Design, synthesis and biological evaluation of 2-(substituted phenyl)thiazolidine-4-carboxylic acid derivatives as novel tyrosinase inhibitors

Article abstract of DOI:10.1016/j.biochi.2011.09.002

Herein we describe the design, synthesis and biological activities of 2-(substituted phenyl)thiazolidine-4-carboxylic acid derivatives as novel tyrosinase inhibitors. The target compounds 2a-2j were designed and synthesized from the structural characteristics of N-phenylthiourea, tyrosinase inhibitor and tyrosine, and l-DOPA, the natural substrates of tyrosinase. Among them, (2R/S,4R)-2-(2,4-dimethoxyphenyl)thiazolidine-4-carboxylic acid (2g) caused the greatest inhibition 66.47% at 20 μM of l-DOPA oxidase activity of mushroom tyrosinase. Kinetic analysis of tyrosinase inhibition revealed that 2g is a competitive inhibitor. We predicted the tertiary structure of tyrosinase, and simulated the docking of mushroom tyrosinase with 2g. These results suggest that the binding affinity of 2g with tyrosinase is high. Also, 2g effectively inhibited tyrosinase activity and reduced melanin levels in B16 cells treated with α-MSH. These data strongly suggest that 2g can suppress the production of melanin via the inhibition of tyrosinase activity.

Full text of DOI:10.1016/j.biochi.2011.09.002

Biochimie 94 (2012) 533e540
Contents lists available at SciVerse ScienceDirect
Biochimie
journal homepage: www.elsevier.com/locate/biochi
Research paper
Design, synthesis and biological evaluation of 2-(substituted phenyl)thiazolidine-
4-carboxylic acid derivatives as novel tyrosinase inhibitors
,
Young Mi Haa, Yun Jung Parkb, Ji Yeon Leeb, Daeui Parka, Yeon Ja Choia, Eun Kyeong Leea c, Ji Min Kima,
**
,
a,
*
Jin-Ah Kimb, Ji Young Parkb, Hye Jin Leeb, Hyung Ryong Moonb , 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
c Research Center, Dongnam Institute of Radiological and Medical Sciences, Busan 619-953, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein we describe the design, synthesis and biological activities of 2-(substituted phenyl)thiazolidine-
4-carboxylic acid derivatives as novel tyrosinase inhibitors. The target compounds 2ae2j were designed
and synthesized from the structural characteristics of N-phenylthiourea, tyrosinase inhibitor and tyro-
Received 17 December 2010
Accepted 1 September 2011
Available online 17 September 2011
sine, and
L-DOPA, the natural substrates of tyrosinase. Among them, (2R/S,4R)-2-(2,4-dimethoxyphenyl)
thiazolidine-4-carboxylic acid (2g) caused the greatest inhibition 66.47% at 20
m
M of -DOPA oxidase
L
Keywords:
activity of mushroom tyrosinase. Kinetic analysis of tyrosinase inhibition revealed that 2g is a compet-
itive inhibitor. We predicted the tertiary structure of tyrosinase, and simulated the docking of mushroom
tyrosinase with 2g. These results suggest that the binding affinity of 2g with tyrosinase is high. Also, 2g
effectively inhibited tyrosinase activity and reduced melanin levels in B16 cells treated with
These data strongly suggest that 2g can suppress the production of melanin via the inhibition of
Melanin biosynthesis
Tyrosinase inhibitor
Hyperpigmentation
Docking analysis
2-(Substituted phenyl)thiazolidine-4-
carboxylic acid derivatives
a-MSH.
tyrosinase activity.
Published by Elsevier Masson SAS.
1. Introduction
tyrosinase, a copper-containing protein (Fig. 1). The first two steps
in the biosynthesis of melanin are catalyzed by tyrosinase, and both
Melanin is a pigment that is ubiquitous in nature, being found in
all organisms. Melanin-like pigments are synthesized by melano-
cytes within specialized organelles called melanosomes. Ultraviolet
(UV) radiation, chronic inflammation, and the release of abnormal
the substrate and the product of tyrosinase have in common a free
carboxylic acid (COOH) and free amine (NH) functionality. On the
other hand, N-phenylthiourea (Fig. 2) has been reported to effi-
ciently inhibit a catechol oxidase enzyme that belongs to the type-3
copper proteins group including tyrosinase. The sulfur atom of
N-phenylthiourea binds to both copper ions in the active site of the
enzyme, resulting in inhibition of tyrosinase activity.
On the basis of these findings, we thought that it would be
interesting to design and synthesize 2-(substituted phenyl)thia-
zolidine-4-carboxylic acid derivatives with an amine (NH), a free
carboxylic acid, and a divalent sulfur atom in a single structure in
order to search for potent skin-whitening agents. The scaffold of
2-(substituted phenyl)thiazolidine-4-carboxylic acid (Fig. 2) is
perfect in view of its structural similarity to the substrate (tyrosine
and DOPA) and the product (DOPA and DOPA quinone) of tyrosinase
and having a sulfur atom as in N-phenylthiourea.
a
-melanocyte stimulating hormone (a-MSH) are well-known trig-
gering factors for hyperpigmentation and inflammatory pigmen-
tation [1,2]. The modulation of melanogenesis is a widely used
strategy to treat abnormal skin pigmentation through medication
and cosmetics [3]. This is not surprising because melanin is prin-
cipally responsible for skin color and plays an important role in the
prevention of skin injury under normal physiological conditions.
On the other hand, abnormal pigmentations such as freckles, age
spots, and melasma can be serious skin problems. Melanogenesis is
greatly increased by tyrosinase, which catalyzes the hydroxylation
of tyrosine to 3,4-dihydroxyphenylalanine (L-DOPA) and L-DOPA to
dopaquinone [4]. Conversion of tyrosine to melanin requires
Over the last few decades, various tyrosinase inhibitors
including kojic acid [5], arbutin [6], and polyphenolic compounds
[7e9] have been studied. Additionally, recent reports show that
tetrahydroxystilbene compounds, which are resveratrol analogs,
potently inhibit tyrosinase [10] and have potent inhibitory effects
against the activities of cyclooxygenase [11,12], rat liver
* 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).
0300-9084/$ e see front matter Published by Elsevier Masson SAS.
doi:10.1016/j.biochi.2011.09.002

534
Y.M. Ha et al. / Biochimie 94 (2012) 533e540
date, not been reported. Our work was designed to characterize the
HO
HO
O
O
effects of 2g on mushroom tyrosinase activity and on tyrosinase
activityand melanin levels in B16 melanoma cells (B16 cells). We also
predicted the tertiary structure of tyrosinase and simulated its
docking with 2g.
Tyrosinase
HO
OH
OH
NH2
NH2
DOPA
Tyrosine
2. Materials and methods
Tyrosinase
O
2.1. Experimental
O
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 deuterated 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), and dd (doublet of
doublets). All the reactions described below were done under an
argon or nitrogen atmosphere and monitored by TLC. All anhydrous
solvents were distilled over CaH2 or Na/benzophenone prior to use.
Melanin
O
OH
NH2
DOPAquinone
Fig. 1. Biosynthetic pathway for melanin production.
mitochondrial ATPase [13], and
L
-DOPA oxidase [11,14]. Oxy-
resveratrol has a potent inhibitory effect on tyrosinase [15].
Recently, we reported the synthesis of a new family of hydroxy
substituted phenyl naphthalenes as analogs of resveratrol [16], and
their potent inhibitory activities against tyrosinase and melano-
genesis [17e20]. We also found several potent tyrosinase inhibitors
in natural materials that have potent inhibitory effects on mela-
nogenesis [16,21,22] and microphthalmia-associated transcription
factor [23].
In the current study, we report the synthesis of various
2-(substituted phenyl)thiazolidine-4-carboxylic acid derivatives
and present an evaluation of their ability too inhibit mushroom
activity. Ten 2-(substituted phenyl)thiazolidine-4-carboxylic acid
2.1.2. General procedure for the synthesis of 2-(substituted phenyl)
thiazolidine-4-carboxylic acid analogs (2ae2j)
A suspension of substituted benzaldehydes (1.53e2.46 mmol)
and L-cysteine (1.0 or 1.5 eq.) in EtOH (5e30 mL) was refluxed for
1.5e48 h. The precipitates generated were filtered and the filter
cakes were washed with EtOH, methylene chloride, water, ethyl
acetate and/or MeOH, depending on the property of the remaining
starting materials, to give the title products (yields: 9.9e78.6%).
2.1.2.1. (2R/S,4R)-2-(4-hydroxyphenyl)thiazolidine-4-carboxylic acid
derivatives were obtained by reaction of L-cysteine with various
(2a). White solid; reaction time, 13 h; yield, 50.0%; melting point,
161.8e164.4 ꢀC; 1H NMR (400 MHz, DMSO-d6)
d 9.48 (br s, 1 H,
substituted benzaldehydes (1ae1j), as described in Scheme 1.
Synthetic reactions for the target compounds proceeded smoothly
under reflux conditions in ethanol giving 2ae2j in 9.9%e78.6% yield
(Table 1). The structures of the synthesized compounds were
determined by 1H and 13C NMR and mass spectroscopic analyses.
All of the 2-(substituted phenyl)thiazolidine-4-carboxylic acid
derivatives 2ae2j were present as an inseparable diastereomeric
mixture. According to 1H NMR experiments, the ratios of the dia-
stereomeric mixtures were almost 1:1.
40-OH), 9.39 (br s, 1 H, 40-OH), 7.27 (d, 2 H, J ¼ 8.8 Hz, 20-H, 60-H),
7.20 (d, 2 H, J ¼ 8.4 Hz, 20-H, 60-H), 6.70 (d, 2 H, J ¼ 8.4 Hz, 30-H, 50-
H), 6.66 (d, 2 H, J ¼ 8.4 Hz, 30-H, 50-H), 5.49 (s, 1 H, benzylic H), 5.36
(s, 1 H, benzylic H), 4.21 (dd, 1 H, J ¼ 4.4, 7.6 Hz, 4-H), 3.79 (dd, 1 H,
J ¼ 7.6, 8.4 Hz, 4-H), 3.30 (dd, 1 H, J ¼ 7.2, 10.0 Hz, 5-Ha), 3.23 (dd, 1
H, J ¼ 7.2, 10.4 Hz, 5-Ha), 3.11 (dd, 1 H, J ¼ 4.4, 10.4 Hz, 5-Hb), 3.00 (t,
1 H, J ¼ 8.4, 10.0 Hz, 5-Hb); 13C NMR (100 MHz, DMSO-d6)
d 173.8
(COOH), 173.0 (COOH), 158.1 (C40), 157.6 (C40), 131.4 (C10), 129.5
(C10), 129.2 (C20, C40), 129.0 (C20, C40), 115.8 (C30, C50), 115.6 (C30,
C50), 72.5 (C2), 72.0 (C2), 65.9 (C4), 65.4 (C4), 39.2 (C5), 38.5 (C5);
LRMS(ESI) m/z 224 (MeH)ꢁ.
Compounds 2d, 2g, and 2i significantly inhibited tyrosinase
activity. Among ten 2-(substituted phenyl)thiazolidine-4-carboxylic
acid derivatives synthesized, 2g exhibited the greatest inhibition of
the
L-DOPA oxidase activity of mushroom tyrosinase: 66.47% inhibi-
tion at a concentration of 20
mM. Compound 2g’s analog with the
2.1.2.2. (2R/S,4R)-2-(2-hydroxyphenyl)thiazolidine-4-carboxylic acid
same substituents at positions 2 and 4 was reported to have a cyto-
protective action even at an anti-secretory dose, and had significant
anti-ulcer activity, comparable to that of cimetidine [24]. However,
effect of compound 2g on tyrosinase or skin melanogenesis has, to
(2b). White solid; reaction time, 6 h; yield, 75.0%; melting point,
173.2e175.1 ꢀC; 1H NMR (500 MHz, DMSO-d6)
d 9.90 (br s, 2 H,
2 ꢂ 20-OH), 7.33 (dd,1 H, J ¼ 1.0, 8.0 Hz, 60-H), 7.28 (d, 1 H, J ¼ 7.5 Hz,
60-H), 7.12 (td, 1 H, J ¼ 1.5, 8.0 Hz, 40-H), 7.05 (td, 1 H, J ¼ 1.5, 8.0 Hz,
R1
H
S
O
S
R2
OH
HN
R
OH
N
H
NH2
R3
NH2
O
R4
N-Phenylthiourea
target compounds
Structure of substrate and
product of tyrosinase
Fig. 2. The rationale for the design of the 2-(substituted phenyl)thiazolidine-4-carboxylic acid derivatives.

Y.M. Ha et al. / Biochimie 94 (2012) 533e540
535
R1
O
R1
R4
H
S
O
R2
R3
R2
R3
H
OH
HS
OH
HN
+
NH2
-Cysteine
-Cysteine HCl monohydrate
O
R4
1a - 1j
L
or
L
2a - 2j
Scheme 1. Synthesis of the target compounds, 2-(substituted phenyl)thiazolidine-4-carboxylic acid analogs (2ae2j); reagents and conditions: EtOH, reflux, 1.5e48 h, 9.9e78.6%.
40-H), 6.81e6.73 (m, 4 H, 2 ꢂ 30-H, 2 ꢂ 50-H), 5.83 (s, 1 H, benzylic
H), 5.64 (s, 1 H, benzylic H), 4.20 (dd, 1 H, J ¼ 5.5, 6.5 Hz, 4-H), 3.82
(dd, 1 H, J ¼ 7.0, 9.0 Hz, 4-H), 3.33 (dd, 1 H, J ¼ 7.0, 10.0 Hz, 5-Ha),
3.19 (dd, 1 H, J ¼ 7.0, 10.5 Hz, 5-Ha), 3.01 (dd, 1 H, J ¼ 5.0, 10.0 Hz,
5-Hb), 2.96 (t, 1 H, J ¼ 10.0 Hz, 5-Hb); 13C NMR (100 MHz, DMSO-d6)
3.75 (s, 3 H, 40-OCH3), 3.73 (s, 3 H, 40-OCH3), 3.33 (dd, 1 H, J ¼ 7.5,
9.5 Hz, 5-Ha), 3.25 (dd, 1 H, J ¼ 7.5, 10.0 Hz, 5-Ha), 3.10 (dd, 1 H,
J ¼ 4.0, 10.0 Hz, 5-Hb), 3.02 (t, 1 H, J ¼ 9.5 Hz, 5-Hb); 13C NMR
(100 MHz, DMSO-d6)
d
173.8 (COOH), 173.1 (COOH), 148.3 (C40),
147.8 (C40), 147.1 (C30), 146.9 (C30), 134.0 (C10), 131.8 (C10), 118.8
(C60), 118.4 (C60), 115.0 (C20), 115.0 (C20), 112.6 (C50), 112.4 (C50), 72.5
(C2), 71.8 (C2), 65.9 (C4), 65.5 (C4), 56.3(40-OCH3), 56.3 (40-OCH3),
39.2 (C5), 38.5 (C5); LRMS(ES) m/z 254 (MeH)ꢁ.
d
173.6 (COOH), 173.1 (COOH), 155.8 (C20), 155.3 (C20), 129.7 (C40),
128.8 (C60), 128.5 (C40), 126.7 (C60), 120.1 (C10), 119.7 (C50), 119.4
(C50), 117.9 (C10), 116.3 (C30), 115.7 (C30), 68.3 (C2), 66.3 (C2), 65.9
(C4), 65.5 (C4), 38.9 (C5), 37.7 (C5); LRMS(ES) m/z 224 (MeH)ꢁ.
2.1.2.5. (2R/S,4R)-2-(4-hydroxy-3-methoxyphenyl)thiazolidine-4-
2.1.2.3. (2R/S,4R)-2-(4-methoxyphenyl)thiazolidine-4-carboxylic
carboxylic acid (2e). White solid; reaction time, 48 h; yield, 42.7%;
acid (2c). White solid; reaction time, 48 h; yield, 40.5%; melting
melting point, 166.6e168.4 ꢀC; 1H NMR (500 MHz, DMSO-d6)
d 9.05
point, 157.6e158.2 ꢀC; 1H NMR (500 MHz, DMSO-d6)
d
7.43 (d, 2 H,
(br s, 1 H, 40-OH), 8.97 (br s, 1 H, 40-OH), 7.10 (d, 1 H, J ¼ 1.5 Hz, 20-H),
7.01 (d, 1 H, J ¼ 2.0 Hz, 20-H), 6.88 (dd, 1 H, J ¼ 2.0, 8.0 Hz, 60-H), 6.83
(dd, 1 H, J ¼ 1.5, 8.0 Hz, 60-H), 6.72 (d, 1 H, J ¼ 8.0 Hz, 50-H), 6.69 (d, 1
H, J ¼ 8.0 Hz, 50-H), 5.52 (s, 1 H, benzylic H), 5.39 (s, 1 H, benzylic H),
4.27 (dd, 1 H, J ¼ 4.0, 7.5 Hz, 4-H), 3.83 (dd, 1 H, J ¼ 7.5, 8.5 Hz, 4-H),
3.76 (s, 3 H, 30-OCH3), 3.75 (s, 3 H, 30-OCH3), 3.32 (dd, 1 H, J ¼ 6.5,
9.5 Hz, 5-Ha), 3.27 (dd, 1 H, J ¼ 7.5, 10.5 Hz, 5-Ha), 3.15 (dd, 1 H,
J ¼ 3.5, 10.5 Hz, 5-Hb), 3.05 (dd, 1 H, J ¼ 9.5 Hz, 5-Hb); 13C NMR
J ¼ 8.5 Hz, 20-H, 60-H), 7.35 (d, 2 H, J ¼ 8.5 Hz, 20-H, 60-H), 6.91 (d, 2
H, J ¼ 8.5 Hz, 30-H, 50-H), 6.87 (d, 2 H, J ¼ 9.0 Hz, 30-H, 50-H), 5.58 (s,1
H, benzylic H), 5.44 (s, 1 H, benzylic H), 4.23 (dd, 1 H, J ¼ 4.0, 7.5 Hz,
4-H), 3.85 (dd, 1 H, J ¼ 2.0, 8.5 Hz, 4-H), 3.74 (s, 3 H, CH3), 3.73 (s, 3
H, CH3), 3.34 (dd, 1 H, J ¼ 7.0, 10.0 Hz, 5-Ha), 3.27 (dd, 1 H, J ¼ 7.0,
10.0 Hz, 5-Ha), 3.14 (dd, 1 H, J ¼ 4.0, 10.0 Hz, 5-Hb), 3.05 (dd, 1 H,
J ¼ 8.5, 10.0 Hz, 5-Hb); 13C NMR (100 MHz, DMSO-d6)
d 173.8
(COOH), 172.9 (COOH), 159.9 (C40), 159.4 (C40), 133.4 (C10), 131.4
(C10), 129.3 (C20, C60), 129.0 (C20, C60), 114.5 (C30, C50), 114.2 (C30,
C50), 72.2 (C2), 71.6 (C2), 66.0 (C4), 65.5 (C4), 55.8 (40-OCH3), 55.8
(40-OCH3), 39.2 (C5), 38.5 (C5); LRMS(ES) m/z 238 (MeH)ꢁ.
(100 MHz, DMSO-d6) d
173.8 (COOH), 172.9 (COOH), 148.2 (C30),
148.0 (C30), 147.3 (C40), 146.8 (C40), 132.0 (C10), 130.2 (C10), 120.6
(C60), 120.3 (C60), 115.8 (C50), 115.7 (C50), 112.2 (C20), 112.0 (C20), 72.8
(C2), 72.2 (C2), 66.1 (C4), 65.4 (C4), 56.4 (30-OCH3), 56.3 (30-OCH3),
39.1 (C5), 38.4 (C5); LRMS(ES) m/z 254 (MeH)ꢁ.
2.1.2.4. (2R/S,4R)-2-(3-hydroxy-4-methoxyphenyl)thiazolidine-4-
carboxylic acid (2d). White solid; reaction time, 8 h; yield, 78.6%;
2.1.2.6. (2R/S,4R)-2-(3-ethoxy-4-hydroxyphenyl)thiazolidine-4-
melting point, 146.9e149.8 ꢀC; 1H NMR (500 MHz, DMSO-d6)
d
9.05
carboxylic acid (2f). White solid; reaction time, 3 h; yield, 77.7%;
(br s, 1 H, 30-OH), 8.96 (br s,1 H, 30-OH), 6.92 (s, 1 H, 20-H), 6.87 (m, 3
H, 20-H, 50-H, 60-H), 6.83 (d, 1 H, J ¼ 8.0 Hz, 60-H), 6.80 (d, 1 H,
J ¼ 8.0 Hz, 50-H), 5.51 (s, 1 H, benzylic H), 5.36 (s, 1 H, benzylic H),
4.20 (dd, 1 H, J ¼ 4.5, 6.5 Hz, 4-H), 3.83 (dd, 1 H, J ¼ 7.5, 8.5 Hz, 4-H),
melting point, 174.3e175.9 ꢀC; 1H NMR (400 MHz, DMSO-d6)
d 8.90
(s, 2 H, 2 ꢂ 40-OH), 7.06 (d, 1 H, J ¼ 2.0 Hz, 20-H), 6.97 (d, 1 H,
J ¼ 2.0 Hz, 20-H), 6.84 (dd, 1 H, J ¼ 2.0, 8.0 Hz, 60-H), 6.79 (dd, 1 H,
J ¼ 2.4, 8.4 Hz, 60-H), 6.70 (d, 1 H, J ¼ 8.0 Hz, 50-H), 6.67 (d, 1 H,
J ¼ 8.0 Hz, 50-H), 5.47 (s, 1 H, benzylic H), 5.34 (s, 1 H, benzylic H),
4.23 (dd, 1 H, J ¼ 3.6, 7.2 Hz, 4-H), 3.99e3.94 (m, 4 H, 2 ꢂ CH2CH3),
3.79 (dd, 1 H, J ¼ 7.2, 8.8 Hz, 4-H), 3.32 (dd, 1 H, J ¼ 7.2, 10.4 Hz,
5-Ha), 3.23 (dd, 1 H, J ¼ 7.2, 10.0 Hz, 5-Ha), 3.12 (dd, 1 H, J ¼ 3.6,
10.0 Hz, 5-Hb), 3.01 (t, 1 H, J ¼ 9.6 Hz, 5-Hb), 1.28 (t, 3 H, J ¼ 7.2 Hz,
CH2CH3), 1.28 (t, 3 H, J ¼ 6.8 Hz, CH2CH3); 13C NMR (100 MHz,
Table 1
Substitution pattern of the synthesized 2-(substituted phenyl)thiazolidine-4-carboxylic
acid analogs 2ae2j.
R1
H
S
DMSO-d6)
d
173.8 (COOH), 172.9 (COOH), 147.6 (C30), 147.3 (C30),
R2
147.1 (C40), 147.1 (C40), 131.9 (C10), 130.1 (C10), 120.7 (C60), 120.4
(C60), 115.9 (C50), 115.7 (C50), 113.5 (C20), 113.3 (C20), 72.8 (C2), 72.2
(C2), 66.1 (C4), 65.4 (C4), 64.6 (CH2CH3), 64.6 (CH2CH3), 39.0 (C5),
38.4 (C5),15.4 (CH2CH3), 15.4 (CH2CH3); LRMS(ES) m/z 268 (MeH)ꢁ.
OH
HN
R3
O
R4
R2
R1
R3
R4
Yield (%)
Reaction
2.1.2.7. (2R/S,4R)-2-(2,4-dimethoxyphenyl)thiazolidine-4-carboxylic
Compound
time (hour)
acid (2g). White solid; reaction time, 1.5 h; yield, 34.7%; melting
2a
2b
2c
2d
2e
2f
2g
2h
2i
H
OH
H
H
H
H
OMe
H
H
H
H
H
H
OH
OMe
OEt
H
OMe
OMe
OMe
OH
H
OMe
OMe
OH
H
H
H
H
H
H
H
H
50.0
75.0
40.5
78.6
42.7
77.7
34.7
57.0
69.7
9.9
13
6
48
8
48
3
1.5
10
4
point, 137.7e139.2 ꢀC; 1H NMR (500 MHz, DMSO-d6)
d 7.39 (d, 1 H,
J ¼ 8.5 Hz, 60-H), 7.29 (d, 1 H, J ¼ 8.0 Hz, 60-H), 6.56 (d, 1 H, J ¼ 1.5 Hz,
30-H), 6.53 (dd, 1 H, J ¼ 1.5, 8.5 Hz, 50-H), 6.52 (d, 1 H, J ¼ 1.5 Hz, 30-
H), 6.48 (dd, 1 H, J ¼ 1.5, 8.5 Hz, 50-H), 5.78 (s, 1 H, benzylic H), 5.62
(s, 1 H, benzylic H), 4.18 (t, 1 H, J ¼ 6.0 Hz, 4-H), 3.79 (dd, 1 H, J ¼ 7.5,
8.5 Hz, 4-H), 3.78 (s, 3 H, OCH3), 3.77 (s, 3 H, OCH3), 3.75 (s, 3 H,
OCH3), 3.74 (s, 3 H, OCH3), 3.31 (dd, 1 H, J ¼ 7.0, 10.0 Hz, 5-Ha), 3.17
(dd, 1 H, J ¼ 7.0, 10.0 Hz, 5-Ha), 2.99 (dd, 1 H, J ¼ 5.0, 10.0 Hz, 5-Hb),
OH
OMe
OMe
OH
OMe
OMe
2j
OMe
5
2.95 (t, 1 H, J ¼ 9.0 Hz, 5-Hb); 13C NMR (100 MHz, DMSO-d6)
d 173.7

536
Y.M. Ha et al. / Biochimie 94 (2012) 533e540
(COOH), 173.1 (COOH), 161.1 (C40), 160.5 (C40), 158.4 (C20), 157.9
(C20), 129.0 (C60), 127.1 (C60), 122.6 (C10), 119.3 (C10), 105.7 (C50),
105.2 (C50), 99.3 (C30), 99.0 (C30), 67.2 (C2), 66.0 (C2), 65.7 (C4), 65.6
(C4), 56.3 (OCH3), 56.2 (OCH3), 55.9 (OCH3), 55.9 (OCH3), 39.0 (C5),
37.9 (C5); LRMS(ES) m/z 268 (MeH)ꢁ.
USA) with 10% fetal bovine serum (FBS; Gibco) and penicillin/
streptomycin (100 IU$50 m
gꢁ1 mlꢁ1) 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.
2.3.2. Cell viability
2.1.2.8. (2R/S,4R)-2-(3,4-dimethoxyphenyl)thiazolidine-4-carboxylic
Cell survival was quantified by a colorimetric assay that used
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
and that measured mitochondrial activity in viable cells. This method
isbasedon the conversion of MTT (Sigma) to MTT-formazan crystals by
a mitochondrial enzyme, as previously described [25]. Briefly, cells
seeded at a density of 3 ꢂ 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
acid (2h). White solid; reaction time, 10 h; yield, 57.0%; melting
point, 173.6e175.4 ꢀC; 1H NMR (500 MHz, DMSO-d6)
d 7.15 (d, 1 H,
J ¼ 2.0 Hz, 20-H), 7.04 (d, 1 H, J ¼ 2.0 Hz, 20-H), 7.01 (dd, 1 H, J ¼ 2.0,
8.0 Hz, 60-H), 6.96 (dd,1 H, J ¼ 2.0, 8.0 Hz, 60-H), 6.90 (d,1 H, J ¼ 8.0 Hz,
50-H), 6.87(d,1 H, J ¼ 8.5 Hz, 50-H), 5.57 (s,1 H, benzylicH), 5.43 (s,1 H,
benzylic H), 4.28 (dd, 1 H, J ¼ 4.0, 7.5 Hz, 4-H), 3.85 (dd, 1 H, J ¼ 7.0,
9.0 Hz, 4-H), 3.75 (s, 3 H, OCH3), 3.74 (s, 3 H, OCH3), 3.74 (s, 3 H, OCH3),
3.72 (s, 3 H, OCH3), 3.33 (dd, 1 H, J ¼ 7.0, 10.0 Hz, 5-Ha), 3.28 (dd, 1 H,
J ¼ 7.5, 10.5 Hz, 5-Ha), 3.16 (dd,1 H, J ¼ 4.0,10.5 Hz, 5-Hb), 3.06 (t,1 H,
at 5 mg mlꢁ1 in phosphate-buffered saline (PBS). Aliquots of 500
ml of
MTT stock solution were added to each well, and the plate was incu-
J ¼ 9.5 Hz, 5-Hb); 13C NMR (100 MHz, DMSO-d6)
d
173.8 (COOH),172.9
bated at 37 ꢀC for 4 h in a humidified 5% CO2 incubator. After 4 h, the
(COOH), 149.4 (C30), 149.3 (C30), 149.2 (C40), 149.0 (C40), 133.6 (C10),
131.8 (C10), 120.3 (C60), 119.9 (C60), 112.1 (C20), 112.0 (C20), 111.7 (C50),
111.5 (C50), 72.6 (C2), 72.0 (C2), 66.2 (C4), 65.5 (C4), 56.2 (3 ꢂ OCH3),
56.1 (OCH3), 39.0 (C5), 38.5 (C5); LRMS(ES) m/z 268 (MeH)ꢁ.
mediumwas removed. To each well, 500 ml of EtOH-DMSO (solution of
a 1:1 mixture) was added to dissolve the formazan. After 10 min, the
optical density of each well was measured spectrophotometrically
using a 560 nm filter. The results from three experiments are shown.
2.1.2.9. (2R/S,4R)-2-(4-hydroxy-3,5-dimethoxyphenyl)thiazolidine-4-
2.3.3. Assay to measure inhibition of mushroom tyrosinase activity
Mushroomwas used as the sourceof tyrosine for the entirestudy.
Tyrosinase activity was determined as described previously with
carboxylic acid (2i). White solid; reaction time, 4 h; yield, 69.7%;
melting point, 139.6e141.0 ꢀC; 1H NMR (500 MHz, DMSO-d6)
d 8.42
(br s, 2 H, 2 ꢂ 40-OH), 6.79 (s, 2 H, 20-H, 60-H), 6.72 (s, 2 H, 20-H, 60-H),
5.52 (s,1 H, benzylic H), 5.39 (s,1 H, benzylic H), 4.30 (dd,1 H, J ¼ 3.5,
7.5 Hz, 4-H), 3.83 (t, 1 H, J ¼ 8.0 Hz, 4-H), 3.75 (s, 6 H, 2 ꢂ OCH3), 3.74
(s, 6 H, 2 ꢂ OCH3), 3.32 (dd, 1 H, J ¼ 7.0, 9.5 Hz, 5-Ha), 3.28 (dd, 1 H,
J ¼ 7.5, 10.5 Hz, 5-Ha), 3.17 (dd,1 H, J ¼ 3.5,10.5 Hz, 5-Hb), 3.06 (t,1 H,
minor modification [26]. Briefly, 20
mushroom tyrosinase (1000 units) was added to a 96-well micro-
plate (Nunc, Denmark), in a 200 l assay mixture containing 1 mM
-tyrosine and 50 mM phosphate buffer (pH 6.5). The assay mixture
ml of an aqueous solution of
m
L
was incubated at 25 ꢀC for 30 min. Following incubation, the amount
of dopachrome produced in the reaction mixture was determined
spectrophotometricallyat 492 nm (OD492) using a microplate reader
(Hewlett Packard, Palo Alto, CA, USA). We determined the inhibition
J ¼ 9.5 Hz, 5-Hb); 13C NMR (100 MHz, DMSO-d6)
d 173.9 (COOH),
172.9 (COOH),148.4 (C30, C50),148.3 (C30, C50),136.2 (C40),135.7 (C40),
131.0 (C10),129.3 (C10),105.7 (C20, C60),105.3 (C20, C60), 73.1 (C2), 72.5
(C2), 66.2 (C4), 65.5 (C4), 56.7 (30-OCH3, 50-OCH3), 56.7 (30-OCH3,
50-OCH3), 39.0 (C5), 38.4 (C5); LRMS(ES) m/z 284 (MeH)ꢁ.
at 20 mM for the synthesized all compounds. In additionally, we
determined the IC50 for kojic acid, resveratrol, and 2g, that is, the
concentration of a compound that causes 50% inhibition. Experi-
mentally, the IC50 value is derived from the X-axis on an inhibitor
concentration versus product formation curve and is determined
from the alignment of the doseeresponse curve on the dependent
Y-axis. In the present study, dose-dependent inhibition experiments
were performed in triplicate to determine the IC50 of test
compounds. Based on the average percentage inhibition for three
doses, log-linear curves and their equations were determined. The
average results from three experiments are shown.
2.1.2.10. Q(2R/S,4R)-2-(3,4,5-trimethoxyphenyl)thiazolidine-4-
carboxylic acid (2j). White solid; reaction time, 5 h; yield, 9.9%;
melting point, 197.0e199.7 ꢀC; 1H NMR (500 MHz, DMSO-d6)
d 6.85
(s, 2 H, 20-H, 60-H), 6.76 (s, 2 H, 20-H, 60-H), 5.57 (s, 1 H, benzylic H),
5.42 (s,1 H, benzylic H), 4.28 (dd,1 H, J ¼ 4.0, 7.0 Hz, 4-H), 3.85 (dd,1
H, J ¼ 6.5, 8.5 Hz, 4-H), 3.77 (s, 6 H, 2 ꢂ OCH3), 3.76 (s, 6 H,
2 ꢂ OCH3), 3.64 (s, 3 H, 40-OCH3), 3.63 (s, 3 H, 40-OCH3), 3.32 (dd, 1
H, J ¼ 6.5, 9.5 Hz, 5-Ha), 3.29 (dd,1 H, J ¼ 7.0, 10.0 Hz, 5-Ha), 3.15 (dd,
1 H, J ¼ 4.0, 10.5 Hz, 5-Hb), 3.07 (t, 1 H, J ¼ 9.5 Hz, 5-Hb); 13C NMR
(100 MHz, DMSO-d6)
d
173.8 (COOH), 172.8 (COOH), 153.4 (C30, C50),
2.3.4. Kinetic analysis of the inhibition of tyrosinase
153.3 (C30, C50), 137.9 (C10), 137.5 (C10), 137.1 (C40), 135.3 (C40), 105.5
(C20, C60), 105.0 (C20, C60), 72.8 (C2), 72.1 (C2), 66.4 (C4), 65.5 (C4),
60.6 (40-OCH3), 60.6 (40-OCH3), 56.6 (2 ꢂ OCH3), 56.5 (2 ꢂ OCH3),
38.2 (C5), 38.5 (C5); LRMS(ES) m/z 298 (MeH)ꢁ.
Varying concentrations (0.25e2 mM) of
substrate, 20 l of an 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 M of 2g) were added to
a 96-well plate in a total volume of 200 l. Using a microplate
L-tyrosine, a tyrosinase
m
m
m
2.2. Materials
reader, the initial rate of dopachrome formation from the reaction
mixture was determined by the increase in absorbance at a wave-
Mushroom tyrosinase,
-alanine (S)-2-amino-3-(4-hydroxyphenyl) propionic acid, kojic
acid [5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one], resveratrol
(3,5,40-trihydroxy-trans-stilbene), and
-MSH (alpha-melanocyte
L
-tyrosine [3-(4-hydroxyphenyl)]-
length of 492 nm per minute (
constant (Km), maximal velocity (Vmax), and the inhibitor constant
(Ki) of the tyrosinase activity were determined by Line-
weavereBurk plot at varying -tyrosine concentrations [26]. Reac-
D
OD492 minꢁ1). The Michaelis
L
a
a
L
stimulating hormone) were purchased from Sigma Chemical Co.
(St. Louis, MO, USA).
tion kinetics required modification of the MichaeliseMenten
equation due to competitive inhibition by the compounds together
with substrate inhibition by
experiments are shown.
L-tyrosine. The results from three
2.3. Methods
2.3.1. Cell culture
2.3.5. Assay of murine tyrosinase activity
B16 cells (obtained fromthe Korean Cell Line Bank) werecultured
in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Carlsbad, CA,
Tyrosinaseactivitywasestimatedby measuringthe rate of L-DOPA
oxidation [27]. Cells were plated in 24-well dishes at a density of

Y.M. Ha et al. / Biochimie 94 (2012) 533e540
537
5 ꢂ104 cells mlꢁ1. B16 cells wereincubated inthepresenceorabsence
skin-whitening agents. For this purpose, we measured tyrosinase
inhibition effects, kinetic analysis, and melanin contents in B16
cells; in addition, we also predicted the tertiary structure of
tyrosinase and simulated its docking with a novel tyrosinase
inhibitor.
The inhibitory activities of the synthesized compounds were
examined using mushroom tyrosinase as described previously with
minor modification [26]. Inhibitoryactivities were compared to those
of kojic acid and resveratrol as reference controls. Inhibition of
tyrosinase with the newly synthesized compounds is shown in Fig. 3.
Our results showed that compounds 2d and 2i exhibit inhibition of
mushroom tyrosinase similar to that of kojic acid which causes 46.1%
of 100 nM a-MSH, and then treated for 24 h with various concen-
trations of 2a or 2d (0e10 mM). Cells were washed and lysed in 100 ml
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 thawing and mixing,
cellular extracts were clarified by centrifugation at 12,000 rpm for
30 min at 4 ꢀC. An 8
ml sample of the supernatant and 20 ml of L-DOPA
(2 mg mlꢁ1) were placed in a 96-well plate, and the absorbance at
492 nm was read every 10 min for 1 h at 37 ꢀC using an ELISA plate
reader. The final activity is expressed as D
O.D. minꢁ1 for each condi-
tion. The results from three experiments are shown.
inhibition at a concentration of 20
mM. In the overall structur-
2.3.6. Determination of melanogenesis in B16 cells
eeactivity relationship study, as shown inTable 1, compound 2d with
two substituents (the hydroxy group at position 3 and the methoxy
groupatposition4)onthephenylringhasasimilarstructuretoDOPA,
whichbears twosubstituents(thehydroxygroupsatposition3and 4)
on the phenyl ring. 2i, with a hydroxyl group at position 4 on the
phenyl ring, has a similar structure to tyrosine. Therefore, the high
anti-tyrosinase effects of 2d and 2i might be attributed to their
structural similarity to tyrosine or DOPA. Compound 2h with two
methoxy substituents at position 3 and 4 on the phenyl ring showed
little activity. But 2g, with two methoxy substituents at positions 2
and 4 showed the most potent inhibitory activity. To the best of our
knowledge, the present study provides the first evidence that 2g has
a potent inhibitory effect on melanogenesis through the modulation
of tyrosinase.
Determination of melanin content of cells was done using
a modification of the method of Bilodeau et al. [28]. In the present
study, the amount of melanin was used as an index of melano-
genesis. B16 cells (5 ꢂ 104) were transferred to 24-well dishes and
incubated in the presence or absence of 100 nM a-MSH. Cells were
then incubated for 24 h with various concentrations of 2g
(0e10
in 100
m
m
M). After washing twice with PBS, samples were dissolved
l of 1 N NaOH. The samples were then incubated at 60 ꢀC 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 three experiments are shown.
2.3.7. Homology modeling of tyrosinase
A three-dimensional model of Lentinula edodes (mushroom)
tyrosinasecomprising618aminoacidswasbuiltusingSWISS-MODEL
[29] based on homology modeling [30]. The SWISS-MODEL program
automatically provides an all-atom model using alignments between
the query sequence and known homologous structures. Known
homologous structures of tyrosinase from the Protein Data Bank
(PDB) (http://www.pdb.org) were used as the structural templates.
SWISS-MODEL selected PDB entry 1BT3 (2.5 Å resolution) as the
suitable structural template (15% sequence identity and 0.00e-1
e-value). To validate the quality of predicted structure model, we
checked Z-valueusing QMEAN server (http://swissmodel.expasy.org/
qmean). The Z-value of the predicted model was ꢁ4.8.
We attempted to ascertain the potential cytotoxicity of 2g on
B16 cells. The cytotoxic effects of 2g were estimated by measuring
cell viability, where no significant cytotoxic effect was found at any
of the concentrations tested. At doses of 0.2,1.0, 2.5, 5.0 and 10.0 mM
of 2g, cell viability registered 98.41, 97.24, 97.01, 96.94 and 96.40%,
respectively, after 24 h treatment. These results indicated that 2g is
relatively non-cytotoxic to cells under the experimental conditions
used. Next, we examined the inhibitory action of 2g on mushroom
tyrosinase activity. Data in Table 2 show that 2g has a strong
suppressive action on the enzyme, with an IC50 value of
5.05 ꢃ 0.51
mM. To assess the relative efficacy of 2g against other
tyrosinase inhibitors, the effects of 2g were compared with the
well-known tyrosinase inhibitors, kojic acid and resveratrol [5].
Interestingly, 2g was found to be significantly stronger than kojic
acid and resveratrol as shown in Table 2. The greater inhibitory
efficacy of 2g may be attributed to chelation of the oxygen atom of
the 2-methoxy moiety on the phenyl ring and the sulfur atom on
2.3.8. In silico docking of tyrosinase and compound 2g
Among the many tools available for in silico protein-ligand dock-
ing, DOCK6.3 and AutoDock4.2 are 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 using
a systemic search technique [31,32]. We described the structure of
compounds in Table 1. To prepare for the docking procedure, we
performed the following steps: (1) conversion of 2D structures into
3D structures, (2) calculation of charges, and (3) addition of hydrogen
atoms using the ChemOffice program (www.cambridgesoft.com).
2.3.9. 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 mean standard error (n ¼ 3). The statistical significance of
the differences among groups was determined by one-factor
analysis of variance (ANOVA) followed by the Fisher’s protected
least significant difference post hoc test. Values of *p < 0.05 were
considered statistically significant.
3. Results and discussion
Fig. 3. Inhibitory effects of compounds on mushroom tyrosinase activity. Values
We designed and synthesized 2-(substituted phenyl)thiazoli-
dine-4-carboxylic acid derivatives in order to search for potent
represent means ꢃ S.E. of three experiments. Inhibitory concentration is 20
mM.
*p < 0.05 and ***p < 0.001, compared to kojic acid. KA, kojic acid; Res, resveratrol.

538
Y.M. Ha et al. / Biochimie 94 (2012) 533e540
Table 2
Inhibitory effects of kojic acid, resveratrol, and 2g on mushroom tyrosinase activity.
Sample
Concentration
M)
Inhibition (%)
Average of
inhibition (%)
IC50 (m
M)a
(
m
Kojic acid
1.25
5.00
20.00
1.25
5.00
20.00
1.25
2.39
27.36
48.74
8.22
25.14
38.13
49.60
43.20
66.00
3.60
25.44
43.87
10.22
24.44
39.99
41.60
56.80
68.40
2.60
28.66
45.78
8.32
24.23
39.33
48.80
47.20
65.00
2.86 ꢃ 0.37
27.15 ꢃ 0.94
46.13 ꢃ 1.42
8.92 ꢃ 0.65
24.61 ꢃ 0.28
39.15 ꢃ 0.55
46.67 ꢃ 2.54
49.07 ꢃ 4.04
66.47 ꢃ 1.01
21.07 ꢃ 0.77
26.82 ꢃ 0.41
5.05 ꢃ 0.51
Resveratrol
2g
5.00
20.00
a
50% inhibitory concentration (IC50).
the thiazolidine ring with copper ion existing in the active site of
tyrosinase. To explore the mechanism of inhibition, a study of the
kinetic behavior of tyrosinase activity was conducted in the pres-
ence of 2g. The results are shown in Table 3 and Fig. 4. As the
concentrations of 2g increased, the Km value of 2g increased
gradually, without changing Vmax, thereby indicating that 2g is
a competitive inhibitor of mushroom tyrosinase. Tyrosinase is
a copper-containing enzyme widely distributed in nature [33], and
most tyrosinase inhibitors that chelate the copper in the active site
of the enzyme show competitive inhibition; examples are tropo-
lone [34] and kojic acid [35]. On the basis of these kinetic results, 2g
might inhibit by binding to the copper-containing active site of
mushroom tyrosinase. To confirm these results, we predicted the
tertiary structure of mushroom tyrosinase and simulated its dock-
ing with 2g. For ligand-protein docking, we used DOCK6.3 and
AutoDock4.2 programs. Also, Chimera program was used for the
preparation of protein such as deleting solvent, adding charge, and
replacing incomplete side chain [36]. For preparing of ligand, we
used ChemOffice program. The docking simulation was successful
with significant scores. The binding energy of 2g was ꢁ22.11 kcal/
mol and the binding energy of kojic acid was ꢁ18.67 kcal/mol in
DOCK6.3 analysis. Also, the binding energy of 2g was ꢁ5.39 kcal/
mol and the energy of kojic acid was ꢁ2.73 kcal/mol in Auto-
Dock4.2 analysis. With the DOCK6.3 results, we predicted that the
ASP 262 and GLU 258 residues of the tyrosinase were mainly
responsible for the hydrogen and/or ionic bonding interaction with
2g. However, there was no hydrogen bonding interaction between
kojic acid and tyrosinase. With the AutoDock4.2 results, we pre-
dicted that the ASP 262 and SER 254 residues of the tyrosinase
could be the hydrogen and/or ionic bonding interaction with 2g.
Also, the ASP 262 residue of the tyrosinase was predicted as the
hydrogen bonding interaction with kojic acid. In order to finding
additional important residues of tyrosinase in inhibitor binding of
2g, we compared the residues of tyrosinase below a 3 Å distance
between 2g and tyrosinase with the residues below a 3 Å distance
between kojic acid and tyrosinase. We found common residues
such as HIS 90, ASP 262, and PRO 277 (Fig. 5). The residues could be
important residues more than other residues in the inhibitor
binding. The docking score between the ligand and the receptor is
represented by various energy terms such as electrostatic energy,
van der Waals energy terms, and the salvation energy. Specially, the
docking simulation using both DOCK6.3 and AutoDock4.2 sup-
ported that compound 2g has higher binding affinity than kojic acid
which was used as a control compound.
We examined the inhibitory effect of 2g on tyrosinase activity of
B16 cells treated with 100 nM
effectively inhibited the tyrosinase activity compared with the
-MSH-only-treated group. After 24 h incubation with 2g, murine-
derived tyrosinase activities were 210.91% at 0.2 M of 2g, 169.73%
at 1.0 M, 107.68 at 2.5 M, 105.44% at 5.0 M, and 103.35% at
10.0 M of 2g, compared with the 100 nM -MSH-only-treated
group (242.98%) and the control group without -MSH (100%). The
a-MSH (Fig. 6) and found that 2g
a
m
m
m
m
m
a
a
inhibitory effect of 2g against murine tyrosinase activity was
stronger than kojic acid and resveratrol, the positive controls at
10.0
mM (Fig. 5). We also evaluated the melanin content of cultured
B16 cells in the presence of 2g (range: 0.2e10
mM), and found that
melanin synthesis is effectively inhibited in a dose-related manner.
The inhibitory influence of 2g on melanin content is shown in Fig. 7.
The melanin content in B16 cells treated with 2g decreased dose-
dependently, showing 201.43% melanin content at 0.2
mM 2g,
187.61% at 1.0 M, 128.97% at 2.5 M, 104.05% at 5.0%, and 109.85%
m
m
Table 3
Kinetic analysis of compound 2g.
Vmax (mM/min)
Km (mM)
Ki (M)
No inhibitor
2.00 ꢂ 10ꢁ2
2.00 ꢂ 10ꢁ2
2.00 ꢂ 10ꢁ2
2.00 ꢂ 10ꢁ2
0.41
0.49
0.65
0.76
e
2g 1.25
2g 5.00
m
m
M
M
2.52 ꢂ 10ꢁ9
7.56 ꢂ 10ꢁ9
2.78 ꢂ 10ꢁ8
2g 20.00 mM
Data are presented as mean values of 1/V, inverse of the increase of absorbance at
a wavelength 492 nm per min ( A492/min), of three independent tests with different
concentrations of -tyrosine as a substrate. The LineweavereBurk plot’s equation is:
D
L
1/V ¼ Km/Vmax ꢂ 1/[S] þ 1/Vmax and the modified MichaeliseMenten equation is:
1/Vmax ¼ 1/Km (1 þ [I]/Ki), where V is the reaction velocity (the reaction rate), Km is
the MichaeliseMenten constant, Vmax is the maximum reaction velocity, [S] is the
substrate concentration, [I] is the inhibitor concentration, and Ki is the inhibitor
constant.
Fig. 4. LineweavereBurk plot of mushroom tyrosinase Data were obtained as mean
values of 1/V, the inverse of the absorbance increase at a wavelength of 492 nm per
min
L-tyrosine as a substrate. Enzyme inhibitors are indicated as follows: 1.25
M (square), 20.0 M (diamond), and no of compound 2g (circle).
(D
A492 minꢁ1), of three independent tests with different concentrations of
m
M (triangle),
5
m
m

Y.M. Ha et al. / Biochimie 94 (2012) 533e540
539
Fig. 5. Computational structure prediction for tyrosinase and docking simulation with 2g and kojic acid Predicted 3D structure of mushroom tyrosinase. Chemical structure of 2g
and kojic acid (magenta color is kojic acid, and blue color is compound 2g). The yellow area is the residues below Z < 3 between 2g and tyrosinase (TYR 68, GLY 91, SER 254, GLU
258, and ASP 276), the red area is the residues between kojic acid and tyrosinase (HIS 261 and HIS 265), and the orange area is common residues of both 2g and kojic acid (HIS 90,
ASP 262, and PRO 277). The docking results were performed by DOCK6.3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version
of this article.)
at 10.0
m
M of 2g when compared to the 100 nM
a
-MSH-only-
value regardless of the concentration of 2g. Therefore, the enzyme
inhibitory activity of 2g might result from binding at the same site as
the copper active site of mushroom tyrosinase. Although we need to
find clearly the inhibition mechanism of 2g through a future study
such as X-ray crystallography, the docking simulation could help to
understand the mechanism of compound 2g. Also, this work
explored the mechanism of compound 2g toward tyrosinase inhi-
bition by utilizing in silico methods. We searched for the residues of
tyrosinase which were close to 2g under a 3 Å distance because the
residues, which are close to inhibitor or activator, could have effect
on binding affinity and could have function as key residues in the
future studies such as mutagenesis study and single nucleotide
polymorphism study.
treated group (242.34%) and the control group (100%). 2g was more
effective than kojic acid (281.13%) and resveratrol (252.44%). These
results of the inhibition of murine-derived tyrosinase and melanin
content support the idea that 2g affects melanin biosynthesis
through the inhibition of tyrosinase. Additionally, our data verify
previous reports by us [21,23] and others [15] that the suppression
of melanogenesis in B16 cells is characterized by the inhibition of
tyrosinase.
In conclusion, we describe the synthesis and tyrosinase inhibi-
tory activity of 2-(substituted phenyl)thiazolidine-4-carboxylic acid
derivatives. High affinity of the scaffold of 2-(substituted phenyl)
thiazolidine-4-carboxylic acid is most likely due to hydrogen and/or
ionic bonding interactions with the active site residues such as SER
254, GLU258, and ASP 262. In a kinetic study, 2g was identified as
a competitive inhibitor of mushroom tyrosinase, with the same Vmax
In the B16 cell culture system, tyrosinase activity and melanin
levels were also significantly reduced by the addition of 2g to the
incubation medium containing cells pretreated with
a-MSH.
Fig. 6. Inhibition of 2g on B16 cells tyrosinase. In the presence of 100 nM MSH, B16
cells were treated with various doses of 2g (0.2e10.0
mM) for 24 h. Results are
Fig. 7. The ability of 2g to inhibit melanogenesis in the presence of 100 nM a-MSH in
expressed as % of control, and each column represents the mean ꢃ S.E. of three
B16 cells. Melanin levels were measured at 405 nm. Values represent the mean ꢃ S.E.
determinations. Data are expressed as % of control. ###p < 0.001 compared to the
of three experiments. Data are expressed as % of control. ###p < 0.001 compared to the
untreated control, ***p < 0.001 compared to the group treated with 100 nM
a
-MSH.
untreated control, ***p < 0.001 compared to the group treated with 100 nM a-MSH.
KA, kojic acid; Res, resveratrol.
KA, kojic acid; Res, resveratrol.

540
Y.M. Ha et al. / Biochimie 94 (2012) 533e540
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