Substituted piperidines with antiparasitic activity

Abstract

The present invention relates to new arylaminoalcohol derivatives of formula (I), and to a method for the preparation of such compounds: I The invention also relates to the use of these compounds as medicaments, and in particular for the prevention and/or the treatment of parasitic diseases caused by apicomplexan parasites such as malaria and toxoplasmosis. Finally, the invention relates to pharmaceutical compositions containing such compounds of formula (I) as active principles. ##STR00001##

Claims

1. A compound of formula (I): ##STR00017## wherein: n is 2; Ar is phenyl or naphthyl, each optionally substituted by CF.sub.3, F, Cl, Br or I; Am is selected from: ##STR00018## R is selected from H, F, Cl, Br, I, NO.sub.2 or CF.sub.3; and R is selected from H, F, Cl, Br, I, NO.sub.2 or CF.sub.3.

2. The compound according to claim 1, wherein Ar is substituted by one F.

3. The compound according to claim 2, wherein Ar is 4-fluorophenyl or 4-fluoro-1-naphthyl.

4. The compound according to claim 3, wherein Ar is 4-fluorophenyl.

5. The compound according to claim 1, wherein Am is: ##STR00019##

6. The compound according to claim 2, wherein Am is: ##STR00020##

7. The compound according to claim 3, wherein Am is: ##STR00021##

8. The compound according to claim 1, wherein R is NO.sub.2 or F.

9. The compound according to claim 2, wherein R is NO.sub.2 or F.

10. The compound according to claim 1, wherein R is H or CF.sub.3.

11. The compound according to claim 1, wherein R is NO.sub.2 and R is CF.sub.3.

12. A pharmaceutical composition comprising at least one compound according to claim 1 as an active principle and at least one pharmaceutically acceptable excipient.

13. The pharmaceutical composition of claim 12, further comprising at least one additional antiparasitic active principle.

14. The pharmaceutical composition of claim 13, wherein the additional antiparasitic active principle is selected from the group consisting of chloroquine, quinacrine, primaquine, artemisinin, atovaquone and pyrimethamine.

15. A method for treating apicomplexan parasitic activity in a mammal, comprising administering to a mammal in need thereof an effective amount of the compound according to claim 1.

16. The method according to claim 15, wherein the apicomplexan parasite is selected from the group consisting of Plasmodium, Babesia, Taxoplasma, Neospora, Cryyptosporidium, Theileria, Sarcosystis and Eimeria.

17. The method according to claim 15, wherein the mammal suffers from a parasitic disease involving an apicomplexan parasite.

18. The method according to claim 17, wherein the parasitic disease involving an apicomplexan parasite is malaria.

19. The method according to claim 17, wherein the parasitic disease involving an apicomplexan parasite is toxoplasmosis.

20. A method for the preparation of a compound of formula (I) according to claim 1 where n is 2, comprising the following steps: (i) reacting a compound of formula (III): ##STR00022## with a compound of formula (IV): ##STR00023## in the presence of dioxolane and acid, to provide a compound of formula (V): ##STR00024## wherein n is 2; and (ii) reacting the compound of formula (V): ##STR00025## wherein n is 2; with sodium borohydride, in the presence of methanol, to provide a compound of formula (I): ##STR00026## wherein n is 2.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the antimalarial activity of Compound 7 tested in vivo in a murine model,

(2) FIG. 2 represents the dockings and binding site alignments of arylaminoalcohols to plasmepsin II. (a) Docking of the most active compound previously reported (A. Mendoza et al., Exp. Parasit. 128(2) (2011) 97-103), and the best conformations found for Compounds 5, 7 and 8 to the active site of plasmepsin II. In white ribbon, the secondary structure of plasmepsin II is shown. (b) Involving the hydroxyl group of each arylaminoalcohol and the residue Asp214, hydrogen bond interactions are coded by dashed lines. In white sticks, catalytic residues Asp214 and Asp34 of plasmepsin II are shown.

EXAMPLES

(3) I/ Chemistry of the Synthesis of Compounds of Formula (I)

(4) The methods used for synthesizing the compounds (1-8) are presented in Schemes 1 and 2. The synthetic method has been published previously (A. Mendoza et al., Exp. Parasit. 128(2) (2011) 97-103).

(5) ##STR00008##

(6) ##STR00009##

(7) A group of 4-nitro-2-trifluromethyl phenyl amines were synthesized using the corresponding BOC-amines and 4-nitro-2-trifluoromethylphenyl as aryl fluoride by an ArS.sub.N reaction via Meisenheimer complex (K. Ersmark et al., Med. Res. Rev. 26 (2006) 626-666) and subsequent removal of the BOC-group with HCl and acetic acid. The products 2-nitro-4-trifluromethyl phenyl piperazine, 4-(4-fluorophenyl)-1,2,3,6-tetrahydropyridine and 4-trifluoromethyl phenyl piperazine were commercially available.

(8) All methyl-ketone precursors (IV) were commercially available. The ketone intermediates (V) were prepared by condensation of the corresponding methyl-ketone (IV) with the different aryl amines via Mannich reaction.

(9) The hydroxyl derivatives (1-8) were obtained by reduction of the corresponding carbonyl group with NaBH.sub.4 in methanol.

(10) II/ Experimental Protocol for the Synthesis of Compounds of Formula (I)

(11) II/1General Methods

(12) Chemicals reagents were purchased from E. Merck (Darmstadt, Germany), Scharlau (F.E.R.O.S.A., Barcelona, Spain), Panreac Quimica S.A. (Montcada i Reixac. Barcelona, Spain), Sigma-Aldrich Qumica S.A. (Alcobendas, Madrid), Acros Organics (Janssen Pharmaceuticals 3a, 2440 Geel, Belgie) and Lancaster (Bischheim-Strasbourg, France).

(13) All of the synthesized compounds were chemically characterized by thin layer chromatography (TLC), melting point (M.P.), infrared (IR) and nuclear magnetic resonance (.sup.1H-NMR) spectra as well as by elemental microanalysis.

(14) .sup.1H NMR spectra were recorded on a Bruker 400 Ultrashield (400 MHz) (Rheinstetten, Germany) using TMS as the internal standard and chloroform (CDCl.sub.3) or dimethyl sulfoxide-d.sub.6 (DMSO-d.sub.6) as solvents. The chemical shifts are reported in ppm () and coupling constant (J) values are given in Hertz (Hz). Signal multiplicities are represented by: s (singlet), bs (broad singlet), d (doublet), t (triplet), q (quadruplet), dd (doublet of doublets), ddd (doublet of doublet of doublets) and m (multiplet). The IR spectra were performed on Thermo Nicolet FT-IR Nexus Euro (Madison, USA) using KBr pellets; the frequencies are expressed in cm.sup.1. Elemental microanalyses were obtained on an Elemental Analyzer (LECO CHN-900, Michigan, USA) from vacuum-dried samples. The analytical results for C. H. and N were within 0.4 of the theoretical values. Alugram SIL G/UV254 (Layer: 0.2 mm) (Macherey-Nagel. Germany) was used for thin layer chromatography and silica gel 60 (0.040-0.063 mm and 0.063-0.200 nm) was used for column flash chromatography (Merck).

(15) Some ketones and hydroxyls were purified by flash chromatography with binary gradient of dichloromethane (synthesis grade SDS-Carlo Erba Reactifs, France) with methanol (Panreac Quimica S.A.) until 99:1 and a UV variable dualwavelength detection. The chromatography was developed in the CombiFlash Rf (Teledyne Isco, Lincoln. USA), with dichloromethanemethanol as solvents and a normal phase of 12 gram Flash Column (RediSep Rf Columns by Teledyne Isco, Inc., USA).

(16) II/2General Method for the Synthesis of Protected Aryl Amines (II)

(17) A mixture of the 2-fluoro-4-nitrobenzo-trifluoride (1 eq), the corresponding BOC-amine (1.2 eq), K.sub.2CO.sub.3 (1.5 eq) and CH.sub.3CN (30 mL) was heated at reflux for 24 hours. The solvent was removed under reduced pressure. The residue was dissolved in CH.sub.2Cl.sub.2 (50 mL) and washed with water (330 mL). The organic phase was dried with anhydrous Na.sub.2SO.sub.4 and filtered. After evaporating to dryness under reduced pressure, the residue was precipitated and washed by adding diethyl ether or petroleum ether, affording the desired protected aryl amine (II).

(18) II/3General Synthesis of Noncommercial Aryl Amines (III)

(19) The protected amine (II) was dissolved in 40 mL of a solution of HCl/AcH (1:1) with stirring for 2 hours at room temperature. The solvent was removed under reduced pressure and the compound was dissolved in water. The aqueous solution was basified with NaOH 2M and stirred for 1 hour. Then the product was extracted with CH.sub.2Cl.sub.2. The organic phase was dried with anhydrous Na.sub.2SO.sub.4 and filtered. After evaporating to dryness under reduced pressure, the crude was purified by column chromatography (SP: silica gel), eluting with dichloromethane (NH.sub.3)/methanol 99:1 (v/v), affording the desired aryl amine (III).

(20) II/4General Method for the Synthesis of Ketone Derivatives (V)

(21) A mixture of the appropriately substituted aryl methyl ketone (IV) (1 eq), the aryl amine (III) (1 eq), dioxolane (1.4%) and concentrated HCl (1 mL) was heated at reflux. Then water was added (50 mL) and the product was extracted with CH.sub.2Cl.sub.2. The organic phase was dried with anhydrous Na.sub.2SO.sub.4, filtered and evaporating to dryness under reduced pressure. The residue was purified by column chromatography (SP: silica gel), eluting with CH.sub.2Cl.sub.2/methanol 95:5 (v/v) or Flash Chromatography eluting with CH.sub.2Cl.sub.2/methanol 99:1 (v/v). In other cases the hydrochloride salt was prepared by adding a hydrogen chloride ethereal solution to the stirred compounds.

(22) II/5General Method for Preparing of Hydroxyl Derivatives (1-8)

(23) Sodium borohydride (3 eq) was added little by little to a pre-cooled suspension (0 C.) of the corresponding ketone (V) (1 eq) in methanol over a period of 30-60 minutes. The solvent was removed under reduced pressure and the residue was dissolved in dichloromethane (40 mL) and then washed with water (330 mL). The organic phase was dried with anhydrous Na.sub.2SO.sub.4 and filtered. After evaporating the solvent to dryness under reduced pressure, the compound was purified by column chromatography (SP: silica gel), eluting with dichloromethane/methanol 99:1 (v/v), by preparative chromatography (SP: silica gel), eluting with dichloromethane/methanol 97:3 (v/v), flash chromatography eluting with dichloromethane/methanol 99:1 (v/v) or preparing the hydrochloride by adding a hydrogen chloride ethereal solution to the stirred compounds.

II/5-1. 3-[4-(4-fluorophenyl)-3,6-dihydropyridin-1(2H)-yl]-1-(naphthalen-2-yl)propan-1-ol (3)

(24) (23% Yield), Mp 127-129 C. .sup.1H NMR (400 MHz, CDCL.sub.3): 1.99-2.12 (m, 2H, CHOHCH.sub.2); 2.64 (s, 2H, H.sub.6 tetrahydropyridine); 2.72-2.96 (m, 4H, H.sub.3+H.sub.5 tetrahydropyridine); 3.31 (d, 2H, CHOHCH.sub.2CH.sub.2, J.sub.CHCH2=16.7 Hz); 5.17 (bs, 1H, CHOH); 6.02 (s, 1H, H.sub.3 tetrahydropyridine); 7.04 (t, 2H, H.sub.3+H.sub.5 phenyl, J.sub.3,2=J.sub.5,6=8.7 Hz); 7.37 (dd, 2H, H.sub.2+H.sub.6 phenyl, J.sub.2,3=J.sub.6,5=8.7 Hz, J.sub.2,F=J.sub.6,F=5.4 Hz); 7.45-7.50 (m, 2H, H.sub.6+H.sub.7 naphtyl); 7.51 (dd, 1H, H.sub.8 naphtyl, J.sub.8,7=7.4 Hz. J.sub.8,6=1.6 Hz); 7.85 (d, 2H, H.sub.3+H.sub.4 naphtyl, J.sub.3,4=J.sub.4,3=8.3 Hz); 7.87 (dd, 1H, H.sub.5 naphtyl, J.sub.5,6=7.8 Hz, J.sub.5,7=2.3 Hz); 7.91 (s, 1H, H.sub.1 naphtyl) ppm. Anal (C.sub.24H.sub.24NFO) C, 77.83; H, 6.62; N, 3.78. Found: C, 77.87; H, 6.82; N, 3.40.

II/5-2. 3-[4-(4-fluorophenyl)-3,6-dihydropyridin-1(2H)-yl]-1-(4-(trifluoromethyl phenyl)propan-1-ol) (4)

(25) (40% Yield), Mp 116-117 C. .sup.1H NMR (400 MHz, DMSO): 1.81 (dd, 2H, CHOHCH.sub.2, J.sub.CHCH2=13.7 Hz, J.sub.CHCHOH=7.3 HZ); 2.45-2.50 (m, 4H, CHOHCH.sub.2CH.sub.2+H.sub.5 tetrahydropyridine); 2.62 (t, 2H, H.sub.6 tetrahydropyridine, J.sub.CHCH=J.sub.CHCH2=5.5 Hz); 3.08 (s, 2H, H.sub.2 tetrahydropyridine); 4.75-4.80 (m, 1H, CHOH); 5.70 (bs, 1H, OH); 6.12 (s, 1H, H.sub.3 tetrahydropyridine); 7.15 (t, 2H, H.sub.5+H.sub.3 phenyl, J.sub.5,F=J.sub.3,F=8.8 Hz, J.sub.5,6=J.sub.3,2=8.8 Hz); 7.47 (dd, 2H, H.sub.6+H.sub.2 phenyl, J.sub.6,5=J.sub.2,3=8.4 Hz, J.sub.6,F=J.sub.2,F=5.6 Hz); 7.57 (d, 2H, H.sub.2+H.sub.6 CF.sub.3-phenyl, J.sub.2,3=J.sub.6,5=7.9 Hz); 7.68 (d, 2H, H.sub.3+H.sub.5 CF.sub.3-phenyl, J.sub.3,2=J.sub.5,6=8.1 Hz) ppm. Anal (C.sub.21H.sub.21NF.sub.4O) C, 66.49; H, 5.54; N, 3.70. Found: C, 66.11; H, 5.67; N, 3.81.

II/5-3. 1-(4-fluoronaphthalen-1-yl)-3-((1-(4-nitro-2-(trifluoromethyl) phenyl) piperidin-4-yl)amino)propan-1-ol (5)

(26) (90% Yield), Mp 93-95 C. .sup.1H NMR (400 MHz. CDCl.sub.3): 1.68-1.83 (m, 2H, H.sub.3ax+H.sub.5ax piperidine); 1.86-2.01 (m, 1H, CHOHCH.sub.2); 2.15-2.23 (m, 2H, H.sub.3ec+H.sub.5ee piperidine); 2.12 (d, 1H, CHOH. J.sub.CHCH2=12.7 Hz); 2.75-2.84 (m, 1H, H.sub.4 piperidine); 2.98 (t, 2H, H.sub.2ax+H.sub.6ax piperidine, J.sub.2ax,2ec=J.sub.6ax,6ec=11.5 Hz); 3.00-3.03 (m, 1H, CHOHCH.sub.2CH.sub.2); 3.08-3.15 (m, 1H, CHOHCH.sub.2CH.sub.2); 3.42 (d, 2H, H.sub.2ec+H.sub.6ec piperidine); 5.71 (dd, 1H, CHOH, J.sub.CH,CH2=8.2 HZ, J.sub.CHCH2=2.3 Hz); 7.17 (dd, 1H, H.sub.3 naphtyl, J.sub.3,F=10.2 Hz, J.sub.3,2=8.1 Hz); 7.27-7.30 (m, 1H, H.sub.6 phenyl); 7.53-7.61 (m, 2H, H.sub.6+H.sub.7 naphtyl); 7.67-7.72 (m, 1H, H.sub.2 naphtyl); 8.04 (d, 1H, H.sub.8 naphtyl, J.sub.8,7=7.8 Hz); 8.16 (dd, 1H, H.sub.5 naphtyl J.sub.5,6=7.2 Hz, J.sub.5,F=3.3 Hz); 8.33 (dd, 1H, H.sub.5 phenyl, J.sub.5,6=9.0 Hz, J.sub.5,3=2.7 Hz); 8.53 (d, 1H, H.sub.3 phenyl, J.sub.3,5=2.7 Hz). ppm. Anal (C.sub.25H.sub.25N.sub.3F.sub.4O.sub.3) C, 61.11; H, 5.43; N, 8.55. Found: C, 60.77; H, 5.43; N, 8.30.

II/5-4. 1-(4-fluoronaphthalen-1-yl)-3-[4-(4-fluorophenyl)-3,6-dihydropyridin-1(2H)-yl]propan-1-ol (6)

(27) (82% Yield), Mp 162-163 C. .sup.1H NMR (400 MHz. DMSO): 2.14-2.20 (m, 2H, CHOHCH.sub.2); 2.73 (bs, 2H, H.sub.6 tetrahydropyridine); 2.84-3.12 (m, 4H, CHOHCH.sub.2CH.sub.2+H.sub.5 tetrahydropyridine); 3.41 (d, 1H, H.sub.2ax tetrahydropyridine, J.sub.2ax,2ec=11.2 Hz); 3.47 (d, 1H, H.sub.2ec tetrahydropyridine); J.sub.2ec,2ax=11.3 Hz); 5.74 (bs, 1H, CHOH); 6.00-6.05 (m, 1H, H.sub.3 tetrahydropyridine); 7.00-7.08 (m, 2H, H.sub.2+H.sub.6 phenyl); 7.18 (dd, 1H, H.sub.3 naphtyl, J.sub.3,F=10.2 Hz, J.sub.3,2=8.1 Hz); 7.35-7.40 (m, 2H, H.sub.6+H.sub.7 naphtyl); 7.54-7.61 (m, 2H, H.sub.5+H.sub.3 phenyl); 7.70 (dd, 1H, H.sub.2 naphtyl, J.sub.2,3=8.0 Hz, J.sub.2,F=5.6 Hz); 8.07 (d, 1H, H.sub.5 naphtyl, J.sub.5,6=8.2 Hz); 8.2 (dd, 1H, H.sub.8 naphtyl, J.sub.8,7=7.0 Hz, J.sub.8,6=2.5 Hz) ppm. Anal (C.sub.24H.sub.23NF.sub.2O) C, 75.95; H, 6.06; N, 3.69. Found: C, 75.45; H, 6.45; N, 3.50.

II/5-5. Hydrochloride of 1-(4-fluorophenyl)-3-[1-(4-nitro-2-trifluoromethylphenyl) piperidin-4-yl)amino]propan-1-ol (7)

(28) (11% Yield), Mp 185-187 C., .sup.1H NMR (400 MHz, DMSO): 1.38 (bs, 2H, H.sub.3ax+H.sub.5ax piperidine); 1.70 (bs, 2H, H.sub.3ec+H.sub.5ec piperidine); 1.90 (d, 2H, CHOHCH.sub.2, J.sub.CHCH=J.sub.CHCH2=11.0 Hz); 2.58 (bs, 1H, H.sub.4 piperidine); 2.63-2.64 (m, 2H, H.sub.2ax+H.sub.6ax piperidine); 2.95 (t, 2H, H.sub.2ec+H.sub.6ec piperidine, J.sub.2ec,2ax=J.sub.6ec,6ax=12.1 Hz); 3.2-3.3 (m, 2H, CHOHCH.sub.2CH.sub.2); 4.69 (t, 1H, CHOH, J.sub.CHOHCHCH=9.0 Hz); 7.13 (t, 2H, H.sub.3+H.sub.5 fluorophenyl, J.sub.3,2=J.sub.5,6=8.1 Hz); 7.36 (bs, 2H, H.sub.2+H.sub.6 fluorophenyl); 7.50 (d, 1H, H.sub.6 phenyl, J.sub.6,5=8.0 Hz); 8.37 (bs, 2H, H.sub.3+H.sub.5 phenyl) ppm. Anal (C.sub.21H.sub.25N.sub.3ClF.sub.4O.sub.3) C, 52.51; H, 5.03; N, 8.80. Found: C, 52.13; H, 4.91; N, 8.49.

II/5-6. 1-(4-fluorophenyl)-3-[4-(4-fluorophenyl)-3,6-dihydropyridin-1 (2H)-yl]propan-1-ol (8)

(29) (12% Yield), Mp 199-201 C. .sup.1H NMR (400 MHz, DMSO): 1.76-1.80 (m, 2H, CHOHCH.sub.2); 2.45 (bs, 4H, CHOHCH.sub.2CH.sub.2+H.sub.5 tetrahydropyridine); 2.57-2.62 (m, 2H, H.sub.6 tetrahydropyridine); 3.06 (bs, 2H, H.sub.2 tetrahydropyridine); 4.66 (t, 1H, CHOH. J.sub.CHOH=6.3 Hz); 5.49 (bs, 1H, OH); 6.12 (bs, 1H, H.sub.3 tetrahydropyridine); 7.14 (dd, 4H, H.sub.3+H.sub.5 phenyl+H.sub.3+H.sub.5 fluorophenyl, J.sub.3,F=J.sub.5,F=J.sub.3,F=J.sub.5,F=12.6 Hz, J.sub.3,2=J.sub.5,6=J.sub.3,2=J.sub.5,6=8.0 Hz); 7.37 (dd, 2H, H.sub.2+H.sub.6 fluorophenyl, J.sub.2,3=J.sub.6,5=7.9 Hz, J.sub.2,F=J.sub.6,F=6.0 Hz); 7.46 (dd, 2H, H.sub.2+H.sub.6 phenyl, J.sub.2,3=J.sub.6,5=7.7 Hz, J.sub.2,F=J.sub.6,F=5.5 Hz) ppm. Anal (C.sub.20H.sub.21NF.sub.2O) C, 72.42; H, 6.34; N, 4.22. Found: C, 72.06; H, 6.33; N, 4.03.

(30) III/ Biological tests

(31) III/1In Vitro Antiplasmodial Drug Assay

(32) Culture of chloroquine-resistant FCR-3 strain of Plasmodium falciparum was carried out at 37 C. in a 5% CO.sub.2 environment on RPMI 1640 medium supplemented with 25 mM Hepes, 5% (w/v) NaHCO.sub.3, gentamicin 0.1 mg/ml and 10% heat-inactivated human serum A.sup.+ (hematocrit 5%), as previously described (W. Trager et al., Science, 193 (1976) 673-675). The drugs dissolved in dimethylsulfoxide (DMSO) were added at final concentrations ranging from 200 to 0.1 M. All experiments were performed in triplicate. The final DMSO concentration was never greater than 0.1%. In vitro antimalarial activity was measured using [.sup.3H]-hypoxanthine (MP Biomedicals, USA) incorporation assay (R. E. Desjardins et al., Antimicrob. Agents Chemother. 16 (1979) 710-718). All experiments were performed in triplicate. Results were expressed as the concentration resulting in 50% inhibition (IC.sub.50) which was calculated by linear interpolation (W. Huber et al., Acta Trop. 55 (1993) 257-261) as follows:
Log(IC.sub.50)=log(X1)+(50Y1)/(Y2Y1)[Log(X2)log(X1)]
X1: concentration of the drug that gives a % inhibition of the parasitemia Y1>50%,

(33) X2: concentration of the drug that gives a % inhibition of the parasitemia Y2<50%,

(34) % Inhibition of the incorporation of labeled hypoxanthine=100(P/T*100),

(35) P: c.p.m. for every concentration, and

(36) T: negative control (red blood cells without drug).

(37) The results are presented in Table 1:

(38) TABLE-US-00001 TABLE 1 In vitro antimalarial activity against Plasmodium falciparum FCR-3 and VERO cytotoxicity of tested compounds Compounds Ar Amine R R IC.sub.50 TC.sub.50 3 2- naphthyl 0 F H 36.15 >100 4 4- trifluoromethyl phenyl F H 5.60 >100 5 4-fluoro-1- naphthyl NO.sub.2 CF.sub.3 0.15 5.5 6 4-fluoro-1- naphthyl F H 0.40 >50 7 4-fluoro-1- phenyl NO.sub.2 CF.sub.3 0.48 30.2 8 4-fluoro-1- phenyl F H 0.66 >100 CQ 0.13 >50 CQ: chloroquine; IC.sub.50 and TC.sub.50 values represent of 50% of Plasmodium falciparum growth or VERO cells survival. Data represents the average of three independent determinations and are expressed in M. Errors for individual measurements differed by less than 50%.

(39) III/2In Vivo Antiplasmodial Drug Assay

(40) The antiplasmodial activity of Compound 7 was tested in vivo in a murine model. The antiplasmodial activity of a Compound A representative of the prior art was also tested:

(41) ##STR00016##

(42) Studies were conducted according to the French and Colombian guidelines on laboratory animal use and care (No 2001-464 and No 008430, respectively). The classical 4-day suppressive test was carried out (W. Peters, Chemotherapy and drug resistance in malaria, Academic Press: London, 1970). Swiss male mice weighing 202 g, were infected with 10.sup.7 P. berghei ANKA parasitized cells (day 0). Two hours after infection and at the same time during 4 consecutive days, batches of three mice were orally treated at dose of 50 mg/kg/day, (drugs were dissolved in vehicle water dimethylsulfoxide 9:1). A control group received the vehicle while a reference group was administered chloroquine diphosphate (CQ) at 3 mg/kg/day (oral route). Survival of the mice was checked daily and the percentage of parasitized erythrocytes was determined on day 4, by Giemsa-stained thin blood smears made from peripheral blood. The percentage of inhibition of parasitaemia was calculated.

(43) The results are presented on FIG. 1.

(44) Compound 7 shows, at 50 mg/kg/day, 76% of inhibition while chloroquine reference dosed at 3 mg/kg/day shows 65% of inhibition and Compound A shows, at 50 mg/kg/day, 17% of inhibition.

(45) III/3Toxicity Assay

(46) VERO cells (African Green Monkey kidney epithelial cells) were seeded (510.sup.5 cells/ml, 100 l/well) in a 96-well flat-bottom plate at 37 C. and with 5% CO.sub.2 in RPMI 1640 without phenol red (Sigma), supplemented with 10% heat-inactivated fetal bovine serum. Drugs were added at different concentrations and the cells were cultured for 48 hours. The effect was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay. Four hours after the addition of MTT, 100 l of lysis buffer (50% isopropanol, 30% water, 20% SDS) were added and the cells were incubated at room temperature for 15 min under agitation. Finally, optical density was read at 590 nm with a 96-well scanner (Bio-Rad). All experiments were performed in triplicate. The TC.sub.50 determined by linear regression analysis was defined as the concentration of test sample resulting in a 50% reduction of absorbance when compared with controls.

(47) The results are presented in Table 1.

(48) III/4Cytoxicity

(49) The cytotoxicity of Compound 7 was compared to the cytotoxicity of Compound 31 of the publication of Silvia Perez-Silanes et al., Molecules 2009, 14, 4120-4135, the compounds only differentiated by the nature of the Ar group (4-fluoro-1-phenyl group vs benzo[b]thiophenyl).

(50) Both compounds were administered at 50 mg.Math.kg.sup.1 in a malaria murine model. No cytotoxicity was detected after administration of Compound 7, while more than half of the treated mice died when exposed to Compound 31 of Silvia Perez-Silanes et al.

(51) The too high cyctotoxicity of Compound 31 of Silvia Perez-Silanes et al. indicates that this compound cannot be administered in human or animal, whereas the lowest cytotoxicity of Compound 7 allows a safe administration in mouse.

(52) IV/ Physicochemical Parameters

(53) Virtual Computational Chemistry Laboratory (http://www.vcclab.org/) (I. V. Tetko et al., J. Comput.-Aided Mol. Des. 19 (2005) 453-463) and Molispiration online property calculation toolkit (http://www.molispiration.com/services/properties.html) were used to calculate Topological Polar Surface Area (P. Ertl et al., J. Med. Chem. 43 (2000) 3714-3717) mi Log P, AlogPS2.1, KOWLog P, Log P (AB/Log P), number of rotable bonds and violations of Lipinski's rule of five (C. A. Lipinski et al., Adv. Drug Deliv. Rev. 23 (1997) 3-25).

(54) Absorption (% ABS) was calculated by: % ABS=109-(0.345TPSA) (Y. Zhao et al., Pharmaceutical Research, 19 (2002) 1446-1457).

(55) The results are presented in Table 2.

(56) TABLE-US-00002 TABLE 2 Physical chemical properties of tested compounds n- Com- % TPSA n- Molecular KOW ALog OHNH n-ON Lipinski's pounds ABS ({acute over ()}.sup.2) ROTB weight miLogP LogP PS 2.1 MLogP donors acceptors violations Rule >500 <5 <5 <5 <4.15 <5 <10 1 3 100.00 23.47 5 361.46 4.76 5.48 4.66 4.57 1 2 0 4 100.00 23.47 6 379.74 4.47 5.26 4.69 4.68 1 2 0 5 80.95 81.32 8 491.47 5.36 5.76 4.65 4.86 2 6 1 6 100.00 23.47 5 379.45 4.85 5.68 4.88 4.95 1 2 0 7 80.95 81.32 8 443.44 4.24 4.59 3.74 4.19 2 6 0 8 100.00 23.47 5 329.39 3.74 4.50 3.95 4.25 1 2 0 CQ 99.30 28.20 8 319.90 5.01 4.50 5.28 3.52 1 3 1 % ABS: percentage of absorption, calculated by: % ABS = 109 (0.345 TPSA); TPSA: topological polar surface area; n-ROTB: number of rotatable bonds; LogP: logarithm of compound partition coefficient between n-octanol and water; n-OHNH: number of hydrogen bond donors; n-ON: number of hydrogen bond acceptors. CQ: chloroquine

(57) It appears that all the compounds respected Lipinski's rules (Log P<5, under 5 H-bond donors and 10H-bond acceptors) (C. A. Lipinski at al., Adv. Drug Deliv. Rev. 23 (1997) 3-25). All the structures have a molecular weight under 500 Daltons with limited lipophilicity. Calculated absorption (% ABS) suggests good aptitudes for oral treatment.

(58) V/ Computational Docking Studies

(59) In previous studies using computational molecular binding tools, the enzyme Plasmodium plasmepsin II was proposed as a potential target for arylaminoalcohols (A. Mendoza et al., Exp. Parasit. 128(2) (2011) 97-103). This study suggested that the activity of arylaminoalcohol derivatives could be due to the formation of a hydrogen bond with one of the catalytic aspartates from the active site (Asp214 and Asp34). This interaction involves the unique hydroxyl group present in the most active compounds and Asp 214 residue.

(60) The molecular docking program ICM (ICM. Version 3.4-8. La Jolla. Calif. Molsoft LLC. 2006) was used to determine the potential binding mode between the most active synthesized compounds and the selected Plasmodium plasmepsin II enzyme target candidate. In order to validate our methodology and check the ability of the program ICM to investigate the binding mode of inhibitors into the binding site of the enzyme, the complex Plasmodium plasmepsin II with EH58 (O. A. Asojo et al., J. Mol. Biol. 327 (2003) 173-181) was computationally re-docked (PDB code 1LF3). As previously reported (W. Cunico et al., Eur. J. Med. Chem. 44 (2009) 1363-1368; W. Cunico et al., Eur. J. Med. Chem. 44 (2009) 3816-3820), RMSD values up to 3.0 were considered correctly docked structures for preparing the input structures for ICM methodology (R. Abagyan et al., J. Comput. Chem., 15(5) (1994) 488-506). The structures of the compounds were sketched by using the ICM software, Molecular Editor (Molecular Editor. Version 2.5. La Jolla. Calif. Molsoft LLC. 2006). Protein and compound structures were converted into ICM objects. During the protein conversion process, hydrogens were added and the modified structure was optimized. Meanwhile, during ligand conversions, two-dimensional (2D) representations were converted into three-dimensional (3D) ones, partial charges were assigned, and rotatable bonds were identified. According to previous studies (A. Mendoza et al., Exp. Parasit. 128(2) (2011) 97-103; W. Cunico et al., Eur. J. Med. Chem. 44 (2009) 1363-1368; W. Cunico et al., Eur. J. Med. Chem. 44 (2009) 3816-3820), the residues involved in the active site were Asp34 and Asp214. IcmPocketFinder (J. An et al., Mol. Cell. Prot. 4(6) (2005) 752-761) was used to identify the active site pocket with a tolerance value of 4.6 . Initial ligand position and orientation and box position and size were maintained in accordance with the values suggested by the program. The most representative binding modes calculated with at least one hydrogen bond with one of the catalytic aspartates were chosen for analysis (V. Kasam et al., J. Chem. Inf. Model. 47(5) (2007) 1818-1828; J. Aqvist et al., Prot. Eng. 7 (1994) 385-391). The docking poses for each ligand were analyzed by examining their relative total energy score. The more energetically favorable conformation was selected as the best pose.

(61) The comparison of the different docking results between Compounds 5, 7 and 8 and the most active compound previously reported, i.e. the 1-(4-fluoronaphthalen-1-yl)-3-[4-(4-nitro-2-trifluoromethylphenyl)piperazin-1-yl]propan-1-ol (Compound 13 of A. Mendoza et al., Exp. Parasit. 128(2) (2011) 97-103), revealed that presumably all compounds adopt the same binding mode (FIG. 2a) and establish a hydrogen bond between the hydroxyl group and Asp214 (FIG. 2b).

(62) This similar binding mode is not surprising since all of the tested compounds contain related scaffolds. In general, all these compounds are situated near the S1, S1 and S3 pockets of the protein. 4-nitro-2-trifluromethyl phenyl and 4-trifluromethyl phenyl groups of these conformations were set near 51 and S3 pocket, and 4-fluoro-1-phenyl and 4-fluoro-1-naphtyl were located in the vicinity of the S2 pocket.