Analogs of 5-(substituted benzylidene)hydantoin as inhibitors of tyrosinase and melanin formation

Article abstract of DOI:10.1016/j.bbagen.2011.03.001

Background: Many tyrosinase inhibitors find application in cosmetics and pharmaceutical products for the prevention of the overproduction of melanin in the epidermis. A series of 5-(substituted benzylidene)hydantoin derivatives 2a-2k were prepared, and th

Full text of DOI:10.1016/j.bbagen.2011.03.001

Biochimica et Biophysica Acta 1810 (2011) 612–619
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journal homepage: www.elsevier.com/locate/bbagen
Analogs of 5-(substituted benzylidene)hydantoin as inhibitors of tyrosinase and
melanin formation
Young Mi Ha a,1, Jin-Ah Kim b,1, Yun Jung Park b, Daeui Park a, Ji Min Kim a, Ki Wung Chung a,
Eun Kyeong Lee a,c, Ji Young Park b, Ji Yeon Lee b, Hye Jin Lee b, Jeong Hyun Yoon a,
Hyung Ryong Moon b, , Hae Young Chung
⁎
a,⁎⁎
a
College of Pharmacy, Pusan National University, Kumjeong-Gu, Busan 609-735, Republic of Korea
Laboratory of Medicinal Chemistry, College of Pharmacy, Pusan National University, Busan 609-735, Republic of Korea
Research Center, Dongnam Institute of Radiological and Medical Sciences, Busan 619-953, Republic of Korea
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Background: Many tyrosinase inhibitors find application in cosmetics and pharmaceutical products for the
prevention of the overproduction of melanin in the epidermis. A series of 5-(substituted benzylidene)
hydantoin derivatives 2a–2k were prepared, and their inhibitory activities toward tyrosinase and melanin
formation were evaluated.
Received 13 January 2011
Received in revised form 28 February 2011
Accepted 2 March 2011
Available online 11 March 2011
Methods: The structures of the compounds were established using 1H and 13C NMR spectroscopy and mass
spectral analyses. All the synthesized compounds were evaluated for their mushroom tyrosinase inhibition
activity.
Keywords:
5-(substituted benzylidene)hydantoin
derivatives
Tyrosinase inhibitory activity
Docking study with mushroom tyrosinase
Antimelanogenesis
Results: The best results were obtained for compound 2e which possessed hydroxyl group at R2 and methoxy
group at R3, respectively. We predicted the tertiary structure of tyrosinase, simulated its docking with
compound 2e and confirmed that this compound interacts strongly with mushroom tyrosinase residues as a
competitive tyrosinase inhibitor. In addition, we found that 2e inhibited melanin production and tyrosinase
activity in B16 cells.
Conclusions: Compound 2e could be considered as a promising candidate for preclinical drug development in
skin hyperpigmentation applications.
General significance: This study will enhance understanding of the mechanism of tyrosinase inhibition and
will contribute to the development of effective drugs for use hyperpigmentation.
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
occurs through a complex pathway involving enzymatic and chemi-
cal reactions that are restricted to melanosomes, the melanocyte-
Human skin pigmentation occurs as a result of the accumulation
of melanin in the epidermis; melanin is produced by melanocytes
within specialized organelles called melanosomes. Melanogenesis
specific organelles containing all the components required to syn-
thesize the pigment. The conversion of tyrosine to melanin requires
tyrosinase, a copper-containing protein (Fig. 1). This tyrosinase
(monophenol monooxygenase, EC 1.14.18.1) plays the central role in
melanogenesis as the key enzyme that catalyzes the hydroxylation of
tyrosine to form 3,4-dihydroxyphenylalanine (L-DOPA), and L-DOPA
to DOPA quinone [1,2]. A large number of dermatological disorders
are characterized by the increased production and accumulation of
melanin; such disorders include abnormal pigmentations such as
freckles, age spots, and melasma, which can be serious [3,4].
Abbreviations: AcOH, Acetic acid; ANOVA, Analysis of variance; CaH2, Calcium
hydride; CO2, Carbon dioxide; CDCl3, Deuterated chloroform; L-DOPA, L-3,4-dihydrox-
yphenylalanine; DMSO, Dimethyl sulfoxide; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide; DMEM, Dulbecco's modified eagle's medium; FBS, Fetal
bovine serum; HCl, Hydrogen chloride; NMDA, N-methyl-D-aspartic acid; NMR, Nuclear
magnetic resonance; NaOAc, Sodium acetate; TLC, Thin-layer chromatography; PBS,
Phosphate-buffered saline; PDB, Protein data bank
Many tyrosinase inhibitors find application in cosmetics and
pharmaceutical products for the prevention of the overproduction of
melanin in the epidermis. Because of its key role in melanogenesis,
tyrosinase is an attractive target in the search for various kinds of
depigmenting agents [5–8]. In recent years, various tyrosinase
inhibitors have been reported such as azelaic acid [9], ascorbic acid
derivatives [10], arbutin [11], kojic acid [12], hydroxystilbene
compounds such as resveratrol [13–15], and polyphenolic compounds
⁎ Correspondence to: H.R. Moon, Laboratory of Medicinal Chemistry, College of
Pharmacy, Pusan National University, Busan 609–735, Republic of Korea. Tel.: +82 51
510 2815; fax: +82 51 513 6754.
⁎⁎ Correspondence to: H.Y. Chung, Molecular Inflammation Research Center for
Aging Intervention (MRCA), College of Pharmacy, Pusan National University, Kum-
jeong-Gu, Busan 609-735, Republic of Korea. Tel.: +82 51 510 2814; fax: +82 51 518
2821.
E-mail addresses: mhr108@pusan.ac.kr (H.R. Moon), hyjung@pusan.ac.kr (H.Y. Chung).
Y.M. Ha and J.-A. Kim contributed equally to this article.
1
0304-4165/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbagen.2011.03.001

Y.M. Ha et al. / Biochimica et Biophysica Acta 1810 (2011) 612–619
613
HO
HO
O
O
O
O
Tyrosinase
OH
OH
Melanin
NH2
NH2
DOPA quinone
DOPA
"Imide"
O
Tyrosinase
O
R1
R1
"Carboxylic acid"
H
S
R2
R3
R2
R3
OH
O
HN
NH
HN
HO
R4
OH
R4
O
I
NH2
""
Previously designed
Designed
hydantoin aderivatives
Tyrosine
thiazolidine-carboxylic acid
Fig. 1. Rationale for the design of the 5-(substituted benzylidene)hydantoin derivatives.
[16–18]. A previous study has shown that 3,4-dihydroxyacetophe-
none and 4,4′-dihydroxybiphenyl have strong anti-tyrosinase activ-
ities that inhibits melanogenesis [19,20]. We have also reported that
hydroxy substituted 2-phenyl-naphthalenes as resveratrol analogs, 5-
(6-hydroxy-2-naphthyl)-1,2,3-benzenetriol, 6-(3-hydroxyphenyl)-2-
naphthol, and 4-(6-hydroxy-2-naphtayl-1,3-bezendiol) inhibit tyros-
inase activity and reduced melanin biosynthesis in B16 cells, which
indicates that these compounds could find use as depigmentation
agents. We have also found several potent tyrosinase inhibitors in
natural materials; these have a potent inhibitory effect on melano-
genesis [18,20,21] and the microphthalmia-associated transcription
factor [19].
Recently, our laboratory synthesized thiazolidine-carboxylic acid
derivatives I on the basis of the structures of the substrates and
products of tyrosinase, and we found that some of them showed
significant inhibitory activities against tyrosinase (Fig. 1). The
carboxylic acid and amine (NH) functionalities in I appeared to
play an important role in the inhibition of tyrosinase activity. Our
interest in an alternative structure capable of replacing thiazolidine-
carboxylic acid led us to study the hydantoin template. We
considered that the imide group of the hydantoin would play the
role of the carboxylic acid of I at the active site of tyrosinase because
the hydrogen atoms of both the imide and the carboxylic acid could
commonly act as acidic protons and because the imide and the
carboxylic acid both possessed a carbonyl group capable of
interacting with amino acid residues present at the active site of
tyrosinase (Fig. 1). Hydantoin derivatives have been identified as
anticancer [22,23], anticonvulsant [24], anti-inflammatory [25,26],
anti-HIV [27], antidiabetic [28], antimuscarinic [29,30], antiulcer
[31], antiarrythmic [32], and antihypertensive agents [33]. They have
also been found to act as serotonin and fibrinogen receptor antago-
nists [34,35], inhibitors of the glycine binding site of the NMDA
receptor [36], and antagonists of leukocyte cell adhesion, acting as
allosteric inhibitors of the protein–protein interaction [37]. Hydan-
toin derivatives also show antiproliferative effects toward human
carcinoma cells [38]. However, their effects on tyrosinase and skin
melanogenesis are not yet known.
2. Materials and methods
2.1. Experimental
2.1.1. General
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 (δH 3.30 and δC 49.0
for CD3OD, δH 7.27 and δC 77.0 for CDCl3). Coupling constants are
reported in Hertz. The abbreviations used are as follows: s (singlet), d
(doublet), t (triplet), dd (doublet of doublets), and br s (broad
singlet). All the reactions described below were performed under an
argon or nitrogen atmosphere and were monitored by thin-layer
chromatography (TLC). All anhydrous solvents were distilled over
CaH2 or Na/benzophenone before use.
2.1.2. Method A: general procedure I for the synthesis of substituted
benzylidene-hydantoin analogs (2a–2d, 2f–2g, and 2i)
A suspension of substituted benzaldehydes (2.42–7.70 mmol) and
hydantoin (1.1 equiv.) in piperidine (1 ml/4 mmol benzadehyde) was
heated at reflux for duration between 30 min and 6 h. The reaction
mixture was cooled, and water (20 times the volume of piperidine
used) was added at 60 °C. Any traces of tarry material were removed
by filtration. The filtrate was acidified with 12 N HCl at room
temperature. The mixture was kept at room temperature for a few
hours. The precipitates generated were then filtered off using a
Buchner funnel and washed with cold water. They were then dried
under reduced pressure, and the substituted benzylidene-hydantoin
products (yields: 9.7–79%) were obtained.
2.1.3. Method B: general procedure II for the synthesis of substituted
benzylidene-hydantoin analogs (2e, 2h, and 2k)
A suspension of substituted benzaldehydes (1.08–1.28 mmol) and
hydantoin (1.1 equiv.) in EtOH (2–4 ml) and H2O (2–4 ml) was
heated to 80 °C. After the reaction mixture had been heated at the
same temperature for 30 to 50 h, the precipitates formed were filtered
off using a Buchner funnel and washed with water to remove excess
hydantoin and methylene chloride or ethyl acetate, depending on the
property of the substituted benzaldehydes remaining, to give the title
products (yields: 11.4–71.4%).
In this study, eleven 5-(substituted benzylidene)hydantoin deri-
vatives, 2a–2k were synthesized, and their mushroom tyrosinase
inhibitory activities were evaluated. Among these derivatives, 2e
exhibited the highest inhibition of the L-DOPA oxidase activity of
mushroom tyrosinase at a concentration of 20 μM. This work was
designed to characterize the effects of 2e on mushroom tyrosinase
activity and on tyrosinase activity and melanin levels in B16
melanoma cells (B16 cells). We also predicted the tertiary structure
of tyrosinase, and simulated its docking with 2e.
2.1.4. Method C: synthesis of (Z)-5-(4-hydroxy-3,5-dimethoxybenzylidene)
imidazolidine-2,4-dione (2j)
A suspension of syringaldehyde (300 mg, 1.65 mmol), hydantoin
(198 mg, 1.98 mmol), and sodium acetate (405 mg, 4.94 mmol) in

614
Y.M. Ha et al. / Biochimica et Biophysica Acta 1810 (2011) 612–619
acetic acid (1.6 ml) was heated at reflux for 24 h. The precipitate
generated was filtered using a Buchner funnel and washed with
methylene chloride and a small amount of water. After being drying
under reduced pressure, the title product (192.9 mg, 44.3%) was
obtained.
(dd, 1H, J=2.0, 8.4Hz, 6′-H), 6.76 (d, 1H, J=8.0 Hz, 5′-H), 6.30 (s, 1H,
vinylic H), 4.06 (q, 2H, J=6.8 Hz, CH3CH2-), 1.30 (t, 3 H, J=6.8 Hz,
CH3CH2-); 13C NMR (100 MHz, DMSO-d6) δ 166.3 (C4), 156.4 (C2),
148.6 (C3′), 147.6 (C4′), 126.1 (C5), 125.0 (C1′), 124.2 (C6′), 116.5
(C5′), 115.3 (C2′), 110.4 (benzylic C), 64.7 (3′-OCH2), 15.4 (CH2CH3);
LRMS(ES) m/z 247 (M-H) −
.
2.1.4.1. (Z)-5-(4-Hydroxybenzylidene)imidazolidine-2,4-dione (2a).
Pale yellowish solid; reaction time, 30 min; yield, 78.5%; melting
point, N300 °C; 1H NMR (500 MHz, DMSO-d6) δ 11.10 (s, 1H, NH),
10.30 (s, 1H, NH), 9.84 (s, 1H, OH), 7.46 (d, 2 H, J=8.0 Hz, 2′-H, 6′-H),
6.77 (d, 2 H, J=8.0 Hz, 3′-H, 5′-H), 6.34 (s, 1 H, vinylic H); 13C NMR
(100 MHz, DMSO-d6) δ 166.3 (C4), 158.7 (C4´), 156.3 (C2), 131.9 (C2′,
C6′), 126.0 (C5), 124.5 (C1′), 116.4 (C3′, C5′), 110.0 (benzylic C);
LRMS(ES) m/z 203 (M-H)–.
2.1.4.8. (Z)-5-(2,4-Dimethoxybenzylidene)imidazolidine-2,4-dione
(2h). White solid; reaction time, 30 h; yield, 71.4%; melting point,
234.1–237.2 °C; 1H NMR (500 MHz, DMSO-d6) δ 11.09 (s, 1H, NH),
10.28 (s, 1H, NH), 7.55 (d, 1H, J=8.5 Hz, 6´-H), 6.60 (s, 1H, vinylic H),
6.59 (d, 1H, J=2.5 Hz, 3′-H), 6.54 (dd, 1H, J=2.0, 8.5 Hz, 5′-H), 3.83
(s, 3H, OCH3), 3.79 (s, 3 H, OCH3); 13C NMR (100 MHz, DMSO-d6) δ
166.3 (C4), 161.8 (C4′), 159.3 (C2′), 156.2 (C2), 130.8 (C6′), 126.7
(C5), 114.9 (C1′), 106.2 (benzylic C), 103.7 (C5′), 98.9 (C3′), 56.4 (2′-
2.1.4.2. (Z)-5-(2-Hydroxybenzylidene)imidazolidine-2,4-dione (2b).
Yellowish solid; reaction time, 2 h; yield, 50.3%; melting point,
265.5–268.4 °C; 1H NMR (500 MHz, DMSO-d6) δ 11.17 (s, 1H, NH),
10.29 (s, 1H, NH), 10.08 (s, 1H, OH), 7.54 (d, 1H, J=8.0 Hz, 6′-H), 7.16
(td, 1H, J=1.0, 8.0 Hz, 4′-H), 6.88 (d, 1H, J=8.0 Hz, 3′-H), 6.82 (t, 1H,
J=8.0 Hz, 5′-H), 6.67 (s, 1H, vinylic H); 13C NMR (100 MHz, DMSO-
d6) δ 166.3 (C4), 156.5 (C2′), 156.1 (C2), 130.6 (C4′), 130.0 (C6′),
127.7 (C5), 120.7 (C1′), 120.0 (C5′), 116.1 (C3′), 104.4 (benzylic C);
LRMS(ES) m/z 203 (M-H)–.
OCH3), 56.1 (4′-OCH3); LRMS(ES) m/z 247 (M-H) −
.
2.1.4.9. (Z)-5-(3,4-Dimethoxybenzylidene)imidazolidine-2,4-dione (2i).
Pale yellowish solid; reaction time, 6 h; yield, 9.7%; melting point,
271.3–273.9 °C; 1H NMR (500 MHz, DMSO-d6) δ 11.15 (s, 1H, NH),
10.48 (s, 1 H, NH), 7.18 (dd, 1H, J=1.5, 8.0 Hz, 6´-H), 7.11 (d, 1H,
J=2.0 Hz, 2′-H), 6.95 (d, 1 H, J=8.5 Hz, 5′-H), 6.37 (s, 1H, vinylic H),
3.81 (s, 3H, OCH3), 3.77 (s, 3H, OCH3); 13C NMR (100 MHz, DMSO-d6)
δ 166.3 (C4), 156.4 (C2), 150.0 (C3′), 149.4 (C4′), 126.8 (C5), 126.3
(C1′), 123.7 (C6′), 113.2 (C5′), 112.4 (C2′), 109.9 (benzylic C), 56.3
2.1.4.3. (Z)-5-(4-Methoxybenzylidene)imidazolidine-2,4-dione (2c).
Yellowish solid; reaction time, 4 h; yield, 17.4%; melting point,
241.8–242.9 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.13 (s, 1 H, NH),
10.40 (s, 1H, NH), 7.55 (d, 2 H, J=8.8 Hz, 2´-H, 6´-H), 6.92 (d, 2 H,
J=8.8 Hz, 3′-H, 5′-H), 6.35 (s, 1H, vinylic H), 3.75 (s, 3 H, 4′-OCH3);
13C NMR (100 MHz, DMSO-d6) δ 166.3 (C4), 160.1 (C4′), 156.3 (C2),
131.8 (C2′, C6′), 126.7 (C5), 126.1 (C1′), 115.0 (C3′, C5′), 109.3
(OCH3), 56.2 (OCH3); LRMS(ES) m/z 247 (M-H)−
.
2.1.4.10. (Z)-5-(4-Hydroxy-3,5-dimethoxybenzylidene)imidazolidine-
2,4-dione (2j). Yellow solid; reaction time, 24 h; yield, 44.3%; melting
point, 266.0–268.5 °C; 1H NMR (500 MHz, DMSO-d6) δ 11.13 (s, 1H,
NH), 10.50 (s, 1H, NH), 8.81 (s, 1H, OH), 6.82 (s, 2H, 2′-H, 6′-H), 6.35
(s, 1H, vinylic H), 3.81 (s, 6H, 3′-OCH3, 5′-OCH3); 13C NMR (100 MHz,
DMSO-d6) δ 166.3 (C4), 156.5 (C2), 148.7 (C3′, C5′), 137.4 (C4′), 126.2
(C5), 123.8 (C1′), 110.8 (benzylic C), 108.0 (C2′, C6′), 56.8 (3′-OCH3,
(benzylic C), 55.9 (4′-OCH3); LRMS(ES) m/z 217 (M-H) −
.
2.1.4.4. (Z)-5-(3,4-Dihydroxybenzylidene)imidazolidine-2,4-dione (2d).
Brown solid; reaction time, 30 min; yield, 68.7%; melting point,
N300 °C; 1H NMR (400 MHz, CD3OD) δ 6.92–6.89 (m, 2H, 2′-H, 6′-H),
6.80 (d, 1H, J=8.8 Hz, 5′-H), 6.43 (s, 1 H, vinylic H); 13C NMR
(100 MHz, CD3OD) δ 166.6 (C4), 156.4 (C2), 146.8 (C4′), 145.6 (C3′),
125.7 (C5), 125.1 (C1′), 121.7 (C6′), 116.4 (C5′), 115.6 (C2′), 111.7
5′-OCH3); LRMS(ES) m/z 263 (M-H)−
.
2.1.4.11. (Z)-5-(3,4,5-Trimethoxybenzylidene)imidazolidine-2,4-dione
(2k). Yellow solid; reaction time, 50 h; yield, 11.4%; melting point,
266.3–267.2 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.19 (br s, 1H, NH),
10.58 (br s, 1H, NH), 6.80 (s, 2H, 2′-H, 6′-H), 6.33 (s, 1 H, vinylic H),
3.80 (s, 6H, 3′-OCH3, 5′-OCH3), 3.64 (s, 3H, 4′-OCH3); 13C NMR
(100 MHz, DMSO-d6) δ 166.2 (C4), 156.5 (C2), 153.7 (C3′, C5′), 138.6
(C4′), 129.2 (C1′), 127.9 (C5), 109.7 (benzylic C), 107.6 (C2′, C6′), 60.7
(benzylic C); LRMS(ES) m/z 219 (M-H) −
.
2.1.4.5. (Z)-5-(3-Hydroxy-4-methoxybenzylidene)imidazolidine-2,4-
dione (2e). Pale green yellow solid; reaction time, 40 h; yield, 36%;
melting point, 250.7–253.4 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.10
(br s, 1H, NH), 10.33 (br s, 1H, NH), 9.01(s, 1 H, OH), 7.05 (dd, 1 H,
J=2.0, 8.4 Hz, 6′-H), 6.97 (d, 1 H, J=2.0 Hz, 2′-H), 6.89 (d, 1 H,
J=8.4 Hz, 5′-H), 6.25 (s, 1 H, vinylic H), 3.77 (s, 3 H, 4′-OCH3); 13C
NMR (100 MHz, DMSO-d6) δ 166.3 (C4), 156.3 (C2), 149.0 (C4′), 147.1
(C3′), 126.9 (C5), 126.4 (C1′), 121.9 (C6′), 117.4 (C2′), 112.7 (C5′),
(4′-OCH3), 56.7 (3′-OCH3, 5′-OCH3); LRMS(ES) m/z 277 (M-H)−
.
2.2. Materials
Mushroom tyrosinase, L-tyrosine [3-(4-hydroxyphenyl)]-L-alanine
(S)-2-amino-3-(4-hydroxyphenyl) propionic acid), kojic acid [5-hy-
droxy-2-(hydroxymethyl)-4H-pyran-4-one], and α-MSH (alpha-mela-
nocyte stimulating hormone) were purchased from Sigma Chemical Co.
(St. Louis, MO, USA).
109.9 (benzylic C), 56.3 (4′-OCH3); LRMS(ES) m/z 233 (M-H)−
.
2.1.4.6. (Z)-5-(4-Hydroxy-3-methoxybenzylidene)imidazolidine-2,4-
dione (2f). Green yellow solid; reaction time, 30 min; yield, 74%;
melting point, 249.2–251.6 °C; 1H NMR (500 MHz, DMSO-d6) δ 11.11
(s, 1H, NH), 10.40 (s, 1H, NH), 9.42 (s, 1H, OH), 7.09 (d, 1H, J=1.5 Hz,
2′-H), 7.06 (dd, 1H, J=1.5, 8.5 Hz, 6′-H), 6.78 (d, 1H, J=8.5 Hz, 5´-H),
6.35 (s, 1H, vinylic H), 3.82 (s, 3 H, 3′-OCH3); 13C NMR (100 MHz,
DMSO-d6) δ 166.3 (C4), 156.4 (C2), 148.4 (C3′), 148.2 (C4′), 126.1
(C5), 125.0 (C1′), 124.1 (C6′), 116.4 (C5′), 113.8 (C2′), 110.5 (benzylic
2.3. Methods
2.3.1. Cell culture
B16 cells (obtained from the Korean Cell Line Bank) were cultured
in Dulbecco's Modified Eagle's Medium (DMEM; Gibco, Carlsbad, CA,
USA) with 10% fetal bovine serum (FBS; Gibco) and penicillin/
streptomycin (100 IU·50 μg−1·ml−1) in a humidified atmosphere
containing 5% CO2 at 37 °C. B16 cells were cultured in 24-well plates
for melanin quantification and enzyme activity assays.
C), 56.4 (3′-OCH3); LRMS(ES) m/z 233 (M-H)−
.
2.1.4.7. (Z)-5-(3-Ethoxy-4-hydroxybenzylidene)imidazolidine-2,4-dione
(2g). Ocherous solid; reaction time, 30 min; yield, 79%; melting point,
253.0–255.4 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.08 (s, 1H, NH),
10.36 (s, 1H, NH), 9.32 (s, 1H, OH), 7.06 (d, 1H, J=2.4 Hz, 2′-H), 7.03
2.3.2. Cell viability
Cell survival was quantified through a colorimetric 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay

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615
that measured the mitochondrial activity in viable cells. This method
is based on the conversion of MTT (Sigma) to MTT-formazan crystals
by a mitochondrial enzyme and has been described previously [39].
Briefly, cells seeded at a density of 3×104/cell in a Corning 48-well
plate (Corning, NY, USA) were allowed to adhere overnight; the
culture medium was then replaced with fresh serum-free DMEM. MTT
was freshly prepared at 5 mg·ml−1 in phosphate-buffered saline
(PBS) solution. 500 μl aliquots of the MTT stock solution were added
to each well, and the plate was incubated at 37 °C for 4 h in a
humidified 5% CO2 incubator. After 4 h, the medium was removed.
500 μl of EtOH-DMSO (1:1 solution) was added to each well to
dissolve the formazan. After 10 min, the optical density of each well
was measured spectrophotometrically using a 560 nm filter.
2.3.5. Assay of murine tyrosinase activity
Tyrosinase activity was estimated by measuring the rate of L-DOPA
oxidation [41]. Cells were placed in 24-well dishes at a density of
5×104 cells·ml−1. B16 cells were incubated in the presence or
absence of 100 nM α-MSH and they were then treated for 24 h with
various concentrations of 2e (0–10 μM). The cells were washed and
lysed in 100 μl of 50 mM sodium phosphate buffer (pH 6.5) containing
1% Triton X-100 (sigma) and 0.1 mM PMSF (phenylmethylsulfonyl
fluoride), and then frozen at −80 °C for 30 min. After being thawed
and mixed, the cellular extracts were clarified by centrifugation at
12000 rpm for 30 min at 4 °C. An 8 μl sample of the supernatant and
20 μl of L-DOPA (2 mg·ml−1) were placed in a 96-well plate, and the
absorbance at 492 nm was recorded every 10 min for 1 h at 37 °C
using an ELISA plate reader. The final activity is expressed as ΔO.
D.·min−1 for each set of conditions. The results from three experi-
ments are shown.
The results from three experiments are shown.
2.3.3. Assay for the measurement of inhibitory effects on mushroom
tyrosinase
Mushroom was used as the source of tyrosine for the entire study.
Tyrosinase activity was determined as described previously with minor
modifications [40]. Briefly, 20 μl of an aqueous mushroom tyrosinase
solution (1000 units) was added to a 96-well microplate (Nunc,
Denmark), in a 200 μl assay mixture containing 1 mM L-tyrosine
solution and 50 mM phosphate buffer (pH 6.5). The assay mixture
was incubated at 25 °C for 30 min. Following incubation, the amount of
dopachrome produced in the reaction mixture was determined
spectrophotometrically at 492 nm (OD492) using a microplate reader
(Hewlett Packard, Palo Alto, CA, USA). IC50, or inhibitory concentration
50, is the concentration of a compound that inhibits a standard 50%
response. IC50 values are derived from the X-axis on a curve of inhibitor
concentration versus product formation and determined from the
alignment of the dose-response curve on the dependent Y-axis. In the
present study, dose-dependent inhibition experiments were performed
in triplicate to determine the IC50 values of the compounds. According to
the inhibition percentages of the three doses in each experiment, the
log-linear curves and their equations were determined. The individual
IC50 values were then calculated as the concentration when the Y-axis
was equal to 50% inhibition. The results from three experiments are
shown.
2.3.6. Determination of melanogenesis in B16 cells
The melanin content was determined using a modification of the
method of Bilodeau et al. [42]. In the present study, the amount of
melanin was used as the index of melanogenesis. B16 cells (5×104)
were transferred to 24-well dishes and incubated in the presence or
absence of 100 nM α-MSH. The cells were then incubated for 24 h
with various concentrations of 2e (0–10 μM). The samples were
washed twice with PBS and were then dissolved in 100 μl of 1N NaOH.
The samples were incubated at 60 °C for 1 h and mixed to solubilize
the melanin. The absorbance at 405 nm was compared with a stan-
dard curve for synthetic melanin. The results from three experiments
are shown.
2.3.7. Homology modeling of tyrosinase
A three-dimensional model of Lentinula edodes (mushroom)
tyrosinase comprising 618 amino acids was built using the SWISS-
MODEL [43] program and on the basis of homology modeling [44]. The
SWISS-MODEL program automatically provides an all-atom model
using alignments between the query sequence and known homolo-
gous structures. Known homologous structures of tyrosinase from the
Protein Data Bank (PDB) (http://www.pdb.org) were used as the
structural templates. The PDB: 1BT3 was a suitable structural template
(15% sequence identity).
2.3.4. Kinetic analysis of tyrosinase inhibition
Various concentration of L-tyrosine (0.25 to 2 mM) as a substrate,
20 μl of aqueous mushroom tyrosinase solution (1000 units), and
50 mM potassium phosphate buffer (pH 6.5), with or without test
samples (1.25 and 20.0 μM of 2e), were added to a 96-well plate in a
total volume of 200 μl. Using a microplate reader, the initial rate of
dopachrome formation from the reaction mixture was determined by
the increase in absorbance at a wavelength of 492 nm per minute
(ΔOD492·min−1). The Michaelis constant (Km) and maximal velocity
(Vmax) of the tyrosinase activity were determined from a Lineweaver–
Burk plot at various L-tyrosine concentrations [34]. A modification of
the Michaelis–Menten equation was required to describe the reaction
kinetics due to competitive inhibition by the compounds together
with substrate inhibition by L-tyrosine. The results from three experi-
ments are shown.
2.3.8. In silico docking of tyrosinase and novel inhibitor candidates
Among the many tools available for in silico protein-ligand
docking, DOCK6 is the most commonly used because of its automated
docking capability. The program performs ligand docking using a set
of predefined 3D grids of the target protein through a systemic search
technique [45].
We describe the structures of the novel inhibitor candidates in
Figs. 1 and 4. To prepare for the docking procedure, we performed the
following steps: (1) conversion of the 2D structures into 3D
structures; (2) calculation of charges; and (3) addition of hydrogen
atoms using the ChemOffice program (www.cambridgesoft.com).
R1
O
R1
O
O
R2
R3
R2
a, b or c
H
+
NH
NH
HN
HN
R3
R4
R4
O
O
Hydantoin
1a - 1k
2a - 2k
Scheme 1. Synthesis of the target compounds, 5-(substituted benzylidene)hydantoin derivatives (2a–2k); reagents and conditions: method A: i) piperidine, reflux, 30 min–6 h;
ii) water at 60 °C; iii) filtration; iv) 12N HCl for 2a–2d, 2f–2g, and 2i; method B: EtOH/H2O (1:1), 80 °C, 30–50 h for 2e, 2 h, and 2k; method C: NaOAc, AcOH, reflux, 24 h for 2j.

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Y.M. Ha et al. / Biochimica et Biophysica Acta 1810 (2011) 612–619
2.4. Statistical analysis
The inhibition of tyrosinase activity is expressed as a percentage of
inhibition, based on the equation 100−[(A×100)/B], where A is
OD492 with a test sample, and B is OD492 without a test sample. The
data collected have a mean standard error (n=3). The statistical
significance of the differences between groups was determined by
one-factor analysis of variance (ANOVA) followed by Fisher's pro-
tected least significant difference post hoc test. Values of *pb0.05
were considered statistically significant.
3. Results and discussion
Fig. 2. Dose-dependent inhibitory effects of 2e and kojic acid on tyrosinase activity.
Tyrosinase activity was measured using L-tyrosine as the substrate. Results are expressed
as percentage of control, and each column represents the mean S.E. of three deter-
minations. The optical density value of control at 492 nm was 0.486 0.007.
Hydantoins and their derivatives constitute a group of pharmaceu-
tical compounds with anticonvulsant and antiarrhythmic properties,
and are also used against diabetes. Eleven 5-(substituted benzylidene)
hydantoin derivatives, 2a–2 k were obtained through three synthetic
methods (Scheme 1). Initially, method A using piperidine as a base, was
employed for the synthesis of some of the target compounds. However,
when using alkoxy-benzaldehydes 1c and 1i (Table 1) as starting
materials, the purification and solidification processes were difficult,
and the yields were low because of gum materials generated. Therefore,
method B, which did not use base, was attempted for the preparation of
2e, 2h, and 2k, and the desired products were obtained more easily
and in higher purity. Both method A and method B were tried for the
synthesis of 2j, but the 1H NMR spectra showed that the product was not
pure, even though it looked like one spot in the TLC results. In this case,
method C, using NaOAc and AcOH, was attempted, and the desired
hydantoin derivative 2j was afforded without any impurities in
moderate yield. For the preparation of 5-(2,4-dihydroxybenzylidene)
hydantoin and 5-(3,5-dihydroxybenzylidene)hydantoin from 2,4-dihy-
droxybenzaldehyde and 3,5-dihydroxybenzaldehyde, the three afore-
mentioned methods were attempted, but none were successful. The
synthetic reactions for the substituted benzylidene-hydantoin analogs
gave 2a–2d, 2f–2 g, and 2i in 9.7–79% yields using method A, 2e, 2h, and
2k in 11.4–71.4% yields using method B, and 2j in 44.% yield using
method C (Scheme 1 and Table 1). The structures of the synthesized
compounds were determined by 1H and 13C NMR spectroscopy and
mass spectroscopic analyses.
The inhibitory activities of the synthesized compounds were
examined using mushroom tyrosinase, as described previously, with
minor modification [40]. The inhibitory activities of these newly
synthesized compounds were compared to that of kojic acid as a
reference control, and the results are shown in Table 1 and Fig. 2. In
terms of the structure-activity relationships, compound 2d with two
substituents (the hydroxy group at R2 and R3, respectively) on the
phenyl ring has a similar structure to DOPA, which also has hydroxyl
groups at R2 and R3 on the phenyl ring, but 2d showed no mushroom
tyrosinase inhibition. Further, compound 2a, with a hydroxyl group at
R3 on the phenyl ring, has a similar structure to tyrosine, one of the
substrates of tyrosinase, but it showed poor mushroom tyrosinase
inhibition. Compound 2e, containing a hydroxyl group at R2 and a
methoxy group at R3, showed the highest inhibitory activity against
mushroom tyrosinase, but 2f, with these groups inverted (a methoxy
group at R2 and a hydroxyl group at R3), and 2i (with methoxy groups
at R2 and R3) showed little inhibitory activity. Among the newly
synthesized compounds, we further investigated the bioactivities of
2e, because this compound exhibited the highest inhibition of mush-
room tyrosinase (Table 1).
Table 1
Substitution pattern of the substituted benzylidene-hydantoin derivatives 2a–2k.
R1
O
R2
NH
To the best of our knowledge, our present study provides the first
evidence that 2e has a potent inhibitory effect on melanogenesis.
First, we attempted to ascertain the potential cytotoxicity of 2e on
B16 cells. The cytotoxic effects of 2e were estimated by measuring
HN
R3
R4
O
Compound R1
R2
R3
R4
Yield Reaction time Tyrosinase
(%)
(hour)
inhibition (%)a
2a
2b
2c
2d
2e
2f
2g
2h
H
OH
H
H
H
H
H
OMe
H
H
H
H
H
OH
OH
OH
H
OMe
OH
OMe
H
H
H
H
H
H
H
H
H
78.5
50.3
17.4
68.7
36.0
74.0
79.0
71.4
9.7
0.5
2
4
0.5
40
0.5
0.5
30
6
38.95 0.36
NI
19.85 0.61
NI
86.54 0.49
17.33 0.41
5.16 0.31
21.61 0.50
17.17 0.40
21.59 0.48
41.79 0.55
41.89 0.95
OMe OH
OEt
H
OMe OMe
OMe OH
OMe OMe OMe 11.4
OH
OMe
2i
2j
OMe 44.3
24
50
2k
Kojic acid
H
Fig. 3. Effect of 2e on the cell viability of B16 cells. Cells were treated with various doses
of 2e (0–10 μM), and the effect of 2e on the viability of B16 cells was examined by MTT
⁎
Values represent means S.E. of three experiments, NI (no inhibition).
assay. Data are expressed as percentage of control. pb0.05 compared to the untreated
control.
a.
Tyrosinase inhibition was measured using L-tyrosine as the substrate at 20 μM.

Y.M. Ha et al. / Biochimica et Biophysica Acta 1810 (2011) 612–619
617
Table 2
Effects on mushroom tyrosinase activity and kinetic analysis of compounds.
a
Compound
IC50 (μM)
Kinetic analysisb
Vmax
Km (mM)
Ki (M)
1.25 μM
0.42
5.0 μM
0.45
–
20.0 μM
0.61
1.25 μM
5.0 μM
1.95×10−7
–
20.0 μM
5.89×10−7
2e
5.73 1.40
1.75×10−2
–
5.46×10−8
Kojic acid
20.99 1.80
a
50% inhibitory concentration (IC50).
b
Lineweaver–Burk plot of mushroom tyrosinase: data are presented as mean values of 1/V, which is the inverse of the increase in absorbance at a wave-length 492 nm/min
(ΔA492/min), for three independent tests with different concentrations of L-tyrosine as the substrate.
cell viability, and no significant cytotoxic effect was found for any of
the concentrations tested, as shown in Fig. 3. These results indicated
that 2e was relatively non-cytotoxic to cells under the experimental
conditions used. As shown in Table 2, we examined the inhibitory
action of 2e on mushroom tyrosinase activity, and found that 2e had
a strong suppressive action on the enzyme, with an IC50 value of
5.73 1.4 μM. Compared with kojic acid (IC50 =20.99 1.8 μM), 2e
appeared to have a more powerful effect. The inhibitory efficacy of
2e may be attributed to its hydroxyl and methoxy groups at R2 and
R3 on the phenyl ring, respectively. The scaffold of 2e was struc-
turally similar to DOPA. To explore the inhibitory mechanism of 2e,
we conducted a study of the kinetic behavior of tyrosinase activity in
the presence of 2e (Table 2). As the concentration of 2e increased,
competitive inhibition; examples of such inhibitors are tropolone
[47] and kojic acid [12]. Thus, the inhibition mechanism of 2e might
involve binding to the copper active site of mushroom tyrosinase.
Therefore, we predicted the tertiary structure of mushroom tyrosi-
nase and simulated its docking with 2e using DOCK6. We searched
for tyrosinase residues that might bind to compound 2e, and found
that the most important binding residues for interaction with 2e are
expected to be THR 195, ASP 262, HIS 265, VAL 266, SER 275, ASP
276, and PRO 277 (Fig. 4). These residues existed within 3 of the
ligand. The docking simulation supported the slope-parabolic
mixed-type inhibition observed, as this type of inhibition is gener-
ally encountered when there are multiple possible binding sites for
an inhibitor. The docking simulation was successful, with signifi-
cant scores using DOCK6 (binding energy: −26.79 kcal/mol), and
suggested that it is mainly the PRO 96 residue that is responsible for
the interaction with 2e.
the Km value of 2e also increased gradually without changing Vmax
,
thereby indicating that 2e acts as a competitive inhibitor of
mushroom tyrosinase. Tyrosinase is a copper-containing enzyme
that is widely distributed in nature [46], and most tyrosinase inhibi-
tors that chelate the copper in the active site of the enzyme show
We also examined the inhibitory effect of 2e on the tyrosinase
activity of B16 cells treated with 100 nM α-MSH, and found that
Fig. 4. Computational prediction of the structure for tyrosinase and docking simulation with 2e. Predicted 3D structure of mushroom tyrosinase. A. Chemical structure of 2e. B. The
box indicates 2e binding sites with tyrosinase residues THR 195, ASP 262, HIS 265, VAL 266, SER 275, ASP 276, and PRO 277. The yellow area interacts with the ligand (Zb3).

618
Y.M. Ha et al. / Biochimica et Biophysica Acta 1810 (2011) 612–619
contribute to the development of effective drugs against hyper-
pigmentation. Considering the tyrosinase inhibitory activities of the 5-
(substituted benzylidene)hydantoin derivatives, the hydantoin tem-
plate was thought to be a perfect surrogate for thiazolidine-carboxylic
acid. This study indicates the possibility of the imide and amine
groups acting as pharmacophores in tyrosinase inhibitors.
Acknowledgments
This work was supported by a grant from the National Research
Foundation of Korea (NRF) funded by the Korea government (MEST,
No. 2009-0083538 and KRF-2008-314-E00292) and by the Basic Science
Research Program through the National Research Foundation of Korea
(NRF) funded by the Ministry of Education, Science and Technology (No.
2010-0012038). We also thank the Aging Tissue Bank for providing
research materials for the study.
Fig. 5. Activity results of 2e on B16 cell tyrosinase. In the presence of 100 nM MSH, B16
cells were treated with various doses of 2e (0.2–0.0 μM) for 24 h. Results are expressed
as percentage of control, and each column represents the mean S.E. of three
determinations. ###pb0.001 compared to the untreated control,
to the group treated with 100 nM α-MSH, KA (kojic acid).
pb0.001 compared
⁎⁎⁎
References
[1] C.J. Cooksey, P.J. Garratt, E.J. Land, S. Pavel, C.A. Ramsden, P.A. Riley, N.P. Smit,
Evidence of the indirect formation of the catecholic intermediate substrate
responsible for the autoactivation kinetics of tyrosinase, J. Biol. Chem. 272 (1997)
26226–26235.
[2] I. Kubo, I. Kinst-Hori, S.K. Chaudhuri, Y. Kubo, Y.T. Ogura, Flavonols from
Heterotheca inuloides: tyrosinase inhibitory activity and structural criteria,
Bioorg. Med. Chem. 8 (2000) 1749–1755.
[3] K. Urabe, J. Nakayama, Y. Hori, in: J.J. Norlund, R.E. Boissy, V.J. Hearing, R.A. King, J.P.
Ortonne (Eds.), The Pigmentary System: Physiology and Pathophysiology, Oxford
University Press, New York, 1998, pp. 909–911.
[4] M.K. Cullen, in: J.J. Norlund, R.E. Boissy, V.J. Hearing, R.A. King, J.P. Ortonne (Eds.),
The Pigmentary System: Physiology and Pathophysiology, Oxford University
Press, New York, 1998, pp. 760–766.
[5] S. Briganti, E. Camera, M. Picardo, Chemical and instrumental approaches to treat
hyperpigmentation, Pigment Cell Res. 16 (2003) 101–110.
[6] D.S. Kim, S.Y. Kim, S.H. Park, Y.G. Choi, S.B. Kwon, M.K. Kim, J.I. Na, S.W. Youn, K.C.
Park, Inhibitory effects of 4-n-butylresorcinol on tyrosinase activity and melanin
synthesis, Biol. Pharm. Bull. 28 (2005) 2216–2219.
[7] S. Parvez, M. Kang, H.S. Chung, H. Bae, Naturally occurring tyrosinase inhibitors:
mechanism and applications in skin health, cosmetics and agriculture industries,
Phytother. Res. 21 (2007) 805–816.
[8] F. Solano, S. Briganti, M. Picardo, G. Ghanem, Hypopigmenting agents: an updated
review on biological, chemical and clinical aspects, Pigment Cell Res. 19 (2006)
550–571.
2e inhibited the tyrosinase activity effectively compared to the
α-MSH-treated group (Fig. 5). As the results show, the inhibitory
effect of 2e on tyrosinase activity was stronger than that of 10 μM
kojic acid with 100 nM α-MSH-treated B16 cells. We also evaluated
the melanin contents of cultured B16 cells in the presence of 2e
(ranging from 0.2 to 10 μM), and found that melanin synthesis was
effectively inhibited in a dose-related manner, thereby supporting
the mechanism by which 2e inhibits the tyrosinase of B16 cells.
The inhibitory effect of 2e on melanin contents was stronger
than that of 10 μM kojic acid with 100 nM α-MSH-treated B16 cells
(Fig. 6). When the clogP values of hydantoin analog and the
corresponding stilbene derivative are compared, hydantoin analog
shows a little lower value. Therefore, hydantoin analogs may have
better beneficial effect on manufacturing cosmetics with moistur-
izing ingredients.
4. Conclusion
[9] L. Lemic-Stoicevic, A.H. Nias, A.S. Breathnach, Effect of azelaic acid on melanoma
cells in culture, Exp. Dermatol. 4 (1995) 79–81.
[10] J.R. Ros, J.N. Rodriguez-Lopes, F. Garcia-Canovas, Effect of L-ascorbic acid on the
monophenolase activity of tyrosinase, Biochem. J. 295 (1993) 309–312.
[11] K. Maeda, M. Fukuda, Arbutin: mechanism of its depigmenting action in human
melanocyte culture, J. Pharmacol. Exp. Ther. 276 (1996) 765–769.
[12] G. Battaini, E. Monzani, L. Casella, L. Santagostini, R. Pagliarin, Inhibition of the
catecholase activity of biomimetic dinuclear copper complexes by kojic acid, J. Biol.
Inorg. Chem. 5 (2000) 262–268.
The 5-(substituted benzylidene)hydantoin derivatives showed
anti-tyrosinase inhibition effects. The best results were obtained for
compound 2e, which possessed a hydroxyl group at R2 and a methoxy
group at R3 on the phenyl ring, respectively. This work will enhance
understanding of the mechanism of tyrosinase inhibition and will
[13] K. Ohguchi, T. Tanaka, T. Ito, M. Iinuma, K. Matsumoto, Y. Akao, Y. Nozawa,
Inhibitory effects of resveratrol derivatives from dipterocarpaceae plants on
tyrosinase activity, Biosci. Biotechnol. Biochem. 67 (2003) 1587–1589.
[14] N.H. Shin, S.Y. Ryu, E.J. Choi, S.H. Kang, I.M. Chang, K.R. Min, Y.S. Kin, Oxyresveratrol
as the potent inhibitor on dopa oxidase activity of mushroom tyrosinase, Biochem.
Biophys. Res. Commun. 243 (1998) 801–803.
[15] Y.M. Kim, J. Yun, C.K. Lee, H. Lee, K.R. Min, Y. Kim, Oxyresveratrol and
hydroxystilbene compounds. Inhibitory effect on tyrosinase and mechanism of
action, J. Biol. Chem. 277 (2002) 16340–16344.
[16] K. Ohguchi, T. Tanaka, I. Iliya, T. Ito, M. Iinuma, K. Matsumoto, Y. Akao, Y. Nozawa,
Gnetol as a potent tyrosinase inhibitor from genus Gnetum, Biosci. Biotechnol.
Biochem. 67 (2003) 663–665.
[17] J.S. Roh, J.Y. Han, J.H. Kim, J.K. Hwang, Inhibitory effects of active compounds
isolated from safflower (Carthamus tinctorius L.) seeds for melanogenesis, Biol.
Pharm. Bull. 27 (2004) 1976–1978.
[18] J.K. No, M.S. Kim, Y.J. Kim, S.J. Bae, J.S. Choi, H.Y. Chung, Inhibition of tyrosinase by
protocatechuic aldehyde, Am. J. Chin. Med. 32 (2004) 97–103.
[19] Y. Kim, J. No, J. Lee, M. Kim, H. Chung, Antimelanogenic activity of 3,4-
dihydroxyacetophenone: inhibition of tyrosinase and MITF, Biosci. Biotechnol.
Biochem. 70 (2006) 532–534.
[20] Y.J. Kim, J.K. No, J.H. Lee, H.Y. Chung, 4,4'-Dihydroxybiphenyl as a new potent
tyrosinase inhibitor, Biol. Pharm. Bull. 28 (2005) 323–327.
[21] Y.M. Ha, S.W. Chung, S.H. Song, H.S. Suh, H.Y. Chung, 4-(6-Hydroxy-2-naphthyl)-
1,3-bezendiol: a potent, new tyrosinase inhibitor, Biol. Pharm. Bull. 30 (2007)
1711–1715.
[22] C. Carmi, A. Cavazzoni, V. Zuliani, A. Lodola, F. Bordi, P.V. Plazzi, 5-benzylidene-
hydantoins as new EGFR inhibitors with antiproliferative activity, Bioorg. Med.
Chem. Lett. 16 (2006) 4021–4025.
Fig. 6. The ability of 2e to inhibit melanogenesis in the presence of 100 nM α-MSH in
B16 cells. Melanin levels were measured at 405 nm. Values are the means S.E. of three
experiments. Data are expressed as percentage of control. ###pb0.001 compared to the
⁎
⁎⁎⁎
untreated control, pb0.05 and
pb0.001 compared to the group treated with
100 nM α-MSH, KA, kojic acid.

Y.M. Ha et al. / Biochimica et Biophysica Acta 1810 (2011) 612–619
619
[23] S.S. Hah, H.M. Kim, R.A. Sumbad, P.T. Henderson, Hydantoinderivativeformation from
oxidation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) and incorporation of
14C-labeled 8-oxodG into the DNA of human breast cancer cells, Bioorg. Med. Chem.
Lett. 15 (2005) 3627–3631.
dioxoimidazolidinyl)-N, N-dimethyltryptamine derivatives: novel antagonists for
the vascular 5-HT(1B)-like receptor, J. Med. Chem. 42 (1999) 2504–2526.
[36] M. Jansen, H. Potschka, C. Brandt, W. Loscher, G. Dannhardt, Hydantoin-
substituted 4,6-dichloroindole-2-carboxylic acids as ligands with high affinity
for the glycine binding site of the NMDA receptor, J. Med. Chem. 46 (2003)
64–73.
[24] J. Chinnappa, P.T. Wong, W. Thenmozhiyal, K. Chui, Anticonvulsant activity of
phenylmethylenehydantoins:
a structure–activity relationship study, J. Med.
Chem. 47 (2004) 1527–1535.
[37] K. Last-Barney, W. Davidson, M. Cardozo, L.L. Frye, C.A. Grygon, J.L. Hopkins,
Binding site elucidation of hydantoin-based antagonists of LFA-1 using multidis-
ciplinary technologies: evidence for the allosteric inhibition of a protein–protein
interaction, J. Am. Chem. Soc. 123 (2001) 5643–5650.
[25] K.I. Ahmed, Synthesis of arylidenehydrazono- and glycopyranosylhydrazino-
sulfonylbenzylidene-2,4-imidazolidinediones as potential antiviral and antitu-
moral agents, Carbohydr. Res. 306 (1998) 567–573.
[26] T.R. Rodgers, M.P. LaMontagne, A. Markovac, A.B. Ash, Hydantoins as antitumor
agents, J. Med. Chem. 20 (1977) 591–594.
[27] R.N. Comber, R.C. Reynolds, J.D. Friedrich, R.A. Manguikian, R.W. Buckheit, J.J.W.
Truss, 5,5-disubstituted hydantoins: syntheses and anti-HIV activity, J. Med.
Chem. 35 (1992) 3567–3572.
[28] L. Somsak, L. Kovacs, M. Toth, E. Osz, L. Szilagyi, Z. Gyorgydeak, Synthesis of and a
comparative study on the inhibition of muscle and liver glycogen phosphorylases
by epimeric pairs of D-gluco- and D-xylopyranosylidene-spiro-(thio)hydantoins
and N-(D-glucopyranosyl) amides, J. Med. Chem. 44 (2001) 2843–2848.
[29] C.W. Bazil, T.A. Pedley, Advances in the medical treatment of epilepsy, Annu. Rev.
Med. 49 (1998) 135–162.
[38] C.S. Ananda Kumar, S.B. Benaka Prasad, K. Vinaya, S. Chandrappa, N.R. Thimme-
gowda, S.R. Ranganatha, S. Sanjay, K.S. Rangappa, Synthesis and antiproliferative
activity of substituted diazaspiro hydantoins: a structure–activity relationship study,
Invest. New Drugs 27 (2009) 131–139.
[39] H. Tada, O. Shiho, K. Kuroshima, M. Koyama, K. Tsukamoto, An improved
colorimetric assay for interleukin 2, J. Immunol. Meth. 93 (1986) 157–165.
[40] J.K. No, D.Y. Soung, Y.J. Kim, K.H. Shim, Y.S. Jun, S.H. Rhee, T. Yokozawa, H.Y. Chung,
Inhibition of tyrosinase by green tea components, Life Sci. 65 (1999) PL241–PL246.
[41] D.S. Kim, S.Y. Kim, J.H. Chung, K.H.H. Kim, C. Eun, K.C. Park, Delayed ERK activation
by ceramide reduces melanin synthesis in human melanocytes, Cell. Signal. 14
(2002) 779–785.
[30] M.S. Luer, Fosphenytoin, Neurol. Res. 20 (1998) 178–182.
[42] M.L. Bilodeau, J.D. Greulich, R.L. Hullinger, C. Bertolotto, R. Ballotti, O.M. Andrisani,
BMP-2 stimulates tyrosinase gene expression and melanogenesis in differentiated
melanocytes, Pigmen. Cell Res. 14 (2001) 328–336.
[43] K. Arnold, L. Bordoli, J. Kopp, T. Schwede, The SWISS-MODEL workspace: a web-
based environment for protein structure homology modeling, Bioinformatics 22
(2006) 195–201.
[44] R. Rodriguez, G. Chinea, N. Lopez, T. Pons, G. Vriend, Homology modeling, model
and software evaluation: three related resources, Bioinformatics 14 (1998)
523–528.
[45] D.T. Moustakas, P.T. Lang, S. Pegg, E. Pettersen, I.D. Kuntz, N. Brooijmans, R.C.
Rizzo, Development and validation of a modular, extensible docking program:
DOCK 5, J. Comput. Aided Mol. Des. 20 (2006) 601–619.
[31] M. Matsukura, Y. Daiku, K. Ueda, S. Tanaka, T. Igarashi, N. Minami, Synthesis and
antiarrhythmic activity of 2,2-dialkyl-1'-(N-substituted aminoalkyl)-spiro-(chro-
man-4,4'-imidazolidine)-2',5'-diones, Chem. Pharm. Bull. 40 (1992) 1823–1827.
[32] J. Knabe, J. Baldauf, A. Ahlhelm, Racemates and enantiomers of basic, substituted
5-phenylhydantoins, synthesis and anti-arrhythmic action, Pharmazie 52 (1997)
912–919.
[33] J.C. Menendez, M.P. Diaz, C. Bellver, M.M. Sollhuber, ChemInform abstract:
synthesis, anticonvulsant and antihypertensive activity of diastereomeric 9,10-
dimethoxy-1,3,4,6,7,11b-hexahydrospiro(benzo(a) quinolizin-2,4′-imidazoli-
dine)-2′,5′-diones, Eur. J. Med. Chem. 27 (1992) 61–66.
[34] G.P. Moloney, A.D. Robertson, G.R. Martin, S. MacLennan, N. Mathews, S.
Dosworth, A novel series of 2,5-substituted tryptamine derivatives as vascular
5HT1B/1D receptor antagonists, J. Med. Chem. 40 (1997) 2347–2362.
[35] G.P. Moloney, G.R. Martin, N. Mathews, A. Milne, H. Hobbs, S. Dosworth, Synthesis
and serotonergic activity of substituted 2, N-benzylcarboxamido-5-(2-ethyl-1-
[46] V.J. Hearing, K. Tsukamoto, Enzymatic control of pigmentation in mammals,
FASEB J. 5 (1991) 2902–2909.
[47] A. Andrawis, V. Kahn, Effect of methimazole on the activity of mushroom tyrosinase,
Biochem. J. 235 (1986) 91–96.