Design and biological evaluation of novel antioxidants containing N-t-Butyl-N-hydroxylaminophenyl moieties

Article abstract of DOI:10.1016/S0960-894X(03)00444-X

In order to develop therapeutic agents against neurodegenerative diseases, we designed novel antioxidants containing N-t-butyl-N-hydroxylaminophenyl moieties and evaluated in vitro and in vivo neuroprotective properties as well as anti-ischemic effects.

Full text of DOI:10.1016/S0960-894X(03)00444-X

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2273–2275
Design and Biological Evaluation of Novel Antioxidants
Containing N-t-Butyl-N-hydroxylaminophenyl Moieties
Kyung-Mi Kim,a Kyung-Hwa Kim,b Tae-Cheon Kang,c Won-Yeon Kim,d
Myung-ryul Lee,a Hyuk-Jun Jung,b In Koo Hwang,c Sung-Bo Ko,b Jae-Young Koh,d
Moo Ho Won,c Eu-gene Ohb,* and Injae Shina,*
aDepartment of Chemistry, Yonsei University, Seoul 120-749, South Korea
bDepartment of Drug Discovery, Samsung Adv. Inst. Tech., Suwon 440-600, South Korea
cDepartment of Anatomy, Hallym University, Chunchon 200-702, South Korea
dDepartment of Neurology, University of Ulsan, Seoul 138-040, South Korea
Received 27 February 2003; revised 27 April 2003; accepted 28 April 2003
Abstract—In order to develop therapeutic agents against neurodegenerative diseases, we designed novel antioxidants containing
N-t-butyl-N-hydroxylaminophenyl moieties and evaluated in vitro and in vivo neuroprotective properties as well as anti-ischemic
effects.
# 2003 Elsevier Science Ltd. All rights reserved.
One of main causative agents of neurologic disorders is
reactive oxygen species (ROS). ROS-mediated oxidative
stress leads to cell damage directly by modifications of
polyunsaturated membrane lipids, proteins and nucleic
acids or indirectly by increased levels of intracellular
free calcium ions.1 Central nervous system is especially
sensitive to cell injury and/or death by ROS due to
increased oxygen consumption rate and low levels of
natural antioxidants, as well as high concentrations of
iron ions and polyunsaturated fatty acids.2
neurodegenerative diseases such as stroke, Parkinson’s
and Alzheimer’s diseases.1a
To date, many efforts have been devoted to the develop-
ment of efficient antioxidants, in particular, phenol-type
antioxidants to protect neuron cells from neurodegen-
erative disorders.3 Recently, novel therapeutic agents to
control multiple events were also developed.4 For
example, it was reported that a dual inhibitor of neuro-
nal nitric oxide synthase and lipid peroxidation was a
useful therapeutic agent to treat neurodegerative dis-
orders.4a Herein we describe design and biological eval-
uation of novel antioxidants containing N-t-butyl-N-
hydroxylaminophenyl moieties.
Human and animals have adopted various defense
mechanisms to regulate intracellular concentration of
ROS.3 Superoxide dismutase, catalase and glutathione
peroxidase are used in enzymatic defense systems, and
a-tocopherol and ascorbic acid are involved in non-
enzymatic defense mechanisms. However, with aging
these intricate defense mechanisms become less efficient,
particularly owing to decreased activity of a large num-
ber of enzymes involved in ROS defense. This explains
why oxidative stress induced by ROS is one of the main
etiological factors of cerebral aging, as well as acute
The N-t-butyl-N-hydroxylaminophenyl derivatives (t-
Bu-NR-OH) have been examined for the development
of molecule-based magnets since their radicals (t-Bu-
NR-OÁ) are stable enough to be employed as organic
magnetic materials.5 Therefore, we reasoned that N-t-
butyl-N-hydroxylaminophenyl derivatives could have
therapeutic potentials as novel antioxidants because of
high stability of radical species generated from abstraction
of hydrogen on N-OH by ROS.
*Corresponding author at present address: COS Biotech, Inc., Daejon
305-390, South Korea. Tel.: +82-2-2123-2631; fax: +82-2-364-7050;
e-mail: injae@yonsei.ac.kr
Target compounds 1–3 were prepared by oxidation of
aldehyde 4 synthesized by a known procedure and
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00444-X

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K.-M. Kim et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2273–2275
at concentrations where their neurotoxicity is low
(below 100 mM). Neurotoxicity induced by ROS was
remarkably decreased in the presence of 1–3 at 10 mM
concentrations, indicating that compounds 1–3 have
excellent neuroprotective activities (Fig. 2).
Finally, we evaluated the anti-ischemic properties of
thiazole-possessing compound 3 which showed the best
LPO inhibition and in vivo neuroprotective activities
using gerbils as animal models. Neuronal cell survival
rate was determined by counting the CA1 neuron cells
in the gerbil hippocampus.11 Neuron cells of gerbils
suffering ischemic injury were almost killed (less than
15% neuronal cell survival relative to sham) as shown in
Figure 3a. However, 3 exhibited 57% and 81% of neu-
ronal cell survival when gerbils were treated with 3
before and after ischemic injury, respectively.12 Ebselen-
treated gerbils as a positive control showed 58% of
neuronal cell survival in both pre- and post-treated
groups. This demostrates that 3 efficiently protects neu-
ronal cell death from ischemic injury, particularly in
post-treated groups. All the results of LPO inhibition
activity, in vivo neuroprotective activity and animal
studies indicate that 3 can be used as a novel therapeutic
agent to treat neurodegenerative disorders that involve
ROS.
Scheme 1. Synthesis of compounds 1–3.
subsequent coupling of the resulting acid to appropriate
amines followed by deprotection of TBS group
(Scheme 1).6 Having these compounds at hand, we
initially examined lipid peroxidation (LPO) inhibition
to evaluate their antioxidant activities in vitro by mea-
suring the extent of the repression of the radical chain
reaction of multilayer liposomes.7 As a positive control,
ebselen, which is a synthetic small molecule mimicking
the action of glutathione peroxidase and one of the
most promising antioxidants, was used.8 Compounds 1–
3 had superior LPO inhibition activities in comparison
to ebselen as shown in Table 1. Interestingly, LPO
inhibition activity depends on the amino group attached
to N-t-butyl-N-hydroxylaminophenyl moieties. Thia-
zole-containing compound 3 showed the best LPO
inhibition effect among three compounds and
2-pyridylamine group (1) exhibits better inhibition than
2-pyridylmethylamine group (2).
We also investigated radical-scavenging ability of N-t-
butyl-N-hydroxylaminophenyl group by incubating 1
with galvinoxyl which is known to be stable at room
temperature.9 EPR spectrum of incubation mixture
showed a triplet structure by nitrogen atom of N–Oꢀ
which was further splitted by hydrogens on 1 (Fig. 1).6
This demonstrates that a hydrogen atom of N-OH is
readily transferred to phenoxide radical of galvinoxyl to
form a more stable N–Oꢀ species. This also suggests that
N-t-butyl-N-hydroxylaminophenyl
remove ROS more efficiently than di-t-butylphenol-type
compounds.
derivatives
can
Figure 1. EPR spectra of (a) galvinoxyl and (b) radical species gener-
ated from incubation of galvinoxyl with compound 1 in DMSO at
25 ꢂC. The EPR spectra were recorded on an X-band Bruker spectro-
meter: microwave frequency, 9.77 GHz; microwave power, 1.00 mW;
modulation frequency, 100.0 kHz; modulation amplitude, 0.52 G.
We then examined neuroprotective effects of the com-
pounds on neuronal cell death induced by Fe2+ ions
that generate ROS by their auto-oxidation to Fe3+
ions.10 For these studies, we first determined the effec-
tive optimal concentration of compounds by measuring
the activity of lactate dehydrogenase (LDH) released by
damaged or destroyed cells into the extracellular fluid.10
It was found that 1–3 showed little neurotoxicity up to
100 mM (data not shown). Based on these observa-
tions, we examined neuroprotective properties of 1–3
Table 1. IC50 values for compounds 1 ꢁ3 and ebselen in inhibition
of lipid peroxidation
Compd
1
2
3
Ebselen
68.9
Figure 2. Neuroprotective effect of compounds 1–3 on neuronal cell
death induced by Fe2+ ions.
IC50 (mM)
4.3
11.1
1.44

K.-M. Kim et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2273–2275
2275
ROS and ischemic injury although in vivo neuroprotec-
tive mechanisms remain open to future studies.
This work (IS) was supported by a grant of the Ministry
of Health and Welfare (02-PJ2-PG3-21503-0008).
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Figure 3. Staining images of the hippocampus of (a) sham (left) and
global ischemic control (right), and gerbils suffering ischemic injury
before (left) or after (right) treatment with (b) ebselen and (c) com-
pound 3. (d) Quantitative data of neuronal cell survival.
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12. Twenty gerbils were treated with a single dose (60 mg/kg
gerbil) of compound 3 prior and posterior to ischemic injury.
In summary, we developed novel antioxidants contain-
ing N-t-butyl-N-hydroxylaminophenyl moieties that
afforded excellent neuroprotective activities against