A novel synthesized tyrosinase inhibitor: (E)-2-((2,4-dihydroxyphenyl) diazenyl)phenyl 4-methylbenzenesulfonate as an azo-resveratrol analog

Article abstract of DOI:10.1271/bbb.120547

We synthesized a novel series of (E)-2-((substituted phenyl)diazenyl)phenyl 4-methylbenzenesulfonate derivatives (2 and 3) and (E)-2-((substituted phenyl)diazenyl) phenol derivatives (4 and 5), and conducted an evaluation in order to determine their inhib

Full text of DOI:10.1271/bbb.120547

Biosci. Biotechnol. Biochem., 77 (1), 65–72, 2013
A Novel Synthesized Tyrosinase Inhibitor:
(E)-2-((2,4-Dihydroxyphenyl)diazenyl)phenyl 4-Methylbenzenesulfonate
as an Azo-Resveratrol Analog
1;
2
2
3
1
Sung Jin BAE,1; Young Mi HA, Jin-Ah KIM, Ji Young PARK, Tae Kwun HA,y Daeui PARK,
*
*
Pusoon CHUN,4 Nam Hee PARK,5 Hyung Ryong MOON,2 and Hae Young CHUNG
1;
1Molecular Inflammation Research Center for Aging Intervention (MRCA), College of Pharmacy,
Pusan National University, Kumjeong-gu, Busan 609-735, Republic of Korea
2Laboratory of Medicinal Chemistry, College of Pharmacy, Pusan National University,
Busan 609-735, Republic of Korea
3Department of Surgery, Busan Paik Hospital, College of Medicine, Inje University, Busan 614-735, Republic of Korea
4College of Pharmacy, Inje University, Gimhae, Gyeongnam 621-749, Republic of Korea
5Department of Nursing, College of Medicine, Inje University, Busanjin-gu, Busan 633-165, Republic of Korea
Received July 19, 2012; Accepted September 26, 2012; Online Publication, January 7, 2013
[doi:10.1271/bbb.120547]
We synthesized a novel series of (E)-2-((substituted
phenyl)diazenyl)phenyl 4-methylbenzenesulfonate de-
rivatives (2 and 3) and (E)-2-((substituted phenyl)dia-
zenyl)phenol derivatives (4 and 5), and conducted an
evaluation in order to determine their inhibitory effects
on mushroom tyrosinase, with the aim of discovering a
tyrosinase inhibitor. Most of the compounds (3–5)
exhibited higher inhibitory effects than kojic acid
(IC50 = 49.08 ꢀM), a representative tyrosinase inhibitor.
A novel synthesized compound, (E)-2-((2,4-dihydroxy-
phenyl)diazenyl)phenyl 4-methylbenzenesulfonate (3),
showed the best results with an IC50 of 17.85 ꢀM, and
showed competitive inhibition on Lineweaver-Burk
plots, as further confirmed by the docking results. In
addition, active compounds 3–5 were not cytotoxic to
cultured B16F10 cells at the concentrations tested, and
inhibited both tyrosinase and melanin synthesis. There-
fore the active compounds (3–5) might be considered
excellent candidates for use in the development of
therapeutic agents for diseases associated with hyper-
pigmentation.
(L-dopa), and oxidizes L-dopa to form dopaquinone.
The former reaction is the rate-limiting step in melanin
biosynthesis.2,3) This O-quinone, a highly reactive com-
pound, can undergo spontaneous polymerization to form
melanin.4)
Numerous naturally occurring and synthetic tyrosi-
nase inhibitors, including arbutin,5) ascorbic acid
derivatives,6) azeleic acid,7) hydroquinone,8) kojic
acid,9) polyphenolic compounds,10–12) retinoids,13) and
tetrahydroxystilbenes,14) have been reported, but most
of these inhibitors either do not have sufficient
potential activity to be of practical use, or are do not
meet safety regulations for use as medicines and
cosmetics. The clinical effectiveness of well-known
active tyrosinase inhibitors, such as kojic acid and
arbutin, has not yet been demonstrated.15,16) Due to its
cytotoxic and mitogenic properties, hydroquinone, a
widely used skin-lightening agent, is still controversial
with regard to biosafety.17) Hence it remains necessary
to search for and develop novel candidates demonstrat-
ing safe and potent activities that are devoid of side
effects.
Key words: tyrosinase inhibitor; resveratrol; azo-
resveratrol; docking analysis; (E)-2-((2,4-
dihydroxyphenyl)diazenyl)phenyl 4-methyl-
benzenesulfonate
Resveratrol, 3,5,40-trihydroxy-trans-stilbene, presents
naturally at high concentrations in grape seeds and
exhibits a wide range of biological and pharmacological
activities, including antifungal, anticarcinogenesis, anti-
aging, cardioprotective, and antioxidant effects.18–21)
Some hydroxystilbene compounds, including oxyres-
veratrol and resveratrol, can also inhibit tyrosinase
activity.22,23)
Hence we have conducted a search for easily
accessible biological substances, including polyphenolic
compounds, that can induce potent inhibition of
tyrosinase activity and potentially prevent abnormal
pigmentation.12,24) In addition, the interesting biological
activities of resveratrol have prompted us to synthesize
azo-resveratrol, and to evaluate its biological activities
and compare them with those of resveratrol (Fig. 1).
The enzyme tyrosinase (EC1.14.18.1), a multifunc-
tional, glycosylated, copper-containing metalloenzyme,
is the key enzyme involved in the biosynthesis of the
large biological pigment melanin.1) Melanin is formed by
a combination of enzymatically catalyzed and chemical
reactions. Tyrosinase exhibits monophenolase activity
and diphenolase activity. That is, it catalyzes two
divergent reactions of melanin biosynthesis, the hydrox-
ylation of a monophenol and the conversion of an O-
diphenol to the corresponding O-quinone. Thereby,
it converts tyrosine to 3,4-dihydroxy-phenylalanine
y
To whom correspondence should be addressed. Hae Young CHUNG, Tel: +82-51-510-2814; Fax: +82-51-518-2821; E-mail: hyjung@pusan.
ac.kr
These authors contributed equally to this work.
*

66
S. J. BAE et al.
OH
OH
OH
O
O
HO
HO
N
N
HO
OH
Resveratrol
OH
Azo-resveratrol
Kojic acid
Fig. 1. Chemical Structures of Resveratrol, Azo-Resveratrol, and Kojic Acid.
The organic layer was dried over anhydrous MgSO4, filtered, and
evaporated under reduced pressure. The resulting residue, which was
purified by silica gel column chromatography using hexane and ethyl
acetate (3.5:1) as the eluent, gave tosylated diazo compound 2 (135 mg,
64%), a yellow oil.
UV (MeOH) ꢁmax 282 nm; 1H NMR (500 MHz, CDCl3) ꢀ 7.64 (d,
2 H, J ¼ 8:5 Hz, 200-H, 600-H), 7.60 (d, 2 H, J ¼ 8:5 Hz, 2-H, 6-H), 7.56
(d, 1 H, J ¼ 8:5 Hz, 30-H), 7.45–7.47 (m, 2 H, 40-H, 60-H), 7.36–7.32
(m, 1 H, 40-H), 7.02 (d, 2 H, J ¼ 8:5 Hz, 3-H, 5-H), 6.91 (d, 2 H,
J ¼ 8:0 Hz, 300-H, 500-H), 5.44 (s, 1 H, 400-OH), 2.22 (s, 3 H, CH3);
13C NMR (100 MHz, CDCl3) ꢀ 159.2 (C400), 147.2, 146.9. 145.5,
145.3, 132.3 (C1), 131.4 (C50), 129.8 (C3, C5), 128.6 (C2, C6), 128.0
(C30), 125.8 (C200, C600), 124.9 (C40), 117.5 (C60), 115.6 (C300, C500),
21.8 (4-CH3); HRMS (ESI) m=z C19H15N2O4S ðM ꢁ HÞꢁ calcd
367.0753, obsd 367.0746.
The aim of the current study was to synthesize
phenolic azo compounds as novel tyrosinase inhibitors,
(E)-2-((substituted phenyl)diazenyl)phenyl 4-methyl-
benzenesulfonate derivatives (2 and 3) and (E)-2-
((substituted phenyl)diazenyl)phenol derivatives (4 and
5), in order to study their potency as tyrosinase
inhibitors and the kinetic mechanical parameters of
inhibition, and to study their effects on melanin
production and tyrosinase activity in the B16 murine
melanoma cell line. In addition, using our compounds,
we simulated the docking of mushroom tyrosinase.
These results indicate that the affinity of the compounds
for binding with tyrosinase is higher than that of kojic
acid, used as control. Docking simulation suggested that
the mechanism of compounds by Glu258, His261, and
Asp276 showed possible hydrogen bonding interactions.
The results should provide a basis for development of
new effective skin-whitening agents with fewer adverse
side effects.
(E)-2-((2,4-Dihydroxyphenyl)diazenyl)phenyl 4-methylbenzenesul-
fonate (3). Compound 1 (150 mg, 0.57 mmol) was converted to
compound 3 (74 mg, 34%), a yellow oil, by a procedure similar to that
used in the preparation of compound 2.
UV (MeOH) ꢁmax 288 nm; 1H NMR (500 MHz, CD3OD) ꢀ 7.63
(dd, 1 H, J ¼ 1:6, 8.0 Hz, 30-H), 7.55 (d, 2 H, J ¼ 8:0 Hz, 2-H, 6-H),
7.55 (d, 1 H, J ¼ 9:0 Hz, 600-H), 7.47 (td, 1 H, J ¼ 1:5, 8.0 Hz, 50-H),
7.42–7.39 (m, 2 H, 40-H, 60-H), 7.13 (d, 2 H, J ¼ 8:0 Hz, 3-H, 5-H),
6.52 (dd, 1 H, J ¼ 2:5, 9.0 Hz, 500-H), 6.33 (d, 1 H, J ¼ 2:5 Hz, 300-H),
2.23 (s, 3 H, 4-CH3); 13C NMR (100 MHz, CD3OD) ꢀ 164.2 (C400),
156.8 (C200), 146.2 (C10), 144.9 (C4), 143.2, 134.8 (C50), 133.3, 131.5,
130.3 (C600), 129.6 (C3, C5), 128.4 (C2, C6), 128.0 (C30), 124.4 (C40),
117.2 (C60), 109.5 (C500), 102.8 (C300), 20.4 (4-CH3); HRMS (ESI) m=z
Materials and Methods
Experimental.
General. Melting points are uncorrected. 1H and 13C NMR spectra
were recorded on Varian Unity INOVA 400 and Varian Unity AS 500
instruments (Varian, Midland, ON). Chemical shifts are reported with
reference to the respective residual solvent or deuterated peaks (ꢀH
3.30 and ꢀC 49.0 for CD3OD, ꢀH 7.27 and ꢀC 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 of the reactions described below were
performed under an argon or a nitrogen atmosphere, and were
monitored by TLC. All anhydrous solvents were distilled over CaH2 or
Na/benzophenone prior to use.
C
19H15N2O5S ðM ꢁ HÞꢁ calcd 383.0702, obsd 383.0707.
(E)-2-((4-Hydroxyphenyl)diazenyl)phenol (4).26,27) Tosylated diazo
compound 2 (52 mg, 0.14 mmol) was dissolved in 5% KOH w/w
solution (1 mL), and the reaction mixture was refluxed for 1 h.
Following the addition of 5% aqueous HCl solution for adjustment of
the pH to 5–7, the reaction mixture was partitioned between ethyl
acetate and water. The organic layer was dried over anhydrous MgSO4,
filtered, and concentrated under reduced pressure. To purify the
resulting residue, silica gel column chromatography was performed
using hexane and ethyl acetate (6.5:1), which afforded diazo compound
5 (28 mg, 92%), a red solid.
Melting point = 113–115 ꢀC; UV (MeOH) ꢁmax 270 nm; 1H NMR
(400 MHz, CD3OD) ꢀ 7.81 (dd, 1 H, J ¼ 1:6, 8.0 Hz, 3-H), 7.78 (d,
2 H, J ¼ 8:8 Hz, 20-H, 60-H), 7.29 (td, 1 H, J ¼ 2:0, 8.0 Hz, 5-H), 7.01
(td, 1 H, J ¼ 1:6, 8.0 Hz, 4-H), 6.94 (dd, 1 H, J ¼ 1:2, 8.4 Hz, 3-H),
6.91 (d, 2 H, J ¼ 8:8 Hz, 30-H, 50-H); 13C NMR (100 MHz, CD3OD) ꢀ
161.2 (C40), 154.5 (C1), 144.4 (C10), 137.5 (C2), 132.1 (C5), 130.7
(C3), 124.1 (C20, C60), 119.7 (C4), 117.6 (C6), 115.9 (C30, C50).
(E)-4-((2-Hydroxyphenyl)diazenyl)benzene-1,3-diol (5).26,28) Tosy-
lated diazo compound 3 (43.8 mg, 0.11 mmol) was converted to
compound 5 (8.0 mg, 30%), an orange solid, by a procedure similar to
that used in the preparation of compound 4.
Melting point = 130.4–133.1 ꢀC; UV (MeOH) ꢁmax 282 nm;
1H NMR (500 MHz, CD3OD) ꢀ 7.72 (dd, 1 H, J ¼ 1:5, 8.0 Hz, 60-
H), 7.62 (d, 1 H, J ¼ 9:0 Hz, 5-H), 7.28 (td, 1 H, J ¼ 1:5, 8.5 Hz, 40-H),
7.01–6.96 (m, 2 H, 30-H, 50-H), 6.50 (dd, 1 H, J ¼ 2:5, 9.0 Hz, 6-H),
6.34 (d, 1 H, J ¼ 2:5 Hz, 2-H); 13C NMR (100 MHz, CD3OD) ꢀ 163.2
(C1), 157.6 (C3), 152.6 (C20), 136.1 (C10), 131.5 (C4), 131.2 (C40),
131.0 (C60), 125.2 (C5), 119.9 (C50), 117.5 (C30), 109.1 (C6), 103.2
(C2) (Table 1).
2-Aminophenyl 4-methylbenzenesulfonate (1).25) Triethylamine
(3.8 mL and 27.26 mmol) and p-toluenesulfonyl chloride (5.24 g,
27.48 mmol) were added subsequently to a stirred solution of 2-
aminophenol (2.00 g, 18.33 mmol) in anhydrous THF (30 mL) at 0 ꢀC,
and the reaction mixture was stirred at the same temperature overnight.
Following partitioning of the reaction mixture between ethyl acetate
and water, the organic layer was dried over anhydrous MgSO4, filtered,
and evaporated. The resulting residue was purified by silica gel column
chromatography using hexane and ethyl acetate (6:1) as eluent to give
O-tosyl product (4.80 g, 99%), an orange solid.
Melting point 100.0–101.5 ꢀC; 1H NMR (500 MHz, CDCl3) ꢀ 7.77
(d, 2 H, J ¼ 8:5 Hz, 2-H, 6-H), 7.32 (d, 2 H, J ¼ 8:0 Hz, 3-H, 5-H),
7.01 (t, 1 H, J ¼ 7:5 Hz, 40-H), 6.79 (d, 1 H, J ¼ 8:0 Hz, 60-H), 6.71 (d,
1 H, J ¼ 7:5 Hz, 30-H), 6.59 (td, 1 H, J ¼ 1:5, 7.5 Hz, 50-H), 3.83 (s,
2 H, NH2), 2.45 (s, 3 H, 4-CH3); 13C NMR (100 MHz, CDCl3) ꢀ 145.8
(C10), 140.0 (C4), 137.1 (C20), 132.8 (C1), 130.1 (C3, C5), 128.7 (C2,
C6), 128.0 (C50), 123.1 (C60), 118.5 (C40), 117.4 (C30), 22.0 (4-CH3).
(E)-2-((4-Hydroxyphenyl)diazenyl)phenyl 4-methylbenzenesulfo-
nate (2). Tosylate 1 (150 mg, 0.57 mmol) was dissolved in a mixed
solvent of THF (2 mL) and 1 M hydrochloric acid (1.5 mL). The
solution was cooled to a temperature of 0–5 ꢀC and diazotized by drop-
wise addition of sodium nitrite (47.2 mg, 0.68 mmol). After stirring for
30 min, the diazonium salt solution that formed was added in drop-wise
Materials. Tyrosinase (EC1.14.18.1) from mushroom, L-tyrosine
(L-Tyr), resveratrol (3,5,40-trihydroxy-trans-stilbene), kojic acid
[5-hydroxy-2-(hydroxymethyl)-4H-pyran-4one], and ꢂ-MSH (alpha-
Melanocyte Stimulating Hormone) were purchased from Sigma (St.
Louis, MO). All other chemicals and solvents were of analytical grade.
fashion to
a
pre-cooled (0–5 ꢀC) solution of phenol (64.3 mg,
0.68 mmol) in 1 M NaOH (1.5 mL, 1.5 mmol). The reaction mixture
was stirred for 3 h at 0–5 ꢀC, followed by extraction with ethyl acetate.

A Novel Synthesized Tyrosinase Inhibitor
67
Table 1. Substitution Pattern and Inhibitory Effects on Mushroom
Tyrosinase of the (E)-2-((Substituted phenyl)diazenyl)phenyl 4-Meth-
ylbenzenesulfonate and (E)-2-((Substituted phenyl)diazenyl)phenol
Derivatives
aqueous mushroom tyrosinase solution (1,000 units), and 50 mM
potassium phosphate buffer (pH 6.5) with and without test samples
(20 and 100 mM) were added to a 96-well plate in a total volume of
200 mL. With a microplate reader, the initial rate of dopachrome
formation from the reaction mixture was determined by the increase in
absorbance at a wavelength of 492 nm per min (ꢀOD492/min). The
Michaelis constant (Km), the maximal velocity (Vmax), and the inhibitor
R1
R2
constant (Ki) of tyrosinase activity were determined using
a
Lineweaver-Burk plot at varying concentrations of L-tyrosine.31) Due
to competitive inhibition by the compounds together with substrate
inhibition by L-tyrosine, reaction kinetics required modification of
the Michaelis-Menten equation. All experiments were performed in
triplicate.
N
O
N
R3
R4
Tyrosinase
inhibitionb (%)
Murine tyrosinase activity assay. Measurement of the rate of
L-DOPA oxidation was done to estimate tyrosinase activity.30) Cells
were plated in 24-well dishes at a density of 5 ꢂ 104 cells/mL. B16
cells were incubated in the presence and the absence of 100 nM
ꢂ-MSH, followed by treatment for 24 h with various concentrations of
compounds 3–5 (0–10 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 centrifuged for clarification at 12,000 rpm for 30 min
at 4 ꢀC.
Compound
R1
R2
R3
R4
2
OH
OH
OH
OH
H
H
H
H
H
OH
H
aTs
Ts
H
34:03 ꢃ 8:52
88:84 ꢃ 3:03
82:50 ꢃ 1:68
75:22 ꢃ 1:08
56:16 ꢃ 0:85
3
4
5
OH
H
Kojic acid
Values indicate means ꢃ SE for three determinations.
ap-Toluenesulfonyl
bTyrosinase inhibition was measured using L-tyrosine as substrate at 50 mM.
An 8 mL sample of the supernatant and 20 mL of L-DOPA (2 mg/
mL) were placed in a 96-well plate, and the absorbance was read at
492 nm every 10 min for 1 h at 37 ꢀC using an enzyme-linked
immunosorbent serologic assay (ELISA) plate reader. Final activity
was expressed as ꢀOD/min for each condition. The results of three
determinations are shown.
Methods.
Cell cultures. B16F10 mouse melanoma cells, obtained from the
Korean Cell Line Bank, were cultured in Dulbecco’s Modified Eagle’s
Medium (DMEM; Gibco, Carlsbad, CA) containing 10% fetal bovine
serum (FBS; Gibco) and penicillin/streptomycin (100 IU/50 mg/mL) in
an incubator under a humidified atmosphere containing 5% CO2/95%
air controlled at 37 ꢀC. The cells were cultured in 24-well plates for use
in assays for cell viability, melanin quantification, and enzyme activity.
Cell viability assay. To determine cell viability, a colorimetric cell
proliferation assay was performed using 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) for measurement of mito-
chondrial activity in the viable cells. This method is based on the
conversion of MTT (Sigma) to MTT-formazan crystals by a mitochon-
drial enzyme by a previously described method.29) Briefly, cells were
seeded at a density of 3 ꢂ 104 cells in a Corning 48-well plate and
allowed to adhere overnight. The culture medium was then replaced
with fresh serum-free DMEM. MTT was freshly prepared at 5 mg/mL
in phosphate-buffered saline (PBS). Aliquots of 500 mL of MTT stock
solution were added to each well, followed by incubation of the plate in
an incubator at 37 ꢀC under a humidified atmosphere containing 5%
CO2 for 4 h. Then, following removal of the medium, 500 mL of EtOH-
DMSO (solution of a 1:1 mixture) was added to each well in order to
dissolve the formazan. After 10 min, spectrophotometric measurement
of the optical density of each well was performed using a 560 nm filter.
The results for three determinations are shown Fig. 4.
Melanin content assay. Melanin content was used as an index of
melanogenesis. The method described by Bilodeau et al., with
modifications, was used to determinate melanin content.32) In the
present study, the amount of melanin was used as an index of
melanogenesis. B16 cells (5 ꢂ 104) were transferred to 24-well dishes
and incubated in the presence and absence of 100 nM ꢂ-MSH. The cells
were then incubated for 24 h with various concentrations of compounds
3, 4, and 5 (0–10 mM). After washing twice with PBS, samples were
dissolved in 100 mL of 1 N NaOH. They were then incubated at 60 ꢀC
for 1 h and mixed in order to solubilize the melanin. The absorbance at
405 nm was compared with a standard curve for synthetic melanin. The
results of three determinations are shown.
Homology modeling of tyrosinase. A three-dimensional model of
Lentinula edodes (mushroom) tyrosinase, comprising 618 amino acids,
was built using SWISS-MODEL based on homology modeling.33) The
SWISS-MODEL program automatically provides an all-atom model
using alignments between the query sequence and known homologous
structures. According to the known homologous structure of tyrosinase
˚
from the Protein Data Bank (PDB), 1BT3 (2.5 A resolution) was used
as structural template (15% sequence identity and e-value of 0.00e-1).
By homology search between the target sequence and the total protein
sequences in the PDB, the automated model of SWISS-MODEL
automatically selected the 1BT3 structure as the best template. In order
to validate the quality of the predicted structure model, we evaluated
the Z-value using the QMEAN server (http://swissmodel.expasy.org/
qmean). To prepare a docking simulation, we performed deletion of
solvent, addition of hydrogen, addition of charge, and replacement of
the incomplete side chain of the structure model using the Chimera
program (http://www.cgl.ucsf.edu/chimera/). For addition of charge,
we used the charge data of AMBER ff99SB for standard residues.
In silico docking of tyrosinase and target compounds. For docking
simulation, we used the AutoDock4.2 program. Among the many tools
available for in silico protein-ligand docking, AutoDock4.2 is the most
commonly used due to its automated docking capability.34) To define
the docking pocket, we used a set of predefined active sites which got
from the tyrosinase structure of Bacillus megaterium in complex with
kojic acid as the inhibitor. We attempted to perform a docking
simulation of tyrosinase and the compounds, with kojic acid as the
reference inhibitor. To prepare of compounds for the docking
simulation, we performed the following steps: (1) conversion of 2D
structures into 3D structures, (2) calculation of charges, and (3) the
addition of hydrogen atoms using the ChemOffice program (http://
www.cambridgesoft.com).
Assay of tyrosinase inhibitory activity. Mushroom tyrosinase was
used as the enzyme source for the entire study. Tyrosinase activity was
determined by a previously described method, with minor modifica-
tions.30) Briefly, 20 mL of an aqueous solution of mushroom tyrosinase
(1,000 units) was added to a 96-well microplate (Nunc, Roskilde,
Denmark), in a 200 mL assay mixture containing 1 mM L-tyrosine and
50 mM phosphate buffer (pH 6.5). The assay mixture was incubated at
25 ꢀC for a period of 30 min. Following incubation, spectrophotometric
measurement was done at 492 nm (OD492) using a microplate reader
(Hewlett Packard, Palo Alto, CA) in order to determine the amount of
dopachrome produced in the reaction mixture. IC50 refers to the
concentration of a substance that inhibits a standard response by 50%
of the activity. The IC50 value is derived experimentally from the X-
axis on an inhibitor concentration versus the product formation curve,
and is determined from the alignment of the dose-response curve on
the dependent Y-axis. In the present study, dose-dependent inhibition
experiments were performed in triplicate in order to determine the IC50
values of the test compounds. Determination of log-linear curves and
their equations was based on the average percentage inhibition for
three doses. Average results for three determinations are shown.
Kinetic analysis of the inhibition of tyrosinase. Varying concen-
trations (0.25 to 2 mM) of L-tyrosine, a tyrosinase substrate, 20 mL of an

68
S. J. BAE et al.
OH
R
OH
R
NH2
NH2
OH
a
b
c
O
N
O
N
O
S
N
N
O
S
O
OH
O
2: R = H
3: R = OH
4: R = H
5: R = OH
1
Scheme 1. Synthesis of Target Compounds.
Reagents and conditions: (a) p-TsCl, Et3N, THF, room temperature, overnight, 99%; (b) NaNO2, 1 M HCl, THF, 0–5 ꢀC, 30 min, and then
phenol, 1 M NaOH, 0–5 ꢀC, 3 h, 64% for 2, 1,3-dihydroxybenzene, 1 M NaOH, 0–5 ꢀC, 3 h, 34% for 3; (c) 5% KOH, reflux, 1 h, 92% for 4, 4 h,
30% for 5.
Shared pharmacophore of the target compounds. Pharmacophore is
an ensemble of ligand features that is necessary for interaction with a
specific receptor in any biological response.35) Because three of the
compounds had similar chemical structures and kinetic responses for
tyrosinase inhibition, we searched the pharmacophore of the target
compounds.
A pharmacophore model was generated using the
LigandScout 3.0 program.36) On the basis of the type of atom, the
chemical features of the target compounds were defined according to
various pharmacophore elements, including hydrogen bond acceptor,
hydrogen bond donor, positive ionizable area, negative ionizable area,
hydrophobic interactions, and aromatic ring. Specifically, we searched
shared pharmacophores by alignment of the predicted pharmacophores
of the target compounds.
Statistical analysis. Inhibition of tyrosinase activity was expressed
as a percentage of inhibition based on 100 ꢁ ½ðAꢄ100Þ=Bꢅ, where
A = OD492 with a test sample and B = OD492 without a test sample.
The data collected had a mean standard error (n ¼ 3). One-factor
analysis of variance (ANOVA) followed by the Fisher’s protected least
significant difference post hoc test was used to determine statistical
significance of differences among groups. Values of ꢄ p < 0:05 were
considered statistically significant.
Fig. 2. Dose-Dependent Inhibitory Effects of Compounds 3, 4, and 5
against Mushroom Tyrosinase.
L-Tyrosine was used as substrate for the measurement of
tyrosinase activity. Compounds 3 (diamonds), 4 (rectangles), and
5 (triangles) were found to be inhibitors of tyrosinase. The effects of
various concentrations of compounds (5–100 mM) on tyrosinase
activity are represented as % inhibition and mean ꢃ SE of three
determinations.
Results
Synthesis of azo compounds (1–5)
A strategy for the synthesis of diaryl azo compounds
is outlined in Scheme 1. It was envisioned that azo
compounds can be synthesized from the aryl diazonium
ion, obtained from the reaction of an aniline derivative
with sodium nitrite under acidic conditions and phenolic
analogs. 20-Hydroxyaniline was selected as the starting
material for the preparation of diazonium salt. In order
to prevent side reactions, an appropriate group must be
used protect the hydroxyl group. Because the diazotiza-
tion reaction proceeds under acidic conditions, acetyl
and silyl, which are unstable in acidic conditions, cannot
be used as a protecting group for the hydroxyl group on
a phenolic compound. Of ether and sulfonate function-
alities, which are stable under acidic conditions, sulfo-
nate was chosen, because in case of protection with ether
functionality, extremely strong acidic conditions were
required in order to deprotect the bond after formation of
the diaryl azo compound. According to the literature,37)
by the proper choice of a base, selective O-tosylation in
the presence of a free amine or N-tosylate in the
presence of a free hydroxyl is possible at good yields.
Treatment of 20-hydroxyaniline with p-TsCl in the
presence of triethylamine in THF gave O-tosylate 2, at
a yield of 99%, a sulfonyl-protected aniline, which in
turn was diazotized with sodium nitrite under acidic
conditions to afford diazonium salt. The reaction of
diazonium salt with phenolic compounds, including
phenol and 2,4-dihydroxybenzene, under weak basic
conditions resulted in the generation of 4-hydroxyphenyl
and 2,4-dihydroxyphenyl azo compounds 2 and 3
respectively, yields of 64% and 34%, indicating that
the para-position of the hydroxyl group on an aromatic
ring is more nucleophilic than its ortho- or meta-
position. Removal of the tosyl group was accomplished
by refluxing compounds 2 and 3 in 5% KOH aqueous
solution, affording 4-hydroxyphenyl and 2,4-dihydro-
xyphenyl azo compounds 4 and 5 respectively.
Inhibitory effects of compounds 2, 3, 4, and 5 on
mushroom tyrosinase
These (E)-2-((substituted phenyl)diazenyl)phenyl 4-
methylbenzenesulfonate and (E)-2-((substituted phe-
nyl)diazenyl)phenol derivatives showed strong tyrosi-
nase inhibition as compared to that of kojic acid, used as
positive control when the enzymatic reactions used
L-tyrosine as substrate. The results of the inhibition of
tyrosinase using these compounds are shown in Table 1.
Our results indicate that three out of the four compounds
(compounds 2, 3, 4, and 5) showed significant inhibition
activity of mushroom tyrosinase greater than that of
kojic acid. As shown in Fig. 2, active compounds 3, 4,
and 5 induced dose-dependent reductions in tyrosinase
activity (5–100 mM). In particular, novel compound 3
showed the most potent inhibitory activity, with an IC50

A Novel Synthesized Tyrosinase Inhibitor
69
Fig. 3. Effects on Mushroom Tyrosinase Activity and Determination of the Inhibitory Mechanism.
The equation for the Lineweaver–Burk plot is 1=V ¼ Km=Vmax ꢂ 1=½Sꢅ þ 1=Vmax, and the modified Michaelis–Menten equation is
1=Vmax ¼ 1=Kmð1 þ ½Iꢅ=KiÞ, where V is the reaction velocity, Km is the Michaelis–Menten constant, Vmax is the maximum reaction velocity, [S]
is the substrate concentration, [I] is the inhibitor concentration, and Ki is the inhibition constant.
Table 2. Kinetic Analysis of Active Compounds
to determine whether the inhibitors would adversely
induce B16F10 melanoma cell death. A cell viability
a
c
IC50
(mM)
Type of
inhibitionb
Km
Kic
Compound
assay using MTT was done to estimate cytotoxic effects,
as shown in Fig. 4A. The results indicate that these
compounds showed no significant cytotoxic effect in
B16F10 melanoma cells at any of the concentrations
tested, indicating that these compounds are relatively
non-cytotoxic under the experimental conditions used
for the examinations of melanin content and tyrosinase
activity.
(mM)
(mM)
2
200:77 ꢃ 2:75
17:85 ꢃ 0:17
44:11 ꢃ 0:88
48:29 ꢃ 0:59
49:08 ꢃ 0:36
59:80 ꢃ 0:98
Competitive
Competitive
Competitive
Competitive
—
1.19
0.93
1.74
1.26
—
27.60
54.40
14.17
26.67
—
3
4
5
Kojic acid
Resveratrol
—
—
—
Lineweaver-Burk plots for inhibition of the compounds on 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, for three
independent tests using various concentrations of L-tyrosine substrate.
a50% inhibitory concentration (IC50).
bLineweaver-Burk plot for mushroom tyrosinase.
cValues were measured at 20 mM of active compounds.
Effects of compounds 3, 4, and 5 on melanin
production in B16F10 melanoma cells
The inhibitory potency of active compounds 3, 4, and
5 against melanin synthesis, we quantified the melanin
contents of cultured B16F10 cells in the presence of 1, 5,
and 10 mM compounds 3–5. ꢂ-MSH, which stimulates
melanogenesis, was used at 100 nM as negative control
and its effect on melanin production was compared with
that of the active compounds in the presence of 100 nM
ꢂ-MSH. Melanin synthesis in the B16F10 melanoma
cells treated with these compounds showed a significant
reduction. The inhibitory influence of these compounds
on melanin content is shown in Fig. 4B. The percentages
of melanin content of compounds 3–5 in the cells were,
respectively, 157.54%, 171.0%, 153.92% at 1 mM,
132.61%, 171.67%, 132.22% at 5 mM, and 131.73%,
113.28%, 121.58% at 10 mM, as compared to 100 nM
ꢂ-MSH alone (269.87%) and the control (100%). Thus
data from the cell viability test (Fig. 4A) indicate
effective suppression of melanin production by active
compounds 3, 4, and 5, with less or no cytotoxicity.
value of 17.85 mM, while those of kojic acid and
resveratrol, used as positive controls, were 49.08 and
59.80 mM respectively (Table 2).
Active compounds inhibitory of mushroom tyrosinase
with L-tyrosine as substrate
Because active compounds were investigated to
identify very strong tyrosinase inhibition activity, analy-
sis of kinetic behavior during the oxidation of L-tyrosine
was done by Lineweaver-Burk plot assay (Fig. 3). Plots
of the initial rates of tyrosinase activity in the presence of
increasing concentrations of L-tyrosine yielded a family
of straight lines of different slopes intersecting one point
on the Y axis. Because the concentration of L-tyrosine
substrate varied, the Km value for tyrosinase showed a
dose-dependent increase without any change in the Vmax
value, regardless of the concentration of compounds 2–5,
indicating that (Table 2). Many tyrosinase inhibitors
have been foune to be competitive inhibitors by chelating
the copper in the active site of the enzyme.38,39) Hence we
assumed that compounds 2–5 can induce inhibition in a
similar manner, but further study is necessary in order
to support this assumption.
Inhibitory effects of tyrosinase activity in B16F10
melanoma cells by active compounds
To determine the inhibitory mechanisms of the
inhibition of melanin biosynthesis by compounds 3, 4,
and 5, the effect of compounds 3–5 on cellular
tyrosinase activity in the B16F10 melanoma cells treated
with ꢂ-MSH was examined. As shown in Fig. 4C, we
found that compounds 3–5 had inhibitory effects on
ꢂ-MSH-stimulated tyrosinase activity. After 24 h of
incubation with compound 3, tyrosinase activity was
154.97% at 1 mM, 125.80% at 5 mM, and 109.24% at
Effects of active compounds 3, 4, and 5 on the
viability of B16F10 melanoma cells
We examined the cytotoxicity of compounds 3, 4, and
5, which exhibited strong tyrosinase inhibition, in order

70
S. J. BAE et al.
A
B
C
Fig. 4. Effects of Compounds 3, 4, and 5 on Cell Viability, Melanin Synthesis, and Tyrosinase Activity in B16F10 Melanoma Cells.
A, Cells were treated with various concentrations of compounds 3, 4, and 5 (0–50 mM), followed by examination by MTT assay. Each value
represents the mean ꢃ SE for three determinations. Data are expressed as % cell viability. B, Measurement of the amount of melanin was done as
described in ‘‘Materials and Methods.’’ C, B16 cells were harvested and tyrosinase activity was measured. Data are expressed as percentages of
control, and values are expressed as mean ꢃ SE for three determinations. ## p < 0:01 vs. the untreated control group; ꢄ p < 0:05, and ꢄꢄ p < 0:01
vs. the 100 nM ꢂ-MSH treatment group.
10 mM, while that of the control group treated with
ꢂ-MSH alone was 165.59%.
pounds, and kojic acid. Only the Asp262 residue of
tyrosinase was mainly responsible for the hydrogen
bonding interactions with kojic acid. However, of the
compounds, the Glu258 (4), His261 (4, 3, 2), and
Asp276 (3, 2) residues of tyrosinase were predicted as
hydrogen bonding interactions with the compounds
(Fig. 5B). The residues functioned, as key residues and
had an effect on binding affinity. Shared pharmacophore
results confirmed the hydrogen bonding interaction of
the docking simulation. The target compounds shared
one donor and three acceptors of hydrogen bonding
(Fig. 5C).
Molecular docking study of active compounds
To the predicted structure model, we evaluated the
Z-score of the 3D structure using the QMEAN server.
Because mushroom tyrosinase showed low sequence
identity (15%) with the template structure (3NQ1), the
Z-score for the predicted model was ꢁ4:8: However, we
evaluated the quality of each residue on the tyrosinase
structure (data not shown). The internal region, which
was bound with kojic acid and compounds, was more
reliable (blue) than the external region (red) of
tyrosinase. The estimated residue error was visualized
using a color gradient from blue (more reliable regions)
to red (potentially unreliable regions, estimated error
Discussion
Melanin plays an important role in the protection of
human skin from the harmful effects of UV radiation
from the sun. Melanin is also a determinant of a person’s
phenotypic appearance. Although it has mainly a photo-
protective function in human skin, accumulation of an
abnormal amount of melanin in different specific parts
of the skin, resulting in a greater number of pigmented
patches, can become an esthetic problem. Hyperpig-
mentation, such as melasma, ephelide, and lentigo in
human skin and enzymatic browning in fruits and
vegetables is not desirable. The occurrence of these
phenomena has encouraged researchers to seek potent
new safe tyrosinase inhibitors for use in anti-browning
in farm products40) and skin whitening for medicinal41)
and cosmetic purposes.42) Recent expansion of the
global market has resulted in an ever-growing demand
for new products for depigmentation, cosmeceutical, and
skin lightening purposes.43)
˚
above 3.5 A). The docking simulation was successful,
with significant scores. The binding energies of com-
pounds were ꢁ5:94 kcal/mol (2), ꢁ5:70 kcal/mol (3),
ꢁ5:10 kcal/mol (4), and ꢁ4:57 kcal/mol (5), and the
binding energy of kojic acid was ꢁ2:73 kcal/mol. The
docking score between the ligand and the receptor is
represented by various energy terms, such as electro-
static energy, van der Waals energy, and salvation
energy. In particular, the docking simulation confirmed
that the binding affinity of compounds 2–5 was higher
than that of kojic acid, which was used as control. In
addition, we determined the differences, in docking
position of kojic acid and the compounds. The distances
˚
between the copper ions and the compounds, were 6.7 A
˚
˚
˚
˚
(kojic acid), 7.5 A (2), 7.7 A (3), 6.3 A (4), and 8.0 A (5)
(Fig. 5A). In addition, we searched for hydrogen
binding interaction between tyrosinase and the com-

A Novel Synthesized Tyrosinase Inhibitor
71
A
C
B
Fig. 5. Docking Simulation between Tyrosinase and Target Compounds.
A, Yellow zone is the active site. Gold ball is the copper ion. Magenta compound is kojic acid, which was used as control compound. Blue,
yellow, cyan, and red compounds are 2, 3, 4, and 5 respectively. B, Possible hydrogen binding interaction between tyrosinase and target
compounds. C, The pharmacophore model was generated using the LigandScout 3.0 program. Red, green, and yellow phores represent a
hydrogen bond acceptor, a hydrogen bond hondor, and a hydrophoic region respectively.
In anticipation of finding an effective new substance
for skin whitening and the prevention of hyperpigmen-
tation, we synthesized (E)-2-((substituted phenyl)diaze-
nyl)phenyl 4-methylbenzenesulfonate and (E)-2-((sub-
stituted phenyl)diazenyl)phenol derivatives (Table 1),
and investigated the effects of our derivatives on
tyrosinase inhibition. Three compounds, 3–5, showed
more potent dose-dependent inhibitory activity against
mushroom tyrosinase than kojic acid and resveratrol,
used as positive controls (Fig. 2 and Table 2). We also
found that compound 3, which was newly synthesized,
gave the best results. A tosyl group as a protecting group
is compatible with both a diazotization reaction and a
sequential coupling reaction with phenolic compounds,
and is easily removed under reflux in basic conditions.
This finding might be helpful in the synthesis of azo-
resveratrol which is our next target compound. The
results of a kinetic study of inhibition of mushroom
tyrosinase by our compounds indicated competitive
inhibition of the enzyme, meaning that the compound
binds to the active site of the enzyme (Fig. 3).44) Due to
structural similarity to tyrosinase substrates, the inhib-
itory mode of phenolic compounds, the largest group
of tyrosinase inhibitors to date, is usually competitive
inhibition.45,46)
is higher than that of kojic acid which was used as
control. A docking simulation suggested that the
mechanism of compounds by Glu258, His261, and
Asp276 had possible hydrogen bonding interactions
(Fig. 5B). A shared pharmacophore model underlined
the features of the four compounds, which had one
donor and three acceptors of hydrogen bond (Fig. 5C).
The docking results a pharmacophore model, and this
clarified the key features required for optimal tyrosinase
inhibition. No cytotoxicity of active compounds 3–5 was
observed in cultured B16F10 melanoma cells up to
50 mM (Fig. 4A). Dose-dependent suppression of tyrosi-
nase activity and melanin synthesis was observed at
concentrations ranging from 1 to 10 mM (Fig. 4B and C),
the proposed mechanism of tyrosinase inhibition of
active compounds. Thus compounds 3–5 with an honest
effort inhibited, melanin production at concentrations
that did not significantly affect cell viability. In a
previous study, we examined the inhibitory effects of
kojic acid on tyrosinase activity and melanin contents in
ꢂ-MSH-treated B16F10 melanoma cells. We also found
that kojic acid did not inhibit tyrosinase activity and did
not affect the level of melanin contents in comparison
with the level in the group treated with ꢂ-MSH only.47)
These cell-based experiments indicate that compounds
3–5 are much better whitening agents than kojic acid.
Based on the results of the present study, we conclude
that a series of azo-resveratrol compounds were synthe-
sized, and that compounds 2–5 were competitive
inhibitors of mushroom tyrosinase. A docking simula-
A homology modeling program was used to predict
the tertiary structure of tyrosinase, and simulated
docking of mushroom tyrosinase was done using our
compounds (Fig. 5A). The results suggest that the
affinity of the compounds for binding with tyrosinase

72
S. J. BAE et al.
16) Halder RM and Richards GM, Skin Therapy Lett., 9, 1–3 (2004).
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tion with mushroom tyrosinase indicated that the bind-
ing affinities of compounds 2–5 were even higher than
that of kojic acid, and a shared pharmacophore model
indicated that the compounds had one donor and three
acceptors for the hydrogen bond in common. These
findings should give researchers help in developing
potent tyrosinase inhibitors. Three azo-resveratrol ana-
logs, 3–5, with potent mushroom tyrosinase inhibitory
activity also induced inhibition of cellular tyrosinase
activity and melanin production in murine B16F10
melanoma cells. In particular, newly synthesized {(E)-2-
((2,4-dihydroxyphenyl) diazenyl)phenyl 4-methylbenze-
nesulfonate} (3), showing an IC50 of 17:85 ꢃ 0:17 mM,
was more effective than kojic acid and resveratrol, and
hence might be considered an excellent candidate for
use as a therapeutic agent for anti-melanogenesis.
Further studies for the development of active com-
pounds 3–5 as promising therapeutic materials for the
control of abnormal skin pigmentation are warranted.
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