A novel synthetic compound, (Z)-5-(3-hydroxy-4-methoxybenzylidene)-2-iminothiazolidin-4-one (MHY773) inhibits mushroom tyrosinase

Article abstract of DOI:10.1080/09168451.2018.1445518

As part of continued efforts for the development of new tyrosinase inhibitors, (Z)-5-(substituted benzylidene)-2-iminothiazolidin-4-one derivatives (1a – 1l) were rationally synthesized and evaluated for their inhibitory potential in vitro. These compound

Full text of DOI:10.1080/09168451.2018.1445518

Bioscience, Biotechnology, and Biochemistry
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20
A novel synthetic compound, (Z)-5-(3-hydroxy-4-
methoxybenzylidene)-2-iminothiazolidin-4-one
(MHY773) inhibits mushroom tyrosinase
Hee Jin Jung, Min Jung Lee, Yeo Jin Park, Sang Gyun Noh, A Kyoung Lee,
Kyoung Mi Moon, Eun Kyeong Lee, Eun Jin Bang, Yun Jung Park, Su Jeong
Kim, Jungho Yang, Sultan Ullah, Pusoon Chun, Young Suk Jung, Hyung Ryong
Moon & Hae Young Chung
To cite this article: Hee Jin Jung, Min Jung Lee, Yeo Jin Park, Sang Gyun Noh, A Kyoung Lee,
Kyoung Mi Moon, Eun Kyeong Lee, Eun Jin Bang, Yun Jung Park, Su Jeong Kim, Jungho Yang,
Sultan Ullah, Pusoon Chun, Young Suk Jung, Hyung Ryong Moon & Hae Young Chung (2018):
A novel synthetic compound, (Z)-5-(3-hydroxy-4-methoxybenzylidene)-2-iminothiazolidin-4-one
(MHY773) inhibits mushroom tyrosinase, Bioscience, Biotechnology, and Biochemistry, DOI:
10.1080/09168451.2018.1445518
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Bioscience, Biotechnology, and Biochemistry, 2018
https://doi.org/10.1080/09168451.2018.1445518
A novel synthetic compound, (Z)-5-(3-hydroxy-4-methoxybenzylidene)-2-
iminothiazolidin-4-one (MHY773) inhibits mushroom tyrosinase
Hee Jin Junga#, Min Jung Leea#, Yeo Jin Parka,b, Sang Gyun Noha,b, A Kyoung Leea,b, Kyoung Mi Moona, Eun
Kyeong Leec, Eun Jin Banga, Yun Jung Parkb, Su Jeong Kimb, Jungho Yangb, Sultan Ullahb, Pusoon Chund,
Young Suk Junga,b, Hyung Ryong Moona,b and Hae Young Chunga,b
amolecular inflammation research center for aging intervention (mrca), college of Pharmacy, Pusan national University, Busan, republic of
Korea; bcollege of Pharmacy, Pusan national University, Busan, republic of Korea; canalytical research center, Korea institute of toxicology,
daejeon, republic of Korea; dcollege of Pharmacy, inje University, gimhae, republic of Korea
ABSTRACT
ARTICLE HISTORY
received 17 october 2017
accepted 6 February 2018
As part of continued efforts for the development of new tyrosinase inhibitors, (Z)-5-(substituted
benzylidene)-2-iminothiazolidin-4-one derivatives (1a – 1l) were rationally synthesized and
evaluated for their inhibitory potential in vitro.These compounds were designed and synthesized
based on the structural attributes of a β-phenyl-α,β-unsaturated carbonyl scaffold template.
Among these compounds, (Z)-5-(3-hydroxy-4-methoxybenzylidene)-2-iminothiazolidin-4-
one (1e, MHY773) exhibited the greatest tyrosinase inhibition (IC50 = 2.87 μM and 8.06 μM for
KEYWORDS
tyrosinase; α-melanocyte-
stimulating hormone(α-
msh); 3-isobutyl-1-
methylxanthine (iBmX);
mhy773
monophenolase and diphenolase), and outperformed the positive control, kojic acid (IC50
=
15.59 and 31.61 μM).The kinetic and docking studies demonstrated that MHY773 interacted with
active site of tyrosinase. Moreover, a melanin quantification assay demonstrated that MHY773
attenuates α-melanocyte-stimulating hormone (α-MSH) and 3-isobutyl-1-methylxanthine
(IBMX)-induced melanin contents in B16F10 melanoma cells. Taken together, these data suggest
that MHY773 suppressed the melanin production via the inhibition of tyrosinase activity.
MHY773 is a promising for the development of effective pharmacological and cosmetic agents
for skin-whitening.
Melanin is synthesized by melanocytes distributed in the
innermost layer of the epidermis [1]. Melanin synthesis
is stimulated by a variety of molecules and conditions,
such as α-melanocyte stimulating hormone (α-MSH),
cyclic AMP (cAMP) elevating agents, including for-
skolin, glycyrrhizin, and 3-isobutyl-1-methylxanthine
(IBMX), UVB radiation, and the placental total lipid
fraction [2–4]. Melanin, can act as a photoprotective
form melanins, which determine the color of mamma-
lian skin and hair [9]. Various dermatological disorders,
such as age spots, freckles, melasma, and ephelide, are
caused by the excessive accumulation of epidermal pig-
mentation [10]. Hence, effective tyrosinase inhibitors
have been reported to potentially improve food quality
and to ameliorate skin hyperpigmentation in medical
applications [11].
Extensive research has been devoted to the search
for effective and harmless tyrosinase inhibitors, and a
number of naturally occurring and synthetic tyrosinase
inhibitors have been reported [9,11,12]. However, these
inhibitors are rarely used in practice owing to their weak
individual activities or safety concerns. Undoubtedly,
it is still necessary to find and develop new tyrosinase
inhibitors with better activity and minimal side effects.
Potential active inhibitors, such as kojic acid and arb-
utin, have not yet been demonstrated to be clinically
efficient inhibitors [13,14]. Hydroquinone, a widely
used skin-lightening agent, is still controversial with
respect to its biosafety owing to its cytotoxic and mito-
genic properties [15]. Hence, there is still a considerable
agent; however, excessive accumulation can cause hyper
-
pigmentation, which reduces the quality of food, and can
cause age spots and melisma spots on the skin [5,6]. Most
skin-whitening products target melanin production, and
tyrosinase inhibitors are the most commonly used agents
[7,8]. When the skin is exposed to UV light, melano-
genesis increase substantially via tyrosinase activation.
Tyrosinase (EC1.14.18.1), a multifunctional copper-con
-
taining enzyme in animals and plants, catalyzes the first
2 steps of melanin biosynthesis from l-tyrosine i.e. the
hydroxylation of l-tyrosine by monophenolase action
and oxidation of l-dihydroxyphenylalanine (l-DOPA)
to o-dopaquinone [7]. ese quinones are highly reac-
tive compounds that can polymerize spontaneously to
CONTACT hyung ryong moon
mhr108@pusan.ac.kr; hae young chung
hyjung@pusan.ac.kr
#these authors contributed equally to this work.
© 2018 Japan society for Bioscience, Biotechnology, and agrochemistry

2
H. J. JUNG ET AL.
demand for novel tyrosinase inhibitors that are safer and
effective.
General procedure for the synthesis of (Z)-5-
(substituted benzylidene)-2-iminothiazolidin-4-
one analogues (1a – 1l)
In this study, considering the important roles of the
β-phenyl-α,β-unsaturated carbonyl skeleton for tyrosi-
nase inhibition [16,17], twelve 2-iminothiazolidin-4-one
derivatives (1a – 1l) were synthesized (Figure 1), and
their inhibitory activity against tyrosinase was evalu-
ated. In addition, we examined the melanin contents in
B16F10 melanoma cells and performed enzyme kinetics
and in silico molecular modeling studies with mush-
room tyrosinase.
e desired compounds, (Z)-5-(substituted ben-
zylidene)-2-iminothiazolidin-4-one analogues (1a
– 1l) (Figure 1), were prepared by Knoevenagel con-
densation. With one exception, refluxing a solution of
appropriate benzaldehydes and pseudothiohydantoin
in acetic acid in the presence of NaOAc produced (Z)-
5-benzylidene-2-iminothiazolidin-4-ones as a single
stereoisomer in yields of 41.4–94.1%. A Knoevenagel
condensation between 2,4-dimethoxybenzaldehyde and
pseudothiohydantoin under the same conditions gave a
mixture of (E)- and (Z)-5-(2,4-dimethoxybenzylidene)-
2-iminothiazolidin-4-ones. ese compounds could not
be easily separated by conventional silica gel column
chromatography. Milder reaction conditions capable
of accomplishing the Knoevenagel condensation were
needed to synthesize only (Z)-stereoisomer. Interestingly,
heating a solution of 2,4-dimethoxybenzaldehyde
and pseudothiohydantoin in ethanol:H2O (1:1) in the
presence of 1.0 equiv. of piperidine as a base catalyst
at 80 °C afforded the corresponding (Z)-stereoisomer
(1l) as a sole product. A suspension of an appropriate
benzaldehyde (300 mg, 1.53–2.46 mmol), pseudothi-
ohydantoin (1.1 eq.), and sodium acetate (3.0 eq.) in
acetic acid (1 mL/1 mmol of benzaldehyde) was refluxed
for 3–7 h. e reaction mixture was cooled and water
was added. e resulting precipitates were filtered, and
washed with water and, if necessary, a small amount
of methylene chloride or ethyl acetate, to produce (Z)-
5-(substituted benzylidene)-2-iminothiazolidin-4-one
products (1a – 1k) in 41.4–94.1% yields. e result-
ing precipitates were filtered, and washed with water,
ethyl acetate and methylene chloride to give (Z)-5-(2,4-
dimethoxybenzylidene)-2-iminothiazolidin-4-one (1l)
as a single isomer in a yield of 21.0%. e structures of
Materials and methods
Chemicals and instrumentation
Mushroom tyrosinase (EC 1.14.18.1), α-MSH, IBMX,
l-tyrosine, l-dihydroxyphenylalanine (l-DOPA), dime-
thyl sulfoxide (DMSO), and kojic acid were purchased
from Sigma-Aldrich (St. Louis, MO, USA.). Dulbecco’s
modified Eagle’s medium (DMEM), fetal bovine serum
(FBS), streptomycin, and amphotericin were purchased
from Gibco Life Technologies Inc. (Carlsbad, CA, U.S.A.).
in-layer chromatography (TLC) and silica gel 60 (mesh
230–400) was carried out on silica gel F254-precoated
plates from Merck Millipore (Darmstadt, Germany). All
anhydrous solvents were distilled over CaH2 or Na/ben-
zophenone prior to use. All melting points were deter-
mined in an open capillary on a Stuart capillary melting
point apparatus (Staffordshire, UK) and are uncorrected.
NMR spectra were recorded using Varian Unity INOVA
400 (400 MHz for 1H, 100 MHz for 13C) and Varian Unity
1
AS 500 (500 MHz for H) instruments. Chemical shif
values (δ) are reported with reference to the respective
residual solvent or deuterated peaks (δH 2.50 and δC
39.51 for DMSO). Low-resolution mass spectrometry
data were obtained on a using an Expression CMS mass
spectrometer (Advion, Ithaca, NY, USA).
Figure 1. the synthetic scheme for (Z)-5-(substituted benzylidene)-2-iminothiazolidin-4-one derivatives 1a – 1l.

BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY
3
1
all final products were confirmed by H and 13C NMR
(Z)-5-(3-Hydroxy-4-methoxybenzylidene)-2-
iminothiazolidin-4-one (1e, MHY773)
spectroscopy and mass spectroscopy.
Brown solid; reaction time, 7 h; yield, 78.9%; melting
point, 282.8–285.7 °C; 1H-NMR (500 MHz, DMSO-d6)
δ 9.38 (s, 1H, OH), 9.30 (br s, 1H, NH), 9.06 (s, 1H,
NH), 7.44 (s, 1H, vinylic H), 7.04 (dd, 1H, J = 1.0,
8.5 Hz, 6′-H), 7.01 (d, 1H, J = 8.5 Hz, 2′-H), 6.99 (d,
1H, J = 1.0 Hz, 5′-H), 3.81 (s, 3H, 4′-OCH3); 13C-NMR
(100 MHz, DMSO-d6) δ 181.3 (C-4), 176.2 (C-2), 149.9
(C-4′), 147.5 (C-3′), 130.1 (benzylic C), 127.4 (C-5),
126.9 (C-1′), 123.1 (C-6), 116.1 (C-2), 113.0 (C-5), 56.3
(4′-OCH3); LRMS (ESI-) m/z 249 (M-H) -.
(Z)-5-(4-Hydroxybenzylidene)-2-iminothiazolidin-
4-one (1a, MHY762)
Orange colored solid; reaction time, 3 h; yield, 61.8%;
melting point, >300 °C; 1H-NMR (500 MHz, DMSO-d6)
δ 10.10 (s, 1H, OH), 9.29 (br s, 1H, NH), 9.04 (s, 1H,
NH), 7.49 (s, 1H, vinylic H), 7.40 (d, 2H, J = 9.0 Hz,
2′-H, 6′-H), 6.88 (d, 2H, J = 8.5 Hz, 3′-H, 5′-H); 13C-
NMR (100 MHz, DMSO-d6) δ 181.4 (C-4), 176.1 (C-2),
159.6 (C-4′), 132.1 (C-2′, C-6′), 130.2 (benzylic C), 126.0
(C-5), 125.5 (C-1′), 116.8 (C-3′, C-5′); LRMS (ESI-) m/z
219 (M-H)-.
(Z)-2-Imino-5-(4-methoxybenzylidene) thiazolidin-
4-one (1f, MHY775)
(Z)-5-(3,4-Dihydroxybenzylidene)-2-
iminothiazolidin-4-one (1b, MHY764)
Yellow solid; reaction time, 3 h; yield, 50.7%; melting
point, 285.9–288.5 °C; 1H-NMR (500 MHz, DMSO-d6)
δ 9.34 (br s, 1H, NH), 9.09 (s, 1H, NH), 7.53 (d, 2H,
J = 8.5 Hz, 2′-H, 6′-H), 7.51 (s, 1H, vinylic H), 7.07 (d,
2H, J = 9.0 Hz, 3′-H, 5′-H), 3.80 (s, 3H, 4′-OCH3); 13C-
NMR (100 MHz, DMSO-d6) δ 181.3 (C-4), 176.1 (C-2),
160.9 (C-4′), 131.9 (C-2′, C-6′), 129.7 (benzylic C), 127.1
(C-5), 127.1 (C-1′), 115.4 (C-3′, C-5′), 56.1 (4′-OCH3);
LRMS (ESI-) m/z 233 (M-H) -.
Brown solid; reaction time, 6 h; yield, 62.0%; melting
1
point,>300 °C; H-NMR (500 MHz, DMSO-d6) δ 9.60
(br s, 1H, OH), 9.33 (br s, 2H, OH, NH), 9.02 (s, 1H,
NH), 7.40 (s, 1H, vinylic H), 6.96 (s, 1H, 2′-H), 6.89
(d, 1H, J = 8.5 Hz, 6′-H), 6.83 (d, 1H, J = 8.5 Hz, 5′-H);
13C-NMR (100 MHz, DMSO-d6) δ 181.4 (C-4), 176.2
(C-2), 148.3 (C-4′), 146.4 (C-3′), 130.6 (benzylic C),
126.0 (C-1′), 125.8 (C-5), 123.5 (C-6′), 116.8 (C-5′),
116.6 (C-2′); LRMS (ESI-) m/z 235 (M-H) -.
(Z)-5-(3,4-Dimethoxybenzylidene)-2-
iminothiazolidin-4-one (1g, MHY777)
(Z)-5-(4-Hydroxy-3-methoxybenzylidene)-2-
iminothiazolidin-4-one (1c, MHY768)
Dark yellow solid; reaction time, 6 h; yield, 82.4%; melting
point, 274.2–276.3 °C; 1H NMR (400 MHz, DMSO-d6) δ
9.32 (br s, 1H, NH), 9.05 (s, 1H, NH), 7.52 (s, 1H, vinylic
H), 7.14 (d, 1H, J = 2.0 Hz, 2′-H), 7.12 (dd, 1H, J = 2.0,
8.4 Hz, 6′-H), 7.06 (d, 1H, J = 8.4 Hz, 5′-H), 3.77 (s,
3H, OCH3), 3.77 (s, 3H, OCH3); 13C-NMR (100 MHz,
DMSO-d6) δ 181.2 (C-4), 176.0 (C-2), 150.7 (C-3′), 149.5
(C-4′), 130.1 (benzylic C), 127.4 (C-1′), 127.3 (C-5), 123.5
(C-6′), 113.4 (C-5′), 112.7 (C-2′), 56.3 (3′-OCH3), 56.1
(4′-OCH3); LRMS (ESI-) m/z 263 (M-H) -.
Orange colored solid; reaction time, 7 h; yield, 92.4%;
1
melting point, 263.9–265.6 °C; H-NMR (400 MHz,
DMSO-d6) δ 9.69 (s, 1H, OH), 9.27 (br s, 1H, NH),
9.00 (s, 1H, NH), 7.48 (s, 1H, vinylic H), 7.11 (d, 1H,
J = 2.0 Hz, 2′-H), 6.99 (dd, 1H, J = 2.0, 8.4 Hz, 6′-H),
6.86 (d, 1H, J = 8.4 Hz, 5′-H), 3.78 (s, 3H, 3′-OCH3); 13C-
NMR (100 MHz, DMSO-d6) δ 181.3 (C-4), 176.0 (C-2),
149.1 (C-3′), 148.6 (C-4′), 130.5 (benzylic C), 126.2
(C-5), 126.0 (C-1′), 123.9 (C-6′), 116.7 (C-5′), 114.1
(C-2′), 56.2 (3′-OCH3); LRMS (ESI-) m/z 249 (M-H) -.
(Z)-5-(3,5-Dihydroxybenzylidene)-2-
iminothiazolidin-4-one (1h, MHY782)
(Z)-5-(3-Ethoxy-4-hydroxybenzylidene)-2-
iminothiazolidin-4-one (1d, MHY770)
Dark brown solid; reaction time, 4 h; yield, 41.4%; melt-
1
ing point,>300 °C; H-NMR (500 MHz, DMSO-d6) δ
9.58 (s, 2H, OH), 9.37 (br s, 1H, NH), 9.13 (s, 1H, NH),
7.35 (s, 1H, vinylic H), 6.42 (s, 2H, 2′-H, 6′-H), 6.27
(s, 1H, 4′-H); 13C-NMR (100 MHz, DMSO-d6) δ 181.1
(C-4), 176.4 (C-2), 159.5 (C-3′, C-5′), 136.3 (C-1′), 130.3
(benzylic C), 129.6 (C-5), 108.2 (C-2′, C-6′), 104.8 (C-4′);
LRMS (ESI-) m/z 235 (M-H) -.
Orange colored solid; reaction time, 7 h; yield, 84.9%;
1
melting point, 248.8–251.2 °C; H-NMR (500 MHz,
DMSO-d6) δ 9.63 (s, 1H, OH), 9.28 (br s, 1H, NH), 9.01
(s, 1H, NH), 7.49 (s, 1H, vinylic H), 7.11 (s, 1H, 2′-H),
7.02 (d, 1H, J = 8.5 Hz, 6′-H), 6.89 (s, 1H, J = 8.0 Hz,
5′-H), 4.06 (q, 2H, J = 7.0 Hz, 3′-OCH2), 1.35 (t, 3H,
J = 7.0 Hz, CH3); 13C-NMR (100 MHz, DMSO-d6) δ
181.3 (C-4), 176.0 (C-2), 149.4 (C-3′), 147.7 (C-4′), 130.5
(benzylic C), 126.1 (C-1′), 126.0 (C-5), 124.1 (C-6′),
116.8 (C-5′), 115.1 (C-2′), 64.5 (3′-OCH2), 15.3 (CH3);
LRMS (ESI-) m/z 263 (M-H) -.
(Z)-5-(2-Hydroxybenzylidene)-2-iminothiazolidin-
4-one (1i, MHY787)
Brown solid; reaction time, 7 h; yield, 69.3%; melting
point, 210.1–211.5 °C; 1H-NMR (500 MHz, DMSO-d6)

4
H. J. JUNG ET AL.
δ 8.13 (s, 1H, vinylic H), 8.02–7.09 (brs, 3H, OH,
2 × NH), 7.72 (d, 1H, J = 6.5 Hz, 6′-H), 7.57 (t, 1H,
J = 7.5 Hz, 5′-H), 7.43 (d, 1H, J = 8.0 Hz, 3′-H), 7.32 (t,
1H, J = 7.0 Hz, 4′-H); 13C-NMR (100 MHz, DMSO-d6)
δ 180.3 (C-4), 176.2 (C-2), 159.2 (C-2′), 152.5 (benzylic
C), 137.8 (C-5), 132.2 (C-4′), 128.5 (C-6′), 125.6 (C-5′),
119.9 (C-1′), 116.8 (C-3′).
inhibition rate (%) was calculated as (1−Abssample /
Abscontrol) × 100%. e IC50 values was determined by
a regression analysis of a dose-response curve at which
50% target activity was lost.
Kinetic analysis of mushroom tyrosinase
To determine the kinetic mechanisms, two complimen-
tary methods based on Lineweaver-Burk and Dixon
plots were used [20–22]. Using Lineweaver-Burk double
reciprocal plots [a plot of 1/enzyme velocity (1 / V) vs. 1/
substrate concentration (1 / [S])], the type of inhibition
was determined using various concentrations of l-tyros-
ine (0.25, 0.5, 1, 2, 4 and 8 mM) and l-DOPA (1, 2, 4, 8
and 16 mM) as a substrate in the absence and presence
of various concentrations of MHY773 (0–20 μM). e
Dixon plot for the inhibition of tyrosinase was obtained
in the presence of various concentrations of l-tyrosine
(0.5, 1, 2 and 4 mM) and l-DOPA (1, 2, 4 and 8 mM).
e concentrations of MHY773 were as follows: 0–20
μM. e enzymatic procedures consisted of the same,
aforementioned tyrosinase assay methods. e inhibi-
tion constant (Ki) was determined using the Dixon plots.
(Z)-5-(3,4,5-Trimethoxybenzylidene)-2-
iminothiazolidin-4-one (1j, MHY790)
Orange colored solid; reaction time, 3 h; yield, 94.1%;
1
melting point, 253.3–254.8 °C; H NMR (500 MHz,
DMSO-d6) δ 9.40 (br s, 1H, NH), 9.11 (s, 1H, NH), 7.55
(s, 1H, vinylic H), 6.91 (s, 2 H, 2′-H, 6′-H), 3.82 (s, 6 H,
3′-OCH3, 5′-OCH3), 3.70 (s, 3 H, 4′-OCH3); 13C-NMR
(100 MHz, DMSO-d6) δ 181.0 (C-4), 176.1 (C-2), 153.8
(C-3′, C-5′), 139.3 (C-4′), 130.3 (C-1′), 130.0 (benzylic
C), 129.1 (C-5), 107.6 (C-2′, C-6′), 60.8 (4′-OCH3), 56.6
(3′-OCH3, 5′-OCH3); LRMS (ESI-) m/z 293 (M-H) -.
(Z)-5-(4-Hydroxy-3,5-dimethoxybenzylidene)-2-
iminothiazolidin-4-one (1k, MHY792)
Dark orange colored solid; reaction time, 6 h; yield,
87.7%; melting point, 254.3–256.6 °C; 1H-NMR
(400 MHz, DMSO-d6) δ 9.30 (br s, 1H, NH), 9.08 (s,
1H, OH), 9.01 (s, 1H, NH), 7.49 (s, 1H, vinylic H), 6.85
(s, 2H, 2′-H, 6′-H), 3.78 (s, 6H, 3′-OCH3, 5′-OCH3); 13C-
NMR (100 MHz, DMSO-d6) δ 181.2 (C-4), 176.0 (C-2),
148.8 (C-3′, C-5′), 138.3 (C-4′), 130.8 (benzylic C), 126.5
(C-5), 124.9 (C-1′), 108.0 (C-2′, C-6′), 56.6 (3′-OCH3,
5′-OCH3); LRMS (ESI-) m/z 279 (M-H) -.
In silico docking simulation of tyrosinase inhibition
To determine the possible molecular mechanism and
the most active groups of MHY773 for the inhibition
of tyrosinase, molecular docking simulations were per-
formed using AutoDock4.2.6. X-ray crystallographic
structures show quaternary ligand binding to aromatic
residues in the active-site gorge of tyrosinase (PDB
ID: 2Y9X) from the RCSB Protein Data Bank (http://
www.rcsb.org/adb) [23]. e 3D structure of three com
-
pounds were created using ChemDraw Ultra. Because
the docking programs use a set of predefined 3D grids
for the target protein and a systematic search technique,
we selected the previously defined 3D grids for tyrosi-
nase interactions with tropolone at the binding pocket
[24]. Aferward, docking simulations were performed
between tyrosinase and three compounds or kojic acid.
A pharmacophore is an ensemble of ligands required for
interactions with a specific receptor. A pharmacophore
model was generated using LigandScout 4.0. [25]. Based
on the atom types, the chemical features of tyrosinase
were defined in terms of pharmacophore elements, such
as hydrogen bond acceptors, hydrogen bond donors,
positive ionizable areas, hydrophobic interactions, and
aromatic rings.
(Z)-5-(2,4-Dimethoxybenzylidene)-2-
iminothiazolidin-4-one (1l, MHY801)
Yellow solid; reaction time, 7 h; yield, 21.0%; melting
point, 249.1–250.7 °C; 1H-NMR (400 MHz, DMSO-d6)
δ 9.13 (br s, 2H, NH), 7.75 (s, 1H, vinylic H), 7.32 (d, 1H,
J = 8.4 Hz, 6′-H), 6.66 (dd, 1H, J = 2.0, 8.0 Hz, 5′-H), 6.62
(d, 1H, J = 2.0 Hz, 3′-H), 3.84 (s, 3H, 2-OCH3), 3.79 (s,
3H, 4-OCH3); 13C-NMR (100 MHz, DMSO-d6) δ 181.0
(C-4), 176.2 (C-2), 162.8 (C-4′), 160.0 (C-2′), 129.8 (ben-
zylic C), 127.0 (C-5), 124.2 (C-6′), 116.0 (C-1′), 106.8
(C-5′), 99.3 (C-3′), 56.5 (2′-OCH3), 56.2 (4′-OCH3);
LRMS (ESI-) m/z 263 (M-H) -.
Tyrosinase inhibitory activity assay
e inhibitory activities of twelve compounds with dif-
ferent concentration ranges (final concentration: 50 μM
for screening; 0.5, 1, and 5 μM for inhibitory concentra-
tion) were evaluated against mushroom tyrosinase (1000
U) using a modified spectrophotometric method devel-
oped by No et al. and Kang et al. [18,19]. e tyrosinase
Cell culture
Mouse melanoma B16F10 cells were obtained from the
Korea Cell Line Bank, cultured in DMEM supplemented
with 10% FBS and 1% streptomycin, and then incubated
at 37 °C with 5% CO2.

BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY
5
additional hydroxyl substituent at R2 also exhibited
moderate inhibitory activity. Interestingly, compound
1h containing a hydroxyl substituent at both R2 and R4
showed moderate inhibitory activity. Compound 1f with
one methoxy group, compounds 1g and 1l bearing two
methoxy groups as substituents, or compound 1j with
three methoxy groups or two methoxy groups and a
hydroxyl group as the substituent of phenyl ring (1k)
showed no activity on tyrosinase. Our results suggest
that the number and location of methoxy and hydroxyl
groups on the phenyl ring are very closely related to the
capacity of these compounds for tyrosinase inhibition.
e tyrosinase inhibitory activity (IC50) and Ki values
are shown in Table 2. Among the tested compounds 1a –
1l, MHY773 (1e) was found to be most effective in inhib-
iting the first step of oxidation. To further determine the
detailed tyrosinase activity of MHY773, we conducted
two step enzyme reaction including l-tyrosine and
l-DOPA as substrates. As observed in Table 2, the IC50
values of MHY773 were estimated to be 2.87 0.19 μM
and 8.06 0.41 μM on the monophenolase and diphe-
nolase, respectively. As observed in Table 2, MHY773
was more effective in inhibiting the first step of oxida-
tion (l-tyrosine oxidation). To obtain insights into the
mechanism by which the MHY773 inhibited tyrosinase,
we performed kinetics analyses using Lineweaver-Burk
and Dixon plots, as summarized in Table 2. According
to Table 2 and Figure 2(A) and (C), a graphical anal-
ysis of the Lineweaver-Burk plot for MHY773 against
tyrosinase indicated competitive inhibition, suggesting
that this compound can competitively bind to the sub-
strate-binding site to inhibit enzyme activity. Moreover,
Assessment of melanin contents
e cellular melanin content was determined as
described previously [26]. B16F10 cells were seeded in
6-well culture plates at 2.5 × 104 cells/well with 5% CO2
for 24 h. To determine the inhibitory effect of MHY773
on melanogenesis, fresh medium was replaced with
medium containing MHY773 at 1, 5, and 10 μM, with
kojic acid at 5 μM as a positive control, and 0.5 μM
α-MSH and 200 μM IBMX for 72 h. Cells were washed
twice with PBS and cells in each well were pooled for
each test. Afer centrifugation, cell pellets were disrupted
in 100 μL of 1N NaOH, and then incubated at 60 °C for
1 h and mixed to solubilize the melanin. Absorbance
at 405 nm was compared with a standard curve for
synthetic melanin. e production of melanin was cal-
culated by normalizing the melanin values against the
protein content (absorbance/μg protein). For this pur-
pose, the protein content was determined using the BCA
protein assay reagent (ermo Scientific, Rockford, IL,
U.S.A.).e results from three individual experiments
are shown.
Statistical analysis
All data are presented as means S.E.M. Data were ana-
lyzed by one-way ANOVA followed by the Bonferroni
*
post hoc test. A p-value of < 0.05 was considered sta-
tistically significant.
Results and discussion
We investigated the inhibitory effects of the iminothi-
azolidine derivatives on the monophenolase activity of
tyrosinase on the substrate l-tyrosine. e results for
tyrosinase inhibition by the newly synthesized deriva-
tives are summarized in Table 1. MHY773 (1e) exhibited
the most potent inhibitory activity against tyrosinase
with 92.93% inhibition at 50 μM, compared to 79.57%
for the positive control kojic acid. Compounds 1a, 1b,
and 1h exhibited moderate mushroom tyrosinase inhib-
itory activity. Compound 1i showed weak mushroom
tyrosinase inhibitory activity. In contrast, 1c, 1d, 1f, 1g,
1j, 1k, and 1l were inactive at the concentrations tested.
Based on the structure-activity relationship, compound
1e, bearing 2-substituents (R2-hydroxyl and R3-methoxy
groups) on the phenyl ring, was a potent inhibitor (Table
1). Notably, the structural modifications to compound
1c, in which the positions of the hydroxyl and methoxy
substituents of 1e are transposed, showed no inhibitory
activity. Additionally, the insertion of an ethoxy group
on the phenyl ring (1d) resulted in a complete loss of
inhibitory activity against tyrosinase. Compound 1i,
with a hydroxyl substituent at R1 on the phenyl ring,
also showed no activity. However, compound 1a bear-
ing a hydroxyl substituent at R3 showed moderate
inhibitory activity. Moreover, compound 1b with an
Table 1. substitution pattern of the substituted (Z)-5-(sub-
stituted benzylidene)-2-iminothiazolidin-4-one derivatives
(1a–1l).
Tyrosinase
Compounds MHYs
R1
R2
R3
R4
inhibition (%)a
1a
1b
1c
1d
1e
1f
1g
1h
762
764
768
770
773
775
777
782
787
790
792
801
h
h
h
h
h
h
h
h
oh
h
h
oh
ome
oet
oh
h
ome ome
oh
h
oh
oh
oh
oh
ome
ome
h
h
h
h
h
h
h
oh
h
44.39 3 .91
48.23 3.15
nib
ni
92.93 1.18
ni
ni
46.24 2.58
7.07 3.49
ni
h
h
1i
1j
1k
ome ome ome
ome
h
h
ome
oh
ome
ome
h
ni
ni
1l
Kojic acidc
79.57 3.53
aValues are expressed as means s.e.m. of triplicate experiments. tyrosi-
nase inhibition was measured using samples concentration at 50 μm.
bni (no inhibition).
cPositive control.

6
H. J. JUNG ET AL.
Table 2. tyrosinase inhibitory activities and enzyme kinetic analysis of mhy773.
l-Tyrosine
l-DOPA
Inhibition typeb (Ki, μM)c
Compounds
IC50 (μM)a
Inhibition typeb (Ki, μM)c
IC50 (μM)a
mhy773
2.87 0.19
15.59 1.61
competitive (2.24)
not tested
8.06 0.41
31.61 1.08
competitive (2.85)
not tested
Kojic acidd
athe 50% inhibitory concentration (ic50) values (μm) were calculated from a log dose inhibition curve and expressed as means s.e.m of triplicate experi-
ments.
bdetermined by dixon plots.
cdetermined by lineweaver-Burk plots.
dPositive control.
Figure 2. lineweaver-Burk anddixonplotsof tyrosinaseinhibitionbymhy773. (a)tyrosinaseinhibitionwith l-tyrosineasasubstrate
at various concentrations of mhy773: 0 μm (filled circles), 1.25 μm (open circles), 2.5 μm (filled triangles) and 5 μm (open triangles).
(B) tyrosinase inhibition by mhy773 at various concentrations of l-tyrosine: 0.5 mm (filled circles), 1 mm (open circles), 2 mm (filled
triangles) and 4 mm (open triangles). (c) tyrosinase inhibition with l-doPa as a substrate at various concentrations of mhy773: 0 μm
(filled circles), 5 μm (open circles), 10 μm (filled triangles) and 20 μm (open triangles). (d) tyrosinase inhibition by mhy773 at various
concentrations of l-doPa: 1 mm (filled circles), 2 mm (open circles), 4 mm (filled triangles) and 8 mm (open triangles).
we used Dixon plots to determine the Ki by obtaining
secondary plots of the slope vs. the concentration of
MHY773, with a Ki value of 2.24 and 2.85 μM, on the
monophenolase and diphenolase, respectively (Table
2; Figure 2(B) and (D)). As the Ki value represents the
concentration needed to form an enzyme–inhibitor
complex, a lower Ki value may indicate more effective
inhibitors against tyrosinase in the development of pre-
ventive and therapeutic agents.
To understand the mechanism underlying the inhi-
bition of tyrosinase by MHY773, the binding modes
of MHY775 and MHY777 were compared to that
of MHY773 were examined at the catalytic sites of
tyrosinase. By docking simulations, we docked the 3D
structure of mushroom tyrosinase respectively with
MHY773, MHY775 and MHY777, supporting the
hypothesis that three compounds interacts with amino
acid residues in the active site (Figure 3(A)).
According to docking results, MHY773 formed
H-bond interactions with two residues, MET280 and
GLU256 (Figure 3(B)), together with one hydropho-
bic interaction with VAL283. Similarly, kojic acid also
found to be interacted via a single H-bond with MET280
(Figure 3(C)). On the other hand, MHY775 and MHY777
did not form H-bond interaction, however, these com-
pounds showed hydrophobic interactions, with VAL248
and PHE264, respectively (Figure 3(B)). Docking results
confirm that MHY773 showed higher binding affinity

BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY
7
Figure 3. computational structure prediction for mushroom tyrosinase and docking simulation with mhy773, mhy775, mhy777
and kojic acid (mhy773, mhy775, mhy777 and kojic acid are shown as blue, green, orange, and pink, respectively) (a). Predicted 3d
structure of mushroom tyrosinase. Possible hydrogen bonding interactions between tyrosinase residues and three compounds (B)
and kojic acid (c). estimated binding free energy of the ligand-receptor complex of three compounds and kojic acid (d). We searched
for h-binding interactions between tyrosinase and kojic acid or three compounds in the simulated docked structures; green lines
indicate h-bonding interactions and yellow curves and lines indicate hydrophobic bonding interactions.
(–6.52 kcal/mol) and formed both H- and hydrophobic
interactions with active site of mushroom tyrosinase,
while MHY775 and MHY777 oriented differently as well
as did not form H-bond interactions and exhibited lower
binding energy (–5.72 and –5.89 kcal/mol, respectively)
than MHY773 (Figure 3(D)). erefore, these might be
the reasons why MHY773 showed higher mushroom
tyrosinase inhibitory activity compared to MHY775 and
MHY777.
us, docking simulations are in agreement and
supporting experimental results that MHY773 was
a potent mushroom tyrosinase inhibitor whereas,
MHY775 and MHY777 were not active against tyrosi-
nase. Furthermore, comparative structure activity
analysis revealed that three compounds have imino-
thiazolidine functional group, while MHY773 has a
hydroxyl group, whereas MHY777 contains a methoxy
group but MHY775 has not any functional group at R2
position. So, these structural differences showed that
substitutions greatly affected the inhibition activity,
which is made clear by the fact that MHY773 is a potent
tyrosinase inhibitor but MHY775 and MHY777 did not
exhibit tyrosinase inhibitory activity. Recently, Wendt
et al. (2016) [27] also reported that presence or lack of
functional groups and its substitutions greatly affected
the inhibition activity against tyrosinase. From above
results, functional groups at R2 position and amino acid
residue in the active site regarding three iminothiazoli-
dine compounds might play an important for tyrosinase
inhibitory activity.
Skin-whitening agents that inhibit tyrosinase or other
enzymes must first penetrate the melanocyte cell wall.
us, MHY773 was further tested in B16F10 melanoma
cells to confirm its activity in melanocytes. Prior to the
investigation of the pharmacological activity of MHY773
on α-MSH and IBMX-induced melanogenesis-potential,
the dose dependence of the cytotoxic effects of MHY773
and kojic acid on B16F10 cells was assessed using the
EZ-Cytox assay. In our study, both MHY773 and kojic
acid at concentrations up to 10 μM did not cause any
cytotoxic effects during incubation for up to 72 h (data
not shown). e effects of MHY773 on α-MSH and
IBMX-stimulated melanin synthesis are summarized in
Figure 4. To evaluate the inhibitory effect of MHY773 on
melanin production, cells were pretreated with the indi-
cated concentrations (1, 5, and 10 μM) of MHY773 for
2 h and then treated with both 0.5 μM α-MSH and 200
μM IBMX for 72 h. As shown in Figure 4, the melanin
content in cells treated with MHY773 in the presence
of α-MSH and IBMX decreased in a concentration-de-
pendent manner, specifically, the content was 216.75%
at 1 μM, 170.39% at 5 μM, and 153.72% at 10 μM, com-
pared to that of the α-MSH and IBMX-induced control
group (261.58%). e results of this study suggested that
MHY773 can inhibit mushroom tyrosinase potential
and also exhibits potent inhibitory effects on melano-
genesis in B16F10 cells.
In this study, we describe the synthesis and tyrosi-
nase inhibitory activity of (Z)-5-(substituted ben-
zylidene)-2-iminothiazolidin-4-one derivatives (1a

8
H. J. JUNG ET AL.
Figure 4. effect of mhy773 on α-msh and iBmX-induced cellular melanin contents in B16F10 melanoma cells. cells were incubated
in the presence of various concentrations of mhy773 (1, 5 and 10 μm) or kojic acid (5 μm) for 2 h, and then stimulated with 0.5 μm
α-msh and 200 μm iBmX for 72 h. Values are given as means s.e.m. of three independent experiments. ###p < 0.001 compared with
the control group and **p < 0.01 and ***p < 0.001 compared with α-msh and iBmX-induced cells.
– 1l). Among them, MHY773 (1e) was approximately
5-fold more potent than kojic acid, which is ofen used
as a positive control. In particular, structure-activity
relationship analysis indicated the introduction of the
β-phenyl-α,β-unsaturated carbonyl skeleton bearing
R2-hydroxyl and R3-methoxy groups was crucial for the
inhibition of tyrosinase activity. In addition, competitive
inhibition and Ki values of 2.24 and 2.85 μM respective
on the monophenolase and diphenolase were observed
for MHY773 on tyrosinase. A docking simulation with
MHY773 confirmed that MHY773 binds strongly to
mushroom tyrosinase and also suggested that GLU256
and MET280 play a key role in the determination of
the binding affinity of MHY773 via hydrogen bonding
interactions. us, enzyme kinetics and docking simu-
lation results suggested that MHY773 binds to the active
site of tyrosinase, thereby inhibiting tyrosinase activity.
MHY773 also inhibited melanin synthesis in B16F10
melanoma cells. However, the results of this study sug-
gest that the mechanisms by which MHY773 inhibits
tyrosinase and melanogenesis should be investigated in
further studies.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
is work was supported by the Pusan National University
[2017 Post-Doctoral Development Program]; National
Research Foundation of Korea (NRF) funded by the Korean
government (MSIP) [grant number 2009-0083538].
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We thank Aging Tissue Bank for providing materials and
research information.

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