Synthesis and Evaluation of Antiplasmodial Activities of Fluorinated 6-Amino-2-Aryl-3H-Indolone-N-Oxides

A series of novel 6-amino-2-aryl-3H-indolone-N-oxides were synthesized at yields of up to 65% and characterized; one was further characterized using X-ray crystallographic analysis. Synthesized compounds were evaluated for their in vitro activity against a chloroquine-resistant (FcB1) strain of Plasmodium falciparum, as well as for the 50% cytotoxic concentration (CC50) on Vero cell lines. The most promising activities were observed for the fluorinated compounds, the most active in vitro being 6-(2-morpholinoacetamido)-2-(4-(trifluoromethoxy) phenyl)-3H-indolone-N-oxide (IC50: 15.5 nM). In addition, these compounds showed weak cytotoxicity leading to selectivity index values of >170, thus warranting further in vitro and in vivo studies.


Introduction
Many organofluorine compounds have been approved by the US Food and Drug Administration (FDA) for medical use, which clearly demonstrates the importance of fluorine in medicinal chemistry [1][2][3][4]; some of these compounds have shown particular efficacy as antimalarial agents (such as mefloquine hydrochloride) ( Figure 1). In the design and development of biologically active compounds, substitution of fluorine for hydrogen and hydroxy group often introduces beneficial properties such as good lipophilicity, metabolic stability, increased binding to target molecules and membrane permeability [5][6][7].
Over the past few years, we have designed and synthesized a series of 2-aryl-3H-indolone-N-oxides (INODs) (Figure 1) that has antiplasmodial properties targeting the asexual blood stage of Plasmodium falciparum, with IC 50 values in the 1 to 100 nM range [8]. In addition to being active at nM concentrations, these compounds have non-toxic and non-hemolytic properties in healthy and parasitized erythrocytes [8] and are bioreducible [9]. The preparation of a large panel of compounds with different oxidation states [10] showed that only the compounds displaying the reducible N=C bond together with a pseudo-quinoid structure were active.
The bioreduction of these INODs produces free radicals [11][12][13]. With further studies on parasitized red blood cells (RBCs), we showed that these free radicals activate a SYK kinase cascade, which induces hyper-phosphorylation of the major protein in RBCs, band 3 anion transport protein [14], which, in the case of parasitized RBCs, causes an oxidative burst that is fatal to the parasite. We have recently shown that the anti-malarial activity of INODs was linked to their ability to serve as quinone reductase 2 substrates [13]. However, these molecules present two disadvantages: a poor water solubility and a rapid metabolization. To overcome their low aqueous solubility in vivo, INODs were formulated as albumin-based nanoparticles, and these strongly inhibited parasitemia in a mouse model infected with Plasmodium berghei (99.1% inhibition) or in humanized mice parasitized with P. falciparum (99.6% inhibition) [15]. Although the strong antimalarial activities have been shown in vivo, the solubility and metabolic stability of these compounds needs to be improved.
Successive structural modifications showed that the groups R 1 or R 2 at the 5-or 6-position had no major effect on the activity, whereas variation of R 3 at the para-position of the aryl group allowed for the most promising activities. The inclusion of a chloro group at the R 3 para-position provided the most active compound in vivo ( Figure 1, Lead 1). On the other hand, introduction of the dimethylamino group at this position gave the most active compound in vitro ( Figure 1, Hit 1); it had better water solubility and good antiplasmodial activity in vitro (CI 50 <3 nM with the chloroquine-resistant FcB1 strain of P. falciparum) but poor inhibition of parasitemia in vivo (15.3%) ( Figure  1). We therefore synthesized new fluorinated INODs with morpholino or piperidine groups in the C-6 position of the INOD scaffold to obtain better water solubility while keeping the antiplasmodial activity. The amino groups were linked to different fluorinated INODs via ether, ester or amide linkage.
used without further purification. Melting points were determined with an Electrothermal 9300 capillary melting point apparatus and are uncorrected. IR spectra were recorded on a Perkin-Elmer PARAGON 1000 FT-IR spectrometer. 1 H and 13 C NMR spectra were recorded on an AC Bruker spectrometer at 300 MHz ( 1 H) and 75 MHz ( 13 C) using (CD 3 ) 2 SO and CDCl 3 as solvents. HRMS were recorded on a Thermofisher scientific LTQ-Orbitrap-XL-ETD spectrometer (PHARMA-DEV, Toulouse, France). Silica Gel 60 (Merck 70-230) was used for column chromatography. All reactions were monitored by TLC aluminum/silica gel plate with UV-light visualization. Compound purity was determined by HPLC-UV and LC-PDA-MS methods and was ranged 96-99%.

Synthesis of o-iodonitroaryls c-i: See Supporting Information.
General procedure for the preparation of 6-(2-amino(ethoxy, acetamido or acetoxy)-2-arylindolone-N-oxides 9-20: Pd(PPh 3 ) 2 Cl 2 (0.1 mmol) was added to a solution of a commercial alkyne (1 mmol) and 2-iodonitroaryl c-i (1 mmol) in freshly distilled triethylamine (20 mL) and DMF (10 mL), and the mixture was de-aerated with argon for 30 min. CuI (0.05 mmol) was added, and the mixture was de-aerated with argon for 10 min and stirred at room temperature for 6 h. The solution was then diluted with ethyl acetate (50 mL) and washed with water (3 × 50 mL). The organic phase was separated, washed with brine, dried over Na 2 SO 4 , and solvents were evaporated under reduced pressure. The residue obtained was added to a solution of Pd(CH 3 CN) 2 Cl 2 (0.05 mmol) in CH 3 CN (15 mL), and the mixture was refluxed for 3 h in an argon atmosphere. The reaction mixture was concentrated, and the residue obtained was purified by column chromatography (ethyl acetate in petroleum ether).  10

Crystallography
The single crystal of compound 14 was obtained in m-xylene solution. Data were collected at low temperature (100 K) on a Bruker Kappa Apex II diffractometer using a Mo-Kα radiation (λ=0.71073 Å) micro-source equipped with an Oxford Cryosystems Cryostream Cooler Device. The structure was solved by Direct Methods using SHELXS97 [16] and refined by means of least-squares procedures using Crystal software package [17]. The Atomic Scattering Factors were taken from International tables for X-Ray Crystallography [18]. All hydrogen atoms were placed geometrically and refined using a riding model. All non-hydrogen atoms were anisotropically refined. Molecule drawings were performed with the ORTEP 32 program [19] with 30% probability displacement ellipsoids for non-hydrogen atoms. The crystal structures have been deposited at the Cambridge Crystallographic Data Centre and allocated the deposition number: CCDC 1557289.  2-(4-Fluorophenyl)-6-(2-morpholinoacetamido)-3-oxo-3H

2-(4-Chlorophenyl)-6-(2-morpholinoacetoxy)-3-oxo-3H-indole
France) were extensively washed with cold RPMI medium to remove remaining plasma and leukocytes. Parasitized RBCs were maintained in 25 cm 2 culture flasks in a controlled atmosphere and synchronized by ᴅ-sorbitol lysis. As control, stock solution of chloroquine (CQ, Sigma (refC6628) was dissolved in culture medium (stock solution: 1 mg/mL). For the drug assays, serial drug dilutions in triplicates (100 µL/well) were made in RPMI medium (final concentration of DMSO is 0.5%) and were added to a P. falciparum culture media (2% hematocrit, 1% parasitemia, 100 µL/well) in a 96-well culture plate. 48 h after the beginning of incubation, plates were washed three times with 150 µL of PBS and 100 μl of a lysis buffer containing 2 × SG (sybr green) I was added to 100 μL of each dilution of the parasitized RBCs + drugs in a black 96-well plate and incubated in the dark at room temperature for 2 h. Subsequently, the fluorescence values were determined using a fluorescence plate reader at 485 nm and 518 nm excitation and emission wavelengths, respectively. The fluorescence (after subtraction of the background fluorescence for nonparasitized RBCs) values were plotted, and the IC 50 values were determined by linear regression analysis.

In vitro cytotoxicity assay: cytotoxicity against the Vero cell line:
The evaluation of the tested molecules cytotoxicity by MTT assay on the Vero cell line was performed according to the following protocol: in brief, cells (1 × 10 4 cells/mL) in 100 μL of complete medium, (MEM supplemented with 10% fetal calf serum (FCS), 2 mM l-glutamine and NEAA 1X) were seeded into each well of 96-well plates and incubated at 37°C and 5% CO 2 . After 24 h incubation, 100 µL of medium with various product concentrations and appropriate controls (DMSO) were added, and the plates were incubated for 72 h at 37°C and 5% CO 2 . Each plate-well was then microscope-examined to detect possible precipitate formation before the medium was pipetted from the wells. 100 μL of MTT solution (0.5 mg/mL in MEM) were then added to each well. Cells were incubated for 2 h at 37°C and 5% CO 2 . The MTT solution was then removed, and DMSO (100 µL/well) 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). CC 50 were calculated by non-linear regression analysis processed on dose-response curves, using GraphPad Prism ® V7 software. CC 50 values represent the mean value calculated from three independent experiments.

Selectivity index (SI):
The selectivity indices presented correspond to the ratios between, respectively, the toxicity on Vero cell line (or MCF-7 line) and the FcB1 antiplasmodial activity. They are calculated as follows: SI=CC 50 (Vero)/IC 50 (FcB1).

Chemistry
The synthetic pathway for 6-amino-2-arylindolone-N-oxides 9-20 is illustrated in Schemes 1 and 2. O-Alkylation of 4-iodo-3-nitrophenol a with 1,2-dibromoethane in refluxing ethanol, in the presence of K 2 CO 3 , produced the bromo ether c [20]. Treatment of bromoacetyl bromide with 4-iodo-3-nitrophenol a or 4-iodo-3-nitrophenylamine b in dichloromethane at -78°C, in the presence of triethylamine, produced the bromo ester and the bromo amide d and e, respectively [21]. The NaI mediated reaction of bromo intermediates c-e gave the tertiary amino compounds f-i after treatment with morpholine or piperidine in the presence of K 2 CO 3 as a base [20]. The synthesis of the indolone-N-oxide derivatives 9-20 is divided into two sub-steps following a previously reported method [22,23].

Biology
The series was evaluated for antiplasmodial activity against the P. falciparum FcB1 through in vitro red blood cell-based culture using a SYBR-Green-I assay. A summary of these assay results is presented in Table 1. New fluorinated indolone-N-oxides were active against P. falciparum FcB1 with IC 50 values of <75 nM. The non-substituted compound 1 (2-phenylindolone-N-oxide; R 1 =R 2 =R 3 =H) had the lowest antiplasmodial activity (IC 50 =889 nM). Replacement of R 2 (H) by a trifluoromethyl or trifluoromethoxy group improved the activity (IC 50 : 2=186 nM, 3=135 nM, 4=133 nM). Replacement of R 3 (H) by a trifluoromethyl, trifluoromethoxy or fluoro group conferred good activity (IC 50 : 5=50 nM, 6=43 nM, 7=20 nM, 8=120 nM). In compounds 9-16, the introduction of morpholino or piperidinyl group at R 2 (the amino groups were linked to different INODs via ether, ester or amide linkage) and a fluoro or trifluoromethoxy group at R 3 led to the best antiplasmodial activities in this series with IC 50 values ranging from 15.5 to 61.5 nM. Activity decreased when the fluoro group was replaced by a chloro group (compounds 9, 11, 13 and 15 versus 17, 18, 19 and 20, respectively). Compounds with amino-amide-INOD structures (the amino groups being morpholino or piperidine) were found to be more active than their ether and ester linked counterparts (IC 50 : 13=39.1 nM, 14=15.5 nM, 15=26.2 nM). The morpholino-linker- INODs (compounds 13, 14, and 19) and the piperidine-linker- INODs  (compounds 15, 16, and 20) exhibited almost identical activity against P. falciparum FcB1, which indicates that the morpholino and piperidine amino groups do not have a notable impact on the antiplasmodial activity.
All the synthesized compounds were further evaluated by MTT assay for cytotoxicity against MCF7 or Vero cell lines (Table 1). All the compounds evaluated showed good selectivity index in the range of 16-720. Compounds 9-20 were the most potent against malarial parasites and also exhibited high selectivity index in the range of 177-720.
synthesized and evaluated for their antimalarial activity. The in vitro evaluation of these compounds against P. falciparum FcB1 displayed activity in the nM range. Fluorinated amino-INODs 9-16 exhibited very good antiplasmodial activity and low toxicity against the Vero cell lines. In addition, these fluorinated-INODs exhibited high selectivity indices.