Synthesis and trypanocidal activity of substituted 2,4-diarylquinoline derivatives

A small library of nine, novel 2,4-diarylquinoline derivatives has been prepared in high yield via convenient one-or two-step routes from a series of substituted 2-aminobenzophenones. None of the products exhibited toxicity at 20 μM against human cervix adenocarcinoma (HeLa) cells, while many of them exhibited encouraging trypanocidal activity against T. brucei brucei (a parasite responsible for African cattle trypanosomiasis) - some with IC 50 values in the range 2.8 – 6.2 μM


Results and Discussion
Access to both sets of 2,4-diarylquinoline derivatives 4a-g and 5h,i was achieved by adopting Friedländer-type methodology, involving the reaction of the common, 2-aminobenzophenones (6) with the variously substituted acetophenones (7) and (8), respectively (Scheme 1).Thus, in an approach similar to that used by Sankaran et al. 17 in their synthesis of biquinoline-and quinoline-bearing chromenes, equal equivalents of the appropriate acetophenones (7) and 2-aminobenzophenones (6) were reacted in refluxing acetic acid in the presence of a catalytic quantity of sulphuric acid to give the desired substituted 2,4-diarylquinolines 4a-g in yields ranging from 80 to 95% (Table 1).
Toxicity studies conducted against HeLa (human cervix adenocarcinoma) cells using a resazurin-based fluorescence assay, indicated that none of the synthesised compounds 4a-g and 5h,i showed any cytotoxicity at a concentration of 20 μM (Table 1 and Figure 2).On testing against T. brucei brucei, however, most of the compounds exhibited a measure of anti-trypanosomal activity -a number of them with very encouraging IC50 values (2.8 -6.2 μM; Table 1 and Figure 3).Scheme 1. Synthesis of 2,4-diarylquinolines 4a-g and 5h,i.
The bioassay data reveal a number of significant structure-activity relationship patterns.
i) The 2-[2-(furoylamino)phenyl]quinoline derivatives 5h and 5i both exhibited encouraging activity against T. brucei brucei at very low micromolar concentrations (IC50: 2.8 -4.5 μM), whereas only one of the 2-phenylquinoline analogues showed comparable activity (4b: IC50: 6.2 μM).ii) Compound 4b is the only 2-phenylquinoline derivative to contain a meta-trifluoromethyl group (R 3 ) on the D-ring; moving the trifluoromethyl group (R 3 ) to the para-position, as in compound 4a, decreases activity, increasing % T. brucei brucei viability from 6% for 4b to 52% for 4a.iii) Changing the B-ring substituent (R 2 ) from Cl in 4a to the strongly electron-withdrawing nitro group in compound 4c effectively nullifies anti-trypanosomal activity with % T. brucei brucei viability increasing from 52% for 4a to essentially 100% for 4c.iv) Compounds 4a, 4b, 4d and 4e, which contain the same B-ring substituent (R 2 = Cl) but differ in the nature of their D-ring substituents (R 4 = CF3, Cl and OCH3, respectively) all show activity against T. brucei brucei, but when R 4 is changed to Br, as in compound 4g, activity is lost altogether.

Conclusions
In conclusion, convenient synthetic access to library of substituted 2,4-diphenylquinolines has been established and, although the mode of action is not known at present, it is apparent that a number of these compounds exhibit very encouraging anti-trypanosomal activity.Interesting structure-activity relationships have been identified, and the 2-[2-(furoylamino)phenyl]quinoline derivatives 5h,i, in particular, are promising lead compounds for the development of novel trypanocidal agents.

Experimental Section
General.All chemicals were used as purchased from Sigma-Aldrich Chemical Co.Analytical thin layer chromatography (TLC) was performed using pre-coated silica gel plates.NMR spectra were recorded on Bruker 300, 400 and 600 MHz NMR spectrometers, chemical shifts were calibrated relative to the residual proton signal in DMSO-d6 (2.5 ppm) and the NMR spectra were analysed using Mestrenova.IR Spectra were recorded on a Perkin Elmer Spectrum 100 FTIR spectrometer with a diamond window.Melting points were determined using a hot-stage apparatus and are uncorrected.HRMS analyses were conducted at Rhodes University and by the Central Analytical Facilities Unit at the University of Stellenbosch.
Representative synthetic methods, full characterisation data for the other novel compounds, and Bioassay protocols are detailed below.Bioassay reports and NMR spectra are provided in the Supplementary Material file.

Bioassay protocols
Cytotoxicity determination.To assess the overt cytotoxicity of the compounds, HeLa (human cervix adenocarcinoma) cells cultured in DMEM medium containing 10% fetal bovine serum and antibiotics (penicillin, streptomycin, amphotericin B) in a 5% CO2 37°C incubator were plated in 96-well plates at a density of 2 x 10 4 cells per well.After an overnight incubation to allow cell adhesion, test compounds were added to a final concentration of 20 μM and incubation continued for 48 hours.Residual cell viability was determined by adding resazurin to a final concentration of 50 μM and measuring fluorescence (Ex560/Em590) in a Spectramax M3 plate reader (Molecular Devices) after a 4-hour incubation.Fluorescence readings were converted to % cell viability in compound-treated wells relative to untreated controls, after subtracting background readings obtained from wells without cells.Compounds were tested in duplicate wells.Emetine was used as a control drug standard.
Anti-trypanosomal assay.To assess trypanocidal activity, T.b. brucei (strain 427) trypomastigotes were seeded in 96-well plates at a density of 2.4 x 10 4 cells per well in medium consisting of IMDM containing 25 mM HEPES, 10% fetal bovine serum, 1 mM hypoxanthine, antibiotics (penicillin, streptomycin) and HMI-9 supplement.After an overnight incubation at 37°C in a 5% CO2 incubator, compounds were added to a final fixed concentration of 20 µM or as 3-fold serial dilutions, and incubation continued for 24 hours.Resazurin was added to a final concentration of 50 μM and, after an additional 24-hour incubation, fluorescence (Ex560/Em590) was measured in Spectramax M3 plate reader.Fluorescence readings were converted to % parasite viability in compound-treated wells relative to untreated controls, after subtracting background readings obtained from wells without cells.Compounds were tested in duplicate wells.Pentamidine was used a control drug standard

Figure 1 .
Figure 1.Structures of selected drugs used for the treatment of trypanosomiasis.

Figure 2 .
Figure 2. Graphical display of % HeLa and T.brucei brucei cell viability data with compounds 4a-g and 5h,i at 20 M.

Figure 3 .
Figure 3. Dose-response curves showing the % viability of T.brucei brucei cells at various concentrations of compounds 4b, 5h and 5i and the control, pentamidine.

Table 1 .
Effect of compounds 4a-g and 5h,i at 20 μM on the viability of HeLa and T.brucei brucei cells a IC50 values in parentheses.b Emetine control: IC50= 0.05 μM.c Standard deviation in parentheses.d Pentamidine control: IC50= 0.005 μM.

-(2-acetylphenyl)furan-2-carboxamide (8).
room temperature for 1 hour, with the reaction progress being monitored by TLC.After the completion of the reaction, the mixture was extracted into DCM (2 × 60 mL), the organic solutions were combined, washed with deionized water (4 × 60 mL), dried with anhydr.potassium sulphate and filtered.The solvent was removed in vacuo and the residue was purified using column chromatography on silica gel [elution with ethyl acetate-hexane (2:1] to afford NWhite solid (1.6 g, 84%), mp 52-54 °C (Found: MH + , m/z 230.0854.C13H12NO3 requires 230.0817); δH/ppm (600 MHz; DMSO-d6) 12.45 The resulting solution was stirred under a reflux condenser at 80 °C for 2 hours, during which the reaction progress was monitored by TLC.After completion of the reaction, the crude mixture was cooled to room temperature and then poured into ice-cold deionized water (150 mL).The resulting precipitate was filtered off, washed with cold deionized water (2 x 100 mL) and dried at room temperature for 24 hours.