Synthesis, radical scavenging activity and structure-activity relationship of uric acid analogs

Article abstract of DOI:10.1039/c2md20287e

Uric acid (UA) is known to play an important role as an endogenous antioxidant. However, its insolubility in the serum is a risk for hyperuricemia. We assume that UA is an equivalent to hydroquinone or p-aminophenol, which can be oxidized to quinone/quinoimine and thus acts as a radical scavenger. Based on this hypothesis, a series of UA analogs was designed and synthesized. In the chemical radical scavenging assay, active compounds were considered as hydroquinone or p-aminophenol equivalents. A highly functionalized UA structure is not essential to have radical scavenging activity. Potent active 5-hydroxyindolinones (1a, 2a, and 3a) showed sufficient activity with high solubility and low cytotoxicity.

Full text of DOI:10.1039/c2md20287e

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CONCISE ARTICLE
Synthesis, radical scavenging activity and
structure–activity relationship of uric acid analogs†
Cite this: Med. Chem. Commun., 2013,
, 527
4
a
a
a
a
Daisuke Yasuda, Kyoko Takahashi, Tomohiro Kakinoki, Yoko Tanaka,
a
b
a
Tomoyuki Ohe, Shigeo Nakamura and Tadahiko Mashino*
Uric acid (UA) is known to play an important role as an endogenous antioxidant. However, its insolubility in
the serum is a risk for hyperuricemia. We assume that UA is an equivalent to hydroquinone or p-
aminophenol, which can be oxidized to quinone/quinoimine and thus acts as a radical scavenger. Based
on this hypothesis, a series of UA analogs was designed and synthesized. In the chemical radical
scavenging assay, active compounds were considered as hydroquinone or p-aminophenol equivalents. A
highly functionalized UA structure is not essential to have radical scavenging activity. Potent active
5-hydroxyindolinones (1a, 2a, and 3a) showed sufficient activity with high solubility and low cytotoxicity.
Received 24th September 2012
Accepted 20th December 2012
DOI: 10.1039/c2md20287e
www.rsc.org/medchemcomm
The imbalance between the production/reduction of reactive
Here, we assumed that a tautomeric isomer of UA is equiv-
oxygen species (ROS), reactive nitrogen species (RNS), or free alent to hydroquinone or p-aminophenol (Scheme 1). Hydro-
radicals results in oxidative stress, which causes aging, cancer, quinone is easily oxidized to benzoquinone with the release of
ischemic injury, inammation, and neurodegenerative two electrons and thus acts as a radical scavenger. UA also has
1
diseases. ROS, which include superoxide anions, hydrogen the potential to release two electrons with two protons, which
peroxides, and hydroxyl radicals, are thought to be generated by can react with various active radicals (R radical in Scheme 1) to
2
reduction of molecular oxygen in aerobic organisms. Nitric give stable RH species. Based on this hypothesis, the series of
oxide is synthesized by nitric oxide synthases (NOSs) and acts as analogs shown in Fig. 1 was designed and synthesized, and the
a chemical mediator in various signal transduction systems. antioxidant activity was investigated.
However, when nitric oxide reacts with a superoxide anion,
The numbering of the carbon and nitrogen positions is
highly active peroxynitrite is produced. Usually, cells and attributed to UA (depicted in Scheme 1) in this manuscript for
tissues are protected against ROS, RNS, or free radicals by an the sake of convenience and to reduce confusion. As shown in
array of defence systems of enzymes, such as superoxide dis- Fig. 1, nitrogen atoms and carbonyl-oxygen were substituted for
mutase, catalase, and glutathione peroxidase or low-molecular
3
antioxidants. An endogenous potent antioxidant uric acid (UA),
the end product of the purine metabolism, can scavenge
4
hydroxyl radical and peroxynitrite. A number of studies found
that patients with multiple sclerosis have low serum levels of
5
UA. UA is thought to play an important role as an antioxidant in
serum, but its insolubility presents the risk of causing hyper-
uricemia or gouty arthritis. UA usually exists as a sodium salt in
À1
serum, and the solubility of sodium urate is ca. 7.0 mg dL in
6
normal serum under pH 7.4. Because the essential functional
group of UA having antioxidant activity is not well known, we
focused on the highly functionalized structure of UA to develop
a new simple UA analog as a useful antioxidant.
a
Department of Pharmaceutical Sciences, Keio University Faculty of Pharmacy, 1-5-30
Shibakoen, Minato-ku, Tokyo, Japan. E-mail: mashino-td@pha.keio.ac.jp; Fax: +81
3
54002691; Tel: +81 354002694
b
Department of Chemistry, Nippon Medical School, 2-297-2 Kosugi-cho, Nakahara-ku,
Kawasaki-shi, Kanagawa, Japan. E-mail: shigeo-nakamura@nms.ac.jp; Fax: +81
4
†
47221231; Tel: +81 447333574
Electronic supplementary information (ESI) available. See DOI:
0.1039/c2md20287e
1
Scheme 1 Proposed mechanism of the radical scavenging pathway of uric acid.
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Table 1 DPPH radical scavenging activity and pK
a
of the uric acid analogs
DPPH radical scavenging
3
À1 À1
Compounds
Uric acid
rate constants (Â 10 M
s
)
pK
a
0.7
71
110
11
8.7
2.6
11
310
4.4
72
5.6
>14
10.9
11.6
12.0
10.0
10.1
—
1
2
3
4
5
6
1
1
a
a
a
0
1
—
7.0
Edaravone
(5) or 9-NH (6) were essential to show the activity because 5 and
6
showed signicant activity. Based on the structure of 5,
Fig. 1 Structure of uric acid analogs.
addition of 8-CO (5 vs. 3a) and 9-NH (3a vs. 2a) sequentially
increased the activity. But additional introduction of 3-N (2a vs.
1
a) decreased the activity. On the other hand, based on the
carbon or hydrogen subsequently in the order of UA > 1a > 2a >
structure of 2a, 9-NH was substitutable to carbon (3a) or oxygen
3a, 4 > 5, 6 > 7, 8, 9 to simplify the UA structure (Fig. 1). Among
(4) but the activity was greatly decreased.
these compounds, 7, 8, and 9 were designed as un-equivalent
analogs to p-aminophenol 10 or hydroquinone 11.
All the active compounds in this series were designed as
hydroquinone or p-aminophenol equivalents. The activity of
compound 6 was an unexpected result because 6 seemed to be
equivalent to m-aminophenol. But in our speculation, 6 can
scavenge DPPH radicals through the conjugation of the
phenolic OH and the aminovinyl group, and transform to 6H-
indol-6-one (Fig. 2) similar to the pathway with p-aminophenol
depicted in Scheme 1.
We assumed that the 8-carbonyl group was not essential but
was needed to increase the electron-releasing ability. UA, 1a, 2a,
3
equivalents of DPPH (data not shown), which suggested that
these compounds scavenged two equivalents of the radicals by
the same pathway with p-aminophenol 10 or hydroquinone 11
depicted in Scheme 1. To establish the most effective position of
the OH group on the aromatic ring, the DPPH radical scav-
enging activities of regioisomer(s) of 2a and 3a were investi-
gated. As shown in Fig. 3, the activities of 2a and 3a were about
All the new benzimidazolinones (1b, 2b, and 2c) were
prepared by a cycloaddition reaction with urea and the corre-
sponding phenylenediamines under microwave irradiation
7
conditions (Scheme 2). Deprotection of the methoxy derivatives
(1b and 2b) gave phenols (1a and 2a) in moderate yields.
The DPPH (1,1-diphenyl-2-picrylhydrazyl) radical assay is the
simplest chemical method to measure the ability of antioxi-
dants to quench free radicals. The DPPH radical scavenging
activity of the compound was evaluated using the second-order
a, 4, 5, 6, 10, and hydroquinone 11 quenched almost two
rate constants in a mixture of ethanol and an MES buffer (pH
8
7
.4). As shown in Table 1, analogs 1a, 2a, 3a, 4, 5, and 6 showed
higher activity than UA. In particular, the activity of 2a was
greater than that of edaravone, a clinically available radical
scavenger used to protect against ischemia/reperfusion-induced
injury. Non-phenolic or methoxy compounds (1b, 2b, 2c, 3b, 8,
and 9) and simple phenolic compound 7 were inactive (data not
shown). These data indicate that both the 2-OH group and 7-NH
1
0–100 times higher than those of regioisomer(s).
UA exists as a mono-anion form under neutral conditions;
therefore, the active form in the radical scavenging mechanism
2
is thought to be an anion form. Similarly, the potent antioxi-
dant ascorbic acid exists as its anion form, and the anion has a
strong electron-donating ability to act as an effective radical
scavenger. Nevertheless, the pK values of synthetic analogs
a
Scheme 2 Preparation of benzimidazolidinones (1a, 1b, 2a, 2b, and 2c).
ꢀ
Conditions: (a) ZnO, DMF, microwave irradiation (150 W), 110 C and (b) HBr.
Fig. 2 Proposed radical scavenging mechanism of compound 6.
5
28 | Med. Chem. Commun., 2013, 4, 527–529
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Conclusions
Among the tested UA analogs in the DPPH radical scavenging
assay, the common structure of active compounds was
hydroxybenzimidazoline or hydroxyindoline. As a result, the
moiety required for the antioxidant activity of UA was 2-CO and
7-NH or 9-NH because 5 and 6 had greater antioxidant activity
than UA. Introduction of 3-NH, 8-CO, and 9-NH moieties to 5
increased the activity. Most active analog was 2a, whose activity
was 150 times higher than that of UA. It is noteworthy that all of
the active compounds in this series were designed as p-ami-
nophenol 10 and hydroquinone 11 derivatives. These data
suggested that the pyrimidine-trione moiety of UA was not
essential and the hydroquinone/p-aminophenol-equivalent
structure was effective to have the antioxidant activity. Among
the synthetic analogs, compounds 1a, 2a and 3a showed
remarkable radical scavenging activity with low cytotoxicity and
sufficient solubility in a neutral buffer solution. These could be
good candidates for preparing a novel potent antioxidant.
Fig. 3 The radical scavenging activity of regioisomer(s) of 2a and 3a. Numbers
3
À1 À1
depict the second-order rate constants (Â 10
M
s
). The relative activity ratio
of 2a and 3a of each isomer(s) was shown in parenthesis.
(Table 1) were within 10–12; thus, we expected that the active
form of these compounds was not an anion.
Although p-aminophenol 10 was most effective on the
radical scavenging ability, most of the phenolic compounds and
1
0 were highly toxic. To show the utility of these synthetic
Acknowledgements
analogs as bioactive antioxidants, cytotoxicity under physio-
logical conditions using human promyeloid leukemia cell lines
This work was supported by Platform for Drug Discovery,
Informatics, and Structural Life Science from the Ministry
of Education, Culture, Sports, Science and Technology,
Japan (MEXT), and High-Tech Research Center project from
MEXT.
9
(HL-60) was investigated. HL-60 cells were exposed to test
compounds for 24 h, and the number of viable cells was
counted using the trypan blue dye method with a Vi-CELL
10
counter. Compounds 1a, 2a, and 3a and UA did not exert
potent cytotoxicity up to 120 mM. In contrast, p-aminophenol 10
and hydroquinone 11 showed remarkable cytotoxicity.
The radical scavenging activity of 1a, 2a, and 3a was higher Notes and references
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1
1 PBS buffer: pH 7.4, aqueous solution containing KCl (200 mg
À1
À1
À1
L
), NaCl (8000 mg L ), KH
2
PO
4
(200 mg L ), and
À1
Fig. 4 Cytotoxicity on the HL-60 cell line.
Na
HPO
(1150 mg L ).
2
4
This journal is ª The Royal Society of Chemistry 2013
Med. Chem. Commun., 2013, 4, 527–529 | 529