Design and synthesis of 5-(substituted benzylidene)thiazolidine-2,4-dione derivatives as novel tyrosinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2012.01.019

In continuing our search for novel tyrosinase inhibitors, a series of 5-(substituted benzylidene)thiazolidine-2,4-diones were rationally designed and synthesized, and their inhibitory effects on mushroom tyrosinase activity were evaluated. Twelve target c

Full text of DOI:10.1016/j.ejmech.2012.01.019

European Journal of Medicinal Chemistry 49 (2012) 245e252
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design and synthesis of 5-(substituted benzylidene)thiazolidine-2,4-dione
derivatives as novel tyrosinase inhibitors
Young Mi Ha a, Yun Jung Park b, Jin-Ah Kim b, Daeui Park a, Ji Young Park b, Hye Jin Lee b, Ji Yeon Lee b,
**
,
a,
*
Hyung Ryong Moon b , Hae Young Chung
a Molecular Inflammation Research Center for Aging Intervention (MRCA), College of Pharmacy, Pusan National University, Kumjeong-Gu, Busan 609-735, Republic of Korea
b Laboratory of Medicinal Chemistry, College of Pharmacy, Pusan National University, Busan 609-735, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
In continuing our search for novel tyrosinase inhibitors, a series of 5-(substituted benzylidene)thiazo-
lidine-2,4-diones were rationally designed and synthesized, and their inhibitory effects on mushroom
tyrosinase activity were evaluated. Twelve target compounds 2ae2l were designed and synthesized
based on the structural characteristics of N-phenylthiourea, a tyrosinase inhibitor, and tyrosine and
Received 12 January 2011
Received in revised form
13 December 2011
Accepted 10 January 2012
Available online 24 January 2012
L
-DOPA, the natural substrates of tyrosinase. Among them, (Z)-5-(4-hydroxybenzylidene)thiazolidine-
2,4-dione (2a) and (Z)-5-(3-hydroxy-4-methoxybenzylidene)thiazolidine-2,4-dione (2f) exhibited much
higher tyrosinase inhibitory activities, with IC50 values of 13.36 and 9.87 M, respectively, than kojic acid
M). Kinetic analysis of tyrosinase inhibition revealed that 2a and 2f are competitive
m
Keywords:
(IC50 ¼ 24.72
m
Tyrosinase inhibitory activity
5-(Substituted benzylidene)thiazolidine-
2,4-dione derivatives
Docking study with potato catechol oxidase
Antimelanogenesis
inhibitors of mushroom tyrosinase. In addition, through prediction of the potato catechol oxidase tertiary
structure and simulation of docking with compounds 2a and 2f using DOCK6, we found that these
inhibitors likely bind to the active site of the enzyme. Docking simulation results suggested that 2a and
2f have high binding affinities with potato catechol oxidase. In addition, compounds 2a and 2f effectively
inhibited tyrosinase activity and reduced melanin levels in B16 cells treated with
lating hormone ( -MSH). These data strongly suggest that compounds 2a and 2f suppress the production
of melanin via the inhibition of tyrosinase activity.
a-melanocyte-stimu-
a
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
[5e7] such as resveratrol analogs [8e11], have been studied.
Recently, we reported the synthesis of a new family of hydroxy
substituted phenyl naphthalenes as analogs of resveratrol [12] and
described their potent inhibitory activities against tyrosinase and
melanogenesis [13e16]. We also found several potent tyrosinase
inhibitors isolated from natural materials that have potent inhibi-
tory effects on melanogenesis [12,17,18] and microphthalmia-
associated transcription factor [19]. Our previous studies explored
the development of new, potent, and safe tyrosinase inhibitors, and
we reported hydroxy substituted phenyl-benzo[d]thiazole [20], 5-
(substituted benzylidene)hydantoin [21], and 2-(substituted
phenyl)thiazolidine-4-carboxylic acid [22] as newly synthesized
derivatives. We also found several potent tyrosinase inhibitors
isolated from natural materials [12,17,18] that have even greater
potencies than the well-known tyrosinase inhibitor kojic acid
[3,12]. We searched for alternatives to the carboxylic acid func-
tionality in order to identify potent tyrosinase inhibitors. Addi-
tionally, N-phenylthiourea (Fig. 2) was reported to inhibit catechol
oxidase enzymes, including tyrosinase [23]. The sulfur atom in N-
phenylthiourea binds to both enzymes’ copper ions in the active
site, inhibiting tyrosinase activity. On the basis of this information,
Tyrosinase is a copper-containing enzyme present in animals
and plants, and it catalyzes the first 2 steps of melanin biosynthesis
from
L-tyrosine: hydroxylation of
L
-tyrosine to
L-dihydrox-
yphenylalanine (
(Fig. 1) [1,2]. Because of its key role in melanogenesis, tyrosinase
is an attractive target for cosmetic and pharmaceutical products.
L
-DOPA) and oxidation of
L
-DOPA to dopaquinone
As shown in Fig. 1, L-tyrosine, L-DOPA, and dopaquinone are
tyrosinase substrates and/or products. These compounds all
possess a common carboxylic acid functional group, indicating that
this group may play an important role in tyrosinase binding at the
active site.
Over the last few decades, various tyrosinase inhibitors,
including kojic acid [3], arbutin [4], and polyphenolic compounds
* Corresponding author. Tel.: þ82 51 510 2814; fax: þ82 51 518 2821.
** Corresponding author. Tel.: þ82 51 510 2815; fax: þ82 51 513 6754.
E-mail addresses: mhr108@pusan.ac.kr (H.R. Moon), hyjung@pusan.ac.kr
(H.Y. Chung).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.01.019

246
Y.M. Ha et al. / European Journal of Medicinal Chemistry 49 (2012) 245e252
which exhibited significant binding energies of ꢀ22.67 kcal molꢀ1
HO
and ꢀ27.46 kcal molꢀ1, respectively. Docking results indicated that
compound 2f binds potato catechol oxidase more strongly than
compound 2a, which is consistent with the results of reaction
velocity determination in kinetic behavior studies, production of
melanin, and inhibition of tyrosinase activity in a B16F10 cell sys-
tem (B16 cells). These findings are important in the development of
food preservatives and therapeutically potent agents against clini-
cally dermatological disorders, such as hyperpigmentation, and
provide information for the further design of novel tyrosinase
inhibitors.
O
O
Tyrosinase
HO
HO
OH
OH
NH2
NH2
L-Tyrosine
Melanin
DOPA
Tyrosinase
O
O
O
2. Chemistry
OH
NH2
DOPAquinone
Twelve 5-(substituted benzylidene)thiazolidine-2,4-dione deriv-
atives (2ae2l, Table 1) were synthesized by refluxing a variety of
substituted benzaldehydes 1ae1l and 2,4-thiazolidinedione in
ethanol in the presence of a catalytic amount of piperidine, as
depicted in Scheme 1. The yields were 20.4e79.2%, and all products
were in a (Z)-configuration. The structures of the synthesized
compounds were determined by 1H and 13C NMR and mass spectral
analyses.
Fig. 1. Biosynthesis of melanin via tyrosinase activity.
we designed a novel potential tyrosinase inhibitor consisting of
a thiazolidinedione (TZD) template possessing a divalent sulfur
atom and an imide functional group. We anticipated that the
divalent sulfur atom could participate in binding copper ions
present in the tyrosinase active site and that the template
imide functional group could serve as a carboxylic acid group to
interact with the enzyme in the active site (Fig. 2). TZDs are potent
antidiabetic agents targeting the nuclear receptor peroxisome
3. Pharmacology
3.1. Inhibitory actions of compounds on mushroom tyrosinase
activity
proliferator-activated receptor-
g (PPARg). TZD derivatives have
been approved by the Food and Drug Administration (FDA) for
treating type 2 diabetes [24]. Recently, TZD derivatives have been
The inhibitory activities of the synthesized compounds were
examined using mushroom tyrosinase as described previously,
with minor modifications [28]. The inhibition of mushroom
tyrosinase by the newly synthesized compounds is summarized in
Table 1. Our results showed that 2f exhibited the most potent
inhibitory activity against mushroom tyrosinase, with 82.27%
reported to be potent inhibitors of phosphatidylinositol 3-kinase-
g
(PI3K ) [25], the Pim kinase family [26], and the human immuno-
g
deficiency virus-1 (HIV-1) entry protein gp41 [27], highlighting
their versatile roles in the treatment of inflammatory diseases and
cancers. However, their effects on tyrosinase and skin melano-
genesis are unknown.
inhibition at 20
mM. Compound 2a was less potent, followed by
Therefore, we designed 5-(substituted benzylidene)thiazoli-
dine-2,4-dione analogs as novel potential tyrosinase inhibitors.
Among the twelve 5-(substituted benzylidene)thiazolidine-2,4-
dione compounds synthesized, compounds 2a, 2d, 2f, 2j, 2k, and
compound 21, with 62.68% and 45.80% inhibition at 20
m
M,
respectively. These compounds were more potent inhibitors of
mushroom tyrosinase than the reference tyrosinase inhibitor,
kojic acid, which exhibited 40.98% inhibition at a concentration
2l significantly inhibited mushroom tyrosinase activity at 20 mM
of 20 mM. Compounds 2d, 2j, and 2k exhibited moderate mushroom
concentration. Compound 2f, which contained an isovanillin
moiety, exhibited the highest mushroom tyrosinase inhibition,
tyrosinase inhibition, and the remaining compounds showed little
of no mushroom tyrosinase inhibitory activity. In the overall
structureeactivity relationship study, compound 2f, bearing 2
substituents (the hydroxy group at R2 and the methoxy group at R3)
on the phenyl ring, was the most potent inhibitor (Table 1). Notably,
the structural modifications to compounds 2g, in which the posi-
tions of the hydroxy and methoxy substituents of 2f are transposed,
and 2c, in which the hydroxy group of 2f at R2 is removed, markedly
decreased the inhibitory activity. Compounds bearing 2 methoxy
suppressing 82.27% of -DOPA oxidase activity. Compound 2a
L
exhibited less inhibitory activity than compound 2f. Kinetic analysis
of tyrosinase inhibition revealed that both 2a and 2f are competitive
inhibitors of mushroom tyrosinase. In addition, we conducted
studies to elucidate the inhibitory mechanisms of 2a and 2f
against tyrosinase using a docking program. In silico proteineligand
docking was successfully performed with compounds 2a and 2f,
"Same actions"
R
R1
Carboxyic acid
O
Imide
H
O
R2
R3
N
NH2
NH
S
S
OH
NH2
O
"Same actions"
R4
Structure of substrates and
products of tyrosinase
N-Phenylthiourea
Designed compounds
Fig. 2. Rationale for the design of 5-(substituted benzylidene)thiazolidine-2,4-dione derivatives.

Y.M. Ha et al. / European Journal of Medicinal Chemistry 49 (2012) 245e252
247
Table 1
A
Compound 2a
Substitution pattern and mushroom tyrosinase inhibition of the synthesized 5-
(substituted benzylidene)thiazolidine-2,4-dione derivatives, 2ae2l.
*
100
80
60
40
20
0
Compound
R1
R2
R3
R4
Tyrosinase inhibition (%)a
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
H
OH
H
H
H
H
H
H
OMe
H
H
H
H
OH
H
OMe
OH
H
OMe
OH
OH
OMe
OMe
OH
H
H
H
H
OH
H
H
H
H
62.68 ꢁ 1.03
NI
24.08 ꢁ 1.19
35.79 ꢁ 0.69
12.07 ꢁ 1.06
82.27 ꢁ 1.85
16.31 ꢁ 0.40
NI
12.17 ꢁ 1.16
31.94 ꢁ 0.79
34.26 ꢁ 1.48
45.80 ꢁ 0.28
40.98 ꢁ 1.35
OH
OH
OH
OMe
OEt
H
OMe
OMe
OMe
Control
0.2
1
5
10
Concentration (µM)
H
OMe
OMe
H
H
B
Compound 2f
OMe
Kojic acid
*
100
80
60
40
20
0
R1
O
R2
R3
NH
S
Control
0.2
1
Concentration (μM)
2.5
5
10
R4
O
Values represent the mean ꢁ S.E. of 3 experiments; NI (no inhibition).
a
Fig. 3. Effects of compounds 2a (A) and 2f (B) on B16 cell viability. Cells were treated
with varying doses of compound 2a or 2f (0e10 M) and examined by MTT assay. Data
Tyrosinase inhibition was measured using L-tyrosine as the substrate at 20 mM.
m
are expressed as a percentage of the control. *p < 0.05 compared with the untreated
control.
substituents on the phenyl ring (eg, 2i and 2j) showed low to
moderate activity. Additionally, the insertion of an ethoxy group,
which is larger than a methoxy group, resulted in a complete loss of
inhibitory activity against mushroom tyrosinase. Compound 2b,
with 1 hydroxyl substituent at R1 on the phenyl ring, showed little
activity. However, compound 2a, containing 1 hydroxy substituent
at R3, showed high inhibitory activity. While compounds bearing 2
hydroxyl substituents on the phenyl ring, such as 2d (containing
hydroxy groups at both R2 and R3), showed moderate inhibitory
activity, 2e (containing 2 hydroxy groups at R2 and R4) exhibited
weak inhibitory activity. Compounds bearing 3 methoxy substitu-
ents (2l) or 2 methoxy substituents and one hydroxyl substituent
(2k) on the phenyl ring showed moderate inhibitory activity. Our
results demonstrate that the number and position of methoxy and
hydroxy groups on the phenyl ring are very closely related to the
ability of these compounds to inhibit tyrosinase activity.
estimated by measuring cell viability, and no significant cytotoxic
effect was found at any concentration tested (Fig. 3). These results
indicated that 2a and 2f were relatively noncytotoxic under the
experimental conditions used.
Next, we examined the IC50 values of 2a and 2f with mushroom
tyrosinase, using kojic acid as a positive reference compound. As
shown in Table 2, kojic acid, 2a, and 2f were found to inhibit
mushroom tyrosinase activity in
manner. The data showed that compounds 2a (IC50 ¼ 13.36
and 2f (IC50 ¼ 9.87 M) were more potent inhibitors of mushroom
tyrosinase than kojic acid (IC50 ¼ 24.72 M).
a
concentration-dependent
mM)
m
m
To explore the inhibitory mechanisms of 2a and 2f, we next
conducted a study of the kinetic behavior of tyrosinase activity in
the presence of 2a or 2f (Table 2). As the concentration of 2a or 2f
increased, the Km value of 2a or 2f gradually increased, but the Vmax
did not change, thereby indicating that 2a and 2f act as competitive
3.2. Bioactivities of 2a and 2f
inhibitors of mushroom tyrosinase. Tyrosinase is
a copper-
We investigated the bioactivities of compounds 2a and 2f in
detail because they exhibited greater activity against mushroom
tyrosinase than kojic acid. Our present study provides the first
evidence that compounds 2a and 2f have significant inhibitory
effects on melanogenesis as well as tyrosinase. We first attempted
to ascertain the potential cytotoxicity of compounds 2a and 2f
on B16 cells. The cytotoxic effects of these 2 compounds were
containing enzyme widely distributed in nature [1]. Most tyrosi-
nase inhibitors that chelate the copper in the active site of the
enzyme show competitive inhibition; examples include tropolone
[29] and kojic acid [30]. Thus, 2a and 2f may exert their inhibitory
activities by binding to copper in the mushroom tyrosinase active
site. The Ki value represents enzyme affinity for the inhibitor;
a larger Ki value indicates a greater inhibitory effect of the drug. As
R1
O
O
R1
O
R2
R3
R2
a
20.4 - 79.2%
H
NH
+
NH
S
S
R3
R4
1a -1l
O
R4
O
Thiazolidine-2,4-dione
2a 2l
-
Substituted benzaldehydes
Scheme 1. Synthesis of the target compounds, twelve 5-(substituted benzylidene)thiazolidine-2,4-dione derivatives (2ae2l); reagents and conditions: (a) piperidine (0.3 eq.), Et
OH, reflux, 7e42 h.

248
Y.M. Ha et al. / European Journal of Medicinal Chemistry 49 (2012) 245e252
Table 2
treatment with 2a or 2f in the presence of 100 nM
a-melanocyte-
Effects and kinetic analysis of 2a, 2f, and kojic acid on mushroom tyrosinase activity.
stimulating hormone ( -MSH) decreased dose-dependently, as
a
a
shown in Fig. 5. Based on cytotoxicity results (Fig. 3), the inhibition
of melanogenesis by compounds 2a and 2f did not appear to be
due to either cytotoxic effects or cell growth inhibition. The
suppression of melanogenesis by compounds 2a and 2f can be
attributed to their inhibition of murine-derived tyrosinase.
Lastly, to analyze the mechanisms by which compounds 2a and
2f inhibit melanin biosynthesis, we examined the inhibitory effects
of these compounds on cellular tyrosinase activity of B16 cells
Compound
IC50
(
m
M)
Kinetic analysisb
c
Vmax (mM/min)
Km (mM)
Kic (M)
3.74 ꢂ 10ꢀ7
4.13 ꢂ 10ꢀ7
e
2a
2f
Kojic acid
13.36 ꢁ 0.48
9.87 ꢁ 0.52
24.72 ꢁ 0.17
0.019
0.020
e
1.28
1.91
e
a
50% Inhibitory concentration (IC50).
b
LineweavereBurk plot of mushroom tyrosinase activity: Data are presented as
mean 1/V values, where 1/V is the inverse of the increase of absorbance at a wave-
length 492 nm per min A492/min), of independent tests with different
concentrations of -tyrosine as a substrate.
Values were measured at a 1.25 M concentration of the active compound for
(D
3
treated with 100 nM
a-MSH. We found that 2a and 2f effectively
L
inhibited -MSH-induced tyrosinase activity (Fig. 6).
a
c
m
Compared to 2a, 2f showed more potent inhibitory activity
against murine-derived tyrosinase and more potent inhibition of
melanogenesis in B16 cells in all experimental results.
the Km (MichaeliseMenten constant) and Ki (inhibition constant).
the concentration of 2a or 2f increased, the Ki value of 2f
(Ki ¼ 1.38
m
M at 20
m
M) was larger than that of 2a (Ki ¼ 0.84
mM at
20 M), indicating that 2f has potent inhibitory activity against
m
4. Conclusion
murine-derived tyrosinase than 2a.
Next, we predicted the tertiary structure of potato catechol
oxidase and simulated the docking of 2a and 2f using DOCK6. We
searched for potato catechol oxidase residues that could bind to
both compounds. The most important residues predicted to
interact with 2a included His90, Gly91, Ser254, Glu258, His261,
Asp262, His265, Ser275, and Pro277, and those that may interact
with 2f included His90, Ser254, Glu258, His261, His265, Val266,
Ser275, and Pro277 (Fig. 4). The residues predicted to interact with
these inhibitors are located within 3 Å of the ligand. Docking results
indicated that 2f (ꢀ27.46 kcal molꢀ1) binds to potato catechol
oxidase more strongly than 2a (ꢀ22.67 kcal molꢀ1).
In this study, we describe the synthesis and tyrosinase inhibi-
tory activity of 5-(substituted benzylidene)thiazolidine-2,4-dione
derivatives. Among these, compounds 2a (IC50 ¼ 13.36
2f (IC50 ¼ 9.87 M) were found to be approximately 1.85- and 2.50-
fold, respectively, more potent than kojic acid (IC50 ¼ 24.72 M),
mM) and
m
m
which is often used as a positive reference compound. Docking
simulation with 2a and 2f confirmed that these compounds bind
strongly to the potato catechol oxidase active site. Both 2a and 2f
were identified in a kinetic study as competitive inhibitors of
mushroom tyrosinase and found to effectively suppress not only
tyrosinase activity but also melanogenesis in B16 cells treated with
To assess the inhibitory effects of 2a and 2f on melanogenesis,
we then quantified the melanin content of B16 cells treated with
2a and 2f (Fig. 5). The melanin content of B16 cells after
a-MSH. The lack of 2a- and 2f-induced cytotoxicity indicates that
the suppression of melanogenesis by these compounds can be
attributed to the inhibition of murine tyrosinase. These data
Fig. 4. Computational structure prediction for potato catechol oxidase and docking simulation with 2a and 2f. Predicted 3D structure of potato catechol oxidase. Boxes indicate
binding sites for compounds 2a and 2f with potato catechol oxidase residues. The yellow area indicates the potato catechol oxidaseeligand interactions (Z < 3). (For interpretation
of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Y.M. Ha et al. / European Journal of Medicinal Chemistry 49 (2012) 245e252
249
Compound 2a
A
α-MSH+10.0 μM
α-MSH+5.0 μM
α-MSH+2.5 μM
α-MSH
***
***
***
###
Control
0
50
100
150
200
250
300
Melanin contents (% of control)
B
Compound 2f
α-MSH+10.0 μM
α-MSH+5.0 μM
α-MSH+2.5 μM
α-MSH
***
***
***
###
Control
0
50
100
150
200
250
300
Melanin contents (% of control)
Fig. 5. Ability of 2a (A) and 2f (B) to inhibit melanogenesis after treatment with 100 nM a-MSH in B16 cells. Melanin levels were measured at 405 nm. Values represent the
mean ꢁ S.E. of 3 experiments. Data are expressed as a percentage of the control. ###p < 0.001 compared to the untreated control; ***p < 0.001 compared to 100 nM
a-MSH
treatment alone.
suggest that both 2a and 2f have strong depigmenting activities
without discernible cytotoxicity in a B16 cell system and that
thiazolidine-2,4-dione can serve as an effective scaffold for novel
tyrosinase inhibitors. Overall, compound 2f, containing R2-OH and
R3-OMe groups, was the most efficacious inhibitor. This work will
contribute to furthering our understanding of the mechanisms of
tyrosinase inhibition and the development of effective insecticides
and drugs against hyperpigmentation.
2,4-dione (approximately 0.7e1.2 eq.) in EtOH (4 mL), and the
reaction mixture was refluxed. Before reaching the boiling point of
ethanol, the reaction mixture generally became a clear solution.
During reflux, precipitates were formed and filtered through
a Buchner funnel after cooling. The filter cake was washed with
ethanol, methylene chloride, and/or water, depending on the
properties of the substituted benzaldehydes used for the reaction,
to obtain products (yields: approximately 20.4%e79.2%).
5. Experimental
5.2.2. (Z)-5-(4-Hydroxybenzylidene)thiazolidine-2,4-dione (2a)
Yellow solid; reaction time, 24 h; yield, 67%; melting point,
5.1. General
299.1e299.7 ꢃC; 1H NMR (500 MHz, DMSO-d6)
d: 12.44 (s, 1H, NH),
10.30 (s, 1H, OH), 7.68 (s, 1H, vinylic H), 7.43 (d, 2H, J ¼ 8.5 Hz, 20-H,
Melting points are uncorrected. 1H and 13C NMR spectra were
recorded on Varian Unity INOVA 400 and Varian Unity AS 500
instruments. Chemical shifts are reported with reference to the
respective residual solvent or deuteriated peaks (dH 3.30 and dC 49.0
for CD3OD, dH 7.27 and dC 77.0 for CDCl3). Coupling constants are
reported in hertz. The abbreviations used are as follows: s (singlet),
br s (broad singlet), d (doublet), t (triplet), q (quadruplet), and dd
(doublet of doublets). All the reactions described below were per-
formed under an argon or nitrogen atmosphere and monitored by
thin layer chromatography (TLC). All anhydrous solvents were
distilled using CaH2 or Na/benzophenone prior to use.
60-H), 6.89 (d, 2H, J ¼ 8.5 Hz, 30-H, 50-H); 13C NMR (100 MHz, DMSO-
d6) d
: 168.7 (C2), 168.2 (C4), 160.6 (C40), 133.0 (C20, C60), 133.0
(benzylic C), 124.6 (C10), 119.6 (C5), 117.0 (C30, C50); LRMS(ES) m/z
220 ([M ꢀ H]ꢀ).
5.2.3. (Z)-5-(2-Hydroxybenzylidene)thiazolidine-2,4-dione (2b)
Yellow solid; reaction time, 21 h; yield, 37.1%; melting point,
254.7e255.9 ꢃC; 1H NMR (500 MHz, DMSO-d6)
d: 12.53 (s, 1H, NH),
10.52 (s, 1H, OH), 8.03 (s, 1H, vinylic H), 7.33 (d, 1H, J ¼ 7.5 Hz, 60-H),
7.31 (t, 1H, J ¼ 8.0 Hz, 40-H), 6.97 (d, 1H, J ¼ 8.0 Hz, 30-H), 6.95 (t, 1H,
J ¼ 7.5 Hz, 50-H); 13C NMR (100 MHz, DMSO-d6)
d: 168.8 (C2), 168.2
(C4), 157.9 (C20), 132.9 (benzylic C), 129.0 (C40), 127.7 (C60), 122.6
(C5), 120.6 (C10), 120.4 (C50), 116.8 (C30); LRMS(ES) m/z 220
([M ꢀ H]ꢀ).
5.2. Chemistry
5.2.1. General procedure for the synthesis of substituted
benzylidene-thiazolidine-2,4-dione analogs (2ae2l)
5.2.4. (Z)-5-(4-Methoxybenzylidene)thiazolidine-2,4-dione (2c)
Piperidine (0.3 eq.) was added to a suspension of substituted
benzaldehydes (approximately 1.44e2.60 mmol) and thiazolidine-
Yellowish green solid; reaction time, 24 h; yield, 33%; melting
point, 217.6e218.3 ꢃC; 1H NMR (400 MHz, CD3OD)
d: 7.72 (s, 1H,

250
Y.M. Ha et al. / European Journal of Medicinal Chemistry 49 (2012) 245e252
A
Compound 2a
α-MSH+10.0 μM
α-MSH+5.0 μM
α-MSH+2.5 μM
α-MSH+1.0 μM
α-MSH+0.2 μM
α-MSH
***
***
***
***
###
Control
0
50
100
150
200
250
300
Tyrosinase activity (% of control)
B
Compound 2f
α-MSH+10.0 μM
α-MSH+5.0 μM
α-MSH+2.5 μM
α-MSH+1.0 μM
α-MSH+0.2 μM
α-MSH
***
***
***
***
###
Control
0
50
100
150
200
250
300
Tyrosinase activity (% of control)
Fig. 6. Inhibition of tyrosinase by 2a (A) and 2f (B) in B16 cells. In the presence of 100 nM a-MSH, B16 cells were treated with varying doses of 2a or 2f (0.2e10.0 mM) for 24 h.
Results are expressed as the percent of control, and each column represents the mean ꢁ S.E. of 3 determinations. ###p < 0.001 compared to the untreated control; ***p < 0.001
compared to 100 nM a-MSH treatment alone.
vinylic H), 7.50 (d, 2H, J ¼ 8.4 Hz, 20-H, 60-H), 7.04 (d, 2H, J ¼ 8.8 Hz,
d
: 168.7 (C2), 168.1 (C4), 150.7 (C40), 147.6 (C30), 132.9 (benzylic C),
30-H, 50-H), 3.84 (s, 3H, 40-OCH3); 13C NMR (100 MHz, DMSO-d6)
d:
126.4 (C10), 124.1 (C60), 120.7 (C5), 116.6 (C20), 113.2 (C50), 56.4 (40-
168.7 (C2), 168.1 (C4), 161.7 (C40), 132.8 (C20, C60), 132.5 (benzylic C),
126.2 (C10), 121.0 (C5), 115.6 (C30, C50), 56.2 (40-OCH3); LRMS(ES) m/
z 234 ([M ꢀ H]ꢀ).
OCH3); LRMS(ES) m/z 250 ([M ꢀ H]ꢀ).
5.2.8. (Z)-5-(4-Hydroxy-3-methoxybenzylidene)thiazolidine-2,4-
dione (2g)
5.2.5. (Z)-5-(3,5-Dihydroxybenzylidene)thiazolidine-2,4-dione (2d)
Yellow solid; reaction time, 18 h; yield, 43%; melting point,
Ash-colored solid; reaction time, 9 h; yield, 24%; melting point,
226.0e226.7 ꢃC; 1H NMR (500 MHz, CD3OD)
d: 7.72 (s, 1H, vinylic
288.4e290.2 ꢃC; 1H NMR (500 MHz, DMSO-d6)
d
: 12.53 (s, 1H, NH),
H), 7.09 (d, 1H, J ¼ 1.5 Hz, 20-H), 7.07 (dd, 1H, J ¼ 2.0, 8.0 Hz,
9.62 (s, 2H, 2ꢂ OH), 7.54 (s, 1H, vinylic H), 6.43 (d, 2H, J ¼ 1.5 Hz, 20-
60-H), 6.90 (d, 1H, J ¼ 8.0 Hz, 50-H), 3.91 (s, 3H, 30-OCH3); 13C
H, 60-H), 6.31 (t, 1H, J ¼ 1.5 Hz, 40-H); 13C NMR (100 MHz, DMSO-d6)
NMR (100 MHz, CD3OD) d
: 168.5 (C2), 168.2 (C4), 149.6 (C30),
d
: 168.8 (C2), 168.2 (C4), 159.6 (C30, C50), 135.2 (C10), 132.9 (benzylic
148.3 (C40), 133.3 (benzylic C), 125.3 (C10), 124.7 (C60), 119.6 (C5),
115.7 (C50), 113.2 (C20), 55.2 (30-OCH3); LRMS(ES) m/z 250
([M ꢀ H]ꢀ).
C), 123.9 (C5), 108.7 (C20, C60), 105.6 (C40); LRMS(ES) m/z 236
([M ꢀ H]ꢀ).
5.2.6. (Z)-5-(3,4-Dihydroxybenzylidene)thiazolidine-2,4-dione (2e)
5.2.9. (Z)-5-(3-Ethoxy-4-hydroxybenzylidene)thiazolidine-2,4-
dione (2h)
Greenish-yellow solid; reaction time, 24 h; yield, 79.2%; melting
point, >300 ꢃC; 1H NMR (400 MHz, CD3OD)
d
: 7.61 (s, 1H, vinylic H),
Greenish-yellow solid; reaction time, 24 h; yield, 29%; melting
6.98 (d,1H, J ¼ 2.0 Hz, 20-H), 6.92 (dd,1H, J ¼ 8.4, 2.0 Hz, 60-H), 6.84 (d,
point, 207.1e208.5 ꢃC; 1H NMR (400 MHz, DMSO-d6)
d: 12.42 (br s,
1H, J ¼ 8.4 Hz, 50-H); 13C NMR (100 MHz, CD3OD)
d: 168.8 (C2), 168.3
1H, NH), 9.84 (s, 1H, OH), 7.67 (s, 1H, vinylic H), 7.11 (d, 1H,
J ¼ 2.0 Hz, 20-H), 7.02 (dd, 1H, J ¼ 2.4, 8.4 Hz, 60-H), 6.90 (d, 1H,
J ¼ 8.0 Hz, 50-H), 4.04 (q, 2H, J ¼ 7.2 Hz, CH3CH2e), 1.33 (t, 3H,
(C4), 148.6 (C40), 145.9 (C30), 133.5 (benzylic H), 125.3 (C10), 124.2
(C60),119.2(C5),116.3 (C50),115.7 (C20);LRMS(ES)m/z236([Mꢀ H]ꢀ);
HRMS calculated for C10H7NO4S: 237.0096, found: 237.0103.
J ¼ 7.2 Hz, CH3CH2e); 13C NMR (100 MHz, DMSO-d6)
d: 168.7 (C2),
168.1 (C4), 150.4 (C30), 147.8 (C40), 133.3 (benzylic C), 125.1 (C10),
124.9 (C60), 119.9 (C5), 117.0 (C50), 116.0 (C20), 64.7 (30-CH2), 15.3
(CH2CH3); LRMS(ES) m/z 264 ([M ꢀ H]ꢀ).
5.2.7. (Z)-5-(3-Hydroxy-4-methoxybenzylidene)thiazolidine-2,4-
dione (2f)
Greenish-yellow solid; reaction time, 20 h; yield, 53%; melting
point, 254.0e257.6 ꢃC; 1H NMR (500 MHz, DMSO-d6)
d
: 12.46 (br s,
5.2.10. (Z)-5-(2,4-Dimethoxybenzylidene)thiazolidine-2,4-dione (2i)
1H, NH), 9.48 (s,1H, OH), 7.63 (s,1H, vinylic H), 7.01e7.06 (m, 3H, 20-
H, 50-H, 60-H), 3.82 (s, 3H, 40-OCH3); 13C NMR (100 MHz, DMSO-d6)
Yellow solid; reaction time, 7 h; yield, 39.6%; melting point,
254.7e255.6 ꢃC; 1H NMR (500 MHz, DMSO-d6)
d: 12.42 (br s, 1H,

Y.M. Ha et al. / European Journal of Medicinal Chemistry 49 (2012) 245e252
251
NH), 7.91 (s, 1H, vinylic H), 7.33 (d, 1H, J ¼ 8.5 Hz, 60-H), 6.69 (d, 1H,
5.3.3. Assay to measure inhibitory effects of the novel compounds
on mushroom tyrosinase
Mushroom tyrosinase was used as the source of tyrosine for
the entire study. Tyrosinase activity was determined as described
J ¼ 8.5 Hz, 50-H), 6.68 (s, 1H, 30-H), 3.88 (s, 3H, OCH3), 3.83 (s, 3H,
OCH3); 13C NMR (100 MHz, DMSO-d6)
d
: 168.9 (C2), 168.2 (C4),
163.7 (C40), 160.5 (C20), 130.7 (benzylic C), 127.1 (C60), 120.6 (C5),
114.9 (C10), 107.2 (C50), 99.3 (C30), 56.6 (20-OCH3), 56.3 (40-OCH3);
LRMS(ES) m/z 264 ([M ꢀ H]ꢀ).
previously, with minor modifications [28]. Briefly, 20
aqueous mushroom tyrosinase solution (1000 U) was added to
a 96-well microplate (Nunc, Denmark) in a 200 L assay mixture
containing 1 mM -tyrosine solution and 50 mM phosphate buffer
mL of
m
5.2.11. (Z)-5-(3,4-Dimethoxybenzylidene)thiazolidine-2,4-dione (2j)
L
Pale yellow solid; reaction time, 23 h; yield, 20.4%; melting
(pH 6.5). The assay mixture was incubated at 25 ꢃC for 30 min.
Following incubation, the amount of dopachrome produced was
determined spectrophotometrically at 492 nm (OD492) using
a microplate reader (Hewlett Packard, Palo Alto, CA, USA). The
half-maximal inhibitory concentration (IC50) is the concentration
of a compound that inhibits a standard 50% response. The IC50 is
derived from the X-axis on an inhibitor concentration versus
product formation curve and is determined from the alignment of
a doseeresponse curve on the dependent Y-axis. In the present
study, dose-dependent inhibition experiments were performed
in triplicate to determine the IC50 of the novel compounds.
According to the inhibition percentage of 3 doses in each
experiment, the log-linear curves and their equations were
determined. Individual IC50 values were then calculated as the
concentration corresponding to 50% inhibition on the Y-axis. The
results from 3 experiments are shown.
point, 214.9e216.7 ꢃC; 1H NMR (500 MHz, DMSO-d6)
d: 12.50 (br s,
1H, NH), 7.74 (s, 1H, vinylic H), 7.18 (s, 1H, 20-H), 7.17 (d, 1H,
J ¼ 8.5 Hz, 60-H), 7.11 (d,1H, J ¼ 8.5 Hz, 50-H), 3.81 (s, 3H, OCH3), 3.80
(s, 3H, OCH3); 13C NMR (100 MHz, DMSO-d6)
d: 168.7 (C2), 168.2
(C4), 151.5 (C30), 149.6 (C40), 132.8 (benzylic C), 126.4 (C10), 124.4
(C60), 121.3 (C5), 114.0 (C50), 112.8 (C20), 56.4 (OCH3), 56.2 (OCH3);
LRMS(ES) m/z 264 ([M ꢀ H]ꢀ).
5.2.12. (Z)-5-(4-Hydroxy-3,5-dimethoxybenzylidene)thiazolidine-
2,4-dione (2k)
Yellow solid; reaction time, 42 h; yield, 47.1%; melting point,
248.0e249.9 ꢃC; 1H NMR (400 MHz, DMSO-d6)
d: 12.42 (br s, 1H,
NH), 9.31 (s, 1H, OH), 7.67 (s, 1H, vinylic H), 6.85 (s, 2H, 20-H, 60-H),
3.78 (s, 6H, 30-OCH3, 50-OCH3); 13C NMR (100 MHz, DMSO-d6) cc:
168.7 (C2),168.0 (C4),148.9 (C30, C50), 139.3 (C40), 133.6 (benzylic C),
123.9 (C10), 120.2 (C5), 108.7 (C20, C60), 56.7 (30-OCH3, 50-OCH3);
LRMS(ES) m/z 280 ([M ꢀ H]ꢀ).
5.3.4. Kinetic analysis of tyrosinase inhibition
Varying concentrations of
a substrate, 20 L of aqueous mushroom tyrosinase solution
(1000 U), and 50 mM potassium phosphate buffer (pH 6.5) with
or without test samples (1.25 and 20.0 M of 2a or 2f, respec-
tively) were added to a 96-well plate for a final total volume of
200 L. Using a microplate reader, the initial rate of dopachrome
formation was determined by the increase in absorbance per
minute at a wavelength of 492 nm ( OD492/min). The Michaelis
constant (Km) and maximal velocity (Vmax) of tyrosinase activity
were determined using a LineweavereBurk plot with varying
L-tyrosine (0.25e2 mM) as
5.2.13. (Z)-5-(3,4,5-Trimethoxybenzylidene)thiazolidine-2,4-dione (2l)
m
Yellow solid; reaction time, 42 h; yield, 38.1%; melting point,
179.1e181.0 ꢃC; 1H NMR (400 MHz, DMSO-d6)
d
: 12.48 (br s, 1H,
m
NH), 7.71 (s, 1H, vinylic H), 6.88 (s, 2H, 20-H, 60-H), 3.79 (s, 6H, 30-
OCH3, 50-OCH3), 3.68 (s, 3H, 40-OCH3); 13C NMR (100 MHz, DMSO-
m
d6) d
: 168.5 (C2), 168.0 (C4), 153.9 (C30, C50), 140.1 (C40), 132.7
(benzylic C), 129.2 (C10), 123.2 (C5), 108.2 (C20, C60), 60.9 (40-OCH3),
D
56.7 (30-OCH3, 50-OCH3); LRMS(ES) m/z 294 ([M ꢀ H]ꢀ).
L
-
5.3. Methods
tyrosine concentrations [28]. Reaction kinetics required modifi-
cation of the MichaeliseMenten equation due to competitive
inhibition by the compounds together with substrate inhibition
5.3.1. Cell culture
B16 cells (obtained from the Korean Cell Line Bank) were
cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco,
Carlsbad, CA) with 10% fetal bovine serum (FBS; Gibco) and
by L-tyrosine. The results of 3 independent experiments are
shown.
penicillin/streptomycin (100 IU/mL, 50
m
g/mL) in a humidified
5.3.5. Assay of murine tyrosinase activity
atmosphere containing 5% CO2 at 37 ꢃC. B16 cells were cultured in
24-well plates for melanin quantification and enzyme activity
assays.
Tyrosinase activity was estimated by measuring the L-DOPA
oxidation rate [32]. Cells were plated in 24-well dishes at a density
of 5 ꢂ 104 cells/mL. B16 cells were incubated in the presence or
absence of 100 nM
a-MSH and treated for 24 h with varying
5.3.2. Cell viability
concentrations of 2a or 2f (0e10
mM). Cells were washed and lysed
Cell survival was quantified by colorimetric 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay that measured mitochondrial activity in viable cells. This
method is based on the conversion of MTT (Sigma) to MTT-
formazan crystals by a mitochondrial enzyme, as previously
described [31]. Briefly, cells were seeded at a density of 3 ꢂ 104 cells
in a Corning 48-well plate (Corning, NY) and allowed to adhere
overnight. The culture medium was then replaced with fresh
serum-free DMEM. MTT was freshly prepared at 5 mg/mL in
in 100 L of 50 mM sodium phosphate buffer (pH 6.5) containing 1%
m
Triton X-100 (Sigma) and 0.1 mM phenylmethylsulfonyl fluoride
(PMSF), then frozen at ꢀ80 ꢃC for 30 min. After thawing and mixing,
cellular extracts were clarified by centrifugation at 12,000 rpm for
30 min at 4 ꢃC. An 8
mL sample of each supernatant and 20 mL of L-
DOPA (2 mg/mL) were placed in a 96-well plate, and absorbance at
492 nm was read every 10 min for 1 h at 37 ꢃC using an enzyme-
linked immunosorbent assay (ELISA) plate reader. Final activity is
expressed as
DOD/min for each condition. The results from 3
phosphate-buffered saline (PBS). Aliquots of 500
mL of MTT stock
experiments are shown.
solution were added to each well, and the plate was incubated
at 37 ꢃC for 4 h in a humidified 5% CO2 incubator. After 4 h, the
5.3.6. Determination of melanogenesis in B16 cells
medium was removed. Then, 500
m
L
of EtOHeDMSO (1:1
Melanin content was determined using a modification of the
method described by Bilodeau et al. [33]. In the present study,
the amount of melanin was used as an index of melanogenesis.
B16 cells were transferred to 24-well dishes (5 ꢂ 104 cells per
mixture solution) was added to each well to dissolve the formazan.
After 10 min, the optical density of each well was measured spec-
trophotometrically using a 560 nm filter. The results from 3
experiments are shown.
well) and incubated in the presence or absence of 100 nM
a-

252
Y.M. Ha et al. / European Journal of Medicinal Chemistry 49 (2012) 245e252
MSH. Cells were then incubated for 24 h with varying concen-
trations of 2a or 2f (0e10 M). After washing twice with PBS,
samples were dissolved in 100 L of 1 N NaOH. The samples were
References
m
[1] V.J. Hearing, K. Tsukamoto, FASEB J. 5 (1991) 2902e2909.
[2] S. Passi, M. Nazzaro-Porro, Br. J. Dermatol. 104 (1981) 659e665.
[3] J.T. Lim, Dermatol. Surg. 25 (1999) 282e284.
[4] K. Maeda, M. Fukuda, J. Pharmacol. Exp. Ther. 27 (1996) 665e769.
[5] K. Ohguchi, T. Tanaka, I. Iliya, T. Ito, M. Iinuma, K. Matsumoto, Y. Akao,
Y. Nozawa, Biosci. Biotechnol. Biochem. 67 (2003) 663e665.
[6] J.S. Roh, J.Y. Han, J.H. Kim, J.K. Hwang, Biol. Pharm. Bull. 27 (2004) 1976e1978.
[7] J.K. No, M.S. Kim, Y.J. Kim, S.J. Bae, J.S. Choi, H.Y. Chung, Am. J. Chin. Med. 32
(2004) 1e7.
[8] K. Ohguchi, T. Tanaka, T. Kido, K. Baba, M. Iinuma, K. Matsumoto, Y. Akao,
Y. Nozawa, Biochem. Biophys. Res. Commun. 307 (2003) 861e863.
[9] H. Takiwaki, S. Shirai, H. Kohno, H. Soh, S. Arase, J. Invest. Dermatol. 103
(1994) 642e646.
[10] S.H. Song, H.J. Lee, Y.G. Jin, Y.M. Ha, S.J. Bae, H.Y. Chung, H.S. Suh, Bioorg. Med.
Chem. Lett. 17 (2007) 461e464.
[11] Y.M. Kim, J. Yun, C.K. Lee, H. Lee, K.R. Min, Y. Kim, J. Biol. Chem. 277 (2002)
16340e16344.
[12] Y.M. Ha, S.W. Chung, S. Song, H. Lee, H. Suh, H.Y. Chung, Biol. Pharm. Bull. 30
(2007) 1711e1715.
[13] S. Okombi, D. Rival, S. Bonnet, A.M. Mariotte, E. Perrier, A. Boumendjel, J. Med.
Chem. 49 (2006) 329e333.
[14] A. Andrawis, V. Kahn, Biochem. J. 235 (1986) 91e96.
[15] I. Kubo, K.I. Nihei, K. Shimizu, Bioorg. Med. Chem. 12 (2004) 5343e5347.
[16] R. Matsuura, H. Ukeda, M. Sawamura, J. Agric. Food Chem. 54 (2006)
2309e2313.
[17] Y.J. Kim, J.K. No, J.H. Lee, H.Y. Chung, Biol. Pharm. Bull. 28 (2005) 323e327.
[18] J.K. No, M.S. Kim, Y.J. Kim, S.J. Bae, J.S. Choi, H.Y. Chung, Am. J. Chin. Med. 32
(2004) 97e103.
[19] Y. Kim, J. No, J. Lee, M. Kim, H. Chung, Biosci. Biotechnol. Biochem. 70 (2006)
532e534.
[20] Y.M. Ha, J.Y. Park, Y.J. Park, D. Park, Y.J. Choi, J.M. Kim, E.K. Lee, Y.K. Han,
J.A. Kim, J.Y. Lee, H.R. Moon, H.Y. Chung, Bioorg. Med. Chem. Lett. 21 (2011)
2445e2449.
[21] Y.M. Ha, J.A. Kim, Y.J. Park, D. Park, J.M. Kim, K.W. Chung, E.K. Lee, J.Y. Park,
J.Y. Lee, H.J. Lee, J.H. Yoon, H.R. Moon, H.Y. Chung, Biochim. Biophys. Acta 6
(2011) 612e619.
[22] Y.M. Ha, Y.J. Park, J.Y. Lee, D. Park, Y.J. Choi, E.K. Lee, J.M. Kim, J.A. Kim, J.Y. Park,
H.J. Lee, H.R. Moon, H.Y. Chung, Biochimie. 94 (2012) 533e540.
[23] T. Klabunde, C. Eicken, J.C. Sacchettini, B. Krebs, Nat. Struct. Biol. 5 (1998)
1084e1090.
[24] S.C. Heidi, Curr. Opin. Invest. Drugs 4 (2003) 406e411.
[25] M. Camps, T. RVuckle, H. Ji, V. Ardissone, F. Rintelen, J. Shaw, C. Ferrandi,
C. Chabert, C. Gillieron, B. Francon, T. Martin, D. Gretener, D. Perrin, D. Leroy,
P.A. Vitte, E. Hirsch, M.P. Wymann, R. Cirillo, M.K. Schwarz, C. Rommel, Nat.
Med. 11 (2005) 936e943.
[26] Z. Xia, C. Knaak, J. Ma, Z.M. Beharry, C. Mclnnes, W. Wang, A.S. Kraft,
C.D. Smith, J. Med. Chem. 9 (2005) 74e86.
[27] A.R. Katritzky, S.R. Tala, H. Lu, A.V. Vakulenko, Q.Y. Chen, J. Sivapackiam,
K. Pandya, S. Jiang, A.K. Debnath, J. Med. Chem. 52 (2009) 7631e7639.
[28] J.K. No, D.Y. Soung, Y.J. Kim, K.H. Shim, Y.S. Jun, S.H. Rhee, T. Yokozawa,
H.Y. Chung, Life Sci. 65 (1999) PL241ePL246.
m
then incubated at 60 ꢃC for 1 h and mixed to solubilize the
melanin. Absorbance at 405 nm was compared with a standard
curve of synthetic melanin. The results from 3 experiments are
shown.
5.3.7. Homology modeling of tyrosinase
A 3-dimensional (3D) model using the structure of Octopus
hemocyanin was built in SWISS-MODEL software [34], based on
homology modeling [35]. The SWISS-MODEL program automati-
cally provides an all-atom model using alignments between the
query sequence and known homologous structures. Known
homologous structures of potato catechol oxidase from the Protein
Data Bank (PDB, http://www.pdb.org) were used as the structural
templates. PDB entry 1BT3 was a suitable structural template (15%
sequence identity).
5.3.8. In silico docking of tyrosinase to novel inhibitor candidates
Among the many tools available for in silico proteineligand
docking, DOCK6 is the most commonly used because of its auto-
mated docking capabilities. The program performs ligand docking
using a set of predefined 3D grids of the target protein using
a systemic search technique [23].
We describe the structures of the novel inhibitor candidates
in Fig. 2. To prepare for the docking procedure, we performed
the following steps: (1) conversion of 2-dimensional (2D) struc-
tures into 3D structures, (2) calculation of charges, and (3) addition
of hydrogen atoms using the ChemOffice program (www.
cambridgesoft.com).
5.3.9. Statistical analysis
Inhibition of tyrosinase activity is expressed as a percentage
of inhibition based on 100 ꢀ [(A ꢂ 100)/B], where A ¼ OD492
with a test sample and B ¼ OD492 without a test sample. Data
collected have a standard error of the mean (S.E.M., n ¼ 3).
Statistical significance of the differences among groups was
determined by one-way analysis of variance (ANOVA) followed
by the Fisher’s protected least significant difference post hoc test.
P-values of less than 0.05 were considered statistically
significant.
[29] S.M. Son, K.D. Moon, C.Y. Lee, J. Agric. Food Chem. 48 (2000) 2071e2074.
[30] G. Battaini, E. Monzani, L. Casella, J. Biol. Inorg. Chem. 5 (2000) 262e268.
[31] H. Tada, O. Shiho, K. Kuroshima, M. Koyama, K. Tsukamoto, J. Immunol.
Methods 93 (1986) 157e165.
[32] D.S. Kim, S.Y. Kim, J.H. Chung, K.H.H. Kim, C. Eun, K.C. Park, Cell Signal. 14
(2002) 779e785.
Acknowledgments
[33] M.L. Bilodeau, J.D. Greulich, R.L. Hullinger, C. Bertolotto, R. Ballotti,
O.M. Andrisani, Pigmen. Cell Res. 14 (2001) 328e336.
[34] K. Arnold, L. Bordoli, J. Kopp, T. Schwede, Bioinformatics 22 (2006)
195e201.
This work was supported by a National Research Foundation of
Korea (NRF) grant funded by the Korea government (MEST, No.
2009-0083538). We thank the Aging Tissue Bank for providing
research materials for the study.
[35] C. Gerdemann, C. Eicken, B. Krebs, Acc. Chem. Res. 35 (2002) 183e191.