NAD, NADH, pyruvate, α-ketoisocaproate sodium salt and N-ethylmaleimide were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Nifurtimox was purchased from Bayer (Munich, Germany) and benznidazole was from Roche (Basel, Switzerland). NPOx and Et-NPOx were synthesized according to previously reported methods . All of the chemicals used in the current study were of the highest purity available.
Synthesis of the benzyl ester of N-propyl oxamate (B-NPOx)
A solution of propylamine (0.1 mol) in ether (50 mL) was added in a dropwise manner to an ice-cold solution of dibenzyl oxalate (0.1 mol) in a 4:1 (v/v) mixture of diethyl ether and chloroform (250 mL), and the resulting mixture was stirred for 2 h at 5°C. The reaction mixture was then warmed to room temperature and stirred overnight. The benzyl alcohol produced during the reaction was subsequently removed by distillation at 50–60°C/1 mmHg to give a cloudy residue, which was dissolved in diethyl ether (10 mL) and held for 12 h at 5°C. A crystalline product formed with a melting point of 64–65°C, which was collected by filtration. The filtrate was evaporated to dryness under reduced pressure and then fractionated under vacuum. B-NPOx distilled at 60–65°C/1 mmHg as a colorless oil, which crystallized on standing. The crystals were purified by recrystallization from chloroform to give the desired product in an overall yield of 86% with a melting point of 50–55°C. The final product was analyzed by IR, 1H-NMR, 13C-NMR and HRMS. IR KBr (Perkin Elmer GX spectrometer; Perkin Elmer, Waltham, MA, USA): 3292 (N-H), 3036, 2966 (C-H, aromatic), 1785 (C = O), 1514 (C = C), 1377 (C-H methyl), 1222 (C-N-C), 898 (C-O-C), 765, 895 (aromatic, monosubstituted) cm-1. 1H-NMR (Jeol JNM-GSX270 spectrometer; Jeol, Pleasanton, CA, USA): (270 MHz, CDCl3) δ 0.91 (t, J = 7.4 Hz, 3H, CH3), 1.53 (s, 2H, CH2), 3.25 (q, J = 7.2 Hz, 2H, CH2), 5.29 (s, 2H, CH2), 7.1 (bs, 1H, NH), 7.32–7.37 (m, 5H, H phenyl). 13C-NMR (Jeol JNM-GSX270): (67.5 MHz, CDCl3) δ 11.37 (CH3), 22.46 (CH2Me), 41.69 (CH2-NH), 68.64 (CH2-O), 128.72 (Co,m phenyl), 128.87 (Cp phenyl), 134.47 (Cipso phenyl), 156.38 (CO-NH), 160.75 (COO). HRMS (Bruker micrOTOF-Q II 10392 spectrometer; Bruker, BioSpin Co. Billerica, MA, USA): Calcd for C12H15NO3: m/z = 221.2524 [M]+; found m/z = 221.1085 [M]+. These results confirmed that our synthesis of B-NPOx was successful.
Determination of the cytotoxicity of NPOx, Et-NPOx and B-NPOx
The cytotoxic activities of NPOx, Et-NPOx and B-NPOx were evaluated against monkey kidney epithelial cells (Vero cell line) using an alamar blue (Invitrogen, Life Technologies, Sacramento, CA. USA) viability assay. The cells were cultured in standard culture medium (MEM with 10% fetal bovine serum) in 24-well plates (7.5 × 103 cells/well) to 80% confluence. Increasing concentrations of NPOx, Et-NPOx and B-NPOx (0.1–3 mM) in 5 μL of ethyl alcohol were added to each well. The medium was replaced after 24, 48 and 72 h of incubation (37°C, 5% CO2 and 95% relative humidity) with 200 μL of fresh medium containing alamar blue, and the fluorescence of each well was measured using a Perkin Elmer LS 55 spectrofluorometer (Perking Elmer, Waltham, MA, USA). Cell viability (%) was measured relative to the control wells (cultured in medium alone) according to the following calculation: Cell viability (%) = (fluorescencetest × 100%)/fluorescencecontrol.
Determination of the toxicity of Et-NPOx and B-NPOx on mice
The median lethal dose (LD50) values of Et-NPOx and B-NPOx were determined in NIH male albino mice (18–20 g), according to the established requirements . Et-NPOx and B-NPOx were orally administered as solutions in a 5% Arabic gum in water at doses of 1.5–3.0 g/kg of body weight. The drugs were administered on a daily basis for 50 days.
Trypanosoma cruzi strains
The T. cruzi strains used in the current study were isolated from triatomines and chagasic patients. The Parra and Compostela strains of T. cruzi were isolated from Triatoma longipennis. The Nayarit strain of T. cruzi was isolated from Triatoma picturata, and the NINOA (MHOM/MX/1994/Ninoa) and INC-5 (MHOM/MX/1994/INC-5) strains were isolated from chronic chagasic patients living in two endemic areas of Mexico . The NINOA and INC-5 strains correspond to the prevalent discrete typing unit (DTU) classification designated as TcI, and it is also likely that the Parra, Compostela and Nayarit strains belong to the same TcI classification  as the other strains of Mexican T. cruzi characterized to date . Following their isolation, the strains were maintained in triatomine bugs (Meccus longipennis) and by serial passages in NIH male albino mice .
Culture of Trypanosoma cruzi epimastigotes
Cardiac blood samples from NIH male albino mice that had been individually infected with the different T. cruzi strains were cultured at 28°C in liver infusion tryptose (LIT) broth supplemented with 10% heat-inactivated fetal calf serum . When the growth of the epimastigotes became exponential, they were collected from the liquid phase by centrifugation at 3000 × g for 15 min. The resulting pellet was then washed three times with 20 volumes (with respect to the pellet) of buffer containing 0.1 M sodium phosphate (NaH2PO4/Na2HPO4) and 0.15 M NaCl (pH 7.4) and resuspended in the same buffer at 1 × 106 epimastigotes/mL. This suspension was then aliquoted (1 mL) in Eppendorf tubes. All of these procedures were carried out at 4°C.
Trypanosoma cruzi homogenates and enzyme preparations
One milliliter samples of the T. cruzi suspensions described above were subjected to three cycles of freezing in liquid nitrogen and thawing at room temperature. The disruption of the parasites was monitored by the microscopic examination of the resulting homogenate, which was frozen at –20°C until it was required for the experiments. There was no discernible decrease in the HADH-isozyme II activity of the homogenates after 2 months in storage at –20°C. The homogenates were thawed at room temperature immediately prior to be used in the enzymatic assays, and centrifuged at 1200 × g for 20 min at 4°C. The supernatant of each homogenate (enzyme preparation) was used to analyze the HADH-isozyme II activity. HADH-isozyme II was purified as previously described . The homogenates were prepared without any protease inhibitor, which could inhibit the aliphatic and aromatic carboxyl esterase activities described in T. cruzi [30,31].
Quantification of α-hydroxy acid dehydrogenase isozyme II activity
HADH-isozyme II activity was measured spectrophotometrically by following the oxidation of NADH (λ = 340 nm, ε = 6220 M-1 cm-1) with α-ketoisocaproate as a substrate . Each assay mixture contained, in a final volume of 3 mL, 0.12 mM NADH, 0.1 M sodium phosphate (NaH2PO4/Na2HPO4) plus 0.15 M NaCl (pH 7.4) buffer, 5 mM neutral sodium salt of α-ketoisocaproate, and the purified enzyme or an enzyme preparation from the homogenate of each one of the five T. cruzi strains. The purified enzyme or enzyme preparation was diluted with the 0.1 M sodium phosphate plus 0.15 M NaCl (pH 7.4) buffer prior to being used to allow for a shift in the absorbance at 340 nm of 0.05–0.08 nm/min with 5 mM α-ketoisocaproate. The assay mixtures were incubated at 37°C either alone or in the presence of NPOx, Et-NPOx or B-NPOx, and changes in the absorption at 340 nm were recorded over a 5 min period. In some cases, 1 mM of N-ethylmaleimide, which is a known inhibitor of T. cruzi carboxylesterases , was added to the assay mixtures. The activity of HADH-isozyme II was subsequently expressed as the percentage of ΔE 340 nm/min, where the velocity in the absence of an inhibitor was considered to be 100%. The results were then plotted against the concentrations of NPOx, Et-NPOx or B-NPOx (0.05–1 mM).
Trypanocidal activity of B-NPOx towards cultured epimastigotes
Epimastigotes (1 × 106 units/mL) of the five T. cruzi strains in the 0.1 M sodium phosphate plus 0.15 M NaCl (pH 7.4) buffer were used to evaluate the trypanocidal activity of B-NPOx. Nine hundred and eighty microliters of the epimastigote suspension was placed in an Eppendorf tube, followed by 20 μL of ethyl alcohol or benzyl alcohol or an ethyl alcohol solution of NPOx, Et-NPOx, B-NPOx, Nx or Bz, at a final concentration in the range of 0.1–3 mM. After being incubated at 28°C for 10, 20, 30, 40, 50 and 60 min, the number of live parasites was determined in a Neubauer haemocytometer  using the trypan blue dye exclusion method. The mean number of live epimastigotes in the control group, which was not subjected to any drug treatment, was taken as 100%. The results were subsequently given as the mean values ± the standard deviations of three independent experiments for the viability of the epimastigotes .
To measure the penetration of B-NPOx into the epimastigotes, a suspension of epimastigotes (1 × 106 units/mL) in 980 μL of the 0.1 M sodium phosphate plus 0.15 M NaCl (pH 7.4) buffer was mixed with 20 μL of the 1 mM solution of B-NPOx in ethyl alcohol. After being incubated at 28°C for 15, 30 and 45 min, the samples were centrifuged at 3000 × g and the supernatants were analyzed spectrophotometrically at 248 nm, which corresponds to the absorbance peak of B-NPOx. A decrease in the absorbance of the supernatants at 248 nm correlated well with the penetration of B-NPOx into the epimastigotes.
Trypanocidal activity of B-NPOx on bloodstream trypomastigotes
Bloodstream trypomastigotes of each one of the five T. cruzi strains were obtained by cardiac puncture from NIH male albino mice at the peak of parasitaemia. One hundred and ninety-five microlitre aliquots of blood containing 1 × 106 trypomastigotes were added to each well of a sterile 96-well plate, followed by 5 μL of NPOx, Et-NPOx, B-NPOx, Nx or Bz at final concentrations in the range of 0.1–3 mM. Stock solutions of NPOx, Et-NPOx, B-NPOx and the reference drugs in DMSO were prepared and subsequently diluted with sterile distilled water to give a final DMSO concentration of 1%. Blood containing 1 × 106 trypomastigotes with 1% DMSO was used as a control. The plates were incubated at 4°C for 24 h and the trypanocidal effects of the compounds were determined by comparing the motilities of the trypomastigotes at the different concentrations with the control. The results were subsequently presented as the mean values ± the standard deviations of the number of mobile (viable) trypomastigotes from three independent experiments.
Effect of B-NPOx on the parasitaemia and amastigote nests in infected mice
NIH male albino mice (18–20 g) were inoculated intraperitoneally with 1 × 103 bloodstream trypomastigotes of each of the five different T. cruzi strains. The mice were divided in five groups (10 mice per group), including (1) those without any treatment (control group, infected/untreated), and those treated with (2) Bz, (3) Nx, (4) Et-NPOx or (5) B-NPOx. The four test compounds were orally administered as solutions in 5% Arabic gum in water at a dose of 100 mg/Kg  per day over a period of 50 days. The first dose was given 24 h after the infection. Levels of parasitemia, beginning 24 h after infection, were determined every other day in a Neubauer hemocytometer using 5 μL of blood, which was collected from the tail vein of the infected mice and diluted at a ratio of 1:10 (v/v) with a saturated solution of ammonium chloride . The reduction in the parasitemia was evaluated by comparing the number of trypomastigotes obtained at each time point after the administration of NPOx, Et-NPOx, B-NPOx, Bz or Nx with the number of trypomastigotes obtained at the same point in infected/untreated mice, which was taken as 100% . The experimental protocols for animal care and use were reviewed and approved by the Bioethics Committee of our Institution according to the “Guide for the care and use of laboratory animals”, which was published by the US National Institute of Health .
To evaluate the activity of B-NPOx towards amastigote nests, we obtained tissue samples from the hearts and left legs (skeletal muscle) of mice from each of the five groups described above either 30 or 50 days after infection. The tissue samples were fixed with formaldehyde, dehydrated and embedded in paraffin. Sections of the samples (3 μm thick) were then stained with hematoxylin and eosin and analyzed by light microscopy. Fifty randomly selected microscopic fields from at least three mice were examined to quantify the number of amastigote nests. The mean number of amastigote nests in the infected/untreated group was taken as 100% .