Synthesis and cytotoxic evaluation of some new[1,3]dioxolo[4,5-g]chromen-8-one derivatives

Background Homoisoflavonoids are naturally occurring compounds belong to flavonoid classes possessing various biological properties such as cytotoxicity. In this work, an efficient strategy for the synthesis of novel homoisoflavonoids, [1,3]dioxolo[4,5-g]chromen-8-ones, was developed and all compounds were evaluated for their cytotoxic activities on three breast cancer cell lines. Methods Our synthetic route started from benzo[d][1,3]dioxol-5-ol which was reacted with 3-bromopropanoic acid followed by the reaction of oxalyl chloride to afford 6,7-dihydro-8H-[1,3]dioxolo[4,5-g]chromen-8-one. The aldol condensation of the later compound with aromatic aldehydes led to the formation of the title compounds. Five novel derivatives 4a-e were tested for their cytotoxic activity against three human breast cancer cell lines including MCF-7, T47D, and MDA-MB-231 using the MTT assay. Results Among the synthesized compounds, 7-benzylidene-6,7-dihydro-8H-[1,3]dioxolo[4,5-g]chromen-8-one (4a) exhibited the highest activity against three cell lines. Also the analysis of acridine orange/ethidium bromide staining results revealed that 7-benzylidene-6,7-dihydro-8H-[1,3]dioxolo[4,5-g]chromen-8-one (4a) and 7-(2-methoxybenzylidene)-6,7-dihydro-8H-[1,3]dioxolo[4,5-g]chromen-8-one (4b) induced apoptosis in T47D cell line. Conclusion Finally, the effect of methoxy group on the cytotoxicity of compounds 4b-4d was investigated in and it was revealed that it did not improve the activity of [1,3]dioxolo[4,5-g]chromen-8-ones against MCF-7, T47D, and MDA-MB-231.


Chemistry
All starting materials, reagents, and solvents were prepared from Merck (Germany). Melting points were determined on a Kofler hot stage apparatus (Vienna, Austria) and are uncorrected. 1 H-NMR spectra were recorded using a Bruker 400 spectrometer (Bruker, Rheinstatten, Germany), and chemical shifts are expressed as δ (ppm) with tetramethylsilane (TMS) as internal standard. The IR spectra were obtained on a Nicolet Magna FT-IR 550 spectrophotometer (potassium bromide disks).

Biological assay
Cell lines and cell culture Human breast cancer cell lines including MDA-MB231, MCF-7 and T47D cells were obtained from the National Cell Bank of Iran, Pasteur Institute, Tehran, Iran. Cancer cell lines were grown in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum, 100 μg/ ml streptomycin and 100 U/ml penicillin at 37°C in a humidified atmosphere with 5% CO 2 .

In vitro cytotoxicity assay
The in vitro cytotoxic activity of [1,3]dioxolo[4,5-g]chromen-8-ones 4a-e was achieved using MTT colorimetric assay. The in-vitro cytotoxic activity of all synthesized compounds were evaluated against three human breast cancer cell lines including MCF-7, T47D and MDA-MB-231 using MTT colorimetric assay according to the method of Mosman [31]. Cancer cell lines were grown in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum (Gibco BRL), 100 μg/ml streptomycin and 100 U/ml penicillin at 37°C in a humidified atmosphere with 5% CO 2 .
Briefly, cultures in the exponential growth phase were trypsinized and diluted in complete growth medium to give a total cell count of 5 × 10 4 cells/ml. 195 μl of the cell suspension was seeded into the wells of 96-well plates (Nunc, Denmark). The plates were incubated overnight in a humidified air atmosphere at 37°C with 5% CO 2 . After overnight incubation, 5 μl of the media containing various concentrations of the compounds was added per well in triplicate (final concentration 1, 5, 10 and 20 μg/ml). The plates were incubated for further 72 h. The final concentration of DMSO in the highest concentration of the applied compounds was 0.1%. In each plate, there were three control wells (cells without test compounds) and three blank wells (the medium with 0.1% DMSO) for cell viability. Etoposide and doxorubicine were used as positive controls for cytotoxicity. After treatment, the medium was removed and 200 μl phenol red-free medium containing MTT (1 mg/ml), was added to wells, followed by 4 h incubation. After incubation, the culture medium was then replaced with 100 μl of DMSO and the absorbance of each well was measured by using a microplate reader at 492 nm. For each compound, the concentration causing 50% cell growth inhibition (IC 50 ) compared with the control was calculated from concentration response curves by regression analysis.

Fluorescence microscopy evaluation
Acridine orange/ethidium bromide (AO/EB) double staining [32] was applied to observe the morphological changes in cell death induced by the most potent compounds 4a and 4b. Acridine orange is taken up by both viable and dead cells and emitting green fluorescence if intercalated into double stranded nucleic acid (DNA) or red fluorescence if bound to single stranded nucleic acid (RNA) due to its accumulation in lysosomes. Ethidium bromide is taken up only by cells with an altered cell membrane and emits red fluorescence by intercalation into DNA. Cells were seeded in 6-well plates (4 × 10 5 cell/well) for 24 h. Then, cells were treated with IC 50 concentration of test compounds for 24 h at 37°C with 5% CO 2 . After treatment, cells were washed twice with phosphate buffer saline (PBS) and then 1 μl of dye mixture (100 μg/ml AO and 100 μg/ml EB in PBS were mixed with 25 μl of cell suspension (0.4 × 10 6 cells/well) on a clean microscope slide. The suspension was immediately examined by Axoscope 2 plus fluorescence micro-scope from Zeiss (Germany) at 40× magnification.
To run successful aldol condensation reaction, acidcatalyzed and base-catalyzed approaches were investigated using various conventional acids and base in different solvents. It was found that the aldol condensation was conducted in better yield in the presence of HCl (g).