . 3 . 1 -(3 , 3 , 3 -T r i f l u o r o -2 , 2 -d i h yd r o xyp r o p yl ) p yr i d i n -1 -i u m b r o mi d e ( 5 )

To a solution of 5.00 g (26.3 mmol, 1.3 eq.) of 3-bromo-1,1,1trifluoropropan-2-one 10 in 20 mL of EtOH were added 1.60 mL (20.2 mmol, 1 eq.) of pyridine. The mixture was heated to reflux for 10 h, before to be cooled to 25 °C. Et 2 O was then added until to obtain a murky solution. The mixture was stirred at 25 °C for 15 h. The solid was then filtered to afford 5 (6.35 g, 72%) as a white solid. m.p. 193 °C; NMR 1 H (300 MHz, D 2 O): δH 1.19 (t, J = 7.0 Hz, 3H), 3.62 (q, J = 7.0 Hz, 2H), 4.99 (s, 2H), 8.18 (t , J = 6.9 Hz, 2H), 8.71 (t, J = 6.9 Hz, 1H), 8.91 (d, J = 6.9 Hz, 2H) ppm ; NMR 13 C (75 MHz, D 2 O): δC 18.4, 58.3, 63.8, 94.9 (q, J = 32.0 Hz), 125.0 (q, J = 291.1 Hz), 128.9, 148.2 ppm; IR  max : 3157, 1300, 1239, 1152, 1087 cm -1 .

. 1 . . 2 -( T r i f l u o r o m et h y l ) -6 -(4-(t r i f l u o r o met h yl ) p h en yl ) i s o n i co t i n i c a ci d ( 11)

In 300 mL of MeOH were added 2.92 g (9.24 mmol, 1 eq.) of 5, 3.38 g (13.9 mmol, 1.5 eq.) of 6 and 5.70 g (73.9 mmol, 8 eq.) of NH 4 OAc. The reaction mixture was heated to reflux for 24 h before to concentrate it in vacuo. The residue was directly purified by flash chromatography (cyclohexane/AcOEt/AcOH 1/1/0.01) to afford 11 (2.92 g, 94%) as an orange solid. m.p. 205 °C; NMR 1 H (300 MHz, CD 3 OD): δH 7.69 (d, J = 8.2 Hz, 2H), 8.10 (s, 1H), 8.16 (d, J = 8.2 Hz, 2H), 8.46 (s, 1H) ppm ; NMR 13 C (75 MHz, CD 3 OD): δC 120.0, 122.4 (q, J = 273.6 Hz), 123.9, 125.6 (q, J = 271.6 Hz), 126.8, 128.6, 132.9 (q, J = 32.4 Hz), 141.5, 143.1, 150.5 (q, J = 35.2 Hz), 158.2, 166.3 ppm; IR  max : 2980, 1714, 1128 cm -1 ; HRMS calcd. for C 14 H 8 F 6 NO 2 [M+H] + 336.0459, found 336.0466.

. 1 . 5 . (2-(T r i f l u o r o me t h yl ) -6 -(4-( t r i f l u o r o m et h y l ) p h en yl )p yr i d i n --yl )m et h a n o l ( 12)

To a solution, at 0 °C and under argon, of 78.0 mg (0.23 mmol, 1 eq.) of 11 in 2 mL of anhydrous THF were added 0.58 mL (1.15 mmol, 5 eq.) of BH 3 .DMS (2M in THF). The solution was stirred at 25 °C for 8 h, cooled to 0 °C and treated with an of excess MeOH. The reactional mixture was stirred for 5 min at 0 °C and 5 min at 25 °C, before concentrated in vacuo. The residue was extracted with Et 2 O. The organic layers were combined, dried over Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by flash chromatography (cyclohexane/AcOEt 2/1) to afford 12 (71 mg, 96%) as a light orange solid. m.p. 83 °C; NMR 1 H (400 MHz, CDCl 3 ): δH 4.90 (s, 2H), 7.66 (s, 1H), 7.74 (d, J = 8.2 Hz, 2H), 7.95 (s, 1H), 8.18 (d, J = 8.2 Hz, 2H) ppm; NMR 13 C (100 MHz, CDCl 3 ): δC 63.1, 116.8 (q, J = 2.7 Hz), 120.2, 121.5 (q, J = 274.5 Hz), 124.0 (q, J = 272.2 Hz), 125.6 (q, J = 3.8 Hz), 127.5, 131.6 (q, J = 32.6 Hz), 141.0, 148.6 (q, J = 34.7 Hz), 153.0, 156.4 ppm; IR  max : 3249, 2918, 2850, 1323, 1116 cm -1 ; HRMS calcd. for C 14 H 9 F 6 NONa [M+Na] + 344.0486, found 344.0496.

p h en yl ) i s o n i co t i n a l d eh y d e ( 13)

To a solution, at 0 °C, of 90.0 mg (0.28 mmol, 1 eq.) of 12 in 2 mL of CH 2 Cl 2 were added 132 mg (0.31 mmol, 1.1 eq.) of Dess-Martin Periodinane. The suspension was stirred at 0 °C for 1.5 h before to be treated with sat. aq. NaHCO 3 . The reaction mixture was stirred at 25 °C for 5 min. The solid was then filtered and the filtrate extracted with CH 2 Cl 2 . The organic layers were combined, dried over Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by flash chromatography (cyclohexane/AcOEt 1/1) to afford 13 (72.0 mg, 81%) as a white solid. m.p. 69 °C; NMR 1 H (300 MHz, CDCl 3 ): δH 7.77 (d, J = 8.2 Hz, 2H), 8.06 (s, 1H), 8.24 (d, J = 8.2 Hz, 2H), 8.35 (s, 1H), 10.21 (s, 1H) ppm; NMR 13 C (75 MHz, CDCl 3 ): δC 118.0, 120.2 (q, J = 272.0 Hz), 121.6, 123.9 (q, J = 274.5 Hz), 126.0, 127.6, 132.1 (q, J = 32.7 Hz), 139.8, 144.2, 150.0 (q, J = 35.9 Hz), 158.0, 189.6 ppm; IR  max : 1711, 1319, 1118 cm -1 .

. 1 . 7 . 2 -( T r i f l u o r o m et h y l ) -6 -(4-(t r i f l u o r o m et h yl ) p h en yl ) --vin y l p yr i d i n e ( )

Method A: Wittig reaction: To a suspension, at 0 °C and under argon, of 729 mg (2.04 mmol, 1.3 eq.) of methyltriphenylphosphonium bromide, 75.0 mg (3.14 mmol, 2 eq.) of LiOH in 30 mL of distilled THF were added 1.96 mL (3.14 mmol, 2 eq.) of n-BuLi (1.6 M in hexane). The reaction mixture was stirred at 0 °C for 10 min, at 25 °C for 10 min and cooled to -78 °C. A solution of 500 mg (1.57 mmol, 1 eq.) of 13 in 10 mL of distilled THF were added dropwise. The solution was stirred for 20 min at -78 °C before to be heated to -30 °C. The reaction mixture was stirred at -30 °C for 6 h, heated to 25 °C in 5 h (0.2 °C/min) and stirred at 25 °C for 15 h. The solution was treated with a MeOH excess before to be extracted with AcOEt. The organic layers were combined, dried over Na 2 SO 4 , filtered and concentrated in vacuo. The residue was purified by flash chromatography (cyclohexane/AcOEt 10/1) to afford 4 (270 mg, 63%) as an oil, solidifying over time into a light yellow solid.

Method B: Suzuki coupling: An argon-purged mixture of 21 (6.0 g, 16.3 mmol, 1 eq.), potassium vinyltrifluoroborate (2.4 g, 17.9 mmol, 1.1 eq.), Na 2 CO 3 (3.5 g, 32.6 mmol, 2 eq.) and Pd(PPh 3 ) 4 (0.94 g, 0.81 mmol, 0.05 eq.) in a mixture of toluene/EtOH/H 2 O (6/1/1, 280 mL) was stirred at 110 °C for 16 h. The reaction mixture was cooled to 25 °C and extracted with AcOEt. The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (cyclohexane/EtOAc 10/1) affording 4 (5.08 g, 98%) as a yellow oil, solidifying over time into light yellow solid. m.p. 37 °C; NMR 1 H (400 MHz, CDCl 3 ): δH 5.66 (d, J = 10.9 Hz, 1H), 6.15 (d, J = 17.6 Hz, 1H), 6.78 (dd, J = 17.6 Hz, 10.9 Hz, 1H), 7.65 (s, 1H), 7.72 (d, J = 8.2 Hz, 2H), 7.83 (s, 1H), 8.15 (d, J = 8.2 Hz, 2H) ppm; NMR 13 C (100 MHz, CDCl 3 ): δC 116.2 (q, J = 2.7 Hz), 120.3, 120.7, 121.3 (q, J = 272.2 Hz), 123.9 (q, J = 272.2 Hz), 125.7 (q, J = 3.4 Hz), 127.4, 131.5 (q, J = 32.5 Hz), 133.7, 141.0, 147.7, 148.9 (q, J = 34.5 Hz), 156.7 ppm; IR  max : 1321, 1108 cm -1 ; HRMS calcd. for C 16 H 10 F 6 N [M+H] + 318.0717, found 318.0725.

. 1 . 8 . 2 , 6 -D i b r o mo p yr i d i n --o l ( 14)

An argon-purged mixture of 2,6-dibromopyridine 7 (2.0 g, 8.44 mmol, 1 eq.), pinacolborane (2.20 mL, 15.2 mmol, 1.8 eq.), [Ir(cod)Cl] 2 (56.7 mg, 0.08 mmol, 0.01 eq.) and 4,4′-di-tert-butyl-2,2′-dipyridyl (45.3 mg, 0.017 mmol, 0.02 eq.) in cyclohexane (15 mL) was stirred at 80 °C for 18 h. The reaction mixture was cooled back to 25 °C and concentrated under reduced pressure. The residue was dissolved in THF (20 mL) before adding dropwise a solution of oxone monopersulfate (2.9 g, 9.3 mmol, 1.1 eq.) in H 2 O (30 mL). The resulting mixture was stirred at 25 °C for 30 min, quenched with 1 M NaHCO 3 aq. and extracted with EtOAc. The combined organic layers were washed with water, brine, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The resulting solid was purified by flash chromatography on silica gel (cyclohexane/EtOAc 4/1) affording 14 (1.75 g, 82%) as a white solid. m.p. 207 °C; 1 H NMR (400 MHz, CD 3 OD): δH 6.97 (s, 2H, H 3 , H 5 ) ppm; 13

. 1 . 9 . 2 , 6 -D i b r o mo --( (-me t h o xyb en z yl )o x y) p yr i d i n e ( 15)

To a solution of 14 (8.0 g, 31.6 mmol, 1 eq.) in DMF (100 mL) were added 4-methoxybenzyl chloride (4.3 mL, 31.6 mmol, 1 eq.) and K 2 CO 3 (5.7 g, 41.1 mmol, 1.3 eq.). The suspension was stirred at 110 °C for 15 h, cooled back to 25 °C, filtered over a celite pad and the filtrate was concentrated under reduced pressure. The residue was placed in EtOAc and washed with 0.5 M NaOH. The aqueous layer was extracted with EtOAc. The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated under reduced pressure.

. 1 . 1 0 . 2 -Br o mo -6 -i o d o --( (-m e t h o xyb en z yl )o x y ) p yr i d i n e ( 16)

To a solution of 15 (4.97 g, 13.1 mmol, 1 eq.) in anhydrous THF (13 mL) were added iPrMgCl.LiCl (1.3 M in THF) (16.2 mL, 21.0 mmol, 1.6 eq.). The reaction mixture was stirred at 25 °C for 2 h and cooled to 0 °C before adding portionwise I 2 (5.33 g, 21.0 mmol, 1.6 eq.). The resulting mixture was stirred at 0 °C for 5 min, at 25 °C for 15 h and quenched with 0.5 M Na 2 S 2 O 3 aq. (3 x 40 mL). The aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (cyclohexane/EtOAc 10/1) affording 16 (4.68 g, 85%) as a white solid. m.p. 96 °C; 1 H NMR (400 MHz, CDCl 3 ): δH 3.83 (s, 3H), 4.99 (s, 2H), 6.93 (d, J = 8.7 Hz, 2H), 7.04 (d, J = 1.9 Hz, 1H), 7.26-7.34 (m, 3H) ppm; 13

. 1 . 1 1 . 2 -Br o mo --( (-met h o xyb e n z yl )o xy ) -6 -(4-(t r i f l u o r o met h yl )p h en yl )p yr i d i n e ( 17)

An argon-purged mixture of 16 (2.74g, 6.52 mmol, 1 eq.), 4-(trifluoromethyl)phenylboronic acid (1.11g, 5.87 mmol, 0.9 eq.), Na 2 CO 3 (1.38 g, 13.0 mmol, 2 eq.) and Pd(PPh 3 ) 4 (377 mg, 0.33 mmol, 0.05 eq.) in a mixture of toluene/EtOH/H 2 O (6/1/1) (109 mL) was stirred at 110 °C for 15 h. The reaction mixture was cooled to 25 °C and concentrated under reduced pressure. The residue was directly purified by flash chromatography on silica gel (cyclohexane/EtOAc 8/1) affording 17 (2.73 g, 95%) as a yellow oil. 1 H NMR (400 MHz, CDCl 3 ): δH 3.83 (s, 3H), 5.08 (s, 2H), 6.95 (d, J = 8.6 Hz, 2H), 7.07 (d, J = 2.0 Hz, 1H), 7.27 (d, J = 2.0 Hz, 1H), 7.36 (d, J = 8.6 Hz, 2H), 7.70 (d, J = 8.2 Hz, 2H), 8.04 (d, J = 8.2 Hz, 2H) ppm; 13 C NMR (100 MHz, CDCl 3 ): δC 55. 3, 70.5, 108.1, 112.7, 114.3, 124.0 (q, J = 272.2 Hz), 125.6 (q, J = 3.8 Hz), 126.8, 127.2, 129.5, 131.3 (q, J = 32.6 Hz), 141.0, 143.1, 157.4, 160.0, 166.6 ppm; IR  max : 2935, 1580, 1321 cm -1 ; MS (ESI + ) m/z: 439 [M+H] + .

. 1 . 1 2 . 2 -Io d o --( (-m e t h o x yb en z yl )o x y) -6 -(4-(t r i f l u o r o met h yl ) p h en yl ) p yr i d i n e ( 18)

To a solution of 17 (453 mg, 1.03 mmol, 1 eq.), CuI (10.0 mg, 0.05 mmol, 0.05 eq.) and NaI (309 mg, 2.06 mmol, 2 eq.) in anhydrous 1,4-dioxane (1.3 mL) were added N,N'dimethylethylenediamine (11.0 µL, 0.10 mmol, 0.1 eq.). The solution was stirred at 110 °C for 28 h, cooled to 25 °C, treated with NH 4 OH (28% in H 2 O) (10 mL) and diluted with water. The mixture was extracted with DCM. The organic layers were washed with brine, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (cyclohexane/EtOAc 8/1) affording 18 (442 mg, 88%) as a colorless oil. 1 H NMR (400 MHz, CDCl 3 ): δH 3.74 (s, 3H), 4.96 (s, 2H), 6.86 (d, J = 8.6 Hz, 2H), 7.17 (d, J = 2.0 Hz, 1H), 7.21 (d, J = 2.0 Hz, 1H), 7.26 (d, J = 8.6 Hz, 2H), 7.59 (d, J = 8.2 Hz, 2H), 7.93 (d, J = 8.2 Hz, 2H) ppm; 13 C NMR (100 MHz, CDCl 3 ): δC 55. 3, 70.3, 108.4, 114.2, 118.8, 119.7, 124.0 (q, J = 272.3 Hz), 125.6 (q, J = 3.7 Hz), 126.9, 127.2, 129.5, 131.2 (q, J = 32.5 Hz), 141.0, 157.9, 160.0, 165.5 ppm; IR  max : 2935, 1575, 1319, 1110, 993, 827 cm -1 ; HRMS calcd. for C 20 H 16 F 3 BrINO 2 [M+H] + 486.0178, found 486.0190.

. 1 . 1 3 . -( (-M e t h o x yb en z y l )o xy ) -2 -(t r i f l u o r o m et h yl ) -6 -(4-(t r i f l u o r o m e t h yl )p h en y l )p yr i d i n e ( 19)

To a solution, under argon, of 18 (2.28 g, 4.70 mmol, 1 eq.), KF (1.64 g, 28.2 mmol, 6 eq.), CuI (0.36 g, 1.88 mmol, 0.4 eq.) and 1,10-phenantroline (0.34 g, 1.88 mmol, 0.4 eq.) in anhydrous DMSO (68 mL) were added B(OMe) 3 (3.14 mL, 28.2 mmol, 6 eq.) and TMSCF 3 (2 M in THF) (14.1 mL, 28.2 mmol, 6 eq.).

The solution was stirred at 60 °C for 24 h. The reaction mixture was treated with NH 4 OH (28% in H 2 O) (20 mL) and extracted with diethyl ether. The combined organic layers were washed with brine, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (cyclohexane/EtOAc 4/1) affording 19 (1.78 g, 89%) as a yellow oil. 1 H NMR (400 MHz, CDCl 3 ): δH 3.75 (s, 3H), 5.07 (s, 2H), 6.88 (d, J = 8.7 Hz, 2H), 7.17 (d, J = 2.0 Hz, 1H), 7.30 (d, J = 8.7 Hz, 2H), 7.35 (d, J = 2.0 Hz, 1H), 7.63 (d, J = 8.2 Hz, 2H), 8.02 (d, J = 8.2 Hz, 2H) ppm; 13 C NMR (100 MHz, CDCl 3 ): δC 55.3, 70.5, 106.6 (q, J = 2.8 Hz), 109.7, 114.3, 121.4 (q, J = 274.6 Hz), 124.0 (q, J = 272.2 Hz), 125.7 (q, J = 3.8 Hz), 126.7, 127.4, 129.5, 131.5 (q, J = 32.6 Hz), 141.2, 150.0 (q, J = 34.5 Hz), 157.9, 160.0, 166.7 ppm; IR  max : 2935, 1604, 1324, 1126 cm -1 ; MS (ESI + ) m/z: 428 [M+H] + .

. 1 . 1 . 2 -( T r i f l u o r o m et h y l ) -6 -(4-t r i f l u o r o m e t h yl ) p h en yl )p yr i d i n --o l (20)

To a solution of 19 (1.78 g, 4.17 mmol, 1 eq.) in DCM (42 mL) were added TFA (1 mL). The reaction mixture was stirred at 25 °C for 7 h and treated with 1 M NaOH aq. (3 x 20 mL). The aqueous layers were acidified with 1 M HCl until pH~6 and concentrated under reduced pressure. The residue was washed with MeOH and the solid filtered. The filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (cyclohexane/EtOAc 1/1) affording 20 (1.03 g, 80%) as a white solid. m.p.: 170 °C; 1 H NMR (400 MHz, CD 3 OD): δH 7.14 (d, J = 2.0 Hz, 1H), 7.48 (d, J = 2.0 Hz, 1H), 7.77 (d, J = 8.2 Hz, 2H), 8.20 (d, J = 8.2 Hz, 2H) ppm; 13 C NMR (100 MHz, CD 3 OD): δC 108.8 (q, J = 2.8 Hz), 111.7, 123.0 (q, J = 273.5 Hz), 125.7 (q, J = 271.3 Hz), 126.7 (q, J = 3.8 Hz), 128.7, 132.4 (q, J = 32.2 Hz), 143.0, 150.8 (q, J = 34.2 Hz), 159.2, 168.1 ppm; IR  max : 3097, 1611, 1582, 1323, 1108, 1061, 849 cm -1 ; HRMS calcd. for C 13 H 8 F 6 NO [M+H] + 308.0510, found 308.0523.

. 1 . 1 5 . -Br o mo -2 -(t r i f l u o r o me t h yl ) -6 -(4-(t r i f l u o r o met h yl )p h en y l )p yr i d i n e ( 21)

To a solution, under argon, of 20 (6.5 g, 21.2 mmol, 1 eq.) in DMF (130 mL) were added POBr 3 (24.3 g, 84.6 mmol, 4 eq.).

The suspension was stirred at 110 °C for 16 h and quenched with H 2 O. The resulting mixture was extracted with EtOAc. The combined organic layers were washed with brine, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (cyclohexane/EtOAc 9/1) affording 21 (6.02 g, 93%) as a white solid. m.p.: 59 °C; 1 H NMR (400 MHz, CDCl 3 ): δH 7.76 (d, 3 J = 8.2 Hz, 2H), 7.83 (s, 1H), 8.12 (s, 1H), 8.16 (d, 3 J = 8.2 Hz, 2H) ppm; 13 C NMR (100 MHz, CDCl 3 ): δC 120.7 (q, 1 J = 275.1 Hz), 122.9 (q, 3 J = 2.8 Hz), 123.9 (q, 1 J = 272.4 Hz), 126.0 (q, 3 J = 3.8 Hz), 126.4, 127.6, 132.2 (q, 2 J = 32.7 Hz), 134.9, 139.7, 149.3 (q, 2 J = 35.4 Hz), 157.5 ppm; IR  max : 1573, 1320, 1119, 1057, 843 cm -1 ..

. 1 . 1 6 . (S ) -1 -(2-(T r i f l u o r o met h yl ) -6 -(4-(t r i f l u o r o met h yl )p h en y l )p yr i d i n --yl )et h a n e -1 , 2 -d i o l ( (S ) -22)

To a suspension of AD-mix α (1.26 g) in a tBuOH/H 2 O mixture (1/1) (22 mL) was added potassium osmate (3.00 mg, 0.09 mmol, 0.01 eq.). The mixture was cooled to 0 °C and 4 (285.0 mg, 0.90 mmol, 1 eq.) was added. The reaction mixture was stirred at 0 °C for 5 min and at 25 °C for 15 h. The reaction was cooled to 0 °C and quenched with Na 2 SO 3 (980 mg). The suspension was stirred for 10 min at 0 °C, 10 min at 25 °C and extracted with EtOAc. The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The residue was precipitated with cyclohexane. The resulting solid was filtered affording (S)-22 (316 mg, 99%) as a white solid. m.p. 101 °C; 1 H NMR (400 MHz, CD 3 OD): δH 3.77 (m, 2H), 4.89 (m, 1H), 7.76 (d, J = 8.3 Hz, 2H), 7.84 (s, 1H), 8.20 (s, 1H), 8.29 (d, J = 8.3 Hz, 2H) ppm; 13 C NMR (100 MHz, CD 3 OD) : δC 67.8, 74.2, 118.7 (q, J = 2.8 Hz), 122.5, 123.2 (q, J = 273.6 Hz), 125.6 (q, J = 271.3 Hz), 126.8 (q, J = 3.8 Hz), 128.7, 132.5 (q, J = 32.3 Hz), 142.7, 149.2 (q, J = 34.3 Hz), 157.0, 157.2 ppm; IR  max : 3293, 1323, 1097 cm -1 ; HRMS calcd. for C 15 H 11 F 6 NO 2 Na [M+Na] + 374.0578, found 374.0583; : +29.1° (c 0.1 ; MeOH); Chiral HPLC 99% ee, Chiralpak IB column, heptane/i-PrOH, 90/10, flow 1 mL/min, tr(R) = 10.6 min, tr(S) = 11.6 min.

. 1 . 1 7 . (R ) -1 -(2-( T r i f l u o r o m et h yl ) -6 -(4-(t r i f l u o r o met h yl )p h en y l )p yr i d i n --yl )et h a n e -1 , 2 -d i o l ( (R ) -22)

To a suspension of AD-mix β (8.85 g) in a tBuOH/H 2 O mixture (1/1) (160 mL) was added potassium osmate (23.2 mg, 0.06 mmol, 0.01 eq.). The mixture was cooled to 0 °C and 4 (2.00 g, 6.30 mmol, 1 eq.) was added. The reaction mixture was stirred at 0 °C for 5 min and at 25 °C for 15 h. The reaction was cooled to 0 °C and quenched with Na 2 SO 3 (7 g). The suspension was stirred for 10 min at 0 °C, 10 min at 25 °C and extracted with EtOAc. The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The residue was precipitated with cyclohexane. The resulting solid was filtered affording (R)-22 (2.21 g, 99%) as a white solid. m.p., 1 H NMR, 13 C NMR and IR spectra are the same that those of (S)-

. 1 . 1 8 . (S ) --( Ox i r a n -2 -y l ) -2 -( t r i f l u o r o m et h y l ) -6 -(4-(t r i f l u o r o me t h yl )p h en y l )p y r i d i n e ( S ) -3

To a solution, under argon, of (S)-22 (1.20 g, 3.42 mmol, 1 eq.) in anhydrous DCM (35 mL) were added trimethylorthoacetate (1.3 mL, 10.25 mmol, 3 eq.) and pTsOH (32.5 mg, 0.17 mmol, 0.05 eq.). The reaction mixture was stirred at 25 °C for 30 min and then concentrated under reduced pressure. The residue was placed under argon, dissolved in anhydrous DCM (35 mL) and cooled to 0 °C. TMSBr (1.35 mL, 10.25 mmol, 3 eq.) was then added dropwise. The solution was stirred at 0 °C for 5 min, at 25 °C for 30 min and concentrated under reduced pressure. The residue was placed under argon and dissolved in anhydrous MeOH (35 mL). K 2 CO 3 (2.36 g, 17.08 mmol, 5 eq.) was then added. The suspension was stirred at 25 °C for 1 h, quenched with sat. aq. NH 4 Cl and extracted with DCM. The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (cyclohexane/EtOAc 6/1) affording (S)-3 ( 13 C NMR (100 MHz, CDCl 3 ): δC 48.1 (q, J = 4.1 Hz), 50.9, 121.9 (q, J = 275.6 Hz), 123.1, 124.0 (q, J = 272.2 Hz), 125.9 (q, J = 3.7 Hz), 127.3, 131.6 (q, J = 32.5 Hz), 132.1, 135.5, 140.6, 145.6 (q, J = 34.6 Hz), 154.3 ppm; IR  max : 2928, 1319, 1105, 1068 cm -1 ; HRMS calcd. for C 15 H 11 F 6 NO 2 Na [M+H] + 334.0667, found 334.0679.; : +23.5° (c 0,1 ; MeOH).

. 1 . 1 9 . (R ) --( Oxi r a n -2 -y l ) -2 -( t r i f l u o r o m et h yl ) -6 -(4-(t r i f l u o r o me t h yl )p h en y l )p y r i d i n e ( (R ) -3 )

To a solution, under argon, of (R)-22 (1.00 g, 2.85 mmol, 1 eq.) in anhydrous DCM (30 mL) were added trimethylorthoacetate (1.10 mL, 8.50 mmol, 3 eq.) and pTsOH (27.0 mg, 0.14 mmol, 0.05 eq.). The reaction mixture was stirred at 25 °C for 30 min and then concentrated under reduced pressure. The residue was placed under argon, dissolved in anhydrous DCM (30 mL) and cooled to 0 °C. TMSBr (1.13 mL, 8.54 mmol, 3 eq.) was then added. The solution was stirred at 0 °C for 5 min, at °C for 30 min and concentrated reduced pressure. The residue was placed under argon and dissolved in anhydrous MeOH (30 mL). K 2 CO 3 (1.97 g, 14.25 mmol, 5 eq.) was then added. The suspension was stirred at 25 °C for 2 h, quenched with sat. aq. NH 4 Cl and extracted with DCM. The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (cyclohexane/EtOAc 6/1) affording (R)- 22 : -20.8° (c 0,1 ; MeOH).

. 1 . 2 0 . Gen e r a l Pr o c ed u r e f o r t h e r i n g -o p en i n g r ea ct i o n s : Pr ep a r a t i o n o f -A H Ps ( 2 )

To a solution of the epoxide 3 in ethanol (0.05 M) was added the desired amine. The reaction mixture was stirred at 130°C using microwaves for 30 min and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel affording the corresponding 4-AHP.

. 1 . 2 1 . (S ) -2 -( Bu t yl a mi n o ) -1 -(2-( t r i f l u o r o me t h yl ) -6 -(4-(t r i f l u o r o me t h yl )p h en y l )p y r i d i n --yl )e t h a n o l (( S ) -2a)

Following the general method for the preparation of 4-AHPs and starting from (S)-3 (200.0 mg, 0.60 mmol, 1 eq.) and nbutylamine (0.30 mL, 3.00 mmol, 5.0 eq.), the residue was purified by flash chromatography on silica gel (DCM/MeOH 9/1) affording (S)-2a (191.4 mg, 78%) as a white solid. m.p. 129 °C; 1 H NMR (400 MHz, CDCl 3 ): δH 0.93 (t, J = 7.3 Hz, 3H), 1.31-1.44 (m, 2H), 1.45-1.56 (m, 2H), 2.61-2.79 (m, 3H), 3.06 (dd, J = 12.3, 3.7 Hz, 1H), 3.07 (s br , 2H), 4.84 (dd, J = 9.2, 3.6 Hz, 1H), 7.67 (s, 1H), 7.73 (d, J = 8.2 Hz, 2H), 7.99 (s, 1H), 8.18 (d, J = 8.1 Hz, 2H) ppm; 13 C NMR (100 MHz, CDCl 3 ) : δC 13.9, 20.2, 32.0, 49.0, 56.2, 69.7, 116.6 (q, J = 2.6 Hz), 120.0, 124.0 (q, J = 272.3 Hz), 124.2 (q, J = 274.8 Hz), 125.6 (q, J = 3.8 Hz), 127.5, 131.5 (q, J = 32.6 Hz), 141.1, 148.5 (q, J = 34.6 Hz), 155.0, 156. 4

. 1 . 2 2 . (R ) -2 -( Bu t yl a m i n o ) -1 -(2-(t r i f l u o r o met h yl ) -6 -(4-(t r i f l u o r o met h yl )p h e n yl )p yr i d i n --y l ) et h a n o l (( R ) -2a)

Following the general method for the preparation of 4-AHPs and starting from (R)-3 (40.0 mg, 0.12 mmol, 1 eq.) and nbutylamine (0.071 mL, 0.72 mmol, 6 eq.), the residue was purified by flash chromatography on silica gel (DCM/MeOH 9/1) affording (R)-2a (47 mg, 96%) as a white solid. m.p., 1 H NMR, 13 C NMR and IR spectra are the same that those of (S)-2a.

. 1 . 2 3 . (S ) -2 -( P en t y l a mi n o ) -1 -(2-( t r i f l u o r o m et h y l ) -6 -(4-(t r i f l u o r o met h yl )p h e n yl )p yr i d i n --y l ) et h a n o l (( S ) -2b)

Following the general method for the preparation of 4-AHPs and starting from (S)-3 (200.0 mg, 0.60 mmol, 1 eq.) and npentylamine (0.36 mL, 3.10 mmol, 5.15 eq.), the residue was purified by flash chromatography on silica gel (DCM/MeOH 9/1) affording (S)-2b (236.6 mg, 94%) as a white solid. m.p. 112 °C; 1 H NMR (400 MHz, CDCl 3 ): δH 0.89 (t, J = 7.0 Hz, 3H), 1.20-1.42 (m, 4H), 1.52-1.62 (m, 2H), 2.60-2.81 (m, 3H), 3.09 (dd, J = 12.2, 3.1 Hz, 1H), 3.87 (s br , 2H), 4.94 (dd, J = 9.2, 3.2 Hz, 1H), 7.68 (s, 1H), 7.73 (d, J = 8.2 Hz, 2H), 8.00 (s, 1H), 8.18 (d, J = 8.2 Hz, 2H) ppm; 13 C NMR (100 MHz, CDCl 3 ): δC 13.9, 22.5, 29.3, 29.6, 49.3, 56.2, 69.7, 116.6 (q, J = 2.5 Hz), 120.0, 124.0 (q, J = 272.2 Hz), 124.2 (q, J = 274.5 Hz), 125.7 (q, J = 3.7 Hz), 127.5, 131.5 (q, J = 32.5 Hz), 141.1, 148.5 (q, J = 34.6 Hz), 155.0, 156.4 ppm; IR  max : 2930, 1327, 1126 cm -1 ; HRMS calcd. for C 20 H 22 F 6 N 2 O [M+H] + 421.1715, found 421.1720; : +23.7° (c 0.1 ; MeOH); Chiral HPLC 99% ee, Chiralpak IB column, heptane/i-PrOH/EDA, 99/1/0.1, flow 1 mL/min, tr(R) = 12.4 min, tr(S) = 14.3 min.

. 1 . 2 . (R ) -2 -( Pen t yl a mi n o ) -1 -(2-( t r i f l u o r o m et h y l ) -6 -(4-(t r i f l u o r o met h yl )p h e n yl )p y r i d i n --y l ) et h a n o l (( R ) -2b)

Following the general method for the preparation of 4-AHPs and starting from (R)-3 (200.0 mg, 0.60 mmol, 1 eq.) and npentylamine (0.36 mL, 3.10 mmol, 5.15 eq.), the residue was purified by flash chromatography on silica gel (DCM/MeOH 9/1) affording (R)-2b (214.0 mg, 85%) as a white solid. m.p., 1 H NMR, 13 C NMR and IR spectra are the same that those of (S)-

Fig. 1 .

 1 Fig. 1. Previous work: mefloquine and 4-AHQs 1a-d.

Fig. 2 .

 2 Fig. 2. Present work: Enpiroline and 4-AHPs 2a-d.

Scheme 2 .

 2 Scheme 2. Preparation of the vinyl 4 by Kröhnke-type cyclization. Reagents and conditions: (a) PPh3, THF, 70 °C. (b) Glyoxylic acid, NEt3, CHCl3/MeOH (1/1), 25 °C. (c) Pyridine, EtOH, 80 °C. (d) NH4OAc, THF, 70 °C. (e) BH3.DMS, THF, 25 °C. (f) DMP, CH2Cl2, 0 °C. (g) 1) BrPPh3CH3, n-BuLi, LiOH, THF, 0°C, 2) 13, -78 °C, 1 h then -30 °C, 6 h.

Scheme 3 .

 3 Scheme 3. Preparation of the vinyl 4 using metal-catalyzed reactions. Reagents and conditions: (a) 1) [Ir(cod)Cl]2, pinacolborane, dtbpy, cyclohexane, 80 °C, 2) Oxone, THF/H2O (1/1), 25 °C. (b) 4-methoxybenzyl chloride, K2CO3, DMF, 110 °C. (c) iPrMgCl.LiCl, I2, THF, 25 °C. (d) 4-(trifluoromethyl)phenyl boronic acid, Na2CO3, Pd(PPh3)4, toluene/H2O/EtOH (6/1/1), 110 °C. (e) CuI, NaI, N,N'-dimethylethylenediamine, 1,4-dioxane, 110 °C. (f) KF, CuI, TMSCF3, 1,10-phenantroline, B(OMe)3, DMSO, 60 °C. (g) TFA, DCM, 25 °C. (h) POBr3, DMF, 110 °C. (i) Potassium (vinyl)trifluoroborate, Na2CO3, Pd(PPh3)4, toluene/EtOH/H2O (6/1/1), 110 °C.

Scheme 4 .

 4 Scheme 4. Synthesis of 4-AHPs 2a-d. Reagents and conditions: (a) AD-mixα or -β, K2OsO2(OH)4, tBuOH/H2O (1/1), 0 °C to 25 °C, 15 h. (b) i) trimethyl orthoacetate, pTsOH, DCM, 25 °C, 7 h, ii) TMSBr, DCM, 25 °C, 15 h, iii) K2CO3, MeOH, 25 °C, 4 h. (c) R 1 -NH2, EtOH, 130 °C (M.W.), 30 min.

  H (400 MHz, CDCl 3 ): δH 6.41 (d, J = 12.1 Hz, 2H), 7.49-7.57 (m, 11H), 7.84-7.93 (m, 6H), 8.49 (d, J = 7.5 Hz, 2H) ppm ; NMR 13 C (100 MHz, CDCl 3 ): δC 38.7 (d, J = 61.3 Hz), 118.4 (d, J = 89.3 Hz), 123.3 (q, J = 272.9 Hz), 125.8 (q, J = 3.6 Hz), 130.1 (d, J = 13.1 Hz), 130.4, 133.4 (d, J = 10.7 Hz), 134.8 (d, J = 2.9 Hz), 135.4 (q, J = 32.7 Hz) 137.6 (d, J = 5.6 Hz), 191.2 (d, J = 10.9 Hz) ppm; IR  max : 3435, 3057, 2722, 1680, 1409, 1316, 1107 cm -1 ; HRMS calcd. for C 27 H 21 F 3 OP [M+H] + 449.1282, found 449.1285.

  HRMS calcd. for C 19 H 21 F 6 N 2 O [M+H] + 407.1558 , found 407.1551; : -31.0° (c 0.1 ; MeOH); Chiral HPLC 99% ee, Chiralpak IB column, heptane/i-PrOH/EDA, 99/1/0.1, flow 1 mL/min, tr(R) = 17.7 min, tr(S) = 21.2 min.

Table 1 .

 1 Yields and enantiomeric purities of the synthesized 4-AHPs 2a-d.

	Entry	Compound	-NHR 1	Yield	%ee a
	1	(S)-2a		78%	99
	2	(R)-2a		96%	99
	3	(S)-2b		94%	99
	4	(R)-2b		85%	99
	5	(S)-2c		67%	99
	6	(R)-2c		65%	98
	7	(S)-2d		60%	99
	8	(R)-2d		85%	99

a determined by chiral HPLC, see experimental part.

Table 2 .

 2 In vitro antiplasmodial activities of the 4-AHPs 2a-d and references CQ and MQ against a chloroquine sensitive Plasmodium falciparum clone (3D7) and a chloroquine-resistant Plasmodium falciparum clone (W2), eudysmic ratio and clogP values.

	Entry	Compound code	-NHR 1	IC 50 ± SD (nM) a,b Pf3D7 e PfW2 f	eudysmic ratio c Pf3D7 PfW2	clogP d
	1 2	(S)-2a (R)-2a		45.8 ± 2.3 56.7 ± 2.3	9.0 ± 0.4 10.0 ± 0.7	1.2	1.1	4.74
	3 4	(S)-2b (R)-2b		52.4 ± 1.7 41.8 ± 5.5	8.6 ± 1.0 7.2 ± 0.5	1.3	1.2	5.26
	5 6	(S)-2c (R)-2c		47.1 ± 13.2 32.3 ± 2.0	N.D. g 8.3 ± 0.8	1.5	N.D.	5.81
	7 8	(S)-2d (R)-2d		17.7 ± 4.7 54.4 ± 3.2	5.6 ± 0.3 3.5 ± 0.5	3.1	1.6	6.08
	9	Mefloquine (MQ)	75.9 ± 3,0	198.8 ± 27.0	-	-	3.91
	10	Chloroquine (CQ)	79.7 ± 8.5	31.8 ± 1.0	-	-	4.40

a in vitro measurements against P. falciparum using the SYBR Green I method, see experimental part. b Results expressed as mean ± standard deviation. c ratio between the IC50 of the more active enantiomer and the less active. d Predicted octanol/water partition coefficient calculated with Maestro Material Sciences 2.9.011 software. e P. falciparum strain with decreased sensibility to MQ and sensitive to CQ. f P. falciparum strain resistant to CQ and sensitive to MQ. g Not determined

  2, 190.3 ppm; IR  max : 2982, 1691, 1665, 1504, 1170, 1134, 1111 cm -1 ; HRMS calcd. for C 11 H 7 F 3 O 3 Na [M+Na] + 267.0245, found 267.0238.

  C NMR (100 MHz, CD 3 OD): δC 116.0 (C 3 , C 5 ), 141.8 (C 2 , C 6 ), 168.6 (C 4 ) ppm; IR  max : 2838, 1540, 1147, 769 cm -1 ; HRMS calcd. for C 5 H 4 NO 79 Br 2 [M+H] + 251.8660, found 251.8663.

  EtOAc 9/1) affording 15 (11.7 g, 99%) as a white solid. m.p. 69 °C; 1 H NMR (400 MHz, CDCl 3 ): δH 3.83 (s, 3H), 5.00 (s, 2H), 6.94 (d, 3 J = 8.7 Hz, 2H), 7.04 (s, 2H), 7.31 (d, 3 J = 8.7 Hz, 2H) ppm; 13 C NMR (100 MHz, CDCl 3 ): δC 55.3, 70.8, 114.0, 114.2, 126.3, 129.5, 141.1, 160.0, 166.7 ppm; IR  max : 2925, 1572, 1514, 982, 826 cm -1 ; HRMS calcd. for C 13 H 11 79 Br 2 NO 2 Na [M+Na] + 395.9054, found 395.9065.

					The residue was
	purified	by	flash	chromatography	on	silica	gel
	(cyclohexane/

  22. HRMS calcd. for C 15 H 11 F 6 NO 2 Na [M+Na] + 374.

	(incomplete separation), Chiralpak IB column, heptane/i-PrOH,
	90/10, flow 1 mL/min, tr(R) = 11.0 min, tr(S) = 12.7 min.
		0578, found
	374.0583;	: -29.3° (c 0.1 ; MeOH); Chiral HPLC >98,5% ee

  1.08 g, 95%) as a white solid. m.p. 81 °C; 1 H NMR (400 MHz, CDCl 3 ): δH 2.84 (dd, J = 5.6, 2.5, 1H), 3.29 (dd, J = 5.6, 4.3, 1H), 4.02 (dd, J = 4.3, 2.5 Hz, 1H), 7.59 (m, 1H), 7.75 d, J = 8.3 Hz, 2H), 7.86 (m, 1H), 8.18 (d, J = 8.3 Hz, 2H);

  (804 mg, 85%) as a white solid. m.p. 81 °C; NMR and HRMS data in accordance with (S)-22; IR  max : 2928, 1319, 1105, 1068 cm -1 ; HRMS calcd. for C 15 H 11 F 6 NO 2 Na [M+H] + 334.0667, found 334.0679.

  ppm; IR  max : 2921, 1328, 1109 cm -1 ; HRMS calcd. for C 19 H 21 F 6 N 2 O [M+H] + 407.1558, found 407.1551; : +31.1° (c 0.1 ; MeOH); Chiral HPLC 99% ee, Chiralpak IB column, heptane/i-PrOH/EDA, 99/1/0.1, flow 1 mL/min, tr(R) = 17.8 min, tr(S) = 20.8 min.

.1 . 6 . 2 -( T r i f l u o r o m et h y l ) -6 -(4-(tr i f l u o r o met h yl )

Acknowledgments

This project was financed in part from DGA (Direction Générale de l'Armement, Ministère de la Défense, France), ANR Astrid (project ANR-12-STR-003), région Picardie and the SATT Nord. G. B. was the recipient of a grant from Région Picardie. The authors thank Philippe Grellier (Muséum National d'Histoire Naturelle) who graciously provided P. falciparum strains. We thank Sophie Da Nascimento for mass spectra recording.

2b. HRMS calcd. for C 20 H 22 F 6 N 2 O [M+H] + 421.1715, found 421.1720; : -27.9° (c 0.1 ; MeOH); Chiral HPLC 99% ee, Chiralpak IB column, heptane/i-PrOH/EDA, 99/1/0.1, flow 1 mL/min, tr(R) = 11.9 min, tr(S) = 14.0 min.

. 1 . 2 5 . (S ) -2 -( He xy l a m i n o ) -1 -(2-(t r i f l u o r o m et h yl ) -6 -(4-(t r i f l u o r o met h yl )p h e n yl )p yr i d i n --y l ) et h a n o l (( S ) -2c)

Following the general method for the preparation of 4-AHPs and starting from (S)-3 (40.0 mg, 0.12 mmol, 1 eq.) and nhexylamine (0.05 mL, 0.36 mmol, 3 eq.), the residue was purified by flash chromatography on silica gel (DCM/MeOH 9/1) affording (S)-2c (35 13 C NMR (100 MHz, CDCl 3 ): δC 13.9, 22.5, 26.7, 29.4, 31.6, 49.3, 56.0, 69.5, 116.5 (q, J = 2.6 Hz,), 120.0, 124.0 (q, J = 272.2 Hz), 124.2 (q, J = 274.6 Hz), 125.7 (q, J = 3.8 Hz), 127.5, 131.6 (q, J = 32.6 Hz), 141.0, 148.5 (q, J = 34.6 Hz), 154.

. 1 . 2 6 . (R ) -2 -( He xyl a m i n o ) -1 -(2-(t r i f l u o r o m et h yl ) -6 -(4-(t r i f l u o r o met h yl )p h e n yl )p yr i d i n --y l ) et h a n o l (( R ) -2c)

Following the general method for the preparation of 4-AHPs and starting from (R)-3 (50.0 mg, 0.15 mmol, 1 eq.) and nhexylamine (0.06 mL, 0.45 mmol, 3 eq.), the residue was purified by flash chromatography on silica gel (DCM/MeOH 9/1) affording (R)-2c (42 mg, 65%) as a white solid. m.p., 1 H NMR, 13 C NMR and IR spectra are the same that those of (S)-2c. HRMS calcd. for C 21 H 25 F 6 N 2 O [M+H] + 435.1871, found 435.1857; : -28.6° (c 0.1 ; MeOH); Chiral HPLC 98% ee, Chiralpak IB column, heptane/i-PrOH/EDA, 99/1/0.1, flow 1 mL/min, tr(R) = 9.6 min, tr(S) = 10.7 min.

. 1 . 2 7 . (S ) -2 -( Hep t yl a m i n o ) -1 -(2-( t r i f l u o r o m et h y l ) -6 -(4-(t r i f l u o r o met h yl )p h e n y l )p yr i d i n --y l ) et h a n o l (( S ) -2d)

Following the general method for the preparation of 4-AHPs and starting from (S)-3 (50.0 mg, 0.15 mmol, 1 eq.) and nheptylamine (0.13 mL, 0.90 mmol, 6 eq.), the residue was purified by flash chromatography on silica gel (DCM/MeOH 9/1) affording (S)-2d (40 mg, 60%) as a white solid. m.p. 76 °C; 1 q, J = 2.6 Hz), 120.0, 124.0 (q, J = 272.2 Hz), 124.2 (q, J = 274.6 Hz), 125.8 (q, J = 3.8 Hz), 127.5, 131.2 (q, J = 32.6 Hz), 141.1, 148.5 (q, J = 34.7 Hz), 154.9, 156.4 ppm; IR  max : 2928, 1324, 1114 cm -1 ; HRMS calcd. for C 22 H 27 F 6 N 2 O [M+H] + 449.2028, found 449.2040; : + 24.9° (c 0.1 ; MeOH); Chiral HPLC 99% ee, Chiralpak IB column, heptane/i-PrOH/EDA, 99/1/0.1, flow 1 mL/min, tr(R) = 11.7 min, tr(S) = 13.5 min.

. 1 . 2 8 . (R ) -2 -( Hep t yl a m i n o ) -1 -(2-( t r i f l u o r o m et h y l ) -6 -(4-(t r i f l u o r o met h yl )p h e n yl )p yr i d i n --y l ) et h a n o l (( R ) -2d)

Following the general method for the preparation of 4-AHPs and starting from (R)-3 (31.0 mg, 0.09 mmol, 1 eq.) and nheptylamine (0.08 mL, 0.54 mmol, 6 eq.), the residue was purified by flash chromatography on silica gel (DCM/MeOH 9/1) affording (R)-2d (34 mg, 85%) as a white solid. m.p., 1 H NMR, 13 C NMR and IR spectra are the same that those of (S)-2d. HRMS calcd. for C 22 H 27 F 6 N 2 O [M+H] + 449.2028, found 449.2040; : -27.4° (c 0.1 ; MeOH); Chiral HPLC 99% ee, Chiralpak IB column, heptane/i-PrOH/EDA, 99/1/0.1, flow 1 mL/min, tr(R) = 11.6 min, tr(S) = 13.5 min.

Biological assay

The in vitro antiplasmodial activities were first tested over concentrations ranging from 39 nM to 40 μM and then, if the molecule efficacy warranted it, further checked over a concentration range of 1 nM to 1 μM. The reference strains used were culture-adapted Plasmodium falciparum 3D7 and W2. The former strain is susceptible to chloroquine and displays a decreased susceptibility to mefloquine, while the latter is susceptible to mefloquine and resistant to chloroquine. Parasites were cultivated in RPMI medium (Sigma-Aldrich, Lyon, France) supplemented with 0.5% Albumax I (Life Technologies corporation, Paisley, United Kingdom), hypoxanthine (Sigma-Aldrich), gentamicin (Sigma-Aldrich), and human erythrocytes. They were incubated at 37°C in a candle jar, as described previously. 33 The P. falciparum drug susceptibility test was carried out in 96-well flat bottom sterile plates under a final volume of 250 μL. After 48-h incubation with the drugs, quantities of DNA in treated and control cultures of parasites in human erythrocytes were compared according to the SYBR Green I (Sigma-Aldrich) fluorescence-based method. 33,35 Briefly, after incubation, plates were frozen at -20°C until use. They were then left to thaw for 2 h at room temperature after which 100 μL of the homogenized culture were transferred to 96-well flat bottom sterile black plates (Nunc Inc.) already containing 100 μL of the SYBR Green I lysis buffer (2xSYBR Green, 20 mM Tris base pH 7.5, 5 mM EDTA, 0.008% w/v saponin, 0.08% w/v Triton X-100). A negative control, controls treated by solvents (DMSO and H 2 O, typically), and positive controls (chloroquine and mefloquine) were added to each set of experiments. Plates were incubated for 1 h at room temperature and the SYBRGreen fluorescence was then read on a fluorescence plate reader (Tecan, Austria) using excitation and emission wavelengths of 485 and 535 nm, respectively. Concentrations inhibiting 50% of the parasite's growth (half maximal inhibitory concentration or IC 50 values) were then calculated from the obtained experimental results using a regression program available on line. 36

Supplementary Material

The supplementary material contains general procedure information, NMR spectra, HPLC analysis about the principal compounds and the X-ray analysis of (S)-3 and 5.

Graphical Abstract

To create your abstract, type over the instructions in the template box below. Fonts or abstract dimensions should not be changed or altered.

Enantiopure substituted pyridines as promising antimalarial drug candidates