Design and Synthesis of π-Extended Resveratrol Analogues and In Vitro Antioxidant and Anti-Inflammatory Activity Evaluation

The research on resveratrol (1) has been conducted intensively over a long time due to its proven antioxidant activity and disease-fighting capabilities. Many efforts have also been made to increase these biological effects. In the present study, six new extended aromatic resveratrol analogues containing naphthalene (2) and its bioisosteres quinoline (3 and 4), isoquinoline (5) quinoxaline (6) and quinazoline (7) scaffolds were designed and synthesized using an annulation strategy. The antioxidant and anti-inflammatory activities of these compounds were investigated. All compounds showed better antioxidant activity than resveratrol in ABTS assay. As for the anti-inflammatory test, 5 and 7 exhibited better activity than resveratrol. It is worth noting that nitrogen substitution on the extended aromatic resveratrol analogues has a significant impact on cell viability. Taking the antioxidant activities and NO inhibition activities into consideration, we conclude that isoquinoline analogue 5 may qualify for the further investigation of antioxidant and anti-inflammatory therapy. Furthermore, our study results suggest that in order to improve the biological activity of polyphenolic compounds, extended aromaticity and nitrogen substitution strategy could be a viable method for the design of future drug candidates.


20
To a stirred solution of compound 33 (0.13 g, 0.24 mmol) in anhyd. CH2Cl2 (5 mL) was added BBr3 (1.0 M in CH2Cl2, 0.95 mL) dropwise at -20 ºC under argon atmosphere. The reaction mixture was warmed to room temperature and stirred for 2 h. After completion of the reaction, cool to 0 ºC, MeOH (2 mL) was added dropwise to quench the excess BBr3, warmed to room temperature in 20 min and the solvent was removed under reduced pressure. H2O (10 mL) was added to the crude residue and extracted with EtOAc (3 x 35 mL). The combined organic layer was washed with H2O (3 x 15 mL) and brine (15 mL), dried over anhyd.

Antioxidant assay using ABTS
The radical cation was prepared by mixing an equal amount of 7mM ABTS (2,2'-azino-bis(3ethylbenzothiazoline-6-sulphonic acid)) stock solution with 2.45 mM potassium persulfate stock solution. This mixture was stored at 0˚C for 12 hours under darkness. The above ABTS solution was diluted with methanol appropriately to give UV absorption value of 1.000 at the 734 nm. The compounds 1-7 were dissolved in methanol to prepare 1000 µM stock solutions.
Then these solutions were diluted with methanol to 500 µM, 250 µM, 125 µM, 62.5 µM, 31.25 µM, 15.63 µM, 7.81 µM, 3.90 µM, 1.95 µM, and 0.97 µM. In different 3 sets of test tubes, 0.9 mL of the ABTS solution and 0.1 mL of the compound solution were mixed under darkness. After 30 min of incubation, the UV absorption at 734 nm was measured. Control was used the mixture of 0.9 mL ABTS and 0.1 mL methanol. The radical scavenging rates were obtained from these UV absorption data and the resulting IC50 values were calculated using Origin 8.

NO assay and cell viability test
Murine macrophage, Raw 264.7 cells were grown in Dulbecco's modified Eagle's medium (Gibco, Carlsbad , CA, USA), supplemented with 2 mM glutamine, antibiotics (100 U/mL of penicillin A and 100 mg/mL of streptomycin) and 10% FBS (Gibco). Cells were cultured in 96-well plates (5×10 4 cells), treated with serial dilutions of compound (3.125-100 M) and stimulated with LPS (1 g/mL) for 24 h at 37°C. After incubation, the supernatant was collected and the amount of nitrite was quantified using the Griess Reagent System (Promega, Fitchburg, WI, USA). For the correction of NO production, cell viability was measured using the CellTiter 96 ® Aqueous one solution cell proliferation assay kit (Promega). Cells were incubated with varying amounts of compound for 24 h, then MTS (tetrazolium salt) reagent was added to the plates. After 1h, the absorbance at 490 nm was read using a microplate reader (Thermo Scientific, Waltham, MA, USA).

Statistical analysis.
Statistical significance was analyzed using the GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA). The comparison of two groups was performed by the unpaired t test. Values of *P less than 0.05, **P less than 0.01 and ***P less than 0.001 were considered statistically significant.