Synthesis and anti-proliferative activity of novel oxepin-annulated coumarins

A series of fused dihydrooxepino[h]and dihydrooxepino[g]coumarins (7 and 8) were synthesized through allylation, Claisen rearrangement, allylation and ring-closing metathesis (RCM), respectively. All the synthesized compounds were characterized by appropriate spectral analysis. The anti-proliferative activities of compound 5a-c, 6a, 6c, 7a-c and 8a-c were evaluated against human colon cancer (Caco-2), liver cancer (HepG2) and breast cancer (SKBR-3) cell lines using tamoxifen (TAM) as the positive control. Compound 7b showed significant anti-proliferative activity against resistant Caco-2 and SKBR-3 cell lines on comparison with all other coumarin derivatives. Interestingly, compound 8b was more potent than TAM against sensitive HepG2 cell line.

The synthesized compounds 5-8, except for 6b, were evaluated for their in vitro antiproliferative activity against three cancer cell lines including colorectal adenocarcinoma (Caco-2), hepatocellular carcinoma (HepG2) and breast carcinoma (SKBR-3) cell lines using MTT assay.The concentration-response studies were performed in order to determine their half-medium inhibitory concentration (IC 50 ) values (Table 2) by incubation of cell lines with 11 compounds at concentrations of 0-100 µg/mL for 48 h at 5% CO 2 and 37C.Tamoxifen was also evaluated as references.
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Scheme 1. Synthesis of fused dihydrooxepino[h]-and dihydrooxepino[g]coumarins (7 and 8).
From the analysis of Table 2, it can be concluded that compound 7b was the most cytotoxic to Caco-2 and SKBR-3 cell lines with IC 50 of 30.05 ± 0.71 and 11.18 ± 1.93 μg/mL, respectively.It also showed potent activity on HepG2 cell with IC 50 values of 15.32 ± 0.30 μg/mL.Interestingly, compound 8b has demonstrated the most promising activity against HepG2 cell line with IC 50 values of 8.78 ± 1.21 μg/mL, which is lower than that of tamoxifen, 5a and 5b (IC 50 values of 9.41 ± 1.81, 10.48 ± 3.44 and 10.42 ± 2.16 μg/mL, respectively).Comparison of the substitution at the C-4 position of the coumarin ring suggested that the propyl group contributed to the anti-proliferative activity enhancement.Compounds 5b, 7b and 8b were more active than compounds 5a,c, 7a,c and 8a,c, respectively against almost all tested cell lines.In addition, the nature of the angularly or linearly fused ring (7 or 8) did not significantly affect the activity.Among the tested cell lines, HepG2 has proven to be the most sensitive cell line.Approximately half of the tested coumarin derivatives presented high cytotoxicity (IC 50 ≤ 20 μg/mL).On the other hand, Caco-2 has proven to be the most resistant cell line, since almost all compounds exhibited moderate cytotoxicity (IC 50 ranged between 21 and 200 μg/mL).

Conclusions
We have demonstrated a simple synthetic strategy for synthesis of fused-dihydrooxepino[g]-and [h]coumarins 7 and 8 via the allylation, Claisen rearrangement, allylation and RCM, respectively.The antiproliferative activity of compound 5a-c, 6a, 6c, 7a-c and 8a-c was screened against Caco-2, HepG2 and SKBR-3 cell lines using tamoxifen (TAM) as the positive control.Compound 7b exhibited similar anti-proliferative activity against resistant SKBR-3 cell to TAM and demonstrated the most potent activity against Caco-2 among the tested coumarin derivatives.Compound 8b displayed the most potent anti-proliferative activity against HepG2 cell line.Our results could be used as a starting point for development of powerful coumarin anticancer therapies.

Experimental Section
General.Melting points ( o C) were measured with the Gallenkamp melting point apparatus and are uncorrected. 1H and 13 C NMR spectra were recorded on a Bruker AV400 spectrometer.Chemical shifts (δ) are given in ppm and refer to TMS or the residual undeuterated solvent as the internal standard.The following abbreviations are used: s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, sext = sextet, m = multiplet, dd = double doublet, br.s = broad singlet.ESI mass spectra were recorded on a Thermo Finnigan LCQ Advantage Mass Spectrometer.High Resolution Mass Spectrometry was performed with a MicroTOFLC, Bruker Daltonics.FTIR spectra were obtained with a Perkin Elmer FT-IR Spectrum GX.Column chromatography was performed on silica gel (Kieselgel 60, 70-230 mesh, Merck) in common glass columns.Preparative TLC was carried out on silica gel plate (Merck silica gel 60 PF254).All chemicals were obtained from commercial suppliers, and were used without further purification.The required coumarin precursors 1a-c were prepared via Pechmann reaction. 39neral procedure for allylation of 1a-c.To a solution of phenol derivative (1, 10 mmol) anhydrous K 2 CO 3 (4.15g, 30 mmol) in acetone (30 mL) was added allyl bromide (1.81 g, 15 mmol).The mixture was heated under reflux and stirring for 16-18 h.After cooling to room temperature, the precipitated solid was filtered and washed with acetone.The filtrate was concentrated under reduced pressure and purified by column chromatography on silica gel using EtOAc/hexane as eluent to afford the corresponding allylic ether 2. 25 General procedure for Claisen rearrangement of 2a-c.A solution of allylic ether (2) (9 mmol) in ethylene glycol (45 mL) was heated under stirring at 190 ⁰C in sand bath for 24 h.After cooling to room temperature, the mixture was quenched with water (50 mL) and the aqueous phase was extracted with EtOAc (3 x 45 mL).
The organic phases were combined, dried over anhydrous Na 2 SO 4 and concentrated in vacuo and the residue was purified by column chromatography on silica gel using EtOAc/hexane as eluent to give a mixture of regioisomers of 3 as a major product and 4 as a minor product. 1 H NMR and 13 C NMR were characterized only the major products. 26General procedure for allylation of the mixtures of 3a-c and 4a-c.The synthesis was carried out from the mixture of 3a-c and 4a-c, anhydrous K 2 CO 3 and allyl bromide in acetone using the above general procedure for the synthesis of compounds 2a-c.The diene products 5a-c and 6a-c were obtained after purification by column chromatography or preparative TLC using EtOAc/hexane as eluent.General procedure for ring-closing metathesis of 5a-c and 6a-c.The solution of Grubbs' first-generation catalyst (4 mg, 0.9 mol%) in dry dichloromethane (DCM) (10 mL) was added to a solution of 5 or 6 (0.5 mmol) in dry DCM (40 mL) under N 2 atmosphere.The solution was stirred at room temperature for 24 h.After the evaporation of the solvent, the residue was separated by CC using EtOAc/hexane as eluent to give the oxepinocoumarins (7 or 8). 25,26ell line maintenance.The Three cancer cell lines used in this study were obtained from ATCC (MD, USA) and are as follows: colorectal adenocarcinoma (Caco-2), hepatocellular carcinoma (HepG2) and breast carcinoma (SKBR-3) cell lines.The Caco-2 and HepG2 cells were cultured in EMEM medium whereas SKBR-3 was cultured in DMEM medium.All media (Gibco, Langley, VA, USA) were supplemented at 10% with fetal bovine serum (Gibco) and streptomycin plus penicillin (100 µg/mL and 100 U/mL, respectively; Sigma Co., Madrid, Spain).All cells were maintained at 37°C, 95% relative humidity with 5% CO 2 atmosphere.Evaluation of cell viability.All the candidates oxepin-annulated coumarins 5-8 were evaluated in vitro for their anti-proliferative activity against three different cancer cell lines, colorectal adenocarcinoma (Caco-2), hepatocellular carcinoma (HepG2) and breast carcinoma (SKBR-3) cell lines using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay as previously reported technique. 44In brief, cells were seeded into 96-well tissue culture plates in appropriated basal medium for each cell line containing 10% FBS to a final volume of 100 µL.The cells were subjected to different treatments after 24 h of seeding.The cells were then incubated for 48 h with test compounds, tamoxifen as a positive control, or vehicle (DMSO).MTT solutions were then added and cells were incubated for 3 h.After that, the supernatants were removed and the precipitated formazan was dissolved by adding 200 µL of DMSO.Absorbance at 570 nm was determined using a microplate reader (Varioskan™ Flash Multimode Reader; Thermo Scientific™).Results were calculated by subtracting blank readings.Data analysis.The IC 50 values were obtained from the curve fitted to the means of the absorbance quotients with respect to the control.

Figure 2 .
Figure 2. Examples of natural products and bioactive compounds containing 7-membered oxygen heterocycle.