Some of the derivatives exhibited potency in the nanomolar range, with one (2-phenoxy-1,4-naphthoquinone, B6 in Figure 1) displaying an ED50 value of 80 nM against Trypanosoma brucei selleck Belinostat rhodesiense, as assessed in experiments using in vitro cultured parasites. It also showed a selectivity index (ratio of the compound’s ED50 values on mammalian cell lines and trypanosomes) of 74 [24], which is very close to the specifications required by WHO/TDR to be considered an anti-trypanosomatid hit [25]. However, the molecular mechanism and the target(s) responsible for the biological profile of this class of compounds have remained undisclosed. Figure 1 Chemical structures. Here, by means of a chemical proteomics approach, we aimed at identifying the putative molecular target(s) of B6.

In particular, using its immobilized derivative 1 (Fig. 1), we isolated its targets from parasite extracts. Then, by means of biochemical experiments, we verified the ability of the molecule to bind to recombinant forms of the identified targets. In light of the general property of naphthoquinones to generate free radicals, we finally analyzed oxygen consumption in permeabilized trypanosomes and production of reactive oxygen species (ROS) in trypanosome mitochondrial cell fractions. This allowed us to elucidate additional B6 potential mechanisms of action, as chemical proteomics is not suited to identify non-protein targets. Methods Chemical synthesis and anti-trypanosomal activity of B6-derivatives 1�C3 Before covalently attaching B6 to the solid support through a linker, we analyzed which site(s) of the molecule were more appropriate for linking purposes.

To this end we synthesized derivatives 1�C3 (Fig. 1), which carry in different positions amino or hydroxyl groups that can be easily exploited as anchor points. The synthesis was carried out according to the procedure reported for B6 [24], and which relies on the substitution of a 2-bromonaphthoquinone with the corresponding phenol. Synthesis of 2-(4-amino-phenoxy)-[1], [4]naphthoquinone (1). To a stirred solution of 4-amino-phenol (0.46 g, 4.2 mmol) in 90 ml dimethylformamide (DMF) potassium carbonate (1.70 g, 12.3 mmol) was added. After stirring for 1 h at room temperature, 2-bromo-[1], [4]-naphthoquinone (1.0 g, 4.2 mmol) was added. After stirring for further 3 h, the reaction was diluted with water and ice (500 ml) and the resulted brownish solid was collected by filtration to give 0.

49 g of crude 1, which was crystallized by EtOH/water (40% yield). IR (Nujol) 3421, 3382, 1680, 1635, 1609, 1508, 1204, 980, 718 cm?1; 1H NMR (300 MHz, CDCl3) �� 8.25-8.22 (m, 1H), 8.12-8.09 (m, 1H), 7.80-7.78 (m, 2H), 6.95 (d, 2H, J=8.5 Hz), 6.76 (d, 2H, J=8.5 Hz), 6.04 (s, 1H), 3.77 (br s, 2H, exchangeable with D2O); HRMS (ES) m/z calculated for C16H11NO3Na 288.0637, found 288.0639 [M++Na+]. Synthesis of 5-hydroxy-2-phenoxy-[1], [4]naphthoquinone Entinostat (2).