Acetamide derivatives with antioxidant activity and potential anti-inflammatory activity

Article abstract of DOI:10.3390/molecules15032028

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

Full text of DOI:10.3390/molecules15032028

Molecules 2010, 15, 2028-2038; doi:10.3390/molecules15032028
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Acetamide Derivatives with Antioxidant Activity and Potential
Anti-Inflammatory Activity
Giuseppina Autore 1, Anna Caruso 1, Stefania Marzocco 1, Barbara Nicolaus 2, Chiara Palladino 1,
Aldo Pinto 1, Ada Popolo 1, Maria S. Sinicropi 3, Giuseppina Tommonaro 2 and
Carmela Saturnino 1,*
1
Department of Pharmaceutical Sciences, University of Salerno, Fisciano (SA), Italy;
E-Mails: autore@unisa.it (G.A.); caruso.anna@inwind.it (A.C.); smarzocco@unisa.it (S.M.);
chiara24@hotmail.it (C.P.); pintoal@unisa.it (A.P.); apopolo@unisa.it (A.P.)
2
Institute of Biomolecular Chemistry, CNR, Pozzuoli (NA), Italy;
E-Mails: gtommonaro@icmib.na.cnr.it (G.T.); bnicolaus@icmib.na.cnr.it (B.N.)
3
Department of Pharmaceutical Sciences, University of Cosenza, Arcavacate di Rende (CS), Italy;
E-Mail: s.sinicropi@unical.it (M-S.S.)
* Author to whom correspondence should be addressed; E-Mail: saturnino@unisa.it;
Tel.: +39-89-969769; Fax: +39-89-969602.
Received: 2 February 2010; in revised form: 9 March 2010 / Accepted: 18 March 2010 /
Published: 23 March 2010
Abstract: 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.
Keywords: amides; antioxidant; antiproliferative activity; 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

Molecules 2010, 15
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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,N1-carbonyldiimidazole (CDI, 2 eq) and 4-dimethylaminopyridine
(DMAP, 2 eq) and the mixture was stirred for 20 minutes at rt. After an appropriate 3-phenyl-
propylamine (2 eq) was added and the mixture further stirred for 5 h at rt.
Scheme 1. Synthesis of compounds 30000-4 and 6.
R
O
3-phenylpropylamine
OH
CDI
DMAP
H
N
DCM
O
R = H 30000; R = Cl 30001; R = Br 30002; R = CH3 30003; R = OCH3 30004; R = NO2 30006.
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.
Scheme 2. Synthesis of 2-(4-hydroxyphenyl)-2-phenyl-N-(3-phenylpropyl)acetamide (30005).
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.
The synthesis of the compounds 40000-4 and 6 (Scheme 4) were done using 2-phenylacetic acid
derivatives (1 eq) dissolved in dry DCM (10 mL); afterwards CDI (2 eq) and DMAP (0.5 eq) were
added to the solution that was stirred for 20 minutes at rt. Thereafter was added 3,3-diphenylpropan-1-
amine (1 eq) and the mixture stirred for 24 h at rt.

Molecules 2010, 15
Scheme 3. Synthesis of 2-(aminophenyl)-2-phenyl-N-(3-phenylpropyl)acetamide (30007).
2030
Scheme 4. Synthesis of compounds 40000-4 and 6.
OH
H2N
O
R
R = H 40000; R = Cl 40001; R = Br 40002; R = CH3 40003; R = OCH3 40004; R = NO2 40006
The synthesis of compound 40005 (Scheme 5) was achieved using N-(3,3-diphenylpropyl)-2-(4-
methoxyphenyl)acetamide (1 eq) and sodium iodide (3 eq) dissolved in acetonitrile (35 mL). To the
solution heated to 23 °C was added chlorotrimethylsilane (2.2 eq) and the mixture was stirred for 16 h
at reflux.
Scheme 5. Synthesis of N-(3,3-diphenylpropyl)-2-(4-hydroxyphenyl)acetamide (40005).
H
N
TMS-Cl
NaI
CH3CN
O
HO
The synthesis of compound 40007 (Scheme 6) was accomplished using N-(3,3-diphenylpropyl)-2-
(4-nitrophenyl) acetamide (1 eq) and zinc powder (10 eq) dissolved in EtOH (40 mL). Conc. HCl
(1 eq) was added to the solution and it was cooled to -10 °C.
Scheme 6. Synthesis of 2-(4-aminophenyl)-N-(3,3-diphenylpropyl) acetamide (40007).

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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 LD50 (in ppm). All
compounds showed good activity in this test, in particular compounds 40006 and 40007, which
showed interesting LD50 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).
Table 1. Biological activity of compounds 30003-30007 and 40003-40007. The
experiments were performed in triplicate.
LD50 (ppm)
SAMPLES % INHIBITION ABTS.+ T.E.A.C. (μM)
[95% confidence intervals]
30003
30004
30005
1%
2%
0.26
0.52
0.20
n.a.
n.a.
0.8%
n.a.
41.4591
30006
30007
40003
40004
40005
40006
40007
1%
10.6%
n.a.
0.26
2.74
/
[84.3018/23.5460]
29.8785
[45.6981/19.8115]
58.3200
[132.3034/32.7936]
17.8661
2%
0.52
1.32
1.03
6.77
[28.9967/11.1125]
17.0035
5.1%
4%
[28.5947/10.2798]
3.0443
[4.7830/1.9283]
10.6444
26.2%
[16.8533/6.6340]
Table 2. Shows the results, expressed as IC50 (M) values, indicating the concentration of
each compound that affords cell growth by 50% as compared to control cells. n.d. = not
detectable.
Cell lines
Compounds
30003
HEK-293
n.d.
1.067 × 10-4
4.170 × 10-5
2.439 × 10-6
WHEI-164
n.d.
1.790 × 10-6
2.235 ×x 10-6
1.913 × 10-4
J774.A1
n.d.
30004
n.d.
30005
n.d..
30006
n.d..

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Table 2. Cont.
Cell lines
Compounds
HEK-293
n.d.
WHEI-164
1.513 × 10-4
2.441 × 10-6
n.d.
1.038 × 10-6
2.05 × 10-5
4.124 × 10-5
J774.A1
n.d.
30007
40003
40004
40005
40006
40007
2.5 × 10-4
n.d.
> 10-4
2.324 × 10-4
> 10-4
2.324 × 10-4
> 10-4
1.665 × 10-4
> 10-4
2.069 × 10-5
Table 3. Effect of compounds 40006 and 40007 on nitrite production by LPS- induced
J774.A1 macrophages. Results are expressed as mean ± S.E.M. % inhibition calculated
versus nitrite produced by J774.A1 treated with LPS alone of at least 3 independents
experiments with three replicates each. Comparison was performed using one way analysis
of variance. ** P < 0.01 vs. LPS.
% inhibition nitrite production vs LPS
Compounds
40006
10 µM
79.66 ± 1.33**
85.00 ± 3.51**
1 μM
86.75 ± 0.65**
89.6 ± 1.46**
0.1 μM
91.73 ± 1.59 **
81.5 ± 12.67 **
40007
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.
Figure 1. Effect of compounds 40006 and 40007 on ROS production by tBOH-induced
J774.A1 macrophages. ROS reaction was determined by measuring the converting reaction
of DCFH2 to DCF. ROS production were expressed as mean ± S.E.M. of relative
fluorescent units (RFU) of at least three independent experiments. ** P < 0.01 vs. tBOH.
100
75
50
**
**
25
0
**
tBOH 0.1µM
1µM
10µM 0.1µM
1µM
10 µM
40006
40007
Experimental
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

Molecules 2010, 15
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(Merck) plates with fluorescence indicators and appropriate solvents. A >95% purity could be inferred
from the 1H-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). 1H-NMR spectra were
recorded with a Bruker Advance 300 MHz spectrometer, using CDCl3 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,N1-
carbonyldiimidazole (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 H2SO4 (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
Compound 30000: Yield: 0.390 g (64%); Melting point: 135 °C; MS: m/z 331 (M+ + 2); 329 (M+); 1H-
NMR δ 8.0 (s, 1H), 7.39-6.94 (m, 15H), 5.58-5.43 (s, 1H), 3.53-3.30 (t, 2H), 2.68-2.56 (t, 2H), 1.89-
1.77 (q, 2H).
Compound 30001: Yield: 0.650 g (70%); Melting point: 125 °C; MS: [M] = 363.9; [M +H+] = 364.9;
1H-NMR δ 7.39-7.04 (m, 14 H), 5.60-5.53 (s, 1H), 4.88 (s, 1H), 3.53-3.37 (q, 2H), 2.68-2.60 (t, 2H),
1.89-1.77 (q, 2H
Compound 30002: Yield: 0.210 g (81%); Melting point: 135 °C; MS: [M – H+] = 407.1; [M + H+] =
1
409.1; H-NMR δ 7.50-7.46 (d, 2H), 7.37-7.10 (m, 12 H), 5.57 (s, 1 H), 4.48 (s, 1 H), 3.40-3.30 (q,
2H), 2.64-2.59 (t, 2H), 1.96-1.82 (p, 2H).
Compound 30003: Yield: 0.640 g (71%); Melting point: 110 °C; MS: (M) 342.2, (m + H+) 345.2; 1H-
NMR δ 7.62-7.21 (m, 12 H), 7.18-7.10 (d, 2 H), 5.57 (s, 1 H), 4.90 (s, 1H), 3.38-3.30 (q, 2 H), 2.63-
2.57 (t, 2 H), 1.88-1.77 (p, 2 H).
Compound 30004: Yield: 0.640 g (78%); Melting point: 110 °C; MS: [M] = 359.5; [M+H+] = 360.5;
1H-NMR δ 7.64-7.20 (m, 12H), 6.92 (d, 2H), 5.58 (s, 1H), 4.88 (s, 1 H), 3.87 (s, 3 H), 3.40-3.30 (q, 2
H), 2.64-3.57 (t, 2 H), 1.89-1.76 (p, 2 H).
Compound 30006: Yield: 0.600 g (78%); Melting point: 117 °C; MS: [M] = 374.4; [M +H+] = 375.4;
1H-NMR δ 8.40 (d, 2H); 7.70 (d, 2H); 7.45-7.10 (m, 10H); 5.5 (s, 1H); 4.87 (s, 1H); 3.92-3.85 (t, 2H);
3.35-3.28 (q, 2H); 2.34-2.22 (q, 2H).

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Synthesis of compounds 30005 and 30007
2-(4-Methoxyphenyl)-2-phenyl-N-(3-phenylpropyl)acetamide (0.480 g; 1.34 mmol; FW = 359.5; 1
eq) and sodium iodide (0.440 g; 2.95 mmol; FW = 149.89; 2.2 eq) were dissolved in acetonitrile
(15 mL). Chlorotrimethylsilane (377 μL; 2.95 mmol; FW = 108.64; d = 0.850; 2.2 eq) was added to
the solution and stirred for 16 h at reflux. The mixture was poured into water and extracted with ethyl
acetate, the organic layer was washed with Na2S3O3 and Brine, dried with anhydrous sodium sulphate,
filtered and evaporated. The residue products were purified by column chromatography on silica
(Pe/EtOAc 8:2; Pe/EtOAc 7:3). After recrystallization with DCM/Pe , white crystals were obtained
(0.310 g; 66%). Melting point: 85 °C. m/z 347.2 (M++ 2); 345.2 (M+). 1H NMR δ 8.80-7.18 (m, 10H);
7.10-7.00 (d, 2H); 6.90-6.77 (d, 2H); 5.67 (s, 1H); 4.82 (s, 1H); 3.89-3.80 (t, 1H); 3.33-3.24 (q, 2H);
2.30-2.20 (q, 2H) (30005) .
2-(4-nitrophenyl)-2-phenyl-N-(3-phenylpropyl) acetamide (0.200 g; 0.53 mmol; FW = 450.5; 1 eq)
and zinc powder (0.346 g; 5.3 mmol; FW = 63.93; 10 eq) were dissolved in EtOH (10 mL). HCl conc.
(3 mL; 0.53 mmol; FW = 36.46; 1 eq) was added to the solution and cooled to -10 °C. The mixture
was stirred and heated at 80 °C for 3h. After was poured into water and NaOH to neutralize the excess
of acid, the organic layer was extracted with Et2O, dried with anhydrous sodium sulphate, filtered and
removed by evaporation. The residue products were purified by column chromatography on silica
(Pe/EtOAc 1:1). A yellow-orange oil was obtained (0.160 g; 88%). m/z 346.4 (M++ 2); 344.4 (M+). 1H
NMR (CDCl3, 300 Mhz) δ 7.37-7.19 (m, 8H); 7.15 (d, 2H); 7.05 (d, 2H), 6.68 (d, 2H); 5.59 (s, 1H);
4.48 (s, 1H); 3.68 (s, 2H); 3.36 (q, 2H); 2.63 (t, 2H); 2.10 (p, 2H) (30007).
Synthesis of compounds 40000-4 and 40006
An appropriate acid was dissolved in DCM dry (10 mL) and N1-carbonyldiimidazole CDI (0.541 g;
4.43 mmol; FW = 162.15; 2 eq) and 4-dimethylaminopyridin DMAP (0.134; 1.10 mmol; FW =
122.17; 0.5 eq) were added to the solution and stirred for 20 minutes at rt. Thereafter was added 3,3-
diphenylpropan-1-amine (0.936 g; 4.43 mmol; FW = 211.3; 1 eq) and stirred for 24h at rt. To the
mixture was added H2SO4 (2N) and extracted with ethyl acetate. The organic layer was dried with
anhydrous sodium sulphate, filtered and removed by evaporation. The residue products were purified
by column chromatography on silica (Pe/EtOAc 9:1; Pe/EtOAc 8:2; Pe/EtOAc 7:3; Pe/EtOAc 6:4).
After recrystallization with DCM/Pe, white crystals were obtained (0.620 g; 85%). [M] = 329; [M+H+]
= 330. 1H NMR (CDCl3, 300 Mhz) δ 8.0 (s, 1H), 7.41-7.06 (m, 15H), 4.10-4.05 (t, 1H), 3.44 (s, 2H),
3.33-3.20 (t, 2H), 2.19-2.11 (q, 2H) (40000).
After recrystallization with DCM/Pe, white crystals were obtained (0.750 g; 71%). [M] = 363;
[M+H+] = 364. 1H NMR ( CDCl3, 300 Mhz) δ 7.64-6.94 (m, 14H), 5.27 (s, 1H), 3.91 (t, 1H), 3.47 (s,
2H), 3.27-3.16 (t, 2H), 2.29-2.18 (q, 2H) (40001).
After recrystallization with DCM/Pe, white crystals were obtained (0.700 g; 75%). [M] = 407; [M +
H+] = 408. 1H NMR (CDCl3, 300 Mhz) β 7.80 (d, 2H), 7.32-7.12 (m, 10H), 6.96 (d, 2H), 5.33 (s, 1H),
3.90 (t, 1H), 3.46 (s, 2H), 3.27-3.16 (t, 2H), 2.29-2.19 (q, 2H) (40002).
After recrystallization with DCM/Pe, white crystals were obtained (0.930 g; 82%). Melting point:
110 °C. [M] = 343.5; [M+H+] = 344.5. 1H NMR (CDCl3, 300 Mhz) δ 7.30-7.16 (m, 14H), 5.30 (s, 1H),
3.87 (t, 1H), 3.50 (s, 2H), 3.21 (q, 2H), 2.25 (q, 2H) (40003).

Molecules 2010, 15
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After recrystallization with DCM/Pe, white crystals were obtained (80%). [M] = 343.5; [M+H+] =
344.5. 1H NMR (CDCl3, 300 Mhz) δ 7.30- 7.16 (m, 14H), 5.30 (s, 1H), 3.87 (t, 1H), 3.50 (s, 2H), 3.21
(q, 2H), 2.25 (q, 2H) (40004).
After recrystallization with DCM/Pe, white crystals were obtained (80%). [M] = 343.5; [M + H+] =
344.5. 1H NMR (CDCl3, 300 Mhz) δ 7.30-7.16 (m, 14H), 5.30 (s, 1H), 3.87 (t, 1H), 3.50 (s, 2H), 3.21
(q, 2H), 2.25 (q, 2H) (40006).
Synthesis of compounds 40005 and 40007
N-(3,3-diphenylpropyl)-2-(4-methoxyphenyl)acetamide (1.2 g; 3.4 mmol; FW = 359.5; 1 eq) and
sodium iodide (1.5 g; 10.2 mmol; FW = 149.89; 3 eq) were dissolved in acetonitrile (35 mL). To the
solution heated to 23 °C was added chlortrimethylsilane (1.1 mL; 10.2 mmol; FW = 108.64; d = 0.850;
2.2 eq) and stirred for 16h at reflux. The mixture was poured into water and extracted with
ethoxyethane, the organic layer was washed with Na2S3O3 and Brine, dried with anhydrous sodium
sulphate, filtered and evaporated. The residue products were purified by column chromatography on
silica (Pe/EtOAc 8:2; Pe/EtOAc 7:3; Pe/EtOAc 6:4). After recrystallization with DCM/Pe, white
crystals were obtained (0.510 g; 45%). Melting point: 85 °C. m/z 347.2 (M++ 2); 345.2 (M+). 1H NMR
(CDCl3, 300 Mhz) δ 8.80-7.18 (m, 10H), 7.10-7.00 (d, 2H), 6.90-6.77 (d, 2H), 5.67 (s, 1H), 4.82 (a,
1H), 3.89-3.80 (t, 1H), 3.33-3.24 (q, 2H), 2.30-2.20 (q, 2H) (40005).
N-(3,3-diphenylpropyl)-2-(4-nitrophenyl) acetamide (1.00 g; 2.67 mmol; FW = 374.4; 1 eq) and
zinc powder (1.75 g; 26.7 mmol; FW = 65.41; 10 eq) were dissolved in EtOH (40 mL). HCl conc.
(5 mL; 0.53 mmol; FW = 36.46; 1 eq) was added to the solution and cooled to -10 °C. The mixture
was stirred and heated at 80 °C for 3h. Thereafter was poured into water and NaOH to neutralize the
excess of acid, the organic layer was extracted with Et2O, dried with anhydrous sodium sulphate,
filtered and removed by evaporation. The residue products were purified by column chromatography
on silica (Pe/EtOAc 1:1). Was obtained a yellow-orange oil (0.900 g; 97%). m/z 346.4 (M+ + 2); 344.4
(M+). 1H NMR (CDCl3, 300 Mhz), δ 7.37-7.19 (d, 2H), 7.15 (d, 2H), 7.05 (m, 8H), 6.68 (d, 2H), 5.59
(s, 1H), 4.84 (s, 1H), 3.68 (s, 2H), 3.36 (q, 2H), 2.63 (t, 2H), 2.10 (q, 2H) (40007).
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:

Molecules 2010, 15
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% inhibition (λ = 734 nm) = ( 1- Absc/Abs0) × 100
where Abs0 was the absorbance of uninhibited radical solution and Absc 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 LD50
values with 95% confidence intervals.
-
Analysis of NO2 Production
The murine macrophage cell line, J774.A1, was obtained from American Tissue Culture Collection
(ATCC). J774.A1 cells were maintained in DMEM supplemented with NaHCO3 (42 mM), hepes
(25 mM), penicillin (100 units/mL), streptomycin (100 units/mL), glutamine (2 mM) and foetal calf
serum (FCS, 10%) at 37 °C in a 95% air and 5% CO2 atmosphere.
J774.A1 (3.5 × 104 cells/well) were plated on 96-well microtiter plates and allowed to adhere at
37 °C in a 5% CO2 atmosphere for 2 h. To stimulate the expression of the inducible form of nitric
oxide synthase (iNOS), E. coli lipopolysaccharide (LPS, 6 × 103 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 (NO2 ) accumulation
in cell culture medium, was evaluated 24 h after LPS stimulation by Griess reagent [8].
MTT Assay for Antiproliferative Activity
Murine macrophage cells (J774.A1) were maintained as previously indicated. Murine fibrosarcoma
cells (WEHI-164) were maintained in adhesion on Petri dishes with DMEM supplemented with 10%
heat-inactivated FCS, hepes (25 mM), penicillin (100 u/mL) and streptomycin (100 μg/mL). Human
embryonic kidney cells (HEK-293) were maintained and grown in adhesion on Petri dishes with
DMEM supplemented with FCS (10 %), hepes (25 mM), penicillin (100 u/mL) and streptomycin
(100 units/mL).
J774.A1, WEHI-164 and HEK-293 (3.5 × 104 cells/well) were plated on 96-well microtiter plates
and allowed to adhere at 37 °C in a 5 % CO2 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,

Molecules 2010, 15
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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 × 103 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 (H2DCF-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.
H2DCF-DA is a non-fluorescent permeant molecule which diffuses passively into cells; the acetates
are then cleaved by intracellular esterases to form H2DCF which is thereby trapped within the cell. In
the presence of intracellular ROS, H2DCF 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.
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Sample Availability: Samples of all compounds cited in the text are available from the authors.
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This article is an open-access article distributed under the terms and conditions of the Creative
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