Synthesis , Characterization , and Anticancer Activity of New Benzofuran Substituted Chalcones

Benzofuran derivatives are of great interest in medicinal chemistry and have drawn considerable attention due to their diverse pharmacological profiles including anticancer activity. Similarly, chalcones, which are common substructures of numerous natural products belonging to the flavonoid class, feature strong anticancer properties. A novel series of chalcones, 3-aryl-1-(5-bromo-1benzofuran-2-yl)-2-propanones propenones (3a–f), were designed, synthesized, and characterized. In vitro antitumor activities of the newly synthesized (3a–f) and previously synthesized (3g–j) chalcone compounds were determined by using human breast (MCF-7) and prostate (PC-3) cancer cell lines. Antitumor properties of all compounds were determined by 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell viability assay for the tested chalcone compounds was performed and the log IC 50 values of the compounds were calculated after 24-hour treatment. Our results indicate that the tested chalcone compounds show antitumor activity against MCF-7 and PC-3 cell lines (p < 0.05).


Introduction
Cancer is one of the most important clinical problems worldwide.Among the wide range of compounds approved as potential anticancer agents, derivatives with functionalities as ,-unsaturated Michael acceptor have attracted great interest [1,2].Previous studies have proposed that anticancer compounds such as alkylating agents bind directly to various cellular nucleophiles, thus lacking selectivity.However, Michael acceptors can be structurally modified so that they can react selectively with target nucleophiles [3].Chalcones, the compounds having 1,3-diaryl-2-propen-1-one system, also have shown a broad spectrum of biological activities including anti-inflammatory [4][5][6][7], antimalarial [8], anti-invasive [9], antibacterial [10][11][12], and anticancer [13][14][15][16] activities.On the other hand, chalcones are capable of inducing apoptosis [17,18].Consequently, these compounds are recognized as promising anticancer agents [19][20][21][22].A number of clinically useful anticancer drugs have genotoxic effects because of their interaction with the amino groups of nucleic acids.However, chalcones have been found not to show such undesired side effects [23].Numerous reports have been published on the interesting anti-breast cancer activity shown by chalcones [24][25][26].Benzofuran derivatives are an interesting class of heterocyclic compounds.Benzofuran derivatives are of great interest in medicinal chemistry and have drawn remarkable attention due to their biological activities with chemotherapeutic properties [27].Some benzofurans bearing various substituents at the C-2 position are greatly distributed in nature; for example, ailanthoidol, a neolignan derivative, has been reported to have antiviral, antioxidant, and antifungal activities [28].Furthermore, most of the compounds prepared from 2acetylbenzofuran have antimicrobial, anticancer, antitumor, anti-inflammatory, and antitubulin activities and are also used for treatment of cardiac arrhythmias [29][30][31][32].The use of the combinations of different pharmacological compounds in the design of new drugs may lead to finding novel drugs with interesting biological activity [33,34].Furthermore, no studies were found in the literature evaluating anticancer properties of benzofuran substituted chalcone derivatives.This encouraged us to synthesize benzofuran substituted chalcone compounds and to investigate anticancer properties of these compounds.
In this study, we aimed at designing and synthesizing new compounds (3a-f) with both benzofuran and chalcone units in one molecule and examining anticancer activity of this newly synthesized chalcones (3a-f) and previously synthesized chalcones [35] (3g-j) bearing no substituent in the benzofuran ring as a different series against human breast cancer cell lines (MCF-7) and human prostate cancer cells (PC-3) (Scheme 1).

Materials and Methods
2.1.Materials.Chemical agents used in the present study included dimethyl sulfoxide (DMSO; Merck, Germany), penicillin-streptomycin, fetal bovine serum (FBS), and DMEM (Dulbecco's Modified Eagle Medium).Doubledistilled water was used at all stages of the experiments.Samples of chalcone compounds for testing were prepared at 1, 5, 25, 50, and 100 M concentrations.

Characterization Techniques.
Melting points were measured using a differential scanning calorimeter (Shimadzu DSC-50) and were uncorrected.NMR spectra were determined on a Bruker AC 400 (400 MHz) spectrometer, with tetramethylsilane (TMS) as the internal standard in DMSOd 6 or CDCl 3 as solvents.FT-Infrared (FT-IR) spectra were recorded as KBr pellets on a Perkin-Elmer Spectrum One FT-IR spectrometer.

General Procedure for Synthesis of Chalcones (3a-f ).
A solution of 1-(5-bromo-1-benzofuran-2-yl)ethanone (1 g, 4.18 mmol) and one of the aldehyde derivatives (2a-f, 4.18 mmol) in MeOH (10 mL) was cooled at 0-5 ∘ C and then 6 mL of aqueous NaOH (1 mol/L) was added to this solution and stirred at room temperature for 3 h.The reaction mixture was poured on crushed ice.The precipitated solid was filtered after neutralization with diluted HCl and was washed several times with water and then dried.The product was recrystallized from ethanol.-7) and human prostate cancer (PC-3) were employed in our study.The PC-3 and MCF-7 cell lines were retrieved from American Type Culture Collection (ATCC).MCF-7 and PC-3 cells were fed with DMEM medium (supplemented with 4500 mg/L glucose, 10% FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin added) in 75 cm 2 culture flasks and RPMI-1640 medium (supplemented 10% FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin added), respectively.A humidified carbon dioxide incubator (5% CO 2 + 95% O 2 ; Panasonic, Japan) was used to keep all cells at 37 ∘ C during the experiments.Before the treatment of chalcone compounds, the viability ratios of the cells were identified by 0.4% trypan blue.If the viability ratios were under 90%, we did not initiate the experiments [36].

MTT Assay.
The synthetic chalcone derivatives were tested for their antitumor activities against different type cancer cell lines (PC-3 and MCF-7) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay method.The pale-yellow tetrazolium salt, MTT, was transformed by active mitochondria to form a dark blue formazan that was determined by a microplate reader [37].The MTT method provides a simple way to detect living and growing cells without using radioactivity.
When the cells were confluent, they were removed from the flasks using trypsin-EDTA solution and were seeded in 96-well plates such that there were 15 × 10 3 cells in each well.The plates were incubated for 24 h at 37 ∘ C.After treatment of these cancer cells with DMSO (for positive control group) and different concentrations (1, 5, 25, 50, and 100 M) of chalcone compounds (D1, 3a-j) in DMSO, the cells then were incubated for 24 h at 37 ∘ C in 5% CO 2 humidified incubator.MTT solution (0.5 mg/mL) was prepared from the MTT stock solution in sterile PBS and was added to each well and the plates were then incubated for 3 h after the incubation for 24 h with chalcone compounds.After that, DMSO and the optical density of the cells were determined by an ELISA reader (Synergy HT, USA) at 550 nm wavelength.The averages of the absorbance values were recorded by reading the control wells that were considered as 100%.The values of absorbance achieved from chalcone compounds and solvent (DMSO) added wells were proportioned to the control values, and the percentages of cell viability were determined.The tests were reiterated ten times at several days [38].

Statistical Analyses.
Quantitative data are expressed as mean ± standard deviation (SD).Normal distribution was confirmed using Kolmogorov-Smirnov test.Quantitative data were analyzed using Kruskal-Wallis  test following Mann-Whitney  test with Bonferroni adjustment as a post hoc test.All  values < 0.05 were considered as statistically significant.All analyses were done by IBM SPSS Statistics 22.0 for Windows.The log IC 50 values were determined by using % cell viability values of compounds by GraphPad Prism 6 program.
The most characteristic signals in 1 H-NMR spectra of the benzofuran substituted chalcones were observed at 8.29-8.03ppm (B3-H at benzofuran ring) and at 7.80-7.40ppm (-H and -H of chalcone moiety) with a coupling constant about 15 Hz which characterized the transconfiguration of the alkene moiety.The signal of -H was found downfield at a lower field than that of -H due to resonance of -electrons between -carbons and -carbons with carbonyl group.The carbonyl carbon was observed at about 178 ppm in the 13 C-NMR spectra of 3a-f.

Anticancer Activity.
The benzofuran substituted chalcone compounds synthesized were tested for their in vitro anticancer activity against two cancer cell lines including MCF-7 and PC-3 at five different concentrations (1,5,25,50, and 100 M) by MTT assay.The cell viability percentages of tested benzofuran substituted chalcone compounds were  1.
The benzofuran substituted chalcones showed anticancer activity on PC-3 and MCF-7 cell lines ( < 0.05).All the compounds at 100 M concentrations significantly reduced the viability percentage of PC-3 and MCF-7 cells ( < 0.001).Structure activity relationships between these chalcone derivatives and starting material (D1) demonstrated that benzofuran substituted chalcones showed more potent activities than the starting material bearing only an unsubstituted benzofuran ring.
Among the synthesized chalcones, compounds 3a, 3h, and 3i were found to be the most potent against MCF-7 and PC-3 cell lines.In general, chalcone derivatives show anticancer activity.We have not run across any study in literature on the synthesis and anticancer properties of   the chalcone compounds containing benzofuran ring.In this study we have firstly synthesized the chalcone compounds containing benzofuran ring.And also we have firstly studied on the anticancer properties of these compounds.We have studied only MCF-7 and PC-3 cells.These results suggested that benzofuran substituted chalcones could be used as lead compounds to develop novel potent anticancer agents.

Conclusions
Synthetic benzofuran chalcone compounds were evaluated in vitro for their anticancer activity by MTT assay.The benzofuran substituted chalcone derivatives showed high antitumor activity against MCF-7 and PC-3 cell lines ( < 0.001).These results displayed that chalcone derivatives bearing benzofuran ring may be useful in the future for anticancer drug development.

Table 1 :
Evaluation of the cytotoxicity and log IC 50 values (M) of chalcone compounds and docetaxel (reference chemotherapeutic drug) of two cancer cell lines.log IC 50 is the concentration of drug that reduces cell growth by 50%.