Antioxidant activity of 3,4,5-trihydroxyphenylacetamide derivatives

Article abstract of DOI:10.1007/s12272-013-0189-0

A series of amide derivatives of 3,4,5-trihydroxyphenylacetic acid was synthesized in two steps. The antioxidant activities were evaluated by using four different in vitro models such as ABTS and DPPH radical scavenging activity and FTC and TBA anti-lipid peroxidation activity. Most of compounds were more powerful radical scavengers than vitamin C and were comparable to Trolox. It was found that there were no direct correlations between radical scavenging and anti-peroxidation activities. The inhibitory activity of compound on lipid peroxidation showed remarkable dependency on both the number of phenolic hydroxyl group and the length of methylene linker in N-arylalkyl group of amide. Compound 14, a conjugate of 3,4,5-trihydroxyphenylacetic acid and dopamine, was found as powerful antioxidant as propyl gallate in all four antioxidant assays. Graphical Abstract: A series of amide derivatives of 3,4,5- trihydroxyphenylacetic acid was synthesized. The antioxidant activities were evaluated by using four different in vitro models such as ABTS and DPPH radical scavenging activity and FTC and TBA anti-lipid peroxidation activity. Compound 14, a conjugate of dopamine, was found as powerful antioxidant as propyl gallate in all four antioxidant assays.[Figure not available: see fulltext.]

Full text of DOI:10.1007/s12272-013-0189-0

Arch. Pharm. Res.
DOI 10.1007/s12272-013-0189-0
RESEARCH ARTICLE
Antioxidant activity of 3,4,5-trihydroxyphenylacetamide
derivatives
•
Jina Kim Victor Sukbong Hong Jinho Lee
•
Received: 4 March 2013 / Accepted: 12 June 2013
ꢀ The Pharmaceutical Society of Korea 2013
Abstract A series of amide derivatives of 3,4,5-trihydr-
oxyphenylacetic acid was synthesized in two steps. The
antioxidant activities were evaluated by using four differ-
ent in vitro models such as ABTS and DPPH radical
scavenging activity and FTC and TBA anti-lipid peroxi-
dation activity. Most of compounds were more powerful
radical scavengers than vitamin C and were comparable to
Trolox. It was found that there were no direct correlations
between radical scavenging and anti-peroxidation activi-
ties. The inhibitory activity of compound on lipid peroxi-
dation showed remarkable dependency on both the number
of phenolic hydroxyl group and the length of methylene
linker in N-arylalkyl group of amide. Compound 14, a
conjugate of 3,4,5-trihydroxyphenylacetic acid and dopa-
mine, was found as powerful antioxidant as propyl gallate
in all four antioxidant assays.
alkoxy radical (RO•), and peroxy radical (ROO•). The
hydroxyl (half-life of 10-9 s) and alkoxy radicals (half-life of
seconds) are very reactive and cause oxidative damage rap-
idly to critical biological molecules such as polyunsaturated
fatty acids, membrane lipids, and nucleotides, resulting in
peroxidation of lipid, and oxidation of carbohydrate, and
protein (Gu¨lc¸in 2012). Also, oxidative damages on genetic
material represent the first step involved in mutagenesis,
carcinogenesis, and ageing (Valko et al. 2007). The biomo-
lecular damage caused by the attack of ROS to vital con-
stituents of living organisms can be effectively quenched by
antioxidants. Antioxidants have emerged as a way of mini-
mizing oxidative stress. The majority of natural antioxidants
are phenolic compounds including tocopherol, flavonoids
and phenolic acids. Phenolic acids have shown the potential
protective role against oxidative damage related diseases
such as ischemic heart disease, inflammation, and cancers
(Valko et al. 2007; Fiuza et al. 2004; Lu et al. 2006; Navarro-
Keywords Antioxidant activity ꢀ
´
Peran et al. 2008; Du et al. 2011).
3,4,5-Trihydroxyphenylacetamide ꢀ Radical scavenging ꢀ
Anti-lipid peroxidation
Siquet et al. (2006) studied the antioxidative activity of a
series of phenolic compounds and found that the number of
hydroxyl groups and the type of the spacer between the
aromatic ring and the carboxylic acid influence the anti-
oxidative activity. According to the result, 3,4,5-trihydr-
oxyphenylacetic acid was the most potent antioxidant
in 2,20-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)
(ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays
but was less potent in lipid peroxidation assay. This was
explained in terms of the physicochemical properties of the
compounds, especially their lipophilicity (log P). Trihydr-
oxyphenyl moiety has been used for the synthesis of anti-
oxidants (C¸ etinkaya et al. 2012; Pal et al. 2012; Belin et al.
2003).
Introduction
Oxidative stress resulted from excessive reactive oxygen
species (ROS) is highly related to the occurrence of diseases
such as cancer, diabetes, inflammation, neurological disor-
ders and cardiovascular disease (Valko et al. 2007). Exam-
ples of ROS include singlet oxygen (1O2), ozone (O3),
superoxide anion radical (O•2-), hydroxyl radical (HO•),
J. Kim ꢀ V. S. Hong ꢀ J. Lee (&)
Department of Chemistry, Keimyung University,
2800 Dalgubeoldaero, Dalseo-Gu, Taegu 704-701, Korea
e-mail: jinho@kmu.ac.kr
Based on the above results, we report here the modifi-
cation of 3,4,5-trihydroxyphenylacetic acid and the
123

J. Kim et al.
evaluation of their antioxidative activity to find a new
potent antioxidant.
magnesium sulfate, filtered and concentrated in vacuo. The
desired product was obtained in quantitative yield.
1H NMR (CDCl3?CD3OD) d(ppm): 7.49 (d, J = 7.6 Hz,
aromatic, 2H), 7.21 (t, J = 7.6 Hz, aromatic, 2H), 7.01 (t,
J = 7.6 Hz, aromatic, 1H), 6.54 (s, aromatic, 2H), 3.76 (s,
methyl, 3H), 3.74 (s, methyl, 6H), 3.56 (s, methylenic, 2H).
Materials and methods
General information
N-Phenyl-2-(3,4,5-trihydroxyphenyl) acetamide (2)
1H and 13C NMR spectra were obtained on an AVANEC 400
(Bruker, Switzerland) and are reported in part per million (d)
relative to TMS as an internal standard. TLC was performed
on E. Merck silica gel 60 F254 plates (0.25 mm). Silica gel
column chromatography was performed using Merck
TA1287685. ESI MS was obtained with Waters AQUITY
UPLC, Micromass Quattro microTM API. Unless otherwise
noted, all starting materials were obtained from commer-
cially available sources and they were used without further
purification. All reactions were performed under nitrogen
atmosphere. Pooled sheep erythrocytes were purchased from
Handok Biotech (Seoul, Korea) and were used for hemolysis
experiments within 2 week.
To a solution of 0.172 g (0.57 mmol) of N-phenyl-2-(3,4,5-
trimethoxyphenyl) acetamide in 5 mL dichloromethane,
cooled with an ice bath, was added 1 mL (1 mmol) of
boron tribromide (as a 1.0 M solution in dichloromethane)
by dropwise. The mixture was allowed to warm to room
temperature and was stirred additional 2 h. After the
reaction mixture was cooled to 0 ꢁC, 50 mL of water was
added by dropwise. The product was precipitated when
dichloromethane was removed from the reaction mixture.
After the precipitate was collected by filtration, the addi-
tional product was obtained by extracting the supernatant
with ethyl acetate (20 mL 9 2). The crude product which
was obtained by pooling precipitate and extract together
was purified on silica gel column chromatography (chlo-
roform: methanol: formic acid, 100:10:1). The desired
product was obtained in 32.0 % yield.
1H NMR (DMSO-d6) d (ppm): 7.58 (d, J = 7.6 Hz,
aromatic, 2H), 7.28 (t, J = 7.6 Hz, aromatic, 2H), 7.02 (t,
J = 7.2 Hz, aromatic, 1H), 6.26 (s, aromatic, 2H), 3.33 (s,
methylenic, 2H). 13C NMR (DMSO-d6) d (ppm): 169.56
(CO), 145.83 (C), 139.03 (C), 131.58 (C), 128.55 (CH),
125.88 (CH), 122.96 (C), 118.94 (CH), 107.71 (CH), 43.14
(CH2). ESI MS: m/z = 260 [M?H]?.
Chemistry
3,4,5-Trihydroxyphenylacetic acid derivatives were syn-
thesized from 3,4,5-trimethoxyphenylacetic acid in two
steps (Scheme 1). Amine substituent was introduced by
amide coupling reaction using EDC and HOBt. Methyl
protecting groups were removed by BBr3 to give the desired
compound. The typical reaction procedure is as follow.
N-Phenyl-2-(3,4,5-trimethoxyphenyl) acetamide
To a solution of 0.20 g (0.88 mmol) of 3,4,5-trimethoxy-
phenylacetic acid in 5 mL N, N-dimethylformamide (DMF)
were added 1.02 mL (0.88 mmol) of aniline, 0.254 g
(1.33 mmol) of N-(3-dimethylaminopropyl)-N’-ethylcar-
bodiimide hydrochloride (EDC), and 0.239 g (1.77 mmol)
of 1-hydroxybenzotriazole monohydrate (HOBt). The reac-
tion mixture was stirred at room temperature for 2 h. The
solvent was removed in vacuo. To the residue was added
saturated aqueous sodium bicarbonate, and the product was
extracted with ethyl acetate two times. The organic layers
were combined, washed with 5 % citric acid and then with
water. The organic layer was dried over anhydrous
N-(4-Methylbenzyl)-2-(3,4,5-trihydroxyphenyl)
acetamide (3)
1
Yield 46.2 %. H NMR (CDCl3 ? CD3OD) d (ppm): 7.08
(d, J = 8.4 Hz, aromatic, 2H), 7.06(d, J = 8.4 Hz, aro-
matic, 2H), 6.31 (s, aromatic, 2H), 4.32 (s, methylenic,
2H), 3.37 (s, methylenic, 2H), 2.31 (s, methyl, 3H). 13C
NMR (CDCl3 ? CD3OD) d (ppm): 172.68 (CO), 145.36
(C), 136.95 (C), 134.71 (C), 131.64 (C), 129.13 (CH),
127.30 (CH), 125.73 (C), 108.27 (CH), 43.07 (CH2), 42.93
(CH2), 20.82 (CH3). ESI MS: m/z = 288 [M ? H]?.
Scheme 1 Synthesis of 3,4,5-
H
H
H
O
O
O
O
O
N
H
H
O
trihydroxyphenylacetic acid
derivatives. (a) R-NH2, EDC,
HOBt, DMF, (b) BBr3, CH2Cl2
N
R
R
(a)
(b)
O
O
O
O
O
O
H
O
1
123

Antioxidant activity of 3,4,5-trihydroxyphenylacetamide
N-(4-Trifluoromethylbenzyl)-2-(3,4,5-trihydroxyphenyl)
N-(4-Hydroxybenzyl)-2-(3,4,5-trihydroxyphenyl)
acetamide (4)
acetamide (8)
1
Yield 14.4 %. H NMR (DMSO-d6) d (ppm): 8.77 (s, OH,
Yield 11.8 %. 1H NMR (DMSO-d6) d (ppm): 8.23 (t,
J = 5.6 Hz, NH, 1H), 7.02 (d, J = 8.6 Hz, aromatic, 2H),
6.68 (d, J = 8.6 Hz, aromatic, 2H), 6.19 (s, aromatic, 2H),
4.11 (d, J = 5.6 Hz, methylenic, 2H), 3.16 (s, methylenic,
2H). 13C NMR (DMSO-d6) d (ppm): 170.94 (CO), 156.64
(C), 146.26 (C), 131.92 (C), 130.07 (C), 129.02 (CH),
126.84 (C), 115.44 (CH), 108.30 (CH), 42.57 (CH2), 42.18
(CH2). ESI MS: m/z = 290 [M?H]?.
2H), 8.48 (t, J = 5.6 Hz, NH, 1H), 7.95 (s, OH, 1H), 7.66 (d,
J = 8.0 Hz, aromatic, 2H), 7.43 (d, J = 8.0 Hz, aromatic,
2H), 6.23 (s, aromatic, 2H), 4.33 (d, J = 5.6 Hz, methylenic,
2H), 3.23 (s, methylenic, 2H). 13C NMR (DMSO-d6) d
(ppm): 170.93 (CO), 145.83 (C), 144.54 (C), 131.53 (C),
127.72 (CH), 126.09 (C), 125.06 (q, CF3) 107.82 (CH), 42.08
(CH2), 41.73 (CH2). ESI MS: m/z = 342 [M?H]?.
N-(3-Trifluoromethylbenzyl)-2-(3,4,5-trihydroxyphenyl)
N-(3, 4-Dihydroxybenzyl)-2-(3,4,5-trihydroxyphenyl)
acetamide (5)
acetamide (9)
1
1
Yield 78.9 %. H NMR (CDCl3 ? CD3OD) d (ppm): 7.84
Yield 10.9 %. H NMR (DMSO-d6) d (ppm): 8.77 (s, OH,
3H), 8.18 (t, J = 5.6 Hz, NH, 1H), 6.63 (m, aromatic, 2H),
(s, aromatic, 1H), 7.62 (d, J = 7.6 Hz, aromatic, 1H), 7.33
(m, aromatic, 2H), 6.38 (s, aromatic, 2H), 3.46 (s, methy-
lenic, 2H). 13C NMR (CDCl3 ? CD3OD) d (ppm):149.33
(CO), 142.50 (C), 135.77 (C), 135.18 (q, CF3), 133.22 (C),
129.72 (C), 129.17 (CH), 127.04 (C), 126.46 (CH), 124.60
(CH), 120.62 (CH) 112.39 (CH), 47.52 (CH2). ESI MS: m/
z = 328 [M ? H]?.
6.46 (dd, J = 8.0 Hz, 1.6 Hz, aromatic, 1H), 4.05 (d,
J = 5.6 Hz, methylenic, 2H), 3.15 (s, methylenic, 2H). 13
C
NMR (DMSO-d6) d (ppm): 170.87 (CO), 146.23 (C),
145.50 (C), 144.57 (C), 131.91 (C), 130.68 (C), 126.82 (C),
118.72 (CH), 115.72 (CH), 115.48 (CH), 108.33 (CH),
42.52 (CH2), 42.39 (CH2). ESI MS: m/z = 305 [M?H]?.
N-(3,4-Dichlorobenzyl)-2-(3,4,5-trihydroxyphenyl)
N-[2-(4-Fluorophenyl)ethyl]-2-(3,4,5-trihydroxyphenyl)
acetamide (6)
acetamide (10)
1
Yield 63.3 %. H NMR (CDCl3 ? CD3OD) d (ppm): 7.35
1
Yield 89.6 %. H NMR (DMSO-d6) d (ppm): 8.71 (s, OH,
(d, J = 8.4 Hz, aromatic, 1H), 7.26 (d, J = 2.0 Hz, aro-
matic, 1H), 7.01 (dd, J = 8.4 Hz, 2.0 Hz, aromatic, 1H),
6.31 (s, aromatic, 2H), 4.29 (s, methylenic, 2H), 3.37 (s,
methylenic, 2H). 13C NMR (CDCl3 ? CD3OD) d (ppm):
172.97 (CO), 145.38 (C), 138.49 (C), 132.36 (C), 131.66
(C), 131.08 (C), 130.39 (CH), 129.20 (CH), 126.67 (CH),
125.74 (C), 108.28 (CH), 42.90 (CH2), 42.11 (CH2). ESI
MS: m/z = 342 [M ? H]?.
2H), 7.86 (t, J = 5.6 Hz, NH, 1H), 7.16 (m, aromatic, 2H),
7.07 (m, aromatic, 2H), 6.17 (s, aromatic, 2H), 3.20 (dt,
J = 7.2 Hz, 5.6 Hz, methylenic, 2H), 3.09 (s, methylenic,
2H), 2.66 (t, J = 7.2 Hz, methylenic, 2H). 13C NMR
(DMSO-d6) d (ppm): 171.01 (CO), 146.27 (C), 131.94 (C),
130.96 (C), 130.88 (C), 126.77 (C), 115.48 (CH), 115.27
(CH), 108.30 (CH), 42.67 (CH2), 40.80 (CH2), 34.67
(CH2). ESI MS: m/z = 306 [M?H]?.
N-Naphthalen-2-ylmethyl-2-(3,4,5-trihydroxyphenyl)
N-[2-(4-Chlorophenyl)ethyl]-2-(3,4,5-trihydroxyphenyl)
acetamide (7)
acetamide (11)
1
Yield 27.3 %. H NMR (DMSO-d6) d (ppm): 8.75 (s, OH,
Yield 98.8 %. 1H NMR (DMSO-d6) d (ppm): 7.89 (t,
J = 5.6 Hz, NH, 1H), 7.30 (d, J = 8.4 Hz, aromatic, 2H),
7.16 (d, J = 8.4 Hz, aromatic, 2H), 6.19 (s, aromatic, 2H),
3.22 (dt, J = 7.0 Hz, 5.6 Hz, methylenic, 2H), 3.10 (s,
2H), 8.43 (t, J = 5.6 Hz, NH, 1H), 8.04 (m, aromatic, 1H),
7.94 (m, aromatic, 2H), 7.84 (d, J = 7.6 Hz, aromatic, 1H),
7.54 (m, aromatic, 2H), 7.42 (m, aromatic, 2H), 6.25 (s,
aromatic, 2H), 4.71 (d, J = 5.6 Hz, methylenic, 2H), 3.23(s,
methylenic, 2H). 13C NMR (DMSO-d6) d (ppm): 171.07
(CO), 146.31 (C), 135.09 (C), 133.73 (C), 131.98 (C), 131.35
(C), 128.95 (CH), 128.01 (CH), 126.78 (C), 126.70 (CH),
126.29 (CH), 125.86 (CH), 123.99 (CH), 108.36 (CH), 42.52
(CH2), 40.79 (CH2). ESI MS: m/z = 324 [M?H]?.
methylenic, 2H), 2.67 (t, J = 7.0 Hz, methylenic, 2H). 13
C
NMR (DMSO-d6) d (ppm): 171.07 (CO), 146.28 (C),
139.00 (C), 131.96 (C), 131.16 (C), 131.07 (CH), 128.62
(CH), 126.76 (C), 108.31 (CH), 55.38 (CH2), 42.68 (CH2),
34.82 (CH2). ESI MS: m/z = 322 [M?H]?.
123

J. Kim et al.
N-[2-(3-Chlorophenyl)ethyl]-2-(3,4,5-trihydroxyphenyl)
108.21 (CH), 42.72 (CH2), 38.66 (CH2), 32.98 (CH2),
31.48 (CH2). ESI MS: m/z = 302 [M?H]?.
acetamide (12)
1
Yield 39.7 %. H NMR (DMSO-d6) d (ppm): 8.70 (s, OH,
N-(4-Phenylbutyl)-2-(3,4,5-trihydroxyphenyl) acetamide
1H), 7.91 (t, J = 5.6 Hz, NH, 1H), 7.25 (m, aromatic, 3H),
7.10 (d, J = 7.2 Hz, aromatic, 1H), 6.16 (s, aromatic, 2H),
3.23 (dt, J = 7.2 Hz, 5.6 Hz, methylenic, 2H), 3.09 (s,
(16)
Yield 73.1 %. 1H NMR (DMSO-d6) d (ppm): 7.83 (t,
J = 5.6 Hz, NH, 1H), 7.26 (m, aromatic, 2H), 7.15 (m,
aromatic, 3H), 6.17 (s, aromatic, 2H), 3.08 (s, methylenic,
2H), 3.03 (dt, J = 7.0 Hz, 5.6 Hz, methylenic, 2H), 2.54 (t,
J = 7.6 Hz, methylenic, 2H), 1.53 (m, methylenic, 2H),
1.39 (m, methylenic, 2H). 13C NMR (DMSO-d6) d (ppm):
170.89 (CO), 146.24 (C), 142.58 (C), 131.89 (C), 128.76
(CH), 128.70 (CH), 126.95 (CH), 126.08 (C), 108.19 (CH),
42.67 (CH2), 38.75 (CH2), 35.24 (CH2), 29.27 (CH2), 28.82
(CH2). ESI MS: m/z = 316 [M?H]?.
methylenic, 2H), 2.69 (t, J = 7.2 Hz, methylenic, 2H). 13
C
NMR (DMSO-d6) d (ppm): 171.04 (CO), 146.27 (C),
142.58 (C), 133.35 (C), 131.94 (C), 130.54 (C), 128.94
(CH), 127.97 (CH), 126.72 (CH), 126.52 (CH), 108.27
(CH), 42.59 (CH2), 35.12 (CH2). ESI MS: m/z = 322
[M?H]?.
N-[2-(4-Hydroxyphenyl)ethyl]-2-(3,4,5-trihydroxyphenyl)
acetamide (13)
1
Yield 46.0 %. H NMR (DMSO-d6) d (ppm): 8.72 (s, OH,
Antioxidation assays
2H), 7.88 (t, J = 5.2 Hz, NH, 1H), 6.95 (d, J = 8.0 Hz,
aromatic, 2H), 6.66 (d, J = 8.4 Hz, aromatic, 2H), 6.18 (s,
aromatic, 2H), 3.15 (dt, J = 7.2 Hz, 5.6 Hz, methylenic,
2H), 3.09 (s, methylenic, 2H), 2.55 (t, J = 7.2 Hz,
methylenic, 2H). 13C NMR (DMSO-d6) d (ppm): 170.91
(CO), 156.06 (C), 146.27 (C), 131.91 (C), 129.97 (C),
129.94 (CH), 126.81 (C), 115.55 (CH), 108.27 (CH), 42.64
(CH2), 41.23 (CH2), 34.91 (CH2). ESI MS: m/z = 304
[M?H]?.
Antioxidant activities of the compounds were studied using
four different methods: DPPH and ABTS radical scav-
enging methods for all compounds and the ferric thiocya-
nate (FTC) and thiobarbituric acid (TBA) methods for the
selected compounds. In addition, the inhibitory activity of
compound on oxyhemolysis was studied.
Scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH)
N-[2-(3, 4-Dihydroxyphenyl)ethyl]-2-(3,4,5-
The free radical scavenging activity of the samples was
measured by DPPH. In its radical form, DPPHꢀ absorbs at
517 nm, but upon reduction by antioxidant or a radical
species, its absorption decreases. This assay was carried out
as described by Erkan et al. (2008) with some modifica-
tions; 800 lL of various dilutions of the samples in ethanol
were mixed with 200 lL of a 0.15 mM ethanolic DPPHꢀ
solution. After an incubation period of 30 min at 25 ꢁC, the
absorbance at 517 nm, the wavelength of maximum
absorbance of DPPHꢀ, was recorded. Antioxidant activities
of the compounds were expressed as EC50, defined as the
concentration of the test material required to cause a 50 %
decrease in initial DPPHꢀ concentration.
trihydroxyphenyl) acetamide (14)
1
Yield 25.8 %. H NMR (DMSO-d6) d (ppm): 8.73 (s, OH,
3H), 7.90 (t, J = 5.6 Hz, NH, 1H), 6.62 (d, J = 8.0 Hz,
aromatic, 1H), 6.56 (d, J = 2.0 Hz, aromatic, 1H), 6.40
(dd, J = 8.0 Hz, 2.0 Hz, aromatic, 1H), 6.17 (s, aromatic,
2H), 3.11 (m, methylenic, 4H), 2.48 (m, methylenic, 2H).
13C NMR (DMSO-d6) d (ppm): 170.88 (CO), 146.27 (C),
145.52 (C), 143.98 (C), 131.91 (C), 130.63 (C), 126.81 (C),
119.65 (CH), 116.33 (CH), 115.94 (CH), 108.27 (CH),
42.61 (CH2), 41.25 (CH2), 35.27 (CH2). ESI MS: m/
z = 320 [M?H]?.
Scavenging of 2,20-azino-bis (3-ethylbenzothiazoline-6-
sulphonic acid) radical (ABTS radical)
N-(3-Phenylpropyl)-2-(3,4,5-trihydroxyphenyl) acetamide
(15)
1
Yield 62.4 %. H NMR (DMSO-d6) d (ppm): 8.73 (s, OH,
The assay was carried out according to the procedure
described by Re et al. (1999). ABTS radical cation was
produced by reacting 7 mM aqueous ABTS with 2.45 mM
(final concentration) potassium persulfate and keeping the
mixture in the dark at room temperature for 16 h. Blue–
green ABTS•? was formed at the end of this period. The
solution was diluted with ethanol to the concentration
which shows an absorbance of 0.70 0.02 at 734 nm.
2H), 7.91 (t, J = 5.6 Hz, NH, 1H), 7.26 (m, aromatic, 2H),
7.16 (m, aromatic, 3H), 6.21 (s, aromatic, 2H), 3.14 (s,
methylenic, 2H), 3.04 (dt, J = 7.3 Hz, 5.6 Hz, methylenic,
2H), 2.54 (t, J = 7.6 Hz, methylenic, 2H), 1.68 (dt,
J = 7.3 Hz, 7.6 Hz, methylenic, 2H). 13C NMR (DMSO-
d6) d (ppm): 171.06 (CO), 146.29 (C), 142.20 (C), 131.92
(C), 128.79 (CH), 128.74 (CH), 126.99 (C), 126.17 (CH),
123

Antioxidant activity of 3,4,5-trihydroxyphenylacetamide
After adding 35 lL of ethanol solution of the compound to
3.5 mL of ABTS•? solution, the absorbance was recorded
up to 6 min in 1 min intervals.
While incubated with shaking at 22 rpm at 37 ꢁC, an aliquot
(100 lL) of the mixture was withdrawn every 60 min up to
3 h, and added to 1.25 mL of either ice-cold PBS or water.
The supernatant was collected after centrifugation at
3859gfor 10 minand the absorbance measured at 540 nm in
a 96-well plate format using Infinity M200 (Tecan, Swit-
zerland). The percentage of hemolysis was determined by
(the absorbance of samples/the absorbance of the water-
treated total hemolysis) 9 100.
Ferric thiocyanate (FTC) method
The antioxidant activity analysis using FTC was performed
according to the procedure reported by Kikuzaki and
Nakatani (1993). A mixture of 1 mL of ethanolic solution
of the synthesized compound (50 lM), 1 mL of 2.52 %
linoleic acid in ethanol, 2 mL of 0.05 M phosphate buffer
(pH 7.0), and 1 mL of water was placed in a vial with a
screw cap and then placed in a dark oven at 40 ꢁC. A
50 lL aliquot was taken from the mixture every 48 h and
diluted with 4.85 mL of 75 % ethanol, followed by the
addition of 50 lL of 30 % ammonium thiocyanate. After
adding 50 lL of 0.02 M ferrous chloride in 3.5 % HCl, the
absorbance for the red color was measured at 500 nm. The
measurement was repeated until the absorbance of control
reached its maximum. The ratio of this absorbance to the
absorbance of a blank without any test material was taken
as a measure of ability of the test material to inhibit lipid
peroxidation which is, in turn, a measure of antioxidant
activity. Propyl gallate and gallic acid were used as posi-
tive controls, while a mixture without compounds was used
as a negative control.
Statistical analysis
The data from the experiments were presented as
mean SD. Statistical analysis was performed using one-
way analysis of variance (ANOVA). Duncan’s multiple
range test was used for multiple comparisons. Values of
p \ 0.05 were regarded as statistically significant.
Results and discussion
Phenolic acids are known as potent antioxidants. However,
their highly hydrophilic property reduces their applicability
as pharmaceutical agents or food additives. Efforts to
improve their hydrophobicity have been made by the
modification of carboxylic acid group to ester or amide
functionality. The well-known antioxidant, propyl gallate
(PG) is the typical example of the ester of gallic acid.
The modification of 3,4,5-trihydroxyphenylacetic acid
by introducing various functionalities through amide link-
age provided the compounds with improved antioxidative
activity. Compound 13 and 14 showed antioxidative
activity comparable with PG in ABTS radical scavenging
assay while other compounds were comparable with Trolox
except compound 12 (Table 1). When aniline and benzyl-
amine were used, the effect of substituents in benzene ring
was very small. However, in the case of 2-phenylethyl-
amine, the presence of phenolic substituent improved the
activity more than twice.
TBA method
The method of Ottolenghi (1959) was followed. A 250 lL
aliquot was taken from the mixture, which was prepared for
FTC method, every 48 h and mixed with 500 lL of 20 %
trichloroacetic acid and 500 lL of 0.8 % TBA. The mix-
ture was placed in a boiling water bath for 20 min and then
cooled to room temperature. It was centrifuged at
3,000 rpm for 10 min. Absorbance of supernatant was
measured at 532 nm. Antioxidant activity was based on the
absorbance on the final day of the FTC method. Propyl
gallate and gallic acid were used as positive controls, while
a mixture without compounds was used as a negative
control.
In DPPH radical scavenging assay, compound 14
showed as good activity as PG and gallic acid. All other
compounds except 9 showed better radical scavenging
activity than vitamin C. An interesting result was the
inversion of activity between benzylamine and phenyleth-
ylamine when the phenolic group was attached. In ben-
zylamine type, the presence of hydroxyl groups reduces the
activity up to sixfold (compounds 8 and 9). While in
phenylethylamine type, one phenolic group did not alter
the activity but two adjacent phenolic group improved
activity significantly. The distance between amino and
phenyl group did not influence on the activity in both
radical scavenging assays.
Inhibition of AAPH-induced hemolysis
The inhibition of AAPH-induced hemolysis was performed
as previously reported (Chan et al. 2010). In brief, purchased
RBCs were washed three times with PBS (ThermoScientific,
USA), and re-suspended in PBS at a final concentration of
20 % (v/v). Six hundred microliter of re-suspended RBC was
incubated with the equal volume of the test compound in PBS
(final concentration of 30 lM) or PBS alone. After pre-
incubation for 10 min with shaking at 22 rpm at 37 ꢁC,
300 lL of 400 mM AAPH in PBS was added to the mixture.
123

J. Kim et al.
Table 1 Antioxidant activity of synthesized compounds, vitamin C,
trolox, gallic acid, and PG
No
R
EC50 (lM)a
DPPH
ABTS
2
3
4
6.0 0.7
6.0 0.8
6.7 1.3
29.0 2.5
21.2 2.4
28.8 6.4
CF3
5
6
6.4 0.9
6.0 0.7
29.5 3.3
36.8 1.5
CF3
Cl
Cl
7
8
6.9 0.9
42.5 6.4
33.5 0.6
Fig. 1 Inhibition of lipid peroxidation by synthesized compounds,
gallic acid, and PG (a FTC assay, b TBA assay). Different letters
above the bars indicate statistically significant differences. p \ 0.05
10.5 1.5
OH
OH
9
31.8 6.7
36.7 0.1
The FTC method was used to measure the amount of
peroxide at the beginning of lipid peroxidation, while the
TBA method was used to measure the extent of lipid per-
oxidation. Five compounds were selected to find the effect
of substituent on both FTC and TBA assays (Fig. 1). The
anti-peroxidative activities of benzylamine type com-
pounds (3: 4-methylphenyl, 7: 2-naphthyl, 8: 4-hydroxy-
phenyl) were not as good as their radical scavenging
activity and did not show any trend of substituent effect. In
phenylethylamine type compounds, the presence of two
phenolic groups (compound 14) caused distinctive
enhancement of activity and made it comparable to PG.
Compound 13, with one phenolic group, did not show high
anti-peroxidative activity even though it had similar radical
scavenging ability to compound 14 and PG. In general,
there was no direct correlation between radical scavenging
activity and the inhibition of lipid peroxidation. For
example, gallic acid was the most potent radical scavenger
for both ABTS and DPPH but did not show any anti-per-
oxidation activity in FTC assay. In contrast, PG and
compound 14 showed strong antioxidative activity in all
four different assays. These results can be explained, in
part, by the difference in the nature of assays. The hydro-
gen atom abstraction is a key mechanism in the inhibition
of lipid peroxidation chain reaction (Halliwell and Chirico
1993; Pratt et al. 2011). FTC and TBA assays are mea-
suring the extent of lipid peroxidation and can be classified
as hydrogen atom transfer (HAT) assays which quantify the
OH
10
11
6.7 1.4
6.4 1.0
31.7 6.0
30.8 3.4
F
Cl
12
13
9.6 1.3
6.7 0.6
75.8 7.9
12.9 1.5
Cl
OH
14
3.8 0.2
17.2 1.6
OH
OH
15
7.1 0.9
7.3 1.3
27.8 5.5
36.4 4.2
16
Gallic acid
2.9 0.5
3.0 0.4
NDb
4.3 1.7
14.7 0.1
28.0 1.9
NDb
Propyl gallate (PG)
Trolox
Vitamin C
28.9 0.2
a
Data represent the mean SD of three independent experiments
Not determined
b
123

Antioxidant activity of 3,4,5-trihydroxyphenylacetamide
antioxidants. The protection of erythrocyte from lysis by
PG and trolox had previously been reported (Wu et al.
1998). In our study, synthesized antioxidant compounds
and gallic acid exhibited similar extents of protection on
sheep erythrocytes against peroxyl radicals generated by
AAPH (Fig. 2b). The pattern of time dependent % inhibi-
tion in Fig. 2a suggests that the protected activities of
synthesized compounds are in between PG and trolox.
In summary, we synthesized a series of 3,4,5-trihy-
droxyphenylacetamides with different substituents at amide
by relatively simple chemical reactions and evaluated their
antioxidative activity. Compound 14 was found as a good
antioxidant which is similar to PG. Because compound 14
is a condensed form of two natural compounds 3,4,5-tri-
hydroxyphenylacetic acid and dopamine, it is expected to
have relatively low toxicity. Also, the stability of amide
linkage possibly makes it more stable than PG. With
additional studies such as toxicity and stability, it can be
used as antioxidant for several purposes.
(A)
(B)
% inhibition
Compound
at 60 min
Gallic acid
88.3
91.7
75.6
84.9
90.1
82.6
88.3
86.1
Propyl gallate
Acknowledgments The research was supported by the Bisa
Research Grant of Keimyung University in 2012.
Trolox
3
7
8
13
14
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