Synthesis of Benzofuran-2-One Derivatives and Evaluation of Their Antioxidant Capacity by Comparing DPPH Assay and Cyclic Voltammetry

The present work aimed to synthesise promising antioxidant compounds as a valuable alternative to the currently expensive and easily degradable molecules that are employed as stabilizers in industrial preparation. Taking into account our experience concerning domino Friedel-Crafts/lactonization reactions, we successfully improved and extended the previously reported methodology toward the synthesis of 3,3-disubstituted-3H-benzofuran-2-one derivatives 9–20 starting from polyphenols 1–6 as substrates and either diethylketomalonate (7) or 3,3,3-trifluoromethyl pyruvate (8) as electrophilic counterpart. The antioxidant capacity of the most stable compounds (9–11 and 15–20) was evaluated by both DPPH assay and Cyclic Voltammetry analyses performed in alcoholic media (methanol) as well as in aprotic solvent (acetonitrile). By comparing the recorded experimental data, a remarkable activity can be attributed to few of the tested lactones.


General Information
Solvents and common reagents were purchased from a commercial source and used without further purification. All reactions were monitored by thin-layer chromatography (TLC) carried out on Merck F-254 silica glass plates and visualized with UV light or by 5% phosphomolibdic acid/ethanol test. Flash chromatography was performed on Sigma-Aldrich silica gel (60, particle size: 0.040-0.063 mm). 1 H-NMR and 13 C-NMR were recorded in CDCl3 (99.8% in deuterium) using a Varian Gemini 300 spectrometer (300 MHz, Varian inc., Palo Alto, CA, USA). All chemical shifts are expressed in parts per million (δ scale) and are referenced to the residual protons of the NMR solvent (CDCl3, δ 7.24 ppm). Coupling constant (J) was expressed in Hz. Infrared spectra (FT-IR) were obtained using a Bruker Vector 22 spectrometer (Bruker, Billerica, MA, USA); data are presented as the frequency of absorption (cm −1 ). High-resolution mass spectrometry (HRMS) spectra were recorded with Micromass Q-TOF micro mass spectrometer (Waters Corporations, Milford, MA, USA) and Micromass LCT (ESI, Waters Corporations, Milford, MA, USA) with Lock-Spray-Injector (Injection Loop-Modus in a HPLC system, Waters, Alliance 2695). UV-Vis measurements were performed with a Shimadzu-UV-2401PC spectrophotometer. Cyclic Voltammetry measurements were acquired on a AMEL552 electrochemical workstation. The standard three-electrode arrangement was employed. In all case, a Pt wire auxiliary electrode was used, the working electrode was a 3 mm diameter glassy carbon, and the solution was degassed with N2.
Melting points were determined on a Mel-Temp apparatus.).

General Procedure for the Lewis-acid-catalysed Friedel-Crafts/lactonization reaction
The alkylating agent (2.2 mmol) was added in one portion to a stirred solution of the appropriate phenol (2.0 mmol) in anhydrous CHCl3 (9 mL), and then TiCl4 (1 M in anhydrous CH2Cl2; 0.4 mL, 10 mol-%) was added.
The system was kept under an argon atmosphere. The clear reddish solution was stirred at the reported temperature until the substrate had been completely consumed (TLC monitoring). Afterwards, the reaction mixture was poured into cold water (18 mL), and the aqueous phase was extracted several times with EtOAc (4 x 20 mL). The combined organic layers were washed with brine, dried with anhydrous Na2SO4, and concentrated under vacuum. The residue was purified by flash chromatography on silica gel to give the products as described below.

Characterization Data for benzofuran 9-11
Following the general procedure, the single product 9 was obtained as a white solid in 70% yield after purification by flash chromatography on silica gel (nHexane/EtOAc=7/3

General Procedure for the acid-catalysed Friedel-Crafts/lactonization reaction
The alkylating agent (5.5 mmol) was added in one portion to a stirred solution of the appropriate phenol (5.0 mmol) in acetic acid (3 mL). The system was kept under an argon atmosphere. The clear reddish solution was stirred at reflux temperature, until the substrate had been completely consumed (TLC and HPLC monitoring). Afterwards, the reaction mixture was concentrated under vacuum and the residue was purified by flash chromatography on silica gel to give the products as described below.

Calibration curves
From a stock solution of 250 μM DPPH • in MeOH or ACN, previously degassed with a N2 flow, ten standards at different concentrations from 0 to 110 μM were prepared. The absorbance (λmax = 517 nm) for each standard was measured in a Cary300 spectrophotometer or Shimadzu-UV-2401PC spectrophotometer and plotted versus the concentration of DPPH • . Figure

DPPH assay in methanol
Once the linearity of the monitored method at 517 nm was evaluate by the calibration curve of DPPH • concentration versus absorbance at 517 nm, 2 mL of 140 μM DPPH • stock solution (70 μM DPPH final concentration) were added to the degassed methanol solutions (2mL) of antioxidant, with a final concentration from 0 to 60 μM. After 60 minutes stirring at room temperature in the absence of light, the absorbance was measured at 517 nm. Figure SM

DPPH assay in acetonitrile
The assay was performed as described above, using as solvent the acetonitrile (ACN). Once the linearity of the monitored method at 517 nm was evaluate in this solvent through the calibration curve of DPPH • concentration versus absorbance at 517 nm, the absorbance measurements of each solution, containing a known concentration of antioxidant and DPPH • , were collected. Figure   Sequentially Table SM-3 and Table SM-4 show the corresponding regression lines with the values of R 2 as well as all the deducible parameters from rIC50.  The procedure used to determine k s was common to all solvents and compounds. A solution of DPPH • and the compound were prepared in nitrogen-purged solvents. Then, the decay of DPPH • in the presence of a known concentration of compound was followed at 517 nm on a Shimadzu-UV-2401PC spectrophotometer. The concentration of DPPH • was 8.5*10 -5 M and the compounds were always used in large excess over [DPPH • ]. The decay of the DPPH • absorbance was analysed as pseudo-first-order processes to yield kex/s -1 , using Sigma plot for Windows. The measurements were performed in triplicate. Plots of kex vs compound concentration is linear and its slopes gave the second-order rate constants, k s . The observed results are reported in Table SM-5.