De novo tyrosinase inhibitor: 4-(6,7-Dihydro-5H-indeno[5,6-d]thiazol-2-yl) benzene-1,3-diol (MHY1556)

Article abstract of DOI:10.1016/j.bmcl.2013.05.029

In this study, we have synthesized and studied de novo tyrosinase inhibitor, MHY1556, which showed significantly better efficacy than other pre-existing tyrosinase inhibitors in vitro experiments. The IC₅₀ value of MHY1556 was 0.50 μM which was

Full text of DOI:10.1016/j.bmcl.2013.05.029

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4172–4176
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
De novo tyrosinase inhibitor: 4-(6,7-Dihydro-5H-indeno[5,6-d]
thiazol-2-yl)benzene-1,3-diol (MHY1556)
June Whan Park a, Young Mi Ha b, Kyung-mi Moon a, So-ra Kim a, Hyoung Oh Jeong a, Yun Jung Park c,
Hye Jin Lee c, Ji Young Park c, Yu Min Song c, Pusoon Chun d, Youngjoo Byun e, Hyung Ryong Moon c,
,
⇑
Hae Young Chung a,
⇑
a Molecular Inflammation Research Centre for Aging Intervention (MRCA), College of Pharmacy, Pusan National University, Kumjeong-Gu, Busan 609-735, Republic of Korea
b Department of Chemistry, Dong-A University, Hadan2-dong, Saha-gu, Busan 604-714, Republic of Korea
c Laboratory of Medicinal Chemistry, College of Pharmacy, Pusan National University, Busan 609-735, Republic of Korea
d College of Pharmacy, Inje University, Gimhae, Gyeongnam 621-749, Republic of Korea
e College of Pharmacy, Korea University, Chungnam 339-700, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, we have synthesized and studied de novo tyrosinase inhibitor, MHY1556, which showed
significantly better efficacy than other pre-existing tyrosinase inhibitors in vitro experiments. The IC50
value of MHY1556 was 0.50 lM which was significantly lower than that of kojic acid (IC50 = 53.95 lM),
which is a well-known tyrosinase inhibitor and was used as a positive control in this study. We predicted
the tertiary structure of tyrosinase, simulated the docking with compound MHY1556 and confirmed that
the compound strongly interacts with mushroom tyrosinase residues. Substitutions with a hydroxy
group at both R1 and R3 of the phenyl ring indicated that these groups play a major role in the high bind-
ing affinity to tyrosinase, especially through the hydrogen bonding interaction of the hydroxyl group at
R1 with GLY281. In addition, MHY1556 showed concentration-dependent inhibitory effects in melanin
Received 12 February 2013
Revised 28 April 2013
Accepted 9 May 2013
Available online 18 May 2013
Keywords:
Skin-whitening product
Melanogenesis
Tyrosinase inhibitor
4-(6,7-Dihydro-5H-indeno[5,6-d]
thiazol-2-yl)benzene-1,3-diol
Kojic acid
content assay where B16F10 melanoma cells were treated with
a-melanocyte stimulating hormone
(
a
-MSH), and also there is no significant cytotoxicity of this compound in cell viability assay conducted
in B16F10 melanoma cells. The tyrosinase activity assay results with MHY1556 also support its potent
inhibitory effects. Therefore, our data strongly suggest MHY1556 suppresses the melanogenesis via a
tyrosinase inhibitory effect.
Ó 2013 Elsevier Ltd. All rights reserved.
Human skin color is primarily determined by the production of
melanin, dark-colored pigments, found in the basal layer of the epi-
dermis.1 The amount of melanin production varies in different eth-
nic groups and determines their skin color.1 Melanin has some
protective effects on skin. In response to sun exposure the body
naturally produces more melanin in the skin and the skin color
darkens.1–3 Melanin is located in the outermost layer of skin and
absorbs UV radiation, which prevents UV radiation from causing
direct and indirect DNA damage to the skin.1–3 However, overpro-
duction of melanin is unwanted and there are several skin-whiten-
ing products that are commercially available for cosmetic purposes
to obtain lighter skin color.2,4 They can also be used for medical
purposes.2,4 A number of dermatological problems are character-
ized by overproduction and accumulation of melanin in skin.5,6
Such hyperpigmentation disorders include Café au lait macules,
ephelides (freckles), solar lentigo (age spots), and melisma.5
Most of skin-whitening products target melanogenesis, and
tyrosinase inhibitors are the most commonly used agents.2,3 Upon
exposure of the skin to UV lights, melanogenesis is greatly in-
creased by the activation of tyrosinase.2 Tyrosinase is a key enzyme
that plays a key role in production of melanin.1–3 It catalyses the
hydroxylation of
-DOPA) and the oxidation of
Most melanin-biosynthesis inhibitors are phenolic compounds
with a hydroxyl group, which are structurally similar to tyrosinase
substrate, tyrosine or
-DOPA.8 Acting as a rate-determining en-
zyme in melanogenesis, tyrosinase is often used as a main target
in research of skin-whitening products. As shown in Figure 1,
tyrosine, -DOPA, and dopaquinone are tyrosinase substrates and/
L
-tyrosine to form
L-3,4-dihydroxyphenylalanine
(L
L
-DOPA to DOPA quinone.7
L
L-
L
or products. These compounds all possess one or more hydroxyl
group, indicating that this group may play an important role in
binding to tyrosinase at the active site.
Currently, there are a number of tyrosinase inhibitors including
ascorbic acid, kojic acid, and polyphenol compounds such as
⇑
Corresponding authors. Tel.: +82 51 510 2815; fax: +82 51 513 6754 (H.R.M.);
tel.: +82 51 510 2814; fax: +82 51 518 2821 (H.Y.C.).
E-mail addresses: mhr108@pusan.ac.kr (H.R. Moon), hyjung@pusan.ac.kr (H.Y.
Chung).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.05.029

J. W. Park et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4172–4176
4173
H2
02
Tyrosinase
DOPA quinone
DOPA
GSH
02
Tyrosinase
leuco DOPA Chrome
GSH-DOPA
H2
DOPA chrome
CO2
Cysteinyl DOPA
quinone
DHI
(5,6-dihydroxyindole)
L-tyrosine
Melanin
Figure 1. Biosynthetic pathway for melanin production.
flavonols and coumarins.9 Several phenolic compounds which
showed significant tyrosinase inhibitory effects have been intro-
duced by our laboratory as well.10,11
benzothiazole product, compound 4 in 58% yield. Therefore, we
tried to synthesize MHY1556, our target compound, from com-
pound 4 via O-demethylation (Scheme 1). Treatment of compound
4 with BBr3 as a Lewis acid gave MHY1556 in a moderate yield. The
structures of MHY1556 and compound 4 were determined by 1H
and 13C NMR and low and high mass spectral analysis (Scheme 1).
In this study, we investigated in greater detail the bioactivities
of MHY1556 in vitro. As shown in Table 1, kojic acid and MHY1556
were found to inhibit mushroom tyrosinase activity in a concentra-
tion-dependent manner. The data showed that MHY1556
As our continuous efforts to find potent tyrosinase inhibitors, 4-
(6,7-dihydro-5H-indeno[5,6-d]thiazol-2-yl)benzene-1,3-diol
(MHY1556) with a hydroxyl group at both R1 and R3 of the phenyl
ring was designed. It was envisioned that compound 3 would be an
appropriate key intermediate for the preparation of MHY1556.
Compound 3 was prepared according to the procedures reported
by Kajino et al. with minor modification (Scheme 1).12 Direct cou-
pling reaction of compound 3 with 2,4-dihydroxybenzaldehyde in
the presence of Na2S2S5 as an oxidizing agent just afforded the
corresponding imine derivative instead of the cyclic product such
as benzothiazolidine or benzothiazole analog, whereas condensa-
tion of compound 3 with 2,4-dimethoxybenzaldehyde in the pres-
ence of Na2S2S5 produced the corresponding cyclized and oxidized
(IC50 = 0.50 lM) is a more potent inhibitor of mushroom tyrosinase
than kojic acid (IC50 = 53.95
lM), which was used as a reference
compound (Table 1).
Through a modeling of docking, we were able to detail the ter-
tiary structure of mushroom tyrosinase and simulate its docking
with MHY1556, supporting the hypothesis that MHY1556 interacts
Scheme 1. Synthesis of MHY1556. Reagents and conditions: (a) NH4SCN, benzoyl chloride, acetone, 2 N-NaOH; (b) –Br2, AcOH, 25% NH4OH; (c) KOH, 2-methoxyethanol, H2O;
(d) Na2S2O5, DMF; (e) BBr3, CH2Cl2.

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J. W. Park et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4172–4176
Table 1
Table 2
Comparing IC50 values of MHY1556 and kojic acid
Amino acid via docking simulation between tyrosinase and active compound
b
Sample
Conc (
l
M)
Tyrosinase
IC50
(lM)
Amino acid (Z <3)
Compound
inhibitiona (%)
HIS61
HIS85
PHE90
HIS94
Kojic acid
Kojic acid
Kojic acid
—
Kojic acid
Kojic acid
Kojic acid
Kojic acid
Kojic acid
Kojic acid
—
Kojic acid
Kojic acid
Kojic acid
Kojic acid
Kojic acid
Kojic acid
—
MHY1556
MHY1556
MHY1556
MHY1556
—
Kojic acid
10
20
30
40
50
5.44 2.50
12.81 5.08
12.71 6.12
37.73 5.40
48.15 1.44
53.95 2.45
HIS244
GLU256
HIS259
ASN260
HIS263
PHE264
ARG268
HIS279
MET280
GLY281
SER282
VAL283
ALA286
PHE292
HIS296
CU400
CU401
—
MHY1556
MHY1556
MHY1556
MHY1556
MHY1556
—
MHY1556
MHY1556
MHY1556
MHY1556
MHY1556
MHY1556
MHY1556
MHY1556
MHY1556
MHY1556
0.1
0.5
0.8
1
22.21 4.31
56.52 0.43
68.34 0.33
73.63 0.96
83.55 0.34
0.50 0.05
2
a
Values represent means S.E. of three experiments.
50% Inhibitory concentration (IC50).
b
with residues in the active site of tyrosinase. In our previous study,
we checked the Z-score of the 3D structure to validate the pre-
dicted structural model using QMEAN server (3A). The docking
simulation was successful with significant scores. The binding en-
ergy of MHY1556, as determined by AutoDock4.2 analysis, was
À7.03 kcal/mol and that of kojic acid was À4.20 kcal/mol (Fig. 2).
We searched for tyrosinase residues that would bind to
MHY1556. The most important residues were predicted to interact
with MHY1556 by AutoDock4.2 analysis, as shown in Table 2.
Additionally, we searched for hydrogen bonding interactions be-
tween tyrosinase and MHY1556 or kojic acid. The Met280 and
Gly281 residues of the tyrosinase were responsible for the
hydrogen bonding interactions with kojic acid and MHY1556,
respectively (Fig. 3). Probably, these residues might function as
key determinants for the efficacy of each inhibitor and have an
important effect on the binding affinity.
—
Kojic acid
Kojic acid
treatment doses of 2, 5, 10, 20 and 40 lM of MHY1556, cell viabil-
ity results were recorded as 97.87%, 94.98%, 98.11%, 95.6%, and
88.42%, respectively. These results indicate that MHY1556 is not
cytotoxic to B16F10 cells at the concentrations used in the follow-
ing melanin content assay.
To evaluate the inhibitory effect of MHY1556 on melanin pro-
duction, we measured the melanin content of B16F10 cells after
treating with MHY1556. As shown in Figure 5A, the melanin con-
tent in the cells treated with MHY1556 in the presence of
decreased concentration-dependently, showing 120.71% at
0.05 M, 100.85% at 0.1 M, 94.49% at 0.2 M, and 88.85% at
M, compared to the control group treated with -MSH alone
strong inhibitory effect on
a-MSH
To evaluate the cytotoxic effect of MHY1556, we used the mur-
ine B16F10 melanoma cell line (B16F10 cells). The results of cell
viability assay using an MTT kit are shown in Figure 4. At the
l
l
l
2
l
a
(163.78%). MHY1556 showed
a
B
A
Kojic acid
C
MHY1556
Figure 2. Computational structure prediction for mushroom tyrosinase and docking simulation with MHY1556 and kojic acid. Predicted 3D structure of mushroom
tyrosinase (A). The boxes indicate kojic acid and MHY1556 binding sites with tyrosinase residues. Kojic acid (B), which was used as a control compound, is shown in magenta
and MHY1556 (C) is shown in navy.

J. W. Park et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4172–4176
4175
A
B
Kojic acid
MHY1556
Figure 3. Possible hydrogen bonding interactions between tyrosinase residues and MHY1556 or kojic acid. We searched for hydrogen binding interactions between
tyrosinase and kojic acid (A) or MHY1556 (B) in the simulated docked structures, the green lines indicate hydrogen bonding interactions.
melanogenesis at concentrations that did not adversely affect the
cell viability. Tyrosinase activity assay was performed to measure
the inhibitory effect of MHY1556 on B16F10 cells. As shown in
Figure 5B, MHY1556 concentration-dependently decreased the
tyrosinase activity by 165.73% at 0.05
and 108.92% at 0.2 M, compared to the control group treated with
-MSH alone (205.70%). The inhibitory effect of MHY1556 was
much more potent than kojic acid; the inhibition of MHY1556 at
0.1 M was superior to that of kojic aicd at 30 M.
In this study, we synthesized, from 5-aminoindan, de novo
lM, 133.14% at 0.1 lM,
l
a
l
l
tyrosinase inhibitor, MHY1556, which showed a potent inhibitory
effect on melanogenesis. MHY1556 was found to be approxi-
mately 108-fold more potent than kojic acid which is often used
as
a positive reference compound. Docking simulation with
MHY1556 confirmed that the compound binds strongly to mush-
room tyrosinase and also suggested that Gly281 plays a key role
in determination of binding affinity of MHY1556 through hydro-
gen bonding interaction. Therefore, docking simulation results
imply that MHY1556 binds to the active site of tyrosinase, result-
ing in inhibiting tyrosinase activity. Due to the highly potent
Figure 4. Effect of MHY1556 on B16F10 cell viability. Cells were treated with
varying doses of MHY1556 (2–40 M) for 48 h and examined using an MTT assay.
Data are expressed as a percentage of the control.
l
Figure 5. Inhibitory effect of kojic acid and MHY1556 after treatment with 100 nM
a
-MSH in B16F10 cells 5A; Melanin contents were measured at 405 nM. Values represent
the mean S.E. 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
(30 M) or MHY1556 (0.05–0.2
<0.001 compared to the untreated control, ⁄⁄p <0.01 and ⁄⁄⁄p <0.001 compared to the group treated with 100 nM
a
-MSH. (B) Effect of the active compounds on tyrosinase activity: in the presence of 100 nM
M) for 24 h. Results are expressed as the percentage of control and each column represents the mean S.E. of three determinations. ###p
-MSH.
a-MSH, B16F10 cells were treated with varying doses of kojic acid
l
l
a

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J. W. Park et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4172–4176
inhibitory effect of MHY1556 on mushroom tyrosinase, further
experiments were conducted to evaluate the efficacy of
MHY1556 on B16F10 cells. The cell viability assay showed that
MHY1556 was non-cytotoxic to B16F10 cells at the concentra-
tions tested in the experiments of tyrosinase activity and melanin
content. Considering the cell viability assay result, MHY1556
effectively inhibited tyrosinase activity and melanogenesis with-
out adversely affecting cell viability. These results provide the
strongest evidence that the newly synthesized compound,
MHY1556 with a hydroxyl group at both R1 and R3 of the phenyl
ring might serve as a potent tyrosinase inhibitor. Based on these
findings, we suggest MHY1556 could be potentially utilized as a
new skin-whitening agent.
Supplementary data
Supplementary data associated with this article can be found, in
the
online
version,
at
http://dx.doi.org/10.1016/
j.bmcl.2013.05.029.
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Acknowledgements
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This work was supported by a Grant from the National Research
Foundation of Korea (NRF) funded by the Ministry of Education,
Science and Technology (No. 2009-0083538). We would like to
thank the Aging Tissue Bank for providing research materials for
the study.