New 8-Nitroquinolinone Derivative Displaying Submicromolar in Vitro Activities against Both Trypanosoma brucei and cruzi

An antikinetoplastid pharmacomodulation study was conducted at position 6 of the 8-nitroquinolin-2(1H)-one pharmacophore. Fifteen new derivatives were synthesized and evaluated in vitro against L. infantum, T. brucei brucei, and T. cruzi, in parallel with a cytotoxicity assay on the human HepG2 cell line. A potent and selective 6-bromo-substituted antitrypanosomal derivative 12 was revealed, presenting EC50 values of 12 and 500 nM on T. b. brucei trypomastigotes and T. cruzi amastigotes respectively, in comparison with four reference drugs (30 nM ≤ EC50 ≤ 13 μM). Moreover, compound 12 was not genotoxic in the comet assay and showed high in vitro microsomal stability (half life >40 min) as well as favorable pharmacokinetic behavior in the mouse after oral administration. Finally, molecule 12 (E° = −0.37 V/NHE) was shown to be bioactivated by type 1 nitroreductases, in both Leishmania and Trypanosoma, and appears to be a good candidate to search for novel antitrypanosomal lead compounds.

water, neutralized with HCl and extracted three times with dichloromethane (3 × 100 mL). The resulting organic layer was washed with brine, dried over anhydrous Na 2 SO 4 and evaporated in vacuo to afford a dark oil. In a second step, the crude residue was reacted with 98% sulfuric acid (40 mL) and stirred at rt for 2 h (monitored by TLC). The reaction mixture was then poured into ice, neutralized with K 2 CO 3 and extracted twice with dichloromethane (2 × 100 mL) and once with ethyl acetate (100 mL). The resulting organic layer was washed with water, dried over anhydrous Na 2 SO 4 and evaporated in vacuo. The crude residue was purified by chromatography on silica gel using ethyl acetate as an eluent. Compound 1 was isolated and recrystallized in isopropanol to yield a white solid (42%, 11.68 mmol, 2.5 g). Preparation of 8-nitro-6-trifluoromethylquinolin-2(1H)-one 2 H 2 SO 4 (98%, 10 mL) were added onto 500 mg of 6-trifluoromethylquinolin-2(1H)-one 1 (2.34 mmol, 1 equiv.), cooled with an ice bath. 0.96 mL of 65% HNO 3 (14.1 mmol, 5 equiv.) were then added dropwise at 0°C and the reaction mixture was stirred at rt for 2 h. The reaction mixture was successively poured into ice, neutralized with K 2 CO 3 , extracted three times with dichloromethane (3 × 50 mL) and once with ethyl acetate (50 mL). The organic layer was washed with water, dried over anhydrous MgSO 4 and evaporated in vacuo. The crude residue was purified by chromatography on silica gel using ethyl acetate as an eluent. Compound 2 was isolated and recrystallized in acetonitrile to yield a pale yellow solid (89%, 2.1 mmol, 540 mg). Preparation of 3-chloro-6-trifluoromethylquinolin-2(1H)-one 3 A solution of 15 mL of hydrochloric acid (37%) was added onto 500 mg of 6-trifluoromethylquinolin-2(1H)-one 1 (2.35 mmol, 1 equiv.). The reaction mixture was then stirred at 100 °C before 300 mg of sodium chlorate (2.8 mmol, 1.2 equiv.) were added with caution (Cl 2 formation). After 7 h under reflux, the reaction mixture was left for 1 h under the hood to evacuate remaining Cl 2 vapor, and then was poured into ice, neutralized with K 2 CO 3 , extracted three times with dichloromethane (3 × 50 mL) and once with ethyl acetate (50 mL). The organic layer was washed with water, dried over anhydrous MgSO 4 and evaporated in vacuo. The crude residue was purified by chromatography on silica gel using dichloromethane/ethyl acetate (90/10) as an eluent. Compound 3 was isolated and recrystallized in isopropanol to yield a white solid (48%, 1.1 mmol, 280 mg). Preparation of 3-bromo-6-trifluoromethylquinolin-2(1H)-one 4 6-Trifluoromethylquinolin-2(1H)-one 1 (1.87 mmol, 400 mg) and 835 mg of N-bromosuccinimide (4.69 mmol, 2.5 equiv.) were added in a sealed flask of 25 mL. Then, 10 mL of acetonitrile were added and the reaction mixture was heated at 140 °C in a microwave reactor during 2 hours. The reaction mixture was poured into water and extracted three times with dichloromethane (3 × 50 mL). The organic layer was washed twice with a sodium carbonate solution (pH = 8) and twice with a diluted hydrochloric acid solution (pH=4), dried over anhydrous Na 2 SO 4 and evaporated in vacuo. The crude residue was purified by chromatography on silica gel using dichloromethane/ethyl acetate (75/25) as an eluent. Compound 4 was isolated and recrystallized in acetonitrile to yield a white solid (54%, 1.0 mmol, 293 mg).

S5
Compound 4 (C 10 13  Under argon atmosphere, 250 mg of 8-nitro-6-trifluoromethylquinolin-2(1H)-one 2 (0.97 mmol, 1 equiv.) were solubilized in 5 mL of dry DMF and were added onto a DMF solution (5 mL) of 77 mg of 60% sodium hydride (1.93 mmol, 2 equiv.). After 10 min of stirring at rt, 120 µL of methyl iodide (1.93 mmol, 2 equiv.) were added dropwise. The reaction mixture was stirred at rt during 3 h, before being poured into ice and extracted four times with ethyl acetate (4 × 25 mL). The organic layer was washed twice with water, once with brine, dried over anhydrous MgSO 4 and evaporated in vacuo. The crude residue was purified by chromatography on silica gel using dichloromethane as eluent. Compound 5 was isolated and recrystallized in isopropanol to yield a white solid (69%, 0.66 mmol, 181 mg).  Preparation of 3-chloro-8-nitro-6-trifluoromethylquinolin-2(1H)-one 6 A solution of 25 mL of hydrochloric acid (37%) was added onto 150 mg of 6-trifluoromethyl-8nitroquinolin-2(1H)-one 2 (0.58 mmol, 1 equiv.). The reaction mixture was then stirred at 100 °C before 186 mg of sodium chlorate (1.74 mmol, 3 equiv.) were added with caution (Cl 2 formation). After 2 h under reflux, the reaction mixture was left for 1 h under the hood to evacuate remaining Cl 2 vapor, and then was poured into ice, neutralized with K 2 CO 3 , extracted three times with dichloromethane (3 × 25 S6 mL) and once with ethyl acetate (25 mL). The organic layer was washed with water, dried over anhydrous MgSO 4 and evaporated in vacuo. The crude residue was isolated and recrystallized in isopropanol to yield a yellow solid (88%, 0.51 mmol, 150 mg). A solution of 20 mL of hydrobromic acid (48%) was added onto 350 mg of 6-trifluoromethyl-8nitroquinolin-2(1H)-one 2 (1.36 mmol, 1 equiv.). The reaction mixture was then stirred at 100 °C before 345 mg of sodium bromate (4.07 mmol, 3 equiv.) were added with caution (Br 2 formation). After 3 h under reflux, the reaction mixture was left for 1 h under the hood to evacuate remaining Br 2 vapor, and then was poured into ice, neutralized with K 2 CO 3 and extracted three times with dichloromethane (3 × 50 mL). The organic layer was washed with water, dried over anhydrous Na 2 SO 4 and evaporated in vacuo. The crude residue was purified by chromatography on silica gel using dichloromethane as an eluent. Compound 7 was isolated and recrystallized in acetonitrile to yield a pale yellow solid (94%, 1.27 mmol, 430 mg).

Preparation of 6-bromo-3-chloro-8-nitroquinolin-2(1H)-one 12
A solution of 20 mL of hydrobromic acid (48%) was added onto 250 mg of 3-chloro-8-nitroquinolin-2(1H)-one (1.11 mmol, 1 equiv.). The reaction mixture was then stirred at 100°C before 700 mg of sodium bromate (4.4 mmol, 4 equiv.) were added with caution (Br 2 formation). After 24 h under reflux, the reaction mixture was left for 1 h under the hood to evacuate remaining Br 2 vapor, and then was poured into ice, neutralized with K 2 CO 3 and extracted three times with dichloromethane (3 × 50 mL). The organic layer S9 was washed with water, dried over anhydrous MgSO 4 and evaporated in vacuo. The crude residue was purified by chromatography on silica gel using dichloromethane as an eluent. Compound 12 was isolated and recrystallized in isopropanol to yield a yellow solid (79%, 0.88 mmol, 266 mg).
Cell lines and culture conditions: The clonal Leishmania donovani cell line LdBOB (derived from MHOM/SD/62/1S-CL2D) was grown as promastigotes at 26 °C in modified M199 media, as previously described. [2] LdBOB promastigotes overexpressing NTR1 (LinJ.05.0660) [3] and NTR2 (LinJ.12.0730) [4] were grown in the presence of nourseothricin (100 µg/mL). To examine the effects of test compounds on growth, triplicate promastigote cultures were seeded with 5 × 10 4 parasites/mL. Parasites were grown in 10 ml cultures in the presence of drug for 72 h, after which 200 μL aliquots of each culture were added to 96-well plates, 50 μM resazurin was added to each well and fluorescence (excitation of 528 nm and emission of 590 nm) measured after a further 4 h incubation [5] . Data were processed using Räz et al. [7] After a 69 h incubation period at 37 °C, 10 µL of Alamar Blue ® was then added to each well, and the plates were incubated for 5h. [8] The plates were read in a PerkinElmer ENSPIRE (Germany) microplate reader using an excitation wavelength of 530 nm and an emission wavelength of 590 nm. EC 50 were calculated by nonlinear regression analysis processed on dose-response curves, using GraphPad Prism software (USA). EC 50 was defined as the concentration of drug necessary to inhibit by 50% the viability of T. brucei brucei compared to the control. EC 50 values were calculated from three independent experiments in duplicate. Then, sterile, 06-well plates were seeded with exponentially growing Vero cells (40,000 cells per cm 2 in 2, 2 mL RPMI with serum per well) harvested from the preceding subcultures, were added to each well. After incubation at 37°C for 2 days in 5% CO 2 in air, the cells were infected with T. cruzi trypomastigotes in ratio 3:1 (parasites : host cells). After 24 hours, the non-infecting trypomastigotes removed by washing twice with HBSS buffer (without Ca 2+ and Mg 2+ ) and the chemical compounds in completed RPMI media were added immediately, to be tested to their inhibitory effects on parasite growth and development. Culture plates were incubated for an additional 120 h at 37°C with 5% CO 2 .Three replicate wells for each condition were done. On days 5 and 6 post-infection, trypomastigotes were released from the cells. On day 6, the culture medium was removed and transferred to a centrifuge tube. Attached infected cells were washed with 5 mL of HBSS buffer. The culture medium and wash containing trypomastigotes were mixed and centrifuged at 1000 g for 15 min at room temperature.

Vero cells (normal kidney epithelial cells of Cercopithecus aethiops) were obtained from the
Subsequently, trypomastigotes re-suspended in 2 mL and counted in a haemocytometer (Kova cells) using a light microscope. For 50% effective concentration (EC 50 ) determinations, compounds were serially diluted 2 to 4-fold in RPMI media, with final assay concentrations ranging from 0,1 to 25 µM.
The 50% inhibiting concentrations (EC 50 ), defined as the drug concentration that resulted in a 50% reduction of trypomastigotes compared to the non-treated controls was estimated by non-linear regression analysis. EC 50 values represent the mean value calculated from two independent experiments that were performed in triplicate.
Cultures were initiated with 1 × 10 5 cells ml −1 and sub-cultured when cell densities approached 1-2 (× 10 6 )/mL. In order to examine the effects of inhibitors on the growth of these parasites, triplicate cultures containing the inhibitor were seeded at 1 × 10 5 trypanosomes/mL. Cells overexpressing NTR were induced with tetracycline 48 h prior to EC 50 analysis. Cell densities were determined after culture for 72 h, as previously described [11] . EC 50 values were determined using GraFit as described above.

Cytotoxic evaluation on HepG2 cell line.
The evaluation of the molecules cytotoxicity on the HepG2 (hepatocarcinoma cell line from ECACC purchased from Sigma-Aldrich, ref 85011430-1VL certificated without mycoplasma) was done according to the method of Mosman with slight modifications. [12] Briefly, cells (1 × 10 5 cells/mL) in 100 µL of complete medium, [Alpha MEM Eagle from PAN BIOTECH supplemented with 10% foetal bovine serum, 2 mM L-glutamine and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin)] were seeded into each well of 96-well plates and incubated at 37 °C and 5% CO 2 . After a 24 h incubation, 100 µL of medium with various product concentrations and appropriate controls were added and the plates were incubated for 72 h at 37 °C and 5% CO 2 . Each plate-well was then microscope-examined for detecting possible precipitate formation before the medium was aspirated from the wells. A MTT solution (100 µL, 0.5 mg/mL in Alpha MEM Eagle) was then added to each well. Cells were incubated for 2 h at 37 °C and 5% CO 2 . After this time, the MTT solution was removed and DMSO (100 µL) was added to dissolve the resulting formazan crystals. Plates were shaken vigorously (300 rpm) for 5 min.
The absorbance was measured at 570 nm with a microplate spectrophotometer (Eon BioTek). DMSO was used as blank and doxorubicin (purchased from Sigma Aldrich) as positive control. CC 50 were S16 calculated by non-linear regression analysis processed on dose response curves, using TableCurve 2D V5 software. CC 50 values represent the mean value calculated from three independent experiments.

Comet assay
The alkaline comet assay was used to detect DNA strand breaks and alkali-labile sites. Trypsinized HepG2 cells were embedded in 0.7% low-melting point agarose (Sigma "Low Gelling Temperature") and laid on pre-cut sheets of polyester film (Gelbond® film) to perform minigel deposits as previously described [13] . Films were then placed in lysis solution (2.5 M NaCl, 0.1 M Na 2 EDTA, 10 mM Tris, 1 % Triton X-100, 10 % DMSO pH 10) for 18 h at 4 °C. Electrophoresis (with a solution which contained 0.3 M NaOH, 1 mM Na 2 EDTA, pH > 13) was processed for 24 min in a tank with a power supply giving 28 V (resulting in 0.8 V/cm). After electrophoresis, films were immersed 2 × 5 min in PBS for neutralization, followed by fixation in 100% ethanol for 1.5 h and drying. After staining with SYBR® Gold (Life Technologies) at 10 000 × dilution for 20 min, films were observed at 20x magnification with an epifluorescence microscope equipped with an automated platform (Nikon NiE) and coupled to a camera (DS-Q1Mc) and the software Nikon NiS Element Advanced Research to automatically capture images. In these images, for each cell, the level of DNA damage was evaluated using a semi-automated scoring system, by measurement of the intensity of all tail pixels divided by the total intensity of all pixels in head and tail of comet, by means of the software "Lucia comet assay" (Laboratory Imaging, Prague Czech Republic). Fifty cells per deposit and two deposits per sample were analyzed. The median from these 100 values was calculated and named "% tail DNA".
Parallel Artificial Membrane Permeability Assay (PAMPA) [14,15] The PAMPA-BBB experiments were conducted using the Pampa Explorer Kit (Pion Inc) according to . Where dose is the initial concentration of product in the sample, AUC ∞ is the area under the concentration-time curve extrapolated to infinity and [microsomes] is the microsome concentration expressed in mg/μL.

Plasma protein binding procedure.
The plasma doped with the tested compound is incubated at 37 °C in triplicate in one of the compartments of the insert, the other compartment containing a phosphate buffer solution at pH 7.2.
After stirring for 4 h at 300 rpm, a 25 μL aliquot of each compartment is taken and diluted; the dilution solution is adapted to obtain an identical matrix for all the compartments after dilution. In parallel, the reprocessing of a plasma doped but not incubated will allow to evaluate the recovery of the study. The LC-MS used for this study is a Waters® Acquity I-Class / Xevo TQD, equipped with a Waters® Acquity S19 BEH C18 column, 50 × 2.1 mm, 1.7 μm. The mobile phases are (A) ammonium acetate 10 mM and (B) acetonitrile with 0.1% formic acid. The injection volume is 1 µL and the flow rate is 600 µL/min. The chromatographic analysis, total duration of 4 min, is made with the following gradient: 0 < t < 0.2 min, 2% (B); 0.2 < t < 2 min, linear increase to 98% (B); 2 < t < 2.5 min, 98% (B); 2.5 < t < 2.6 min, linear decrease to 2% (B); 2.6 < t < 4 min, 2% (B). Carbamazepine, oxazepam, warfarine and diclofenac are used as reference drugs and Propranolol is used as internal standard. The unbound fraction (fu) is calculated according to the following formula: The percentage of recovery Four mice received 50 µL of the 12 suspension by oral route at 25 mg/kg (the tolerated dose). One hundred microliters of blood were taken from tail and collected into micro tubes with heparin at 0.5, 1, 3, 6, 9, 12, and 24h after administration. Blood samples were stored frozen until required for assay.

Sample preparation for spectrometry analysis
Megazol was used like the internal standard (IS). LC-MS Optima grade Acetonitrile (ACN) and Methanol (MeOH), acetic acid and formic acid (FA) were purchased from Fisher Scientific. Ready-touse QuEChERS salts (6 g MgSO4/1.5 g NaCl/1.5 g sodium citrate dihydrate/750mg sodium citrate sesquihydrate) were supplied by VWR.
samples. The mixture was vortexed during 30 sec. After 10 min, 40 mg of QuEChERS salts were added.
Samples were briefly vortexed and centrifuged at 16,000g for 10 min. Ten microliters of the upper layer was directly transferred in an injection vial before being diluted (1/10; v/v) in a 0.1 % formic acid in water. Finally, 5μL was injected in the LC-MS-MS system.
Calibrations standards (8 levels, from 10 to 1,000ng/ml) and quality controls (QC) (75 and 625 ng/ml) were obtained by adding appropriate 20X working standard solutions in blank whole blood. [17,18] LC-MS/MS conditions  Table 1. All parameters (collision energy, Q1/Q3 pre-bias) were optimized from standard flow injection analysis. Dwell time was set at 100ms per transition. Validation procedure for whole blood Validation protocol and the set of acceptance criteria were as follows: -Linearity: Calibration curve was generated by plotting the peak area ratios (analyte/internal standard) vs the expected concentration. Linearity of the calibration curve was evaluated by a quadratic regression analysis using a 1/x² weighting. A value greater than 0.99 was expected for the coefficient of determination (r 2 ).
-Precision and accuracy of the method were assessed at lower limit of quantitation (LLOQ; 10ng/ml) and at the two quality control concentrations (75 and 625 ng/ml). Precision is calculated as the coefficient of variation (CV%) within a single run (intra-assay; n=5) and between different assays (interassay; n=5), and accuracy is the percentage of deviation between nominal and found concentration with the established calibration curve. Acceptance criteria were intra-assay and inter-assay precision (CV%) and an accuracy (bias) less than 20 %.
-The lower limit of quantification (LLOQ) was estimated to be the minimal concentration with accuracy and precision within +/-20%. The lower limit of detection (LLOD) was calculated based on a signal-tonoise ratio >3.
with their extracted blank whole blood counterparts spiked at the correct concentration after extraction (n=3). CV% in the extraction recovery had to be less than 20%.
-The effect of dilution was investigated on samples spiked at 5-fold (5,000 ng/ml) and 20-fold (20,000 ng/ml) of ULOQ then re-analyzed after ten-and forty-fold dilutions. Precision CV and bias were set less than 20% to successfully validate.
-The absence of carryover was checked by injecting blank samples just after the analysis of the most concentrated sample (1,000 ng/ml).     Results are mean ± SD of three independent experiments. Statistical analysis was performed by one-way ANOVA followed by Dunnetts's multiple comparison for each time (***P < 0.001 versus control).

In vivo pharmacokinetic study
Following the developed extraction procedure, the preparation of a classical batch that includes 9 calibration standards, 2 internal quality controls, and 20 whole blood samples required less than 1.5 hour. The chromatographic separation of the compound and its internal standard was obtained in 8 min.
As presented in Figure S16, acceptance criteria were obtained for both the intra-assay and the interassay precision and accuracy. The CV% values in the extraction recovery were also less than 20%. Using quadratic regression with a 1/x² weighting, the coefficients of determination of the calibration curves between 10 and 1,000 ng/ml were higher than 0.99. According to these results, the LLOQ was considered to be 10 ng/ml and the LLOD was determined at 5 ng/ml. Figure S16: Main parameters of the validation protocol