Antioxidant and anticancer activities of novel p-alkylaminophenols and p-acylaminophenols (aminophenol analogues)

Article abstract of DOI:10.1016/j.bmc.2006.05.013

Novel compounds were designed based on fenretinide, N-(4-hydroxyphenyl)retinamide (2), which is a synthetic amide of all-trans-retinoic acid (1) that is a potent antioxidant and anticancer agent. Our recent findings indicated that antioxidant and anticancer activities were due to p-methylaminophenol moiety (8) in 2, and that p-octylaminophenol (7), which has an elongated alkyl chain, was more potent than 8. This finding lets us to investigate whether compounds containing alkyl or acyl chains linked to an aminophenol residue as long as 2 and 1, would show activities greater than 2. For this purpose, we prepared p-dodecanoylaminophenol (3), p-decanoylaminophenol (4), p-dodecylaminophenol (5), and p-decylaminophenol (6). The p-alkylaminophenols, 5 and 6, exhibited superoxide scavenging activities, but not p-acylaminophenols, 3 and 4. Elongation of the alkyl chain length reduced superoxide trapping capability (8 > 7 > 6 > 5). In contrast, lipid peroxidation in rat liver microsomes was reduced by 5 and 6 in dose-dependent manner. Compounds 3 and 4 were poor lipid peroxidation inhibitors, being approximately 400- to 1300-fold lower than 5 and 6. In addition, all compounds inhibited cell growth of human leukemia cell lines, HL60 and HL60R, in dose-dependent manners (5 > 6 > 3 = 4). The HL60R cell line is resistant against 1. Growth of both cell lines was suppressed by 5 and 6 in a fashion dependent on the length of the aminophenol alkyl chain, but not by 3 and 4. These results indicate that 5, a potent anticancer agent greater than 2, may potentially have clinical utility, and that its anticancer activity is correlated with inhibitory potency against lipid peroxidation, but not with superoxide scavenging activity.

Full text of DOI:10.1016/j.bmc.2006.05.013

Bioorganic & Medicinal Chemistry 14 (2006) 6089–6096
Antioxidant and anticancer activities of novel p-alkylaminophenols
and p-acylaminophenols (aminophenol analogues)
a,
a
b
b
*
Noriko Takahashi, Toshihiro Ohba, Takayasu Yamauchi and Kimio Higashiyama
a
Laboratory of Physiological Chemistry, Institute of Medicinal Chemistry, Hoshi University, Shinagawa-ku, Tokyo 142-8501, Japan
b
Synthetic Organic Chemistry Research Laboratory, Institute of Medicinal Chemistry, Hoshi University,
Shinagawa-ku, Tokyo 142-8501, Japan
Received 6 April 2006; revised 1 May 2006; accepted 2 May 2006
Available online 24 May 2006
Abstract—Novel compounds were designed based on fenretinide, N-(4-hydroxyphenyl)retinamide (2), which is a synthetic amide of
all-trans-retinoic acid (1) that is a potent antioxidant and anticancer agent. Our recent findings indicated that antioxidant and anti-
cancer activities were due to p-methylaminophenol moiety (8) in 2, and that p-octylaminophenol (7), which has an elongated alkyl
chain, was more potent than 8. This finding lets us to investigate whether compounds containing alkyl or acyl chains linked to an
aminophenol residue as long as 2 and 1, would show activities greater than 2. For this purpose, we prepared p-dodecanoylaminophe-
nol (3), p-decanoylaminophenol (4), p-dodecylaminophenol (5), and p-decylaminophenol (6). The p-alkylaminophenols, 5 and 6,
exhibited superoxide scavenging activities, but not p-acylaminophenols, 3 and 4. Elongation of the alkyl chain length reduced
superoxide trapping capability (8 > 7 > 6 > 5). In contrast, lipid peroxidation in rat liver microsomes was reduced by 5 and 6 in
dose-dependent manner. Compounds 3 and 4 were poor lipid peroxidation inhibitors, being approximately 400- to 1300-fold lower
than 5 and 6. In addition, all compounds inhibited cell growth of human leukemia cell lines, HL60 and HL60R, in dose-dependent
manners (5 > 6 > 3 = 4). The HL60R cell line is resistant against 1. Growth of both cell lines was suppressed by 5 and 6 in a fashion
dependent on the length of the aminophenol alkyl chain, but not by 3 and 4. These results indicate that 5, a potent anticancer agent
greater than 2, may potentially have clinical utility, and that its anticancer activity is correlated with inhibitory potency against lipid
peroxidation, but not with superoxide scavenging activity.
ꢀ
2006 Elsevier Ltd. All rights reserved.
1
. Introduction
types. Compound 2 currently is in clinical trials for the
treatment of breast, bladder, renal, and neuroblastoma
4
–10
N-(4-Hydroxyphenyl)retinamide (2, fenretinide), a
synthetic amide of all-trans-retinoic acid (1, Fig. 1), is
an effective chemopreventive and antiproliferative
–4
agent, which is used against a wide variety of tumor
malignancies.
It induces apoptosis in HL60 and
NB4 human leukemia cell lines, as well as in C33A, a
human cervical carcinoma cell line, meningioma, and
8,11–17
neuroblastoma.
1
Previous studies have shown that 2 exhibits antioxidant
activities that include scavenging a,a-diphenyl-b-
picrylhydrazyl radicals, inhibiting linoleic acid peroxida-
tion initiated by hydroxyl radicals, and reducing lipid
peroxidation in rat liver microsomes to the same extent
Abbreviations: 1, retinoic acid; 2, N-(4-hydroxyphenyl)retinamide,
0
fenretinide; 3, p-dodecanoylaminophenol, 4 -hydroxydodecanilide, N-
(p-hydroxyphenyl)dodecananamide, N-(4-hydroxyphenyl) dodecanana-
0
mide; 4, p-decanoylaminophenol, 4 -hydroxydecanilide, N-(p-hydroxy-
phenyl) decananamide, N-(4-hydroxyphenyl)decananamide; 5, p-
dodecylaminophenol, 4-(dodecylamino)phenol; 6, p-decylaminophenol,
1
8,19
as or greater than vitamin E.
The p-methylamin-
ophenol moiety (8, Fig. 1) contributes most significantly
to the inhibition of lipid peroxidation of 2 as compared
with the 4-aminophenol and p-aminoacetophen moie-
4
-(decylamino)phenol; 7, p-octylaminophenol, 4-(octylamino)phenol; 8,
p-methylaminophenol, 4-(methylamino)phenol; 4-HBR, 4-hydroxyben-
zylretinone; PBS, phosphate-buffered saline (1.5 mM KH PO , 8.1 mM
Na HPO , and 136.9 mM NaCl, pH 7.2); RAR, retinoic acid nuclear
18,20
2
4
ties.
This is derived from the side-chain amido por-
2
4
tion of 2 that distinguishes it from 1, which itself is an
antioxidant. In addition, 7, which has an elongated
methylene chain in 8, increases the inhibitory activity
receptor; EDTA, ethylenediaminetetraacetic acid; ROS, reactive
oxygen species.
Keywords: Aminophenol; Antioxidant; Anticancer; Retinoid.
2
1
*
Corresponding author. Tel./fax: +81 3 5498 5950; e-mail: t-noriko@
hoshi.ac.jp
of lipid peroxidation as compared with 8, while 7
decreases the superoxide scavenging activity (Fig. 1).
0
968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.05.013

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N. Takahashi et al. / Bioorg. Med. Chem. 14 (2006) 6089–6096
O
O
O
On the other hand, growth of various cancer cell lines
including human leukemia cell lines, HL60 and
HL60R, which is resistant against 1, was suppressed
by 7, p-hexylaminophenol, p-butylaminophenol, and 8
in fashions dependent on the alkyl chain length linked
to aminophenol, with 7 being the most potent inhibitor
of cell growth in HL60R cells among these p-alkylamin-
OH
1
H
N
2
3
4
5
2
1
ophenols tested, including 2.
OH
In the present study, we synthesized novel four
compounds, p-dodecanoylaminophenol (3), p-decanoyl-
aminophenol (4), p-dodecylaminophenol (5), and
p-decylaminophenol (6) (Fig. 1), which bear carbon
side-chain lengths similar to those of 1 and 2, and we
examined their antioxidant and anticancer properties.
H
N
OH
OH
OH
O
H
N
H
N
2. Results
2.1. Scavenging of superoxides by p-alkylaminophenols,
p-acylaminophenols, and retinoids
H
N
6
7
The superoxide scavenging activities of compounds were
evaluated by measuring the absorbance of blue forma-
zan produced from the yellow dye NBT . Blue forma-
zan is formed by the reaction of remaining superoxide
produced by a hypoxanthine/xanthine oxidase system.
The absorbance of control was approximately 0.8. At
OH
OH
2
+
H
N
1
and 5 (C ), scavenged superoxide to the extent of
approximately 82% and 70%, respectively (Fig. 2A).
This indicated little effect on xanthine oxidase activity
(Fig. 2B). Compound 6, which has shorter alkyl chain
than 5, was a more potent superoxide scavenger. In con-
0 lM concentration, p-alkylaminophenols, 6 (C )
10
H
N
12
8
OH
Figure 1. Chemical structures of retinoids (1 and 2), p-acylaminophe-
nols (3 and 4), and p-alkylaminophenols (5, 6, 7, and 8).
A
B
Figure 2. Superoxide scavenging activities and effects of the formation of uric acid by retinoids, p-acylaminophenols, and p-alkylaminophenols. (A)
Reaction mixtures containing compounds (1, 2, 3, 4, 5, and 6) at the concentration of 10 lM or DMSO (C) were mixed, and then xanthine oxidase
was added as described under Section 4. After the incubation at 25 ꢁC for 20 min, CuCl was added to the reaction mixture. The subsequent extent of
2
NBT reduction was determined at 560 nm by spectrophotometer. The SD of each data point was ꢀ8% of the mean. N: none. (B) Reaction mixtures
containing compounds (1, 2, 3, 4, 5, and 6) at the concentration of 10 lM or DMSO (C) without NBT solution were mixed, and then xanthine
2
oxidase was added as described under Section 4. After the incubation at 25 ꢁC for 20 min, CuCl was added to the reaction mixture. Absorbances of
uric acid were determined at 290 nm by spectrophotometer. The SD of each data point was ꢀ8% of the mean. Experiments were repeated at least four
times. N: none.

N. Takahashi et al. / Bioorg. Med. Chem. 14 (2006) 6089–6096
6091
1
20
00
120
100
1
8
6
4
2
0
0
0
0
80
60
40
20
0
0
0
0.01
0.1
1
10
Concentration (μM)
0
0.1
1
10
Figure 4. Inhibition of ascorbate-dependent lipid peroxidation by p-
acylaminophenols and p-alkylaminophenols. Microsomes (0.5 mg
protein/ml) and various concentrations (0.01–10 lM) of 3 ( ), 4 ( ),
Concentration (μM)
5
FeCl
(h), and 6 (s) in 100 mM Tris–HCl (pH 7.5) containing 15 lM
Figure 3. Superoxide scavenging activity by various concentrations of
p-alkylaminophenols (5, 6, 7, and 8). Reaction mixture containing
various concentrations of compounds (5 (h), 6 (s), 7 (Æ), and 8 (n))
was mixed, and then xanthine oxidase was added as described under
3
, and 4 mM ADP, were preincubated at 37 ꢁC for 1 min. To
reaction mixtures was added 1 mM ascorbic acid and incubation was
continued at 37 ꢁC for 20 min. TBA reagent was added, then the
mixtures were heated in a boiling water bath for 15 min and
centrifuged as described in Section 4. Supernatant absorbance was
2
Section 4. After the incubation at 25 ꢁC for 20 min, CuCl was added
to the reaction mixture. The subsequent extent of NBT reduction was
determined at 560 nm by spectrophotometer. The SD of each data
point was ꢀ8% of the mean. Experiments were repeated at least three
times.
ꢁ
1
ꢁ1
measured at 535 nm (e = 156,000 cm
M ). The SD of each data
point was ꢀ8% of the mean. Experiments were repeated at least three
times.
trast, retinoids (1 and 2) and p-acylaminophenols (3 and
Table 1. Comparison of IC50 values of p-acylaminophenols and
p-alkylaminophenols on MDA production derived from lipids
4) were inactive on both scavenging of superoxides and
production of superoxides by the xanthine oxidase sys-
tem (Fig. 2). In addition, p-alkylaminophenols, 8 (C1),
7 (C ), 6 (C ), and 5 (C ), eliminated superoxide in
8 10 12
dose-dependent manners with IC50 values of approxi-
Compound
IC50 (lM)
Fold
1
6
5
4
3
0.015
0.0155
6.1
1.03
407
1333
mately 0.6 lM for 8, 1.5 lM for 7, 2 lM for 6, and
20
6
lM for 5 (Fig. 3). These results indicate that p-alkyl-
IC50 values were obtained from Figure 4. Fold (ratio) of the IC50
values for 3, 4, 5, and 6 were calculated according to the following
formula: (IC50 of 3, 4, 5, and 6)/(IC50 of 6).
aminophenols, 6 and 5, exhibit superoxide scavenging
activity, and that elongation of the alkyl chain, which
increases hydrophobicity, reduces the capability to trap
superoxide. Compounds 4 and 3, which inserted carbon-
yl moieties into 6 and 5, were inactive.
(Table 1). Thus, antioxidant activities of 5 and 6 were
approximately 400- and 1300-fold higher than those of
3 and 4, respectively (Fig. 4, Table 1). Compounds 5
and 6 inhibited lipid peroxidation to a similar extent
as 7 (IC50 value approximately 0.014 lM), but not to
the same extent as 8 (IC50 value approximately
4.6 lM). These results indicate that the insertion of
carbonyl moieties into p-alkylaminophenols affected
their ability to inhibit lipid peroxidation greater than
did differences in alkyl chain length.
2.2. Inhibition of liver microsomal lipid peroxidation by
p-alkylaminophenols and p-acylaminophenols
The extent of lipid peroxidation in vitro was measured
by lipid-derived malondialdehyde (MDA) production.
A reaction mixture containing the vehicle DMSO was
identical to a control mixture in the absence of com-
pounds. As shown in Figure 4, analogues 3, 4, 5, and
6
inhibited lipid peroxidation in dose-dependent fash-
ions in the range of 0.01–10 lM. The p-alkylaminophe-
nols, 5 and 6 showed marked inhibition of lipid
peroxidation and were more efficient than the p-acylam-
inophenols, 3 and 4. Estimation of IC50 values from Fig-
ure 4, indicated values of approximately 0.015 lM for 6
2.3. Growth inhibition of HL60 and HL60R cells by
p-alkylaminophenols, p-acylaminophenols, and 2
HL60 and HL60R cells, which were resistant to 1, were
grown in medium containing 10% FBS in the presence
of the new aminophenols (Fig. 5). The parent compound
2 was used as an internal standard for measuring
(
C ) and 0.0155 lM for 5 (C ), while IC values for 4
10 12 50
and 3 were approximately 6.1 and 20 lM, respectively

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N. Takahashi et al. / Bioorg. Med. Chem. 14 (2006) 6089–6096
A 120
B 120
1
00
100
80
60
40
20
0
8
6
4
2
0
0
0
0
0
0
0.1
1
10
0
0.1
1
10
Concentration (μM)
Concentration (μM)
5
Figure 5. Growth of HL60 and HL60R cells in the presence of 2, p-acylaminophenols, and p-alkylaminophenols. Cells (1 · 10 cells/ml) were grown
with various concentrations of 2 (n), 3 ( ), 4 ( ), 5 (h), and 6 (s) in RPMI medium containing 10% FBS. Growth was measured at 94 h for HL60
(A) or 65 h for HL60R (B) as described under Section 4. Net growth % of control is shown for each compound. Each point is the mean of at least four
measurements. The SD of each point was ꢀ8% of the mean.
inhibition of cell growth. Shown in Figure 5 is the per-
cent net cell growth in the presence of the aminophenol
analogues with values adjusted by subtracting the initial
cell concentrations of experimental cultures from the ini-
tial concentrations of control cultures, which were de-
fined as 100% at each time period. Growth of HL60
and HL60R cells was inhibited by various concentra-
tions of p-alkylaminophenols (5 and 6), p-acylamin-
ophenols (3 and 4), and 2 in dose-dependent manners
These results may indicate that the hydrophobic proper-
ties of an dodecanyl residue linked to the aminophenol
may be significant for antitumor potency.
3. Discussion
Analogue 5 exhibited the most potent anticancer and
lipid peroxidation inhibitory activity as compared to 2,
a cancer chemopreventive and antiproliferative agent,
and a potent inducer of apoptosis. In the current study,
a series of aminophenols (3, 4, 5, and 6) having p-acyl
and p-alkyl residues of similar lengths to 1 and 2 were
prepared and examined for two kinds of antioxidant
activities and anticancer efficacy. Compound 5 sup-
pressed growth of both HL60 and HL60R cells to an
extent greater than 2 and the other aminophenols exam-
ined. Cell growth inhibition by the p-alkylaminophenols
was elevated according to the elongation of the alkyl
chains. The extent of lipid peroxidation reduction in
rat liver microsomes by p-alkylaminophenols (5, 6, 7,
and 8) depended on the length of the alkyl chain
appended to the aminophenol residue with positive cor-
relation. Compounds 6 and 5 were potent inhibitors of
lipid peroxidation, being of approximately 400- and
1300-fold higher potency than 4 and 3, respectively.
In addition, 6 and 5 exhibited superoxide scavenging
ability dependent on the length of alkyl chain with neg-
ative correlation. The p-acylaminophenols, 3 and 4, were
either inactive or weakly active. Thus, the presence of
carbonyl moieties in p-alkylaminophenols diminished
their capabilities as anticancer agents and antioxidants.
(
Fig. 5). In HL60 cells, at low concentrations in the
range of 0.1–1 lM, 5 and 6 inhibited cell growth more
potently than 2 (Fig. 5A). In contrast, 3 and 4 were poor
inhibitors of cell growth as compared to 2, 5, and 6. On
the other hand, growth of HL60R cells was inhibited
more strongly in the order 5, 6, 2, than by 3 or 4
(
Fig. 5B). Cell growth in the presence of aminophenol
analogues at a concentration of 1 lM indicated that 5
approximately 65% inhibition) was more potent than
(
6
3
(approximately 47% inhibition), 2 (approximately
3% inhibition), 3 and 4 (approximately 3% inhibition)
in HL60 cells. In contrast, 5 (approximately 82% inhibi-
tion) was more potent than 6 (approximately 42% inhi-
bition), 2 (approximately 32% inhibition), 3 and 4
(
(
approximately 10% inhibition) in HL60R cells
Fig. 5). In both HL60 and HL60R cells, elongation of
the alkyl chain attached to the aminophenol residue (5
and 6) increased antiproliferative activities, while elon-
gation of the acyl chain attached to the aminophenol
residue (3 and 4) had no effect. Inhibition of cell growth
by p-alkylaminophenols was approximately 10- to 50-
fold greater than by p-acylaminophenols, having acyl
residues instead of alkyl chains. These results indicate
that the inhibition of cell growth by these compounds
cannot be attributed to cell necrosis, and that leukemia
cell growth is suppressed by 5 more strongly than by
other aminophenols and retinoids. Compound 5 was
the most potent inhibitor among the seven p-alkylamin-
ophenols examined in previous study, including 7 and 8.
Previous reports have shown that alkylaminophenols
containing 7 and 8 inhibited lipid peroxidation (MDA
2
0,22
formation).
inhibitory activity in dose-dependent fashion in the
Compound 8 showed lipid peroxidation
range of 1–5 lM, and 7, p-hexylaminophenol, and

N. Takahashi et al. / Bioorg. Med. Chem. 14 (2006) 6089–6096
7–29
6093
2
p-butylaminophenol showed marked inhibition of lipid
peroxidation. At concentrations higher than 5 lM, 2
significantly increased MDA formation, while 10 lM
of 1 inhibited lipid peroxidation with an IC50 value of
approximately 3.2 lM. IC50 values were approximately
ery of retinol to eye.
It is generally accepted that 2
may act via pathways which are independent of the
nuclear retinoid receptors, RARs and RXRs, since 2
exhibits extremely poor binding to the nuclear retinoid
receptors and shows anticancer activity against cells
resistant to 1. Therefore, 2 may act on cells directly rath-
er than through hydrolysis to free 1. Recently, in order
to overcome side effects of 2, several non-hydrolyzable
compounds were designed and evaluated. 4-Hydroxy-
benzylretinone (4-HBR), a stable C-linked analogue of
2 that cannot be hydrolyzed to 1, was synthesized and
shown to exhibit antitumor effects similar to 2, without
4
(
0
.6 lM for 8 (C ), 0.3 lM for p-butylaminophenol
1
C ), 0.033 lM for p-hexylaminophenol (C ), and
4
6
.014 lM for 7 (C ). Thus, the elongation of the p-alkyl-
8
aminophenol chains affects ability to inhibit lipid
peroxidation. The longer the alkyl chains of the p-alkyl-
aminophenols, the higher was the antioxidant potency.
In the current study, IC50 values for 6 (C ) and 5
10
2
5,30
(
respectively. These values were not significantly different
C ) were approximately 0.015 and 0.0155 lM,
night blindness side effects.
In addition, 2-C-glucu-
1
2
ronide (2CG), a C-linked analog of 2-glucuronide that
cannot be hydrolyzed to 2, is an effective chemopreven-
tive agent with almost no side-effects (e.g., reduction of
retinol level and elevation of triglyceride in blood,
from the 0.014 lM, IC50 value of 7 (C ). The extent of
8
inhibitory activity against lipid peroxidation incurred
by elongation of the p-alkylaminophenol chains
increased until compound 7 and remained at this
maximum value with compounds 6 and 5. These results
suggest that novel compounds 6 and 5 as well as 7 are
extremely effective antioxidants.
3
1,32
etc.).
However, as both 4-HBR and 2CG contain
the same aromatic ring as retinol and 1 and 2, they
may affect endogenous retinol levels in the body,
because retinol binding protein recognizes and binds
the aromatic ring of retinol. In the current study, we
designed compounds without aromatic rings (3, 4, 5,
and 6) that have markedly different structures from
retinol and 1 and 2. Compound 5 was the most potent
antiproliferative agent among these compounds
Mitochondrial lipid peroxidation was inhibited by reti-
noids (retinol, retinol acetate, 1, retinol palmitate, and
2
3
retinal) only at high concentrations (0.1 ꢂ 10 mM).
In contrast, 13-cis-retinoic acid and 1 suppressed micro-
2
4
somal lipid peroxidation with IC values of 10 lM
including 2. Compound 5 inhibited cell growth
5
0
2
0,22
and 3.2 lM, respectively.
less effective than 8 and that elongation of the methyl
It was found that 2 was
gradually from low to high concentrations as compared
to 2, which was toxic at high concentrations. Compound
5 was more potent than 2 by approximately 5-fold in
HL60 cells and approximately 3-fold in HL60R cells.
Therefore, it is possible that 5 may be an effective
anticancer drug without the side effects observed with
2. It would be interesting to further examine anticancer
activities of 5 against various cancer cell lines.
residue of 8 to an octyl residue increased the efficacy
2
0
of lipid peroxidation inhibition. In the current study,
and 4, p-acylaminophenols containing carbonyl resi-
3
dues, which exhibited structures similar to 2 and other
retinoids, showed much less activity than the p-alkylam-
inophenols 5 and 6. These results indicate that the addi-
tion of oxygen into the alkyl residue may be critical for
lipid peroxidation inhibitory activity. The p-alkylamin-
ophenols are excellent antioxidants as compared with
p-acylaminophenol and retinoids.
In the current study, 5 inhibited cell growth more
potently than 2 against HL60 and HL60R cells, which
are resistant to 1 (Fig. 5). In cytodifferentiation therapy
of promyeloid leukemia, a high percentage of patients in
complete remission induced by 1 alone relapse within a
It has been reported previously that p-alkylaminophe-
nols (8, p-butylaminophenol, p-hexylaminophenol, and
7) exhibit superoxide radical scavenging activity, which
is another antioxidant activity different from lipid per-
oxidation inhibitory activity. Compound 8 was the
most potent scavenger of superoxide radicals (8 > p-
butylaminophenol > p-hexylaminophenol > 7). In the
current study, compound 6 showed superoxide radical
scavenging activity greater than 5, but less than 7 and
3
3–35
few months.
to further treatment with 1.
Most relapsed patients are resistant
5–37
3
The clinical outcome
of patients treated with 1 may be modified by adminis-
tration of 5. Studies are presently underway to deter-
mine the toxicity of 5 in animals and to measure the
effectiveness of 5 on tumors growing in animals.
2
1
Free radicals can cause certain forms of cancer, cardio-
vascular and cerebrovascular diseases and ischemia/re-
perfusion injuries. Antioxidants, including vitamins
8
. In contrast, the p-acylaminophenols, 3 and 4 were
inactive. The hydrophobic nature of the compounds
was not related to superoxide trapping ability. Elonga-
tion of the alkyl chain of p-alkylaminophenols correlat-
ed negatively with superoxide scavenging activity and
positively with inhibitory activity against lipid
peroxidation.
3
8–41
and flavonoids, can prevent these diseases.
Our
study showed that novel 5 and 6 exhibited antioxidative
and anticancer activities. Superoxide scavenging activity
may be due to the phenolic hydroxyl hydroxylÆ In con-
trast, inhibition of lipid peroxidation and anticancer ac-
tion may be due to the three-dimensional structure
formed by the phenolic hydroxyl and the alkyl residues.
The alkyl residue may be particularly significant for
associations to membranes in cells, showing inhibition
of lipid peroxidation or anticancer action. Compounds
5 and 6 may be effective in diseases concerned with hy-
droxy radicals and may affect diseases involving super-
Even though 2 is a potent anticancer drug, clinical stud-
ies have shown that treatment of patients with 2 is
accompanied by a side effect of night blindness due to
2
5,26
decreasing of serum retinol levels.
It appears that
this side effect is due to the displacement of retinol from
serum retinol binding protein, resulting in reduced deliv-

6094
N. Takahashi et al. / Bioorg. Med. Chem. 14 (2006) 6089–6096
oxide. The antioxidant and antiproliferative activities of
and 6 may influence various physiological processes,
including immunostimulation, enhancement of cell com-
munication, and inhibition of metabolic activation.
J = 7.3 Hz), 6.65 (2H, d, J = 8.9 Hz), 7.33 (2H, d,
1
3
5
J = 8.9 Hz), 9.11 (1H, s), 9.56 (1H, s). C NMR (DMSO)
13.9, 22.0, 25.1, 28.6, 28.6, 28.7, 28.8, 31.2, 36.1, 114.8,
120.7, 130.9, 152.9, 170.4. IR (KBr) 3310, 1650, 1550,
ꢁ
520, 1250, 830 cm . Anal. Calcd for C H NO : C,
16 25 2
2.96; H, 9.57; N, 5.32. Found: C, 73.02; H, 9.30; N, 5.26.
1
1
7
4. Experimental
4
.2.4. p-Dodecanoylaminophenol (3). 4-Aminophenol
4
.1. General
(3.27 g, 30 mmol) converted to the title compound by
the procedure described for amide 4 using dodecanoic
anhydride. Compound 3 (86% yield) as a colorless solid
0
Adenosine 5 -diphosphate (ADP), 1, nitroblue tetrazoli-
um (NBT), ethylenediaminetetraacetic acid (EDTA),
bovine serum albumin (BSA, fraction V), and dimethyl-
sulfoxide (DMSO) were obtained from Sigma Chemical
Co. (St. Louis, MO, USA). 2 was provided by Dr. R. C.
Moon, University of Illinois, Chicago, IL, USA. Dithi-
othreitol (DTT), trichloroacetic acid (TCA), and 8 were
purchased from Nacalai Tesque, Inc. (Kyoto, Japan). 4-
Aminophenol, decanoic anhydride, dodecanoic anhy-
was stored under N and could be used directly without
2
further purification: mp 133–134 ꢁC. MS m/z EI, 291
+
1
(M ). H NMR (DMSO) 0.85 (3H, t, J = 6.4 Hz), 1.24
(16H, m), 1.55 (2H, m), 2.22 (2H, t, J = 7.4 Hz), 6.66
(2H, d, J = 8.9 Hz), 7.34 (2H, d, J = 8.9 Hz), 9.12 (1H,
1
3
s), 9.57 (1H, s). C NMR (DMSO) 14.0, 22.1, 25.2,
28.7, 28.7, 28.8, 28.9, 29.0, 29.0, 31.3, 36.2, 114.9,
120.8, 131.0, 153.0, 170.5. IR (KBr) 3310, 1650, 1550,
ꢁ
1520, 1250, 830 cm . HRMS Calcd for C H NO :
18 29 2
1
dride, tetrahydrofuran, and NaBH were obtained from
4
Aldrich Chemical Co. (Milwaukee, WI, USA). Ascorbic
acid and 2-thiobarbituric acid (TBA) were purchased
from Wako Pure Chemicals Industries, Co. Ltd (Osaka,
Japan).
291.2198. Found: 291.2204.
4.2.5. p-Decylaminophenol (6). NaBH (0.946 g, 25 mmol)
4
was added to 4 (2.63 g, 10 mmol) in dry THF (30 ml). To
the suspension treated dropwise iodine (2.54 g, 10 mmol)
4
.2. Synthesis of compounds
in dry THF (20 ml) under N at 0 ꢁC for 1 h. After the
2
reaction mixture was refluxed for 3 h, it was cooled to
room temperature, treated by careful addition of 3 M
HCl (6 ml), neutralized by 1 M NaOH, and extracted
twice with ethylacetate. The combined organic extract
4.2.1. Chemistry. The synthesis of 3, 4, 5, and 6 began by
treating commercially available 4-aminophenol with the
corresponding acyl anhydride. The N-acylation provided
2
4
the desired amide (3, 4) in excellent yield. Reduction of
the amide (3, 4) using NaBH and I in refluxing tetrahy-
was washed with H O and brine, dried over anhydrous
2
Na SO , and evaporated under reduced pressure. The
2
4
2
4
43
drofuran (THF) gave p-alkylaminophenol (5, 6).
residue was purified by column chromatography on silica
gel (ether/hexane = 1:2) to give 6 (2.43 g, 98%); mp
+
1
4
.2.2. Analysis. Melting points were measured with a
82–83 ꢁC. MS m/z EI, 249 (M ). H NMR (CHCl ) 0.88
3
Yanagimoto micro melting point apparatus (Yanagim-
oto Manufacturing Co., Tokyo, Japan) without correc-
tion. IR spectra were recorded on a JASCO FT/IR- 200
(3H, t, J = 6.9 Hz), 1.26 (14H, m), 1.58 (2H, m), 3.03
(2H, t, J = 6.9 Hz), 4.08 (2H, br), 6.53 (2H, d,
J = 8.6 Hz), 6.66 (2H, d, J = 8.6 Hz). C NMR (CHCl3)
14.1, 22.6, 27.1, 29.3, 29.4, 29.5, 29.6, 29.6, 31.9, 45.5,
114.8, 116.2, 142.3, 148.1. IR (KBr) 3280, 1520, 1250,
1
3
ꢁ
1
1
13
and major absorption was listed in cm . H and
C
NMR spectra were obtained on a JEOL GSX 270
instrument (Tokyo, Japan), and chemical shifts were
ꢁ
1
820 cm . Anal. Calcd for C H NO: C, 77.06; H,
1
6
27
reported in ppm on the d-scale from internal Me Si.
10.91; N, 5.62. Found: C, 76.90; H, 10.67; N, 5.70.
4
MS spectra were measured with a JEOL JMS 600
spectrometer by using the electron impact (EI) methods.
Elemental analyses were performed on a Perkin-Elmer
4.2.6. p-Dodecylaminophenol (5). Compound 3 (2.91 g,
10 mmol) converted to the title compound by the proce-
dure described for aminophenol 6. Purification by column
chromatography on silica gel (ether/hexane = 1:1) affor-
ded the product (87% yield) as a colorless needle; mp
2
40-B instrument (Perkin-Elmer Inc., MA, USA).
Column chromatography was performed on silica gel
45–75 lm, Wakogel C-300). TLC was carried out on
(
+
1
glass plates coated with silica gel F254 (Merk, Germany).
Spot detection was performed with UV 254 nm or iodine
vapor. THF was distilled over potassium metal.
86–88 ꢁC. MS m/z EI, 277 (M ). H NMR (CHCl ) 0.88
3
(3H, t, J = 6.7 Hz), 1.26 (18H, m), 1.59 (2H, m), 3.04
(2H, t, J = 7.4 Hz), 6.53 (2H, d, J = 8.7 Hz), 6.69 (2H, d,
J = 8.7 Hz). C NMR (CHCl ) 14.1, 22.6, 27.1, 29.3,
1
3
3
4
(
.2.3. p-Decanoylaminophenol (4). Decanoic anhydride
10.78 g, 33 mmol) was added portionwise to 4-amino-
29.4, 29.5, 29.6, 29.6, 31.9, 45.5, 114.8, 116.2, 142.3,
148.1. IR (KBr) 3370, 1520, 1240, 820 cm 1. HRMS
ꢁ
phenol (3.27 g, 30 mmol) in dry THF (200 ml) at 0 ꢁC.
After being stirred at room temperature for 16 h, the reac-
tion mixture was evaporated under reduced pressure. The
Calcd for C H NO: 277.2406. Found: 277.2396.
8
1
31
4.3. Measurement of microsomal lipid peroxidation
residue was added CHCl (200 ml), stirred for another
3
1
which was stored under N and could be used directly
h, and filtered to give 4 (7.18 g, 91%) as a colorless solid
Lipid peroxidation in rat liver microsomes was quanti-
tated by measurement of malondialdehyde (MDA)
using ADP-chelated ion and ascorbate as described pre-
2
without further purification: mp 129–130 ꢁC. MS m/z
EI, 263 (M ). H NMR (DMSO) 0.84 (3H, t,
J = 6.3 Hz), 1.24 (12H, m), 1.54 (2H, m), 2.21 (2H, t,
+
1
24
viously. Rat liver microsomes were prepared by the
2
method as described previously. Reaction mixtures
0

N. Takahashi et al. / Bioorg. Med. Chem. 14 (2006) 6089–6096
6095
consisting of microsomes (0.5 mg protein/ml) and com-
pound (1, 2, 3, 4, 5, 6, 7 or 8 dissolved in DMSO) in
ed by an electric particle counter (Coulter Electronics,
Hialeah, FL) and viability by Trypan blue dye exclu-
sion. The percentage of net growth is shown with values
adjusted by subtracting the initial cell concentration of
experimental cultures from the initial concentrations of
control cultures which were defined as 100%. Values
for percent net growth were calculated with the follow-
ing formula: [(cell concentration of experimental cul-
ture) ꢁ (initial cell concentration)/(cell concentration of
control culture) ꢁ (initial cell concentration)] · 100.
1
00 mM Tris–HCl (pH 7.5) containing 15 lM FeCl3,
and 4 mM ADP, were preincubated at 37 ꢁC for
min. Reaction mixtures in which ascorbic acid was
1
added at a final concentration of 1 mM were incubated
at 37 ꢁC 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). Superna-
tant absorbance was measured spectrophotometrically
-
1
ꢁ1
at 535 nm (e = 156,000 cm M ).
Acknowledgments
4
.4. Measurement of superoxide anion scavenging activity
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.
The superoxide radicals were generated in vitro by the
hypoxanthine/xanthine oxidase system. The scavenging
activities of compounds were determined by the NBT
Åꢁ
reduction method. In this method, O reduces the yel-
2
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