Enhancement of antioxidant activity of p-alkylaminophenols by alkyl chain elongation

Article abstract of DOI:10.1016/S0968-0896(03)00300-6

The initial finding that the p-methylaminophenol (6) exhibited antioxidant activity led us to investigate whether the length of alkyl chains linked to the aminophenol residue might affect antioxidative activity. Therefore, we synthesized p-butylaminophenol (5), p-hexylaminophenol (4), p-octylaminophenol (3), and p-methoxybenzylaminophenol (7). All p-alkylaminophenols quenched α,α-diphenyl-β-picrylhydrazyl (DPPH) radicals, with 7 being the most potent DPPH radical scavenger. Lipid peroxidation by rat liver microsomes was reduced by p-alkylaminophenols in dose- and aminophenol alkyl chain length-dependent fashion (3>4>5>6), with 3 being the most potent lipid peroxidation inhibitor, at approximately 350-fold higher potency than 6. These results indicate that elongation of alkyl chains in p-alkylaminophenols may increase antioxidative activity, and that p-alkylaminophenols may potentially be useful in the development of antioxidants.

Full text of DOI:10.1016/S0968-0896(03)00300-6

Bioorganic & Medicinal Chemistry 11 (2003) 3255–3260
Enhancement of Antioxidant Activity of p-Alkylaminophenols by
Alkyl Chain Elongation
Noriko Takahashi,* Kayoko Tamagawa, Yoshinori Kubo, Tetsuya Fukui,
Hitoshi Wakabayashi and Toshio Honda
Faculty of Pharmaceutical Sciences, Hoshi University, 4-41, Ebara 2-Chome, Shinagawa-ku,
Tokyo 142-8501, Japan
Received 29 January 2003; accepted 23 April 2003
Abstract—The initial finding that the p-methylaminophenol (6) exhibited antioxidant activity led us to investigate whether the
length of alkyl chains linked to the aminophenol residue might affect antioxidative activity. Therefore, we synthesized p-butylamino-
phenol (5), p-hexylaminophenol (4), p-octylaminophenol (3), and p-methoxybenzylaminophenol (7). All p-alkylaminophenols
quenched a,a-diphenyl-b-picrylhydrazyl (DPPH) radicals, with 7 being the most potent DPPH radical scavenger. Lipid peroxi-
dation by rat liver microsomes was reduced by p-alkylaminophenols in dose- and aminophenol alkyl chain length-dependent fash-
ion (3>4>5>6), with 3 being the most potent lipid peroxidation inhibitor, at approximately 350-fold higher potency than 6. These
results indicate that elongation of alkyl chains in p-alkylaminophenols may increase antioxidative activity, and that p-alkylamino-
phenols may potentially be useful in the development of antioxidants.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
various lengths for the methyl group attached to amino-
phenol. Accordingly, in the present study, we synthe-
sized four compounds, p-butylaminophenol (5),
p-hexylaminophenol (4), p-octylaminophenol (3), and
p-methoxybenzylaminophenol (7) (Fig. 1) and examined
their antioxidant properties.
Previous studies have shown that fenretinide [N-(4-
hydroxyphenyl)retinamide, 2], a synthetic retinoid,
exhibits antioxidant activities that include scavenging
DPPH radicals, inhibiting linoleic acid peroxidation
initiated by hydroxyl radicals, and reducing lipid per-
oxidation in rat liver microsomes to the same extent or
greater than vitamin E.1,2 Recently, we found that the
structure of p-methylaminophenol (6) rather than the 4-
aminophenol and p-aminoacetophen moieties con-
tributed provided most significantly to the antioxidant
activities of 2.2,3 These moieties are derived from the
side chain amido portion of 2 that distinguishes it from
retinoic acid (1), which itself is an antioxidant. In order
to develop potent antioxidants, we investigated effects
on antioxidant activity of substituting alkyl chains of
Results
Scavenging of DPPH radicals by aminophenols having
alkyl chains of various length
Assessment of antioxidant potency using a DPPH radi-
cal assay has been studied previously.4,5 The stoichio-
metry of reaction of DPPH radicals with antioxidants as
measured by quenching to the non-radical form of
DPPH. In the case of cysteine or vitamin E, this has
been shown to be one molecule per one or two DPPH
radicals respectively.4,5 With the absorbance of control
being approximately 0.91, in the presence of vitamin E,
6, 5, 4, 3 and 7 at 20 mM concentration, absorbances at
517 nm were approximately 0.54, 0.50, 0.52, 0.48, 0.5,
and 0.09, respectively. Relative to control, vitamin E
showed an approximate 41% decrease in the absorbance
Abbreviations: 6, p-methylaminophenol, 4-(methylamino)phenol; 5, p-
butylaminophenol, 4-(butylamino)phenol; 4, p-hexylaminophenol, 4-
(hexylamino)phenol; 3, p-octylaminophenol, 4-(octylamino)phenol; 7,
p-methoxybenzylaminophenol, 4-(p-methoxybenzylamino)phenol; 1,
retinoic acid; 2, N-(4-hydroxyphenyl)retinamide, fenretinide.
*Corresponding author. Tel./fax: +81-3-5498-5950; e-mail: t-noriko@
hoshi.ac.jp
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00300-6

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N. Takahashi et al. / Bioorg. Med. Chem. 11 (2003) 3255–3260
of DPPH radicals at 517 nm (ÁOD517=0.37) (Fig. 2A).
In contrast, cysteine and reduced absorbance
approximately 50% relative to vitamin
2
E
(ÁOD517=0.19), with 1 being the same as control (data
not shown). Test compounds, 6, 5, 4, and 3 exhibited
the same level of antioxidant activity relative to vitamin
E (Fig. 2A) and showed antioxidant activity (ÁOD517
)
in dose dependent manner in the range of 0–20 mM (Fig.
2B). At all concentrations, the level of antioxidant
activity of 7 was approximately 2-fold that of vitamin E
and other aminophenols (Fig. 2A and B). These results
indicate that 6, 5, 4, and 3 are antioxidants with poten-
cies equal to vitamin E, in that one molecule of com-
pound scavenges two DPPH radicals. 7 was the most
potent among the five aminophenols tested, with one
molecule of 7 scavenging four molecules of DPPH
radicals. This indicated that phenyl residues may be
critical for scavenging DPPH radicals.
Inhibition of liver microsomal lipid peroxidation by
alkylaminophenols and retinoids
The extent of lipid peroxidation in vitro was measured
by lipid-derived MDA production. A reaction mixture
containing the vehicle DMSO was identical to a control
mixture in the absence of compounds. As shown in
Figure 3, 2, 1, and 6 inhibited lipid peroxidation in
dose-dependent fashion in the range of 1–5 mM, with 1
exhibiting an IC50 value of approximately 3.2 mM. At
Figure 1. Chemical structures of 1, 2, 3, 4, 5, 6 and 7.
Figure 2. Effects of 6, 5, 4, 3, 7 and vitamin E on DPPH radicals. Ethanol (2 mL) and a 500 mM solution of DPPH radicals in ethanol (1 mL) were
added to 0.1 M acetic acid buffer, pH 5.5 (2 mL). Vitamin E, 6, 5, 4, 3, and 7 were added, and mixtures were incubated at room temperature for
30 min as described under Experimental. Absorbance was measured spectrophotometrically at 517 nm. (A) Antioxidant activity in the presence of
DMSO (Control, C), vitamin E (VE), 6, 5, 4, 3, and 7 at the concentrations of 20 mM, expressed as ÁOD517 calculated by the following formula;
(OD517 of control) ꢀ(OD517 of compound) and expressed as % of vitamin E calculated according to the following formula: (control absorban-
ceꢀabsorbance in the presence of 20 mM compound)/(control absorbanceꢀabsorbance in the presence of 20 mM vitamin E) ꢁ 100. (B) Antioxidant
activity (ÁOD517) at various concentrations of vitamin E (~), 6 (*), 5 ( ), 4 (&), 3 (&), and 7 (^). The meansꢂSD of at least four measurements
are shown. Experiments were repeated at least four times.

N. Takahashi et al. / Bioorg. Med. Chem. 11 (2003) 3255–3260
3257
concentrations higher than 5 mM, 2 significantly
increased MDA formation, while 10 mM 1 inhibited
lipid peroxidation (Fig. 3). Other alkylaminophenols
(5, 4, 3, and 7) showed marked inhibition of lipid
peroxidation at 1 mM concentration and decreased
microsomal lipid peroxidation more efficiently than 2,
1, and 6. These results indicate that elongation of
alkylaminophenol chain length affects ability to inhibit
lipid peroxidation.
Marked reduction of lipid peroxidation by 6, 5, 4, 3, and 7
The finding that 5, 4, 3, and 7 were efficient antioxidants
as compared with 2, 1, and 6 contains a methyl residue
linked to an aminophenol, led us to investigate how
effective at inhibiting MDA formation these agents
would be at low concentrations. As shown in Figure 4,
dose–effect curves were displaced from high concen-
tration to low concentration as a function of increasing
number of carbon units in the alkyl chain. 7 also inhib-
ited lipid peroxidation in a dose-dependent fashion to
a similar extent as 5. Estimation of IC50 values from
Figure 4 indicated approximately 4.6 mM for 6 (C1),
0.3 mM for 5 (C4), 0.033 mM for 4 (C6), 0.014 mM for
3 (C8), and 0.4 mM for 7 (Table 1). Antioxidant activity
exhibited by 3 (C8) was approximately 330-fold higher
than by 6 (C1) (Fig. 4, Table 1), and it was approxi-
Figure 3. Inhibition of ascorbate-dependent lipid peroxidation by 1, 2,
6, 5, 4, 3, and 7. Microsomes (0.5 mg protein/mL) and 1 (~), 2 ( ), 6
(*), 5 ( ), 4 (&), 3 (&), and 7 (^) at concentrations of 1, 2, 5, and
10 mM in 100 mM Tris–HCl (pH 7.5) containing 15 mM FeCl3, and
4 mM ADP, were preincubated at 37 ꢃC for 1 min. To reaction mix-
tures was added 1 mM ascorbic acid and incubation was continued at
37 ꢃC for 20 min. TBA reagent was added, then the mixtures were
heated in a boiling waterbath for 15 min, and centrifuged as described
in Experimental. Supernatant absorbance was measured at 535 nm
mately 229-fold and 357-fold higher than by
1
(IC50=3.2 mM) and 2 (IC50=5 mM), respectively (Fig.
3). These results suggest that 3 (C8) is an extremely
effective antioxidant.
(e=156,000 cmꢀ1
M
ments are shown. Experiments were repeated at least three times.
ꢀ1). The meansꢂSD of at least four measure-
Figure 4. Marked reduction of lipid peroxidation by 6, 5, 4, 3, and 7. Microsomes (0.5 mg protein/mL) and various concentrations (0.01–10 mM) of
6 (*), 5 ( ), 4 (&), 3 (&), and 7 (^) in 100 mM Tris–HCl (pH 7.5) containing 15 mM FeCl3, and 4 mM ADP, were preincubated at 37 ꢃC for 1 min.
To reaction mixtures was added 1 mM ascorbic acid and incubation was continued at 37 ꢃC for 20 min. TBA reagent was added, then the mixtures
were heated in a boiling waterbath for 15 min, and centrifuged as described in Experimental. Supernatant absorbance was measured at 535 nm
(e=156,000 cmꢀ1
M
ꢀ1). The meansꢂSD of at least four measurements are shown. Experiments were repeated at least three times.

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N. Takahashi et al. / Bioorg. Med. Chem. 11 (2003) 3255–3260
Table 1. Comparison of IC50 values of 6, 5, 4, 3, and 7 on MDA
production derived from lipidsa
higher was their antioxidant potency (Fig. 4). On
the other hand, retinoids (retinol, retinol acetate, 1,
retinol palmitate, and retinal) inhibited mitochondrial
lipid peroxidation only at high concentrations (0.1–10
mM).7 However, 13-cis-retinoic acid suppressed 97% of
microsomal lipid peroxidation, with an IC50 value of 10
mM,8 and 1 at a concentration of 10 mM inhibited MDA
formation almost completely using microsomal lipids. It
was found that 2 was a more effective antioxidant than
vitamin E while being less potent than 1 (Fig. 3).2,3
These results indicated that alkylaminophenols are
excellent antioxidants as compared with retinoids, and
suggested that aminophenols with alkyl residues longer
than present in 3 may be more effective antioxidants.
Alkyl residues linked to aminophenols may be more
critical for antioxidant activity than with retinoyl resi-
dues. Further investigations are necessary to clarify this
issue.
IC50 (mM)
Fold
6
5
4
3
7
4.6
0.3
0.033
0.014
0.4
1
15.3
139.4
328.6
11.5
aIC50 values were obtained from Figure 4. Fold (ratio) of the IC50
values for 5, 4, 3, and 7 were calculated according to the following
formula: (IC50 of 6)/(IC50 of 5, 4, 3, and 7).
Discussion
The 6 moiety is an essential structural component of 2,
which is a potent antioxidant. In the current study, a
series of p-alkylaminophenols (5, 4, 3, and 7) either
having alkyl residues of various lengths or a phenyl
residue were synthesized and examined for antioxidant
activity. It was observed that 6, 5, 4, 3, 7 and vitamin E
exhibited DPPH radical quenching activity in either 1:2
or 1:4 ratios. In contrast, 2 exhibited a 1:1 ratio relative
to DPPH radicals, while 1 was inactive. The extent of
reduction of lipid peroxidation in rat liver microsomes
by 6, 5, 4, and 3 depended on the length of the alkyl
chain bound to the aminophenol. 3 was the most potent
inhibitor of lipid peroxidation, being approximately
350-fold higher than either 6 or 2.
Lipid peroxidation involves several steps, including the
abstraction by hydroxy radical of a hydrogen atom
from an unsaturated fatty acid; the formation of lipid
radicals, including peroxyl radicals; the uptake of oxy-
gen, and rearrangement of double bonds in unsaturated
lipids. The destruction of membrane lipids, and the for-
mation of MDA as a breakdown product can result
from these processes. Previous reports showed that
inhibition of lipid peroxidations by 13-cis-retinoic acid,
b-carotene and phenolic antioxidants are due to their
reactivity with peroxyl radicals.8ꢀ10 In the current study,
the level of inhibition of lipid peroxidation in rat liver
microsomes by 6, 5, 4, and 3 depended on the length of
the alkyl chain attached to the aminophenol. It was
found that 3 was the most potent inhibitor of lipid per-
oxidation, being approximately 350-fold higher than 6.
Further studies are warranted to examine the effects of
various lengths of alkylaminophenol on inhibition of
lipid peroxidation, quenching of hydroxy radicals, reac-
tivity with lipid radicals and solubility into microsomal
membrane.
A previous study has shown that 1 and retinoids act as
antioxidants that quench DPPH radicals.6 However,
except for 2, no quenching effect on DPPH radicals was
observed for retinoid at concentrations below 500
mM.2,6 At 20 mM, 2 exhibited one half of the scavenging
activity of vitamin E, and 1 showed no effects.2,3 This is
in agreement with our results as described above. On the
other hand, 6, and 4-aminophenol or p-acetaminophen,
which are structural components of 2, scavenged DPPH
radicals in a 1:2 ratio to the same extent as vitamin E or
in a 1:0.5 ratio respectively (Fig. 2).3 In the current
study, 6, 5, 4, and 3 also quenched DPPH radicals to the
same extent as vitamin E, with one molecule of 6, 5, 4,
or 3 reacting with two molecules of DPPH radical,
quenching it to the non-radical DPPH (Fig. 2). It
should be noted that one molecule of 7, which has a
methoxyphenyl group, scavenges four molecules of
DPPH radical. Thus, antioxidant actions of alkylamino-
phenols against DPPH radicals are much greater than
that of other retinoids including 1 and 2. However, the
length of alkyl chain may not be related to the level of
alkylaminophenol DPPH radical scavenging activity.
In conclusion, antioxidant vitamins and flavonoids are
very effective in preventing several diseases that are
considered to involve free radicals, including cardio-
vascular and cerebrovascular diseases, certain forms of
cancer, and ischemia/reperfusion injuries.11ꢀ14 Anti-
oxidant activities of alkylaminophenols may influence
many physiological processes such as immunostimula-
tion, enhancement of cell communication, and inhibition
of metabolic activation, rendering alkylaminophenols of
potential clinical use for treatment of various diseases
for which antioxidants are currently administrated.
There are no reports regarding inhibition of lipid per-
oxidation (MDA formation) by alkylaminophenols
except for 6.3 At a concentration of 5 mM 6, a partial
structure of the side chain of 2, suppressed microsomal
lipid peroxidation by approximately 53%, which is the
same extent as 2 (Fig. 3). The IC50 values of 6 and 2
were both approximately 5 mM, with the IC50 values of
lipid peroxidation by 5, 4, 3, and 7 being extremely poor
as compared with 1, 6 or 2 (Figs 3 and 4, and Table 1).
The longer the alkyl chains of alkylaminophenpls, the
Experimental
General
1, vitamin E (a-tocopherol), a,a-diphenyl-b-picryl-
hydrazyl (DPPH), adenosine 50-diphosphate (ADP) and
dimethylsulfoxide (DMSO) were obtained from Sigma
Chemical Co. (St. Louis, MO, USA). 2 was provided by
Dr. R. C. Moon, University of Illinois, Chicago, IL,

N. Takahashi et al. / Bioorg. Med. Chem. 11 (2003) 3255–3260
3259
USA. Dithiothreitol (DTT), trichloroacetic acid (TCA),
6, n-butylaldehyde, n-hexylaldehyde, n-octylaldehyde, p-
anisaldehyde, and sodium borohydride were purchased
from Nacalai Tesque, Inc. (Kyoto, Japan). Ascorbic
acid and 2-thiobarbituric acid (TBA) were purchased
from Wako Pure Chemicals Industries, Co. Ltd. (Osaka,
Japan). EIMS spectrometry were performed on JEOL
JMS-D360 (Tokyo, Japan), and 1H and 13C NMR spec-
tra were acquired at 270 MHz by JEOL JMS GX-270
(Tokyo, Japan) in CDCl3. Chemical shifts (d value, ppm)
were reported relative to tetramethylsilane internal stan-
dard and coupling constants were reported in Hz.
5 ꢃC. After addition of sodium borohydride (0.76 g, 20
mmol), the mixture was stirred for an additional 30 min
at the same temperature. The mixture was treated with
H2O (50 mL) and the organic solvent was removed
under reduced pressure. The aqueous solution was
extracted with dichloromethane (3ꢁ30 mL). The com-
bined extracts were dried (Na2SO4), and evaporated in
vacuo to give a residue, which was purified by column
chromatography on silica gel (hexane/ethyl acetate=6:1)
to afford pure 5 (200 mg, 12%).
1
p-Butylaminophenol (5). H NMR d: 0.94 (3H, t, J=7.3
Hz, 4-CH3), 1.40 (2H, m, 3-CH2), 1.57 (2H, m, 2-CH2),
3.05 (2H, t, J=7.1 Hz, 1-CH2), 3.80–5.10 (1H, br, NH),
6.54 (2H, d, J=8.8 Hz, Ar–H), 6.68 (2H, d, J=8.8 Hz,
Ar–H); 13C NMR d: 13.8, 20.2, 31.5, 45.4, 115.3, 116.3,
141.8, 148.5; EI–MS m/z (%): 165 (M+) (84), 122 (100),
108 (28); HRMS calcd for C10H15NO (M+) 165.1154,
found 165.1168.
DPPH radical analysis
DPPH radical analysis was performed as described pre-
viously.4,5 Ethanol (2 mL) and a 500 mM DPPH in eth-
anol solution (1 mL) were added to 0.1 M acetic acid
buffer, pH 5.5 (2 mL). To this mixture (5 mL) were
added 100 mL of a 1 mM solution of vitamin E, 1, 2, 6,
5, 4, 3, or 7 dissolved in DMSO along with cysteine
dissolved in 0.1 M acetic acid buffer, pH 5.5. The final
concentration of each compound was 20 mM. After
incubation at room temperature for 30 min, absorbance
was measured spectrophotometrically at 517 nm (Shi-
mazu UV-1600, Shimazu, Kyoto, Japan). Acetic acid
buffer or DMSO (100 mL) were used in place of com-
pounds as blank controls.
p-Hexylaminophenol (4). 1H NMR d: 0.89 (3H, t, J=6.7
Hz, 6-CH3), 1.25–1.45 (7H, m, 3-, 4-, 5-CH2 and OH),
1.54–1.64 (2H, m, 2-CH2), 3.04 (2H, t, J=7.0 Hz, 1-
CH2), 6.54 (2H, d, J=8.6 Hz, Ar–H), 6.69 (2H, d,
J=8.6 Hz, Ar–H); 13C NMR d: 14.0, 22.6, 26.8, 29.6,
31.6, 45.2, 114.4, 116.2, 142.7, 147.7; EI–MS m/z (%):
193 (M+) (30), 171 (15), 149 (27), 133 (15), 122 (100),
113 (16), 99 (12); HRMS calcd for C12H19NO (M+)
193.1467, found 193.1442.
Measurement of microsomal lipid peroxidation
1
p-Octylaminophenol (3). H NMR d: 0.88 (3H, t, J=6.7
Lipid peroxidation in rat liver microsomes was quanti-
tated by measurement of malondialdehyde (MDA)
using ADP-chelated ion and ascorbate as described
previously.8 Rat liver microsomes were prepared by the
method as described previously.3 Reaction mixtures
consisting of microsomes (0.5 mg protein/mL) and
compounds (vitamin E, 1, 2, 6, 5, 4, 3, or 7 dissolved in
DMSO) in 100 mM Tris–HCl (pH 7.5) containing 15
mM FeCl3, and 4 mM ADP, were preincubated at 37 ꢃC
for 1 min. Reaction mixtures in which ascorbic acid was
added at a final concentration of 1 mM, were incubated
at 37 ꢃC for 20 min, then equal volumes of TBA reagent
(0.375% TBA and 15% TCA in 0.25 N hydrochloride)
were introduced. Mixtures were heated in boiling water
for 15 min, and centrifuged at 1000g (10 min). Super-
natant absorbance was measured spectrophoto-
Hz, 8-CH3), 1.25–1.45 (10H, m, 3-, 4-, 5-, 6-, 7-CH2),
1.54–1.64 (2H, m, 2-CH2), 3.04 (2H, t, J=7.1 Hz, 1-
CH2), 3.50–4.60 (1H, br, NH), 6.54 (2H, d, J=8.7 Hz,
Ar–H), 6.68 (2H, d, J=8.7 Hz, Ar–H); 13C NMR d: 14.1,
22.6, 27.2, 29.2, 29.4, 29.6, 31.8, 45.3, 114.6, 116.2, 142.5,
147.8; EI–MS m/z (%): 221 (M+) (35), 213 (25), 189 (6),
171 (22), 167 (18), 157 (14), 149 (75), 122 (100); HRMS
calcd for C14H23NO (M+) 211.1780, found 221.1750.
p-Methoxybenzylaminophenol (7). 1H NMR d: 3.80 (3H,
s, OMe), 4.19 (2H, s, NHCH2), 6.55 (2H, ddd, J=2.2,
3.4, and 8.7 Hz, Ar–H), 6.68 (2H, ddd, J=2.1, 3.5, and
8.7 Hz, Ar–H), 6.87 (2H, ddd, J=2.0, 3.0, and 8.6 Hz,
Ar–H), 7.28 (2H, brd, J=8.6 Hz, Ar–H); 13C NMR d:
48.8, 55.3, 114.0, 114.4, 116.2, 128.8, 131.6, 142.4, 147.8,
158.8; EI–MS m/z (%): 229 (M+) (25), 184 (2), 167 (43),
150 (12), 149 (100), 132 (5), 122 (11), 121 (82), 113 (11),
104 (11); HRMS calcd for C14H15NO2 (M+) 229.1103,
found 229.1117.
metrically at 535 nm (e=156,000 cmꢀ1
M
ꢀ1).
Presentation of results
Each experiment was performed at least three times,
and most experiments were repeated at least four times
with consistent results.
Acknowledgements
We thank Dr. Terrence Burke, Jr. for helpful comments.
This investigation was supported in part by the Ministry
of Education, Science, Sports, and Culture of Japan.
Synthesis of alkylaminophenols
5, 4, 3, or 7 were synthesized as described previously.15
Typical experimental procedures were as follows: To a
stirred solution of n-butyraldehyde (0.72 g, 10 mmol) in
absolute tetrahydrofuran (10 mL) were successively added
molecular sieves 3A (4 g) and 4-aminophenol (1.1 g, 10.1
mmol), and the resulting mixture was stirred for 4 h at
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