Eco-Friendly Synthesis and Antiproliferative Evaluation of Some Oxygen Substituted Diaryl Ketones

A broad variety of oxygen-substituted diaryl ketones has been synthesized by solar energy-induced Friedel Crafts acylations of 1,4-benzo- and 1,4-naphthoquinones with benzaldehydes. The in vitro antiproliferative properties of the photoproducts were assessed on prostate (DU-145), bladder (T24) and breast (MCF7) human-derived tumor cell lines and compared to non-tumor mouse fibroblasts (Balb/3T3). Among the tested compounds, it was found that those containing a 3,4,5-trimethoxyphenyl A-ring, such as 12 and 22 are more active on DU-145, with EC50 values of 1.2 and 5.9 μM, respectively. By comparing their effects on the three cancer cell lines, the analogue 22 has the best mean selective index (2.4).

Among the broad variety of synthetic diaryl ketones the oxygen-substituted members, named phenstatin [35] and naphthylphenstatin [36] (Figure 1), stand out by their biological activity as microtubule-targeting agents.Based on these precedents we wanted to examine the synthetic flexibility of the eco-friendly solar photoacylation of 1,4-benzo-and 1,4-naphthoquinone with substituted benzaldehydes to the synthesis of diverse oxygen-substituted diaryl ketones.Taking advantage of this potentially simple access to oxygen-substituted diaryl ketones we were also interested in evaluating the series for in vitro antiproliferative activity on cancer cells.The aim of this study is mainly directed towards broadening the use of simple and eco-friendly methodologies in the synthesis of new oxygen-substituted diaryl ketones as well as to contribute to the search for new biological active members of this series.

Chemistry
Based on our experience on the synthesis of heteroaroylhydroquinones by solar photoacylation of 1,4-benzo-and 1,4-naphthoquinone 1 and 2 with heteroarylcarbaldehydes in benzene [33], we initially examined the reaction of 1 and 2 with mono-substituted benzaldehydes.The reactions were carried out by using a 6.5 molar excess of the aldehyde with respect to the quinone.It is interesting to point out that molar excess of aldehyde is used to inhibit the dimerization of the quinone [30].To avoid the use of hazardous benzene as the solvent, both the reaction of 1 and 2 with benzaldehyde and the liquid isomers of methyl-and methoxybenzaldehydes were accomplished using the appropriate aldehyde in excess as the solvent.In these experiments, performed by solar irradiation for 30 hours, the reaction mixtures were further submitted to column chromatography to give the respective photoproducts 3-8 and 14-18 (Scheme 1) in the 34%-77% yield range (Table 1).The formation yields of the products were determined on the basis of the initial and the amounts of the respective quinones recovered.
In parallel experiments, the above reactions were run in benzene in order to compare the yields of the photo-acylation reactions with and without this solvent.The results of these assays are collected in Table 1.The data indicate that the photoacylation reactions of quinones 1 and 2 give higher yields in benzene solvent than in excess aldehyde.Scheme 1. Photoacylation of quinones 1 and 2 with substituted benzaldehydes.
The photoacylation of quinones 1 and 2 with the solid di-and trisubstituted benzaldehydes were carried out in benzene under the above mentioned solar irradiation conditions.The treatment provides access to the corresponding photoproducts 9-13 and 19-23 in good yields (Table 1).The new diaryl ketones 7, 9-13, 15, 19-21 and 23 were fully characterized by IR, 1 H-, 13 C-NMR and HRMS.
The synthesis of compounds 4 (65%), 6 (79%), 8 (77%), 14 (57%) and 16 (70%) have been previously reported by photoacylation of 1 and 2 with the respective aldehydes in the presence of catalytic amounts of benzophenone and using artificial light irradiation [29].According to the data in Table 1 better yields on these compounds are achieved by using the solar chemical procedure (method B).Selected indoor photoacylation experiments performed in benzene by using irradiation with fluorescent lamps indicate that the photoproducts were generated in low yields.a Isolated by column chromatography; b Method A: the reaction was carried out using 1 or 2 (1 equiv.)and the aldehyde (7.5 equiv.)without benzene; c Method B: the reaction was carried out using 1 or 2 (1 equiv.), the aldehyde (7.5 equiv.)and benzene as the solvent.

In Vitro Antiproliferative Activity of Diaryl Ketones 3-23 again Select Cancer Cell Lines
The oxygen-substituted diaryl ketones 3-23 were evaluated for their in vitro antiproliferative activity on a panel of four cell lines, including non-tumor fibroblasts (Balb/3T3) and three human-derived tumor cell lines, namely DU-145 (prostate), T24 (bladder) and MCF7 (breast), using the conventional microculture tetrazolium reduction assay [37].
Table 2.In vitro inhibitory effect of compounds 3-23 on the proliferation of the human-derived tumor cell lines: T24 (bladder), DU-145 (prostate) and MCF7 (breast) and the non-tumor fibroblasts (Balb/3T3).Table 2 summarizes the data from these evaluations: it shows the EC 50 values (µM) of the respective benzo-and naphthohydroquinone derivatives.These values were calculated from their effects on MTT reduction in three cancer cell lines and Balb/3T3 non transformed mouse fibroblasts as a function of their concentration during 48 h of incubation.All three cancer cell lines were similarly sensitive to these compounds.With rather few exceptions, the members containing the dihydroxyphenyl fragment, such as 3-13, were in general less active than their corresponding naphthyl analogues 14-23 when tested against cancer cells, but just the opposite was observed in non-transformed fibroblasts.The data in Table 2 showed that compounds 12 and 22 appear as the most potent members of the series with lower EC 50 values against T24 and DU-145 with respect to the reference drug mitomycin C. The biological activity differences of compounds 12 and 22 with respect to their analogues could be attributed to the 3,4,5-trimethoxy substitution on the A-phenyl ring.According to literature precedents, the 3,4,5-trimethoxyphenyl ring is considered essential for the antitubulin activity of a broad variety of antimitotic compounds [36,[38][39][40][41][42][43][44][45].Nevertheless, it should be noted that the EC 50 values of compounds 12 and 22 are two orders of magnitude lower than to that reported to phenstatin when tested in the NCI screen [35] showing a mean panel GI 50 (growth inhibitory) value of 6.01 × 10 −8 M. In addition to the tendency showing that dihydroxynaphthyl analogues are more active than the dihydroxyphenyl derivatives, the vast majority of compounds did not have an adequate selectivity (Table 2), that is they affect both cancer and non-tumor cells in a similar way.In this context, only compound 22 have a good mean selectivity index (2.45).

General
All reagents were commercially available reagent grade and were used without further purification.Melting points were determined on a Stuart Scientific SMP3 apparatus and are uncorrected.The IR spectra were recorded on an FT Bruker spectrophotometer using KBr disks, and the wave numbers are given in cm −1 . 1 H-NMR spectra were run on Bruker AM-200 and AM-400 instruments in deuterochloroform (CDCl 3 ) and dimethyl sulfoxide-d 6 (DMSO-d 6 ).Chemical shifts are expressed in ppm downfield relative to tetramethylsilane (TMS, δ scale), and the coupling constants (J) are reported in Hertz. 13C-NMR spectra were obtained in CDCl 3 + DMSO-d 6 at 50 and 100 MHz.Chemical shifts are reported in δ ppm downfield from TMS, and J-values are given in Hertz.HRMS data were obtained on Thermo Finnigan mass spectrometer, model MAT 95XP and LTQ-Orbitrap mass spectrometer (Thermo-Fisher Scientific) with the analysis performed using an APCI source operated in positive mode.Silica gel Merck 60 (70-230 mesh) was used for preparative column chromatography and TLC aluminum foil 60F 254 for analytical TLC.The solar irradiation experiments were performed at the Canchones Experimental Center in Iquique/Chile (latitude 20°26′43.80"S,990 m above sea level), located in the Atacama Desert.

Chemistry
General Procedure for Photoacylation of 1 and 2 with Substituted Benzaldehydes in the Absence of Benzene (Method A) Quinone 1 or 2 (1 mmol) and the liquid aldehyde (7.5 mmol), were placed into a test tube, nitrogen was bubbled through the solution for 2 min and then the tube was sealed with a septum.The mixture was irradiated for six days (total illumination time of 30 h), under solar radiation conditions in the range 800-1100 Watts/m 2 (November-March).The mixture reaction was chromatographed on silica gel (3:1 petroleum ether/ethyl acetate) to give pure samples of the corresponding diaryl ketones.The remaining precursors were recovered to be used in further preparations.
General Procedure for Photoacylation of 1 and 2 with Substituted Benzaldehydes in Benzene (Method B).
A 100 mL benzene solution of the required quinone 1 or 2 (1 mmol) and the substituted benzaldehyde (7.5 mmol), was placed into the outer jacket of a Liebig condenser type.The solution was bubbled with nitrogen (2 min), the flask was sealed with a septum and then irradiated for six days (total illumination time of 30 h), under solar radiation conditions in the range 800-1100 Watts/m 2 (November-March).The solvent was evaporated under reduced pressure and the residue was chromatographed on silica gel (3:1 petroleum ether/ethyl acetate).The starting aldehyde and the solvent were recovered and employed in the next batches.

Cell Lines and Culture Conditions
Human cancer cell lines (T24, DU-145, MCF7) were cultured in high-glucose Dulbecco's modified Eagle medium (Gibco, Grand Island, NY, USA) supplemented with 10% foetal calf serum, penicillin (100 U/mL), and streptomycin (100 μg/mL).Balb/3T3 cells (normal mouse fibroblasts) were cultured in the same medium, except that the foetal calf serum was replaced by 10% newborn calf serum.All cultures were kept at 37 °C in 95% air/5% CO 2 at 100% humidity.Phosphate-buffered saline (PBS) was purchased from Gibco.Cells were incubated at the indicated times at 37 °C with or without hydroquinones at various concentrations.

Cellular Assays
The cytotoxicity of the bis aryl ketones was assessed by following the reduction of MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) to formazan blue [37].Briefly, cells were seeded into 96-well plates at a density of 10,000 cells/well for 24 h and then incubated for 48 h with or without the compounds.Cells were then washed twice with warm PBS and incubated with MTT (0.5 mg/mL) for 2 h at 37 °C.Incubation medium was thereafter discarded and the blue formazan crystals were solubilized by adding 100 μL DMSO/well.The colour solution was subsequently read at 550 nm.Results are expressed as % of MTT reduction compared to untreated control conditions.The calculation of EC 50 values was performed by using GraphPad Prism software (San Diego, CA, USA).

Conclusions
We have extended the photo-Friedel Crafts acylation of 1,4-benzo-and 1,4-naphthoquinone with aldehydes to the synthesis of a significant number of oxygen-substituted diaryl ketones.The main advantages of this general procedure respect to other methods to construct oxygen-substituted diaryl ketone framework are the atom economy, simplicity, cheap and the chemical stability of the oxygen substituent of precursors and/or products.From the antiproliferative screening of the oxygen-substituted

Table 2 . Cont. EC 50 ± SEM a (μM) N°
a Data represent EC 50 mean values ± SEM of at least three different experiments; b MSI: Mean Selective Index = EC 50 values fibroblasts/EC 50 values tumor cells; c DOX: doxorubicin; d MIT: mitomycin C.