Synthesis and cytotoxic evaluation of some new[1,3]dioxolo[4,5-g]chromen-8-one derivatives
© Alipour et al.; licensee BioMed Central Ltd. 2014
Received: 25 December 2013
Accepted: 16 April 2014
Published: 2 May 2014
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.
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.
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.
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.
KeywordsHomoisoflavonoids [1,3]dioxolo[4,5-g]chromen-8-one Cancer Cytotoxic activity
Homoisoflavonoids, naturally occurring compounds belong to flavonoid classes and possess a wide spectrum of biological properties such as anti-inflammatory , antioxidant , antiproliferative , antifungal , antiviral , and antimutagenic activities . They mainly include a chromanone, chromone, or chromane skeleton and are ubiquitous in plants such as Ophiopogon, Polygonatum, Scilla, Eucomis, and Muscari. Recently, several homoisoflavonoids have been successfully isolated from plants and evaluated for their bioactivities [12, 13].
Chalcones have been the center of attention owing to their significant biological activities [14–18]. Also they are the most important precursors for the formation of α, β-unsaturated carbonyl system in flavonoid classes. Homoisoflavonoids including chalcone system have shown selective biological activities . The isolated natural homoisoflavonoids having 3-benzylidenechroman-4-one skeleton were found to be potent and selective MAO-B inhibitors. Compounds involving benzylidene chromanone have depicted significant medicinal properties such as antioxidant , anticancer , anti-inflammatory , anti-human-immune deficiency virus (HIV-I) activities .
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. 1H-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).
General procedure for the synthesis of [1,3]dioxolo[4,5-g]chromen-8-one derivatives 4
Dry hydrogen chloride gas was passed through an ice-cold solution of 6,7-dihydro-8H-[1,3]dioxolo[4,5-g]chromen-8-one 3 (0.5 mmol) and benzaldehyde derivative (0.7 mol) in absolute EtOH (3 mL) for 2 min. The reaction mixture was allowed to stand at room temperature for 48 h. The precipitated product was filtered off, dried, and recrystallized from ethanol and water.
Yield: 48%, mp 141–144°C. IR (KBr): 1664 (C = O) cm-1. 1H-NMR (CDCl3, 400 MHz) δ: 7.83 (s, 1H, benzylidene), 7.43 (s, 1H, H9), 7.42-7.25 (m, 5H, Ph), 6.41 (s, 1H, H4), 6.00 (s, 2H, H1, CH2), 5.29 (s, 2H, CH2). Anal. Calcd. for C17H12O4: C, 72.85; H, 4.32. Found: C, 72.68; H, 4.18.
Yield: 31%, mp 160–163°C. IR (KBr): 1660 (C = O) cm-1. 1H-NMR (CDCl3, 400 MHz) δ: 7.961 (s, 1H, benzylidene), 7.39 (s, 1H, H9), 7.04-6.94 (m, 4H, H3′, H4′, H5′, H6′), 6.40 (s, 1H, H4), 6.00 (s, 2H, H1, CH2), 5.17 (s, 2H, CH2), 3.86 (s, 3H, OCH3). Anal. Calcd. for C18H14O5: C, 69.67; H, 4.55. Found: C, 69.52; H, 4.41.
Yield: 42%, mp 161–163°C. IR (KBr): 1662 (C = O) cm-1. 1H-NMR (CDCl3, 400 MHz) δ: 7.79 (s, 1H, benzylidene), 7.38 (s, 1H, H9), 7.04-6.93 (m, 4H, H2′, H4′, H5′, H6′), 6.41(s, 1H, H4), 6.00 (s, 2H, H1, CH2), 5.29 (s, 2H, CH2), 3.84 (s, 3H, OCH3). Anal. Calcd. for C18H14O5: C, 69.67; H, 4.55. Found: C, 69.83; H, 4.72.
Yield: 31%, mp 169–172°C. IR (KBr): 1665 (C = O) cm-1. 1H-NMR (CDCl3, 400 MHz) δ: 7.79 (s, 1H, benzylidene), 7.38 (s, 1H, H9), 7.26 (d, J = 8.4 Hz, 2H, H2΄, H6΄), 6.96 (d, J = 8.4 Hz, 2H, H3΄, H5΄), 6.42 (s, 1H, H4), 6.00 (s, 2H, H1, CH2), 5.32 (s, 2H, CH2), 3.86 (s, 3H, OCH3). Anal. Calcd. for C18H14O5: C, 69.67; H, 4.55. Found: C, 69.53; H, 4.82.
Yield: 42%, mp 198–200°C. IR (KBr): 1667 (C = O) cm-1. 1H-NMR (CDCl3, 400 MHz) δ: 7.73 (s, 1H, benzylidene), 7.37 (s, 1H, H9), 6.86-6.67 (m, 3H, H3′, H4′, H6′), 6.41 (s, 1H, H4), 6.03 (s, 2H, H1′,CH2), 6.00 (s, 2H, H1, CH2), 5.29 (s, 2H, CH2). Anal. Calcd. for C18H12O6: C, 66.67; H, 3.73. Found: C, 66.48; H, 3.55.
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% CO2.
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 . 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% CO2.
Briefly, cultures in the exponential growth phase were trypsinized and diluted in complete growth medium to give a total cell count of 5 × 104 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% CO2. 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 (IC50) compared with the control was calculated from concentration response curves by regression analysis.
Fluorescence microscopy evaluation
Acridine orange/ethidium bromide (AO/EB) double staining  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 × 105 cell/well) for 24 h. Then, cells were treated with IC50 concentration of test compounds for 24 h at 37°C with 5% CO2. 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 × 106 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.
Results and discussions
Chemical structures and in vitro cytotoxic activity (IC 50 , μg/ml) a of compounds 4a-4e against breast cancer cell lines
6.2 ± 0.1
4.6 ± 0.1
9.3 ± 2.1
5.7 ± 0.07
27.3 ± 7.1
18.8 ± 2.3
29.05 ± 1.7
9.2 ± 2.9
0.002 ± 0.002
0.03 ± 0.002
0.006 ± 0.004
7.5 ± 0.32
7.9 ± 0.45
11.9 ± 0.87
To run successful aldol condensation reaction, acid-catalyzed 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).
Then, various derivatives including 7-(2-methoxybenzylidene)-6,7-dihydro-8H-[1,3]dioxolo[4,5-g]chromen-8-one (4b) and 7-(3-methoxybenzylidene)-6,7-dihydro-8H-[1,3]dioxolo[4,5-g]chromen-8-one (4c) possessing methoxy (OMe) group at ortho and meta positions were prepared to compare their bioactivities against the studied cell lines with that of the control. Also other two derivatives, 7-benzylidene-6,7-dihydro-8H-[1,3]dioxolo[4,5-g]chromen-8-one (4a) and 7-(benzo[d][1,3]dioxol-5-ylmethylene)-6,7-dihydro-8H-[1,3]dioxolo[4,5-g]chromen-8-one (4e) were prepared to investigate the effect of methxoy substituent on the cytotoxicity (Table 1).
The in vitro cytotoxic activity of [1,3]dioxolo[4,5-g]chromen-8-one derivatives 4, were tested against three human breast cancer cell lines including MCF-7, T47D, and MDA-MB-231. The 50% growth inhibitory concentration (IC50) for all derivatives were calculated and depicted in Table 1.
According to MTT assay results in Table 1, 7-benzylidene-6,7-dihydro-8H-[1,3]dioxolo[4,5-g]chromen-8-one (4a) showed the highest activity against MCF-7, T47D, and MDA-MB-231 cell lines with IC50 values of 6.2 ± 0.1, 4.6 ± 0.1, and 9.3 ± 2.1 μg/ml, respectively. In contrast, 7-(benzo[d][1,3]dioxol-5-ylmethylene)-6,7-dihydro-8H-[1,3]dioxolo[4,5-g]chromen-8-one (4e) did not show any cytotoxicity at the concentrations used. It seems that the presence of benzo[d][1,3]dioxole in benzylidene moiety decreases the cytotoxic activity of the corresponding compound. As can be seen in Table 1 (Entries 2–4), by introduction of OMe into the ortho, meta or para positions of benzylidenemoiety (compounds 4b, 4c, and 4d), different results were observed. All of them were inactive against MCF-7 cell line (IC50 > 100 μg/ml), whereas they exhibited good activity against T47D cell line with IC50 values of 5.7 ± 0.07, 18.8 ± 2.3, and 9.2 ± 2.9 μg/ml, respectively. It should be noted that compounds 4b and 4c were active against MDA-MB-231 cell line and 4d did not show any activity in this cell line. Presence of OMe in benzylidene moiety did not play crucial role on the improvement of cytotoxicity effects.
In conclusion, novel [1,3]dioxolo[4,5-g]chromen-8-one derivatives were synthesized and tested for their cytotoxic activity against three human breast cancer cell lines including MCF-7, T47D, and MDA-MB-231 using the MTT assay. 7-Benzylidene-6,7-dihydro-8H-[1,3]dioxolo[4,5-g]chromen-8-one (4a) showed the highest activity against the three studied cell lines. Also the analysis of acridine orange/ethidium bromide staining results revealed that the cytotoxic effect of 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) may be due to inducing apoptosis in cancer cell lines.
The authors are thankful for financial support from the Research Council of Islamic Azad University and Iran National Elite Foundation (INEF).
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