Phenotypic Drug Discovery for Human African Trypanosomiasis: A Powerful Approach.

The work began with the screening of a library of 700,000 small molecules for inhibitors of Trypanosoma brucei growth (a phenotypic screen). The resulting set of 1035 hit compounds was reviewed by a team of medicinal chemists, leading to the nomination of 17 chemically distinct scaffolds for further investigation. The first triage step was the assessment for brain permeability (looking for brain levels at least 20% of plasma levels) in order to optimize the chances of developing candidates for treating late-stage human African trypanosomiasis. Eleven scaffolds subsequently underwent hit-to-lead optimization using standard medicinal chemistry approaches. Over a period of six years in an academic setting, 1539 analogs to the 11 scaffolds were synthesized. Eight scaffolds were discontinued either due to insufficient improvement in antiparasitic activity (5), poor pharmacokinetic properties (2), or a slow (static) antiparasitic activity (1). Three scaffolds were optimized to the point of curing the acute and/or chronic T. brucei infection model in mice. The progress was accomplished without knowledge of the mechanism of action (MOA) for the compounds, although the MOA has been discovered in the interim for one compound series. Studies on the safety and toxicity of the compounds are planned to help select candidates for potential clinical development. This research demonstrates the power of the phenotypic drug discovery approach for neglected tropical diseases.


Ethyl 3-(3-Benzylureido) Benzofuran-2-Carboxylate
A solution of ethyl 3-aminobenzofuran-2-carboxylate (5.15 g, 25.1 mmol) in toluene (60 mL) was used to reflux in a 500 mL 3-neck flask fitted with a condenser and addition funnel. A solution of benzyl isocyanate (4.78 g, 3.9 mmol) in toluene (120 mL) was added dropwise over 2.5 hours. The addition funnel was removed, and the reaction mixture was concentrated using a Dean-Stark trap inserted between the 3-neck flask and the condenser, until precipitation of the product began. After eight hours, the reaction mixture was allowed to cool to room temperature. The precipitate was filtered off, giving the title compound (4.64 g, 55%, HPLC 95.9 area %) as a white solid. The filtrate was treated as above with more benzyl isocyanate (3.55 g, 26.7 mmol) to give a second crop (1.11 g, total recovery 5.75 g, 68%). 1

2-Chloro-11-Methyl-1,8,12-Triazatricyclo[7.3.0.0³,⁷] Dodeca-2,7,9,11-Tetraene
A total of 8.0 g (42.3 mmol) of compound 6.1 in 30 mL POCl3 was heated at 110 °C for 4 h. The reaction progress was monitored by TLC (10% MeOH in DCM). A small aliquot was taken from the reaction mixture poured into water, the formed precipitate was centrifuged, dissolved in EtOAc, and the solution placed on TLC. After the reaction was completed, the reaction mixture was poured into crushed ice (500 g). The formed precipitate was collected, washed with water, and dried in vacuum. Obtained 8.5 g (97.3%) of product 6.2, which was used in the next step without any further purification.

Ethyl 2-{[3-(3-Chloro-4-Methoxyphenyl)-4-Oxo-3,4-Dihydrophthalazin-1-Yl] Formamido} Acetate
Acid 7.3 (1 g, 3 mmol) and thionyl chloride (5 mL) was reflexed for 1 h. The reaction mixture became clear within 10 min. After that, the thionyl chloride was completely removed under vacuum and dried. The crude product was used next without any purification. To a suspension of glycine ethyl ester hydrochloride (0.54 g, 3.9 mmol) in DCM (20 mL) was added triethylamine (0.45 g, 4.5 mmol). The resulting solution was then cooled to 0° C and a solution of acid chloride in DCM (10 mL) was added dropwise. The reaction mixture was stirred at 0 °C for 2 hours and then at room temperature for 18 hours. The resulting solution was diluted with DCM (50 mL) and washed with 10 % HCl (2 x 25 mL). The organic phase was separated, dried (MgSO4), and concentrated in vacuo to afford the title compound 7.4. Further purification with 3% DCM in MeOH will give pure ester (7.4) with a yield of 1.0 g (80%) as a white solid.

3-(3-Chloro-4-Methoxyphenyl)-N-[2-(2-Methylpiperidin-1-Yl)-2-Oxoethyl]-4-Oxo-3,4-Dihydrophthalazine-1-Carboxamide
LiOH (0.103 g, 4.30 mmol) was added to a stirred solution of ester 7.4 (0.6 g, 1.44 mmol) in THF:MeOH:H2O (3:1:1,10 mL) and the resulting mixture was stirred at room temperature for five hours. The MeOH and THF were then evaporated and the residue was diluted with water (2 mL), acidified with 20% aq HCl, and extracted with EtOAc. The organic layer was washed with saturated brine solution, dried over Na2SO4, and concentrated under reduced pressure to afford acid in a quantitative yield as a white solid. The resulting acid was dissolved in dry CH2Cl2 and sequentially treated with HOBt (0.29 g, 2.16 mmol) and EDCI (0.413 g, 2.16 mmol) at 0 °C under a nitrogen atmosphere. After 10 minutes, 2-methylpiperidine (0.214 g, 2.16 mmol) was followed by DIPEA (0.28 g, 2.16 mmol). The reaction mixture was allowed to reach room temperature and stirred further for 10 h under a nitrogen atmosphere. After that, the reaction mixture was diluted with CHCl3, washed with 1 N HCl, water, and aq. saturated NaHCO3 solution and aq. NaCl solution. The organic layer was dried (Na2SO4) and evaporated to give the crude, which was purified by column chromatography with 5% DCM in MeOH, affording 7.5 in a yield of 0.473 g (70%) as a white solid. 1

2-(4-Nitrophenyl)-1H-1,3-Benzodiazole
A total of 2.0 g (18.5 mmol) of o-phenylenediamine, 3.09 g (18.5 mmol) of p-nitrobenzoic acid, and two drops 4 N HCl were mixed and subjected to microwave irradiation at 140 °C for 3 h. The reaction progress was monitored by TLC (40% EtOAc in hexane). Water was added to the mixture and pH was adjusted to 5-6 with 10% NaOH. The product was extracted with DCM. The solvent was removed and the crude product recrystallized from ethanol-water, yielding 4.2 g (95%) of pure compound 10.1

4-(1H-1,3-Benzodiazol-2-Yl) Aniline
A total of 800 mg (3.37 mmol) of nitro-compound 10.1 was dissolved in 50 mL of EtOAc. To this solution, 3.79 g (16.87 mmol) of tin(II)chloride monohydrate was added. The reaction mixture was stirred under reflux for 6 h. Next, it was cooled down and poured into aq. NaHCO3 and extracted with EtOAc. The organic layers were combined, washed with brine, and dried over Na2SO4. The solvent was removed by rotovap. The crude product 10.2 was used in the next step without further purification.

1-(2-Fluorobenzyl)-1H-Indole-3-Carbaldehyde
Potassium carbonate (5.28 g, 4.00 mmol) was added to a solution of indole-3-carboxaldehyde (1.00 g, 6.89 mmol) in acetone (40 mL). The mixture was stirred for one hour at room temperature. A solution of 2-fluorobenzyl bromide (1.37 g, 7.23 mmol) in acetone (10 mL) was added and the mixture was refluxed for 3.5 hours until the reaction was complete by HPLC. The cooled mixture was filtered. The filtrate was evaporated, dissolved in CH2Cl2, and washed with water. The organic layer was dried (MgSO4), filtered, and evaporated. The crude product was purified on a silica gel column eluting with gradient from neat hexane to hexane/EtOAc (3:2), to give the title compound as a yellow solid (1.50 g, 86%). 1