Synthesis and evaluation of 4-hydroxyphenylacetic acid amides and 4-hydroxycinnamamides as antioxidants

Article abstract of DOI:10.1016/S0960-894X(02)00479-1

4-Hydroxyphenylacetic acid amides and 4-hydroxycinnamamides were synthesized and their antioxidant and neuroprotective activities were evaluated. Among the prepared compounds, 6f, 6g, 8b, and 9 exhibited potent inhibition of lipid peroxidation in rat brain homogenates, and marked DPPH radical scavenging activities. Furthermore, 6f, 6g, and 9 exhibited neuroprotective action against the oxidative damage induced by the exposure of primary cultured rat cortical cells to H₂O₂, xanthine/xanthine oxidase, or Fe²⁺/ascorbic acid. Based on these results, we found that 6f was the most potent antioxidant among the compounds tested.

Full text of DOI:10.1016/S0960-894X(02)00479-1

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2599–2602
Synthesis and Evaluation of 4-Hydroxyphenylacetic Acid Amides
and 4-Hydroxycinnamamides as Antioxidants
Young-Sik Jung,a Tae-Souk Kang,a Joong-Ho Yoon,a Bo-Young Joe,a
Hee-Jong Lim,a Churl-Min Seong,a Woo-Kyu Park,a Jae-Yang Kong,a
Jungsook Chob and No-Sang Parka,*
aMedicinal Science Division, Korea Research Institute of Chemical Technology, PO Box 107, Yusong, Taejon 305-606, Republic of Korea
bDepartment of Pharmacology, College of Medicine, Dongguk University, Kyongju, Kyongbuk 780-714, Republic of Korea
Received 19 April 2002; accepted 17 June 2002
Abstract—4-Hydroxyphenylacetic acid amides and 4-hydroxycinnamamides were synthesized and their antioxidant and neuropro-
tective activities were evaluated. Among the prepared compounds, 6f, 6g, 8b, and 9 exhibited potent inhibition of lipid peroxidation
in rat brain homogenates, and marked DPPH radical scavenging activities. Furthermore, 6f, 6g, and 9 exhibited neuroprotective
action against the oxidative damage induced by the exposure of primary cultured rat cortical cells to H2O2, xanthine/xanthine
oxidase, or Fe2+/ascorbic acid. Based on these results, we found that 6f was the most potent antioxidant among the compounds
tested. # 2002 Elsevier Science Ltd. All rights reserved.
Reactive oxygen species have been recognized to play an
important role in the initiation and/or progression of
various diseases such as ischemia-reperfusion injury,
atherosclerosis, and inflammatory injury.1 There is a
growing interest in natural and unnatural antioxidants
as a protective strategy against these diseases by block
or removal of oxidative stresses.2 a-Tocopherol is the
most important and widely studied natural, lipid-solu-
ble, and chain-breaking antioxidant. Recent studies
provide evidences of protective effects of a-tocopherol
against atherosclerosis3 and reperfusion injury.4 AM-36,
a mixed antioxidant and sodium channel blocker, sig-
nificantly reduced infarct volume and ischemia-induced
increases in reactive oxygen species.5 Intravenous
administration of BN 80933, a dual inhibitor of neuro-
nal nitric oxide synthase and lipid peroxidation, sig-
nificantly reduced brain damage induced by head
trauma in mice, global ischemia in gerbils, and transient
focal ischemia in rats.6
In the course of the development of new antioxidants as
neuroprotective agents, we have been interested in novel
4-hydroxyphenylacetic acid amides and 4-hydroxy-
cinnamamides based on our preliminary findings that a
4-hydroxyphenylacetic acid amide 1 has an inhibitory
activity on lipid peroxidation. The structure of 1 is clo-
sely related with capsaicin, a vanilloid receptor agonist.
Our previous studies suggested that 1 is a centrally act-
ing agent with potent analgesic activity.7 These findings
prompted us to investigate the antioxidant and neuro-
protective properties of a series of 4-hydroxyphenylacetic
acid amides.
*Corresponding author. Tel.: +82-42-860-7130; fax: +82-42-861-1291; e-mail: nspark@krict.re.kr
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00479-1

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Y.-S. Jung et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2599–2602
The present paper deals with the synthesis and the biolog-
ical evaluation of a series of N-{3-(3,4-dimethylphenyl)-
propyl}-4-hydroxyphenylacetic acid amides substituted
at the C-3 position with various alkoxy groups and
several N-{3-(3,4-dimethylphenyl)propyl}-4-hydroxy-
cinnamamides.
4a–e with aqueous sodium hydroxide solution in MeOH
at 0 ꢀC followed by acidic aqueous workup provided the
phenylacetic acids 5a–e in high yields (64–86%). Detailed
synthetic procedures from 2 to 5 were described in our
previous report.8 Coupling of the acids 5 with 3-(3,4-
dimethylphenyl)propylamine using 1,3-dicyclohexycarbo-
diimide (DCC) and 1-hydroxybenzotriazole (HOBT) in
acetonitrile provided the phenylacetic acid amides 6 in 77–
96% yield after flash column chromatography. Demethyl-
ation of the amide 1 and 6e with BBr3 in CH2Cl2 afforded
benzenediol 6f (85%) and benzenetriol 6g (77%), respec-
tively. 4-Hydroxycinnamamides (8a, 8b) were also
obtained from the corresponding acids (7a, 7b) and
3-(3,4-dimethylphenyl)propylamine in 74 and 75% yields,
and the demethylation of the amide 8a with BBr3 in
CH2Cl2 afforded the amide 9 in 65% yield (Scheme 2).
Chemistry
In the course of our research aimed at the development
of new 4-hydroxyphenylacetic acid amides as anti-
oxidants, we needed 4-hydroxyphenylacetic acids, which
contain various alkoxy groups on the C-3 position of
the ring. To the best of our knowledge, the synthesis
of 3-alkoxy-4-hydroxyphenylacetic acids had not been
reported, even though 3-methoxy-4-hydroxyphenyl-
acetic acid (homovanillic acid) is commercially avail-
able. Thus, a general synthetic method of 3-alkoxy-4-
hydroxyphenylacetic acids from methyl 3-alkox-
ybenzoates has been developed (Scheme 1).
Results and Discussion
In order to evaluate antioxidant properties of the newly
synthesized amides, we first examined the effects on lipid
peroxidation in rat brain homogenate by thiobarbituric
acid reactive substances (TBARS) assay9 according to
the method of Stocks.10 The formation of lipid per-
oxides was significantly inhibited by the test com-
pounds. The IC50 values are shown in Table 1.
Regarding the alkoxy substituents at the C-3 position of
amides (1, 6a–6c), longer alkoxy groups enhanced the
inhibition although the potency is still lower than that
of 2,6-di-tert-butyl-4-methylphenol (BHT) or a-toco-
pherol (Table 1). However, phenoxy substituent (6d)
retreated the activity. In the comparison of –OMe
Methyl 3-alkoxybenzoates 2 were easily obtained from
the alkylations of methyl 3-hydroxybenzoate with the
corresponding alkyl bromides or alkyl iodides for 2a–c
or the esterification of 3-phenoxybenzoic acid with
methanol for 2d. Reduction of methyl 3-alkoxy-
benzoates 2a–e with potassium metal under Birch con-
ditions, followed by alkylation with methyl
bromoacetate resulted in the dienes 3a–e in 64–100%
yields. Allylic oxidation of the dienes 3a–e with pyr-
idinium dichromate (PDC) in benzene provided the die-
nones 4a–e in moderate yields (43–66%). Treatment of
Scheme 1. (a) K, t-BuOH, NH3, THF, À78 ꢀC, 1 h; (b) BrCH2CO2CH3, piperylene, rt, 30 min; (c) PDC, Celite, 90% t-BuOOH, C6H6, rt, 6 h; (d)
NaOH, MeOH, 0 ꢀC to rt, 4 h; (e) 3-(3,4-dimethylphenyl)propylamine, HOBT, DCC, acetonitrile, 0 ꢀC to rt; (f) BBr3, CH2Cl2, 0 ꢀC.
Scheme 2. (a) 3-(3,4-Dimethylphenyl)propylamine, HOBT, DCC, acetonitrile, 0 ꢀC to rt; (b) BBr3, CH2Cl2, 0 ꢀC.

Y.-S. Jung et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2599–2602
2601
versus –(OMe)2 (1 vs 6e, 8a vs 8b), the compounds with
two methoxy groups at C-3 and C-5 showed better
inhibition than those with one methoxy group at the C-
3 position. In general, 4-hydroxycinnamamides (8, 9)
exhibited higher potency of inhibition than phenylacetic
acid amides 6. Among the tested compounds, the
amides with –(OH)2 or –(OH)3 (6f, 6g, 9) inhibited lipid
peroxidation most potently.
0.1ꢁ30 mM. However, 9 and 6g were no more protec-
tive at 100 mM (Fig. 1B), implying their intrinsic toxicity
at this concentration. In contrast, 6f was highly potent
and seemed to be nontoxic at this concentration because
its neuroprotective effect was still maintained (Fig. 1B).
The IC50 values were calculated from the data in Figure
1 by nonlinear regression using Prism (Table 2). The
IC50 values of Trolox were included for comparisons.
All the other compounds did not show any significant
neuroprotective action, except that 6b, 6c and 8b were
shown to inhibit the Fe2+/AA-induced toxicity with the
IC50 values of 34.2, 4.2 and 5.4 mM, respectively.
The radical scavenging effects were also examined in the
present study using radicals generated by 1,1-diphenyl-
2-picryl hydrazyl (DPPH).11 As shown in Table 1, the
amides with –(OH)2 or –(OH)3 (6f, 6g, 9) were the most
potent radical scavengers in this series.
Taken together, 6f, 6g, 8b, and 9 were found to exhibit
potent inhibition of lipid peroxidation. Among these
compounds, 6f, 6g, and 9, the amides with –OH)2 or
–(OH)3, were the potent DPPH radical scavengers.
Although all the three compounds exhibited protective
action against oxidative stress-induced neurotoxicity in
cortical cultures, 6f was the most potent and appeared
to be the most promising compound as a potential neu-
roprotective antioxidant.
Given the suggested roles of lipid peroxides and free
radicals in neuronal death after ischemia or in neurode-
generative disorders,12 we next examined whether the
test compounds protect neurons from oxidative stress
evoked by H2O2, xanthine/xanthine oxidase (X/XO), or
Fe2+/ascorbic acid (Fe2+/AA)13 in primary cultured rat
cortical cells.14 These oxidative insults generate reactive
oxygen species including superoxide and hydroxyl radi-
cals, which ultimately induces 70–90% neuronal
damage measured by the leakage of cytoplasmic enzyme
lactate dehydrogenase (LDH). The H2O2- and X/XO-
induced neurotoxicity was dramatically inhibited by the
simultaneous exposure to various concentrations of 6f,
6g, and 9 during the oxidative insults (Fig. 1A). The
order of potency was 9 >6f >6g. As illustrated in Fig-
ure 1B, these three compounds also protected neurons
from the toxicity induced by Fe2+/AA. The neuropro-
tection was concentration-dependent at the range of
Table 1. Inhibition of lipid peroxidation by the newly synthesized
amides and their DPPH radical scavenging activities
Compd
R
R1
Inhibition of DPPH radical
lipid peroxidation scavenging activity
(IC50, mM)
(IC50, mM)
1
6a
6b
6c
6d
6e
6f
6g
25.90
23.96
19.38
9.28
32.01
11.77
2.60
1.81
9.94
3.41
1.11
178.6
182.1
>300
161.7
>300
50.1
31.0
21.3
82.2
89.1
34.3
—
Et
iPr
nBu
Ph
Me OMe
H
H
Me
Me OMe
H
H
H
H
H
H
OH
H
8a
8b
9
H
a-Tocopherol
BHT
4.68
4.03
—
Table 2. IC50 values determined from inhibition of the H2O2-, X/XO-,
or Fe2+/AA-induced oxidative neuronal damage in primary cultured
rat cortical cells
Figure 1. Inhibition of the oxidative neuronal damage induced by
H2O2 or X/XO (A), and by Fe2+/AA (B) in primary cultured rat cor-
tical cells. Primary cultured rat cortical cells were exposed to H2O2, X/
XO, or Fe2+/AA in the absence or presence of various concentrations
of test compounds, and the neuronal damage was determined as
described.13 Data were calculated as percent of control LDH activity
Compd
IC50, mM
(H2O2)
IC50, mM
(X/XO)
IC50, mM
(Fe2+/AA)
6f
6g
9
Troloxa
6.03
34.99
5.91
3.30
34.82
2.54
2.40
26.80
23.70
released into the culture medium treated with H2O2, X/XO, or Fe2+
/
329.61
201.84
430.53
AA, respectively. Each point represents the mean value from 4–6
measurements.
a6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid.

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Y.-S. Jung et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2599–2602
In conclusion, we have synthesized 4-hydroxy-
phenylacetic acid amides and 4-hydroxycinnamamides,
and evaluated their antioxidant activities. Compounds
exhibiting potent inhibition of lipid peroxidation as well
as DPPH radical scavenging activity were selected, and
their structure–activity relationships were discussed.
Furthermore, we examined the ability to protect neu-
rons from various oxidative stresses in primary cultured
rat cortical cells. We found that 6f is the most potent
and promising antioxidant exerting prominent neuro-
protective action. Based on these results, 6f may provide
a useful therapeutic potential for the treatment of neu-
rodegenerative disorders caused by oxidative stress.
6. Chabrier, P.-E.; Auguet, M.; Spinnewyn, B.; Auvin, S.;
Cornet, S.; Demerle-Pallardy, C.; Guilmard-Favre, C.; Marin,
J.-G.; Pignol, B.; Gillard-Roubert, V.; Roussillot-Charnet, C.;
Schulz, J.; Viossat, I.; Bigg, D.; Moncada, S. Proc. Natl. Acad.
Sci. U.S.A. 1999, 96, 10824.
7. Park, N.-S.; Ha, D.-C.; Kim, H.-S.; Choi, J.-K. Kor. J.
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8. Jung, Y.-S.; Kang, T.-S.; Lee, J.-C.; Seong, C.-M.; Ham,
W.-H.; Park, N.-S. Synth. Commun. 2001, 31, 455.
9. TBARS: Brain tissue from male Sprague–Dawley rats
(about 10-weeks old) was obtained after decapitation and
homogenated in ice-cold 10 mM Tris–HCl buffer (pH. 7.4)
using a Teflon-glass homogenizer. The homogenate was cen-
trifuged for 10 min at 1000 Â g, and the supernatant was used
in the test. Lipid peroxidation was stimulated in assays con-
taining 250 mL rat brain homogenate by additions of 0.02 mM
FeCl2 and 0.25 mM ascorbic acid, and the mixture was incu-
bated for 30 min at 37 ꢀC. The reaction was stopped by the
addition of 0.05 mL of 35% perchloric acid. After centrifuga-
tion for 10 min at 1000 Â g, 200 mL of the resulting super-
natant was added to 100 mL of an aqueous solution containing
0.5% TBA and reacted at 80 ꢀC for 1 h. Then, the reaction
mixture was cooled to room temperature, and its absorbance
at 532 nm was measured. Typical TBARS formation in brain
homogenate was calculated from the absorbance at 532 nm
using 1,1,3,3-tetraethoxypropane as an external standard.
10. Stocks, J.; Gutteridge, J. M. C.; Sharp, R. J.; Dormandy,
T. L. Clin. Sci. Mol. Med. 1974, 47, 215.
Acknowledgements
This work was supported by grants from Korea Science
& Engineering Foundation (R01–2001–00160), from
Plant Diversity Research Center of 21st Century Fron-
tier Research Program (PF002103–04), and from the
Ministry of Science and Technology in Korea.
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