Acetamide Derivatives with Antioxidant Activity and Potential Anti-Inflammatory Activity

This study reports the synthesis and antioxidant activity of some new acetamide derivatives. The compounds’ structures were elucidated by NMR analysis and their melting points were measured. The in vitro antioxidant activity of these compounds was tested by evaluating the amount of scavenged ABTS radical and estimating ROS and NO production in tBOH- or LPS-stimulated J774.A1 macrophages. All compounds were tested for their effect on cell viability by an MTT assay and by a Brine Shrimp Test.


Introduction
The antioxidant activity is related with compounds capable of protecting a biological system against the potential harmful effects of oxidative processes. In the last years antioxidant compounds have OPEN ACCESS received increased attention from nutritionists and medical researchers for their potential activities in preventing cancer, cardiovascular disorders, as well as aging [1]. Several papers have been published on structure-activities analysis of new antioxidant compounds. In this study we report for the first time the synthesis and the in vitro antinflammatory and antioxidant activities of some new acetamide derivatives. The syntheses of the compounds 30000-4 and 6 (as reported in Scheme 1) were accomplished using 2-phenylacetic acid derivatives (1 eq) dissolved in dry dichloromethane (DCM, 10 mL). To this solution were added N,N 1 -carbonyldiimidazole (CDI, 2 eq) and 4-dimethylaminopyridine (DMAP, 2 eq) and the mixture was stirred for 20 minutes at rt. After an appropriate 3-phenylpropylamine (2 eq) was added and the mixture further stirred for 5 h at rt. The synthesis of compound 30005 (Scheme 2) was achieved using 2-(4-methoxyphenyl)-2-phenyl-N- (3-phenylpropyl)acetamide (1 eq) and sodium iodide (2.2 eq) dissolved in acetonitrile (15 mL). Chlorotrimethylsilane (2.2 eq) was added to the solution which was stirred for 16 h at reflux. The synthesis of compound 30007 (Scheme 3) was accomplished using 2-(4-nitrophenyl)-2-phenyl-N- (3-phenylpropyl) acetamide (1 eq) and zinc powder (10 eq) dissolved in EtOH (10 mL). Conc. HCl (1 eq) was added to the solution and cooled to -10 °C.

Results and Discussion
The brine shrimp lethality bioassay is an efficient, rapid and inexpensive test developed as a prescreen to evaluate biological activities of natural or synthetic compounds. In Table 1 we report the biological activity of the samples in the brine shrimp assay, expressed as LD 50 (in ppm). All compounds showed good activity in this test, in particular compounds 40006 and 40007, which showed interesting LD 50 values of 3.0443 and 10.6444 ppm, respectively. None of the newly synthesized compounds were cytotoxic on macrophage cell line J774.A1, as revealed by a MTT test ( Table 2). Among the tested compounds we choose to further investigate the pharmacological activity of 40006 and 40007 because of their good results in the ABTS and brine shrimp test. LPS increased NO production in the medium of J774.A1 macrophages, addition to the culture medium of graded concentration of 40006 and 40007 (0.1-10 µM) 1 h before and simultaneously to LPS significantly (P < 0.001) reduced NO production ( Table 3).   85.00 ± 3.51** 89.6 ± 1.46** 81.5 ± 12.67 ** As reported in Figure 1, compound 40006 also significantly (P < 0.01) reduced ROS production in J774.A1 macrophages stimulated with terz-butyl hyproperoxide (tBOH), while compound 40007 have no effect on ROS inhibition.

General
All reagents were purchased from Sigma-Aldrich s.r.l. (Milan, Italy). The reactions were monitored by thin-layer chromatography (TLC), using Whatman K6F silica gel on aluminum and alumina (Merck) plates with fluorescence indicators and appropriate solvents. A >95% purity could be inferred from the 1 H-NMR spectra. Melting points were taken on a Gallenkamp melting point apparatus and are uncorrected. The organic extracts were dried over sodium sulphate dry (Merck). 1 H-NMR spectra were recorded with a Bruker Advance 300 MHz spectrometer, using CDCl 3 as solvent. Mass spectrometry analysis ESI-MS was cwrried out on a Finningan LCQ Deca ion trap instrument.

Synthesis of compounds 30000-4 and 6
An appropriate acid was dissolved in dry DCM (10 mL). To this solution were added N,N 1carbonyldiimidazole (CDI, 0.612 g; 3.78 mmol; FW = 162.15; 2 eq) and 4-dimethylaminopyridine (DMAP, 0.115 g; 0.94 mmol; FW = 122.17; 2 eq) and the resulting mixture was stirred for 20 minutes at rt. After 3-phenylpropylamine (0.511 g; 3.78 mmol; FW = 135.21; 2 eq) was added and the mixture stirred for 5 h at rt [2]. To the mixture was added H 2 SO 4 (2N) and it was extracted with ethyl acetate. The organic layer was dried with anhydrous sodium sulphate, filtered and the solvent removed by evaporation. The residue was purified by column chromatography on silica (Pe/EtOAc 6:4;Pe/EtOAc 4:6). After recrystallization from DCM/Pe, the products were obtained as white crystals

Antioxidant activity assay by ABTS method
The antioxidant activity of compounds was determined by ABTS method, as previously described [3][4]. This method is based on the capacity of different components to scavenge the ABTS radical cation (ABTS • +) compared to standard antioxidant (Trolox) in dose response curves. Briefly ABTS • + radical cation is obtained by reacting ABTS (7 mM) with potassium persulphate; this mixture was stored in the dark for 12-16 h before use. Before the assay, the mixture was diluted in ethanol at a ratio 1:100 to give an absorbance at λ = 734 nm of 0.70 + 0.02. TROLOX was used as standard at a concentration of 1 mg/mL and aliquots of Trolox (0.5 μL, 1 μL, 2 μL, 3 μL, 5 μL and 10 μL ) were added to 1 mL of ethanolic ABTS • + to have a standard curve to which all data are referred. All compounds were dissolved in dichloromethane at a concentration of 20 mg/mL and 5 μL were added to ethanolic ABTS • + to measure absorbance after 1 min.
Antioxidant activity was carried out in triplicate and expressed as percentage of the absorbance of the uninhibited radical solution according to the equation: % inhibition (λ = 734 nm) = ( 1-Abs c /Abs 0 ) × 100 where Abs 0 was the absorbance of uninhibited radical solution and Abs c is the absorbance measured 1 min after addition of compound to assay. The antioxidant activity of samples was expressed also as T.E.A.C. (Trolox Equivalent Antioxidant Capacity -μM) [5].

Brine Shrimps Test
The brine shrimps (Artemia salina) assay was performed in triplicate with appropriate amounts of samples dissolved in DMSO to reach final concentrations of 1, 10 and 100 ppm, using 10 freshly hatched larvae suspended in 5 mL of artificial sea water [6]. Briefly, for each dose tested, surviving shrimps were counted after 24 h, and the data analyzed by the Finney program [7], which affords LD 50 values with 95% confidence intervals.
J774.A1 (3.5 × 10 4 cells/well) were plated on 96-well microtiter plates and allowed to adhere at 37 °C in a 5% CO 2 atmosphere for 2 h. To stimulate the expression of the inducible form of nitric oxide synthase (iNOS), E. coli lipopolysaccharide (LPS, 6 × 10 3 u/mL) was added to fresh culture medium of J774.A1 with graded concentrations of tested compounds (1-100 μg/mL) added 1h before and simultaneously with LPS challenge. Nitric oxide release, evaluated as nitrite (NO 2 -) accumulation in cell culture medium, was evaluated 24 h after LPS stimulation by Griess reagent [8].
J774.A1, WEHI-164 and HEK-293 (3.5 × 10 4 cells/well) were plated on 96-well microtiter plates and allowed to adhere at 37 °C in a 5 % CO 2 atmosphere for 2 h. Thereafter, the medium was replaced with fresh one (50 μL) and a 75 μL aliquot of each tested compound was added and then the cells incubated for further 72 h. 6-mercaptopurine was used as reference drug. Mitochondrial respiration, an indicator of cell viability, was assessed by the mitochondrial-dependent reduction of [3-(4,5-dimethylthiazol-2-yl)-2,5-phenyl-2H-tetrazolium bromide] (MTT) to formazan and cells viability was assessed accordingly to the method of Mosmann [9]. Briefly 5 μL of MTT (5 mg/mL) were added and the cells were incubated for an additional 3 h. Thereafter, cells were lysed and the dark blue crystals solubilised with 100 μL of a solution containing 50% (v:v) N, N-dimethylformamide, 20 % (w:v) SDS with an adjusted pH of 4.5 [10]. The optical density (OD) of each well was measured with a microplate spectrophotometer (Titertek Multiskan MCC/340) equipped with a 620 nm filter. The viability of each cell line in response to treatment with tested compounds and 6-mercaptopurine was calculated as: % dead cells = 100−(OD treated/OD control) × 100.

Measurement of Reactive Oxygen Species (ROS)
The accumulation of ROS was evaluated by means of the probe 2',7'-dichlorofluorescein (DCF) according to the method described by Le Bel et al. [11]. Briefly, J774.A1 cells were seeded at a density of 5 × 10 3 cells/well into 96-well plates and allowed to adhere for 2 h. After cell adhesion, examined compounds (1-100 μg/mL) were added to the culture medium 24 h before and always simultaneously to dichlorofluorescein-diacetate. 2',7'-Dichlorofluorescein-diacetate (H 2 DCF-DA, Sigma) was added directly to the growth medium at a final concentration of 5 µM and the cells incubated for 1 h at 37 °C. H 2 DCF-DA is a non-fluorescent permeant molecule which diffuses passively into cells; the acetates are then cleaved by intracellular esterases to form H 2 DCF which is thereby trapped within the cell. In the presence of intracellular ROS, H 2 DCF is rapidly oxidized to the highly fluorescent DCF. Therefore, cells were washed twice with phosphate-buffered saline (PBS), placed in fresh medium and treated with terz-butyl hyproperoxide (tBOH) 3 mM for 30 min and than were placed in a fluorescent microplate reader (LS 55 Luminescence Spectrometer; Perkin Elmer, Beaconsfield, Bucks, UK). Fluorescence was monitored using an excitation wavelength of 490 nm and an emission wavelength of 520 nm. Results were expressed as Relative Fluorescence Units (RFU).

Conclusions
Results show that these acetamide derivatives, obtained by simple and rapid chemical synthesis, had interesting biological activities and that none of the compounds are toxic on J774.A1, the cell line chosen for the other tests. Among the tested compounds, 40006 showed the more interesting activity because of its inhibition of nitrite and ROS. Taken together, our data let us hypothesized that the inhibitory effect of compound 40006 on t-BuOH-induced NO formation in J774.A1 macrophages could be due to its free radical scavenger properties. Moreover, to have an antioxidant activity, is possible to modify the aromatic function binding to the amidic group, instead is necessary to keep unvaried the aromatic function binding to the alkylic chain. In fact the compound 40006 is more active than the compound 30006. Further studies need to characterize its pharmacological activity.