A newly synthesized, potent tyrosinase inhibitor: 5-(6-Hydroxy-2-naphthyl)- 1,2,3-benzenetriol

Article abstract of DOI:10.1016/j.bmcl.2010.06.087

In searching for new agents with a depigmenting effect, we synthesized a derivative of resveratrol, 5-(6-hydroxy-2-naphthyl)-1,2,3-benzenetriol (5HNB) with a potent tyrosinase inhibitory activity. 5HNB inhibited mushroom tyrosinase with an IC₅₀ value of 2.95 μM, which is more potent than the well-known anti-tyrosinase activity of kojic acid (IC₅₀ = 38.24). The results of the enzymatic inhibition kinetics by Lineweaver-Burk analysis indicated 5HNB inhibits tyrosinase non-competitively when l-tyrosine was used as the substrate. Based on the strong inhibitory action of 5HNB, it is expected that 5HNB can suppress melanin production in which tyrosinase plays the essential role. Our expectation was confirmed by the experimentations with B16 melanoma cells in which 5HNB inhibited melanin production. We propose that 5HNB might have skin-whitening effects as well as therapeutic potential for treating skin pigmentation disorders.

Full text of DOI:10.1016/j.bmcl.2010.06.087

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4882–4884
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A newly synthesized, potent tyrosinase inhibitor:
5-(6-Hydroxy-2-naphthyl)-1,2,3-benzenetriol
Jehun Choi a, Sung Jin Bae a, Young Mi Ha a, Jae Kyung No b, Eun Kyeong Lee a, Jun Sik Lee c, Suhee Song d,
Hyojin Lee d, Hongsuk Suh d, Byung Pal Yu e, Hae Young Chung a,
*
a Department of Pharmacy and Molecular Inflammation Research Center for Aging Intervention (MRCA), Pusan National University, Gumjung-gu, Busan 609-735, Republic of Korea
b Department of Cosmetology, Pusan Women’s College, Busan, Republic of Korea
c Department of Microbiology and Immunology, College of Medicine, BK21 Medical Science Education Center, Pusan National University, Busan 609-739, Republic of Korea
d Department of Chemistry and Chemistry Institute for Functional Materials, Pusan National University, Busan 609-735, Republic of Korea
e Department of Physiology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229-3900, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
In searching for new agents with a depigmenting effect, we synthesized a derivative of resveratrol,
Received 4 March 2010
Revised 14 June 2010
Accepted 15 June 2010
Available online 19 June 2010
5-(6-hydroxy-2-naphthyl)-1,2,3-benzenetriol (5HNB) with
5HNB inhibited mushroom tyrosinase with an IC50 value of 2.95
the well-known anti-tyrosinase activity of kojic acid (IC50 = 38.24). The results of the enzymatic inhibi-
tion kinetics by Lineweaver–Burk analysis indicated 5HNB inhibits tyrosinase non-competitively when
a
potent tyrosinase inhibitory activity.
M, which is more potent than
l
L
-tyrosine was used as the substrate. Based on the strong inhibitory action of 5HNB, it is expected that
Key words:
5HNB can suppress melanin production in which tyrosinase plays the essential role. Our expectation
was confirmed by the experimentations with B16 melanoma cells in which 5HNB inhibited melanin
production. We propose that 5HNB might have skin-whitening effects as well as therapeutic potential
for treating skin pigmentation disorders.
5-(6-Hydroxy-2-naphthyl)-1,2,3-
benzenetriol (5HNB)
Tyrosinase inhibitor
Melanin
Ó 2010 Elsevier Ltd. All rights reserved.
Tyrosinase inhibitors have become increasingly important in
medication,1 cosmetic industry2 and food industry3 due to decreas-
ing the excessive accumulation of pigmentation resulting from the
enzyme action.4–7 Although a number of tyrosinase inhibitors are
reported from both natural and synthetic sources, only a few of
them are used as skin-whitening agents, primarily due to various
safety concerns. For example, linoleic acid, hinokitiol, kojic acid,
all naturally occurring hydroquinones, and catechols inhibit en-
zyme activity but also exhibit harmful side effects.1 Thus, melanin
synthesis inhibitors may help treating localized hyper-pigmenta-
tion in humans. Epidermal and dermal hyper-pigmentation can
be dependent either on an increased number of melanocytes or
on melanogenic enzyme activity.8
key role in melanogenesis, tyrosinase is an attractive target in
the search for various kinds of depigmenting agents.12–15 Thus,
the present study has focused on tyrosinase activity and melanin
production.
The previous study showed 3,4-dihydroxyacetophenone and
4,40-dihydroxybiphenyl have strong anti-tyrosinase that inhibits
melanogenesis.16,17 Recently, our laboratory reported that naph-
thalene-type resveratrol analogs, 6-(3-hydroxyphenyl)-2-naphthol
and 4-(6-hydroxy-2-naphtayl-1,3-bezendiol inhibited tyrosinase
activity and reduced melanin in B16 cells, which indicated that
these compounds could be depigmentation reagents. Furthermore,
some hydroxystilbene compounds, including oxyresveratrol and
resveratrol, can also inhibit tyrosinase activity.11,18
Tyrosinase (monophenol monooxygenase, EC 1.14.18.1), a mul-
tifunctional type-3 copper-containing metalloenzyme, is a key en-
zyme in the undesirable browning of fruits and vegetables and in
the coloring of skin, hair, and eyes in animals.9 The tyrosinase is
a rate-limiting enzyme, catalyzing step in melanin biosynthesis,
which causes abnormal accumulation of melanin pigments.10 This
We have therefore searched for easily accessible biological sub-
stances, including polyphenolic compounds, that potently inhib-
ited tyrosinase activity and could potentially prevent abnormal
pigmentation.19,20 Therefore, we synthesized a series of novel res-
veratrol derivatives successfully through our constant efforts,
including 5-(6-hydroxy-2-naphthyl)-1,2,3-benzenetriol (5HNB), a
polyphenolic compound, to identify tyrosinase inhibitors. The syn-
thesis of compounds including 5HNB has been described in our
previous report.21
enzyme plays a role in the oxidation from L-tyrosine to 3,4-dihy-
droxyphenylalanine (L-DOPA) and from DOPA to DOPA quinone,
which is the initial step in melanin synthesis.11 Because of its
Here we report the characterization and evaluation of new syn-
thesized reveratrol analog, 5HNB. We investigated the inhibitory
effects of 5HNB on tyrosinase activity and melanin production in
* Corresponding author. Tel.: +82 51 510 2814; fax: +82 51 518 2821.
E-mail address: hyjung@pusan.ac.kr (H.Y. Chung).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.087

J. Choi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4882–4884
4883
Table 1
OH
OH
Inhibitory effects of hydroquinone, kojic acid, resveratrol, and 5-(6-
hydroxy-2-naphthyl)-1,2,3-benzenetriol (5HNB) on mushroom tyros-
inase activity
HO
Compounds
IC50 (l
M)a,b
HO
OH
OH
Hydroquinone
Kojic acid
Resveratrol
5HNB
33.48 ( 1.70)
38.24* ( 1.47)
55.61 ( 2.77)
2.95** ( 1.26)
5-(6-Hydroxy-2-naphthyl)-1,2,3-benzenetriol
(5HNB)
Hydroquinone
a
OH
IC50: refers to the concentration of compound that caused 50%
inhibition.
HO
O
b
Values are means of three experiments, standard deviation is
given in parentheses.
HO
*
P <0.05, compared to the hydroquinone.
O
**
OH
P <0.01, compared to the hydroquinone.
OH
Kojic Acid
Resveratrol
Figure 1. Comparison of chemical structures of 5-(6-hydroxy-2-naphthyl)-1,2,3-
benzenetriol (5HNB), hydroquinone, kojic acid, and resveratrol.
Figure 3. Lineweaver–Burk plot of 5HNB on mushroom tyrosinase. Data were
obtained as mean values of 1/V, inverse of the increase of absorbance at a wave-
length 492 nm per min (
concentrations of -tyrosine as a substrate. Inhibitors of the enzyme were 5HNB
0.5 M (triangle), 0.125 M (rectangle), and 5HNB (diamond shape). The modified
Michaelis–Menten equation is: 1/Vmax = 1/Km (1 + [I]/Ki). V denotes the velocity of
the reaction, S for -tyrosine concentration, Ki for inhibitor constant.
DA492/min), of three independent tests with different
L
l
l
L
Figure 2. Dose-dependent inhibitory effects of 5HNB (diamond shape) on tyros-
inase activity. Tyrosinase activity was measured using L-tyrosine as the substrate.
Values are mean S.E. of n = 3 determinations.
tively, after 24 h (Fig. 4A). We next tested whether 5HNB could in-
hibit melanin production in B16 melanoma cells after treatment
the B16 murine melanoma cell line. Furthermore, we report here
on the tyrosinase inhibitory potential of 5HNB for use in the treat-
ment of tyrosinase-related skin disorders.
with
25 M, 45.83% at 100
trol (the -MSH-treated group at 100%) (Fig. 4B). Similarly, 5HNB
reduced tyrosinase activity to 26.18% at 25 M, 14.09% at
100 M, and 2.05% at 400 M compared to controls (Fig. 4C).
In the present study, we found that 5HNB clearly reduced both
tyrosinase activity and melanin synthesis in a dose-dependent
manner. During our search for potent tyrosinase inhibitors, we
found that 5HNB was more effective for tyrosinase and melanin
synthesis than kojic acid and resveratrol (Table 1). A kinetics study
of 5HNB inhibition against mushroom tyrosinase showed that it
a
-MSH. 5HNB decreased melanin content to 50.67% at
l
l
M, and 34.67% at 400 M compared to con-
l
a
5HNB (Fig. 1) dose-dependently inhibited mushroom tyrosinase
l
by 7.53 6.43% at 0.5
2.5 M, and 86.12 5.47% at 5
tometric assay (Fig. 2). 5HNB thus inhibited the oxidation of L-
DOPA by mushroom tyrosinase with an IC50 value of 2.95 M.
l
M, 18.66 3.16% at 1
lM, 39.38 2.46% at
l
l
l
lM, as monitored by a spectropho-
l
Kojic acid, a well-known tyrosinase inhibitor, one of the most
popular tyrosinase inhibitors and it has been widely used as whit-
ening.22 Using hydroquinone, kojic acid, and resveratrol as positive
controls, these inhibitors exhibited IC50 values of 33.48, 38.24, and
behaves as a non-competitive inhibitor of the enzyme with
sine as the substrate.
L-tyro-
55.61 lM, respectively (Fig. 2 and Table 1). Therefore, 5HNB was a
more potent inhibitor of tyrosinase activity than kojic acid.
We next measured the inhibitory mechanism of 5HNB on
mushroom tyrosinase for oxidation of L-DOPA was determined
from Lineweaver–Burk double-reciprocal plots. Figure 3 showed
the double-reciprocal plots of the enzyme inhibited by 5HNB.
The result displayed that the plot of 1/V versus 1/[S] gave three
straight lines with different slopes, but intersected on the horizon-
tal axis. Accompanying the increase of the concentration of com-
pound, the value of Vmax descended but the values of Km
remained the same, which suggested that 5HNB was a non-com-
petitive inhibitor of the tyrosinase.
It would be interesting to know something about the mode of
the interaction between 5HNB and tyrosinase. 5HNB shows non-
competitive inhibition of tyrosinase, based on Michaelis–Menten
kinetics (Fig. 3), implying that 5HNB binds to the enzyme at a site
other than the substrate-binding site. Thus, it allosterically reduces
the maximum rate of the catalytic reaction (i.e., Vmax) without
affecting the apparent binding affinity of the enzyme for the sub-
strate. This 5HNB binding could change the shape or the structure
of tyrosinase, reducing substrate binding to the enzyme and inhib-
iting the enzymatic reaction.
Considering cytotoxicity, the cell permeability of 5HNB would
be important to determine. Although we have no quantitative
information on cell permeability, 5HNB can inhibit tyrosinase
5HNB was not toxic to B16 melanoma, producing cell viability
values of 98.32%, 98.08%, and 95.14% at 25, 50, and 100 lM, respec-

4884
J. Choi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4882–4884
Figure 4. The effect of 5HNB on cell viability, melanin production, and tyrosinase activity in B16 melanoma cells. Cells were treated with 5HNB (25–100 lM) and were
examined by the MTT assay (A). Melanin content was measured at 405 nm (B). The cells were harvested and tyrosinase activity was measured (C). Data are expressed as % of
control, and each column represents the mean S.E. of three determinations. This result is representative of three experiments that gave similar results. #P <0.01 versus
**
control, P <0.01 versus the group treated by
a-MSH alone.
and melanin synthesis in B16 melanoma cells, indicating sufficient
membrane permeability. In addition, 5HNB shows negligible cyto-
toxicity (Fig. 4A). 5HNB could dose-dependently inhibit tyrosinase
activity and melanin synthesis in B16 melanoma cells pretreated
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.06.087.