The tyrosinase-inhibitory effects of 2-phenyl-1,4-naphthoquinone analogs: importance of the (E)-β-phenyl-α,β-unsaturated carbonyl scaffold of an endomethylene type

Article abstract of DOI:10.1007/s00044-018-2267-9

In order to investigate the effect of the (E)-β-phenyl-α,β-unsaturated carbonyl scaffold of an endomethylene type on tyrosinase inhibition, 2-phenyl-1,4-naphthoquinone derivatives were synthesized by Michael addition of substituted benzenes to 1,4-naphtho

Full text of DOI:10.1007/s00044-018-2267-9

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-2267-9
ORIGINAL RESEARCH
The tyrosinase-inhibitory effects of 2-phenyl-1,4-naphthoquinone
analogs: importance of the (E)-β-phenyl-α,β-unsaturated carbonyl
scaffold of an endomethylene type
1
Sultan Ullah1 Jinia Akter1 Su J. Kim1 Jungho Yang1 Yujin Park1 Pusoon Chun2 Hyung R. Moon
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Received: 27 June 2018 / Accepted: 22 November 2018
© Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
In order to investigate the effect of the (E)-β-phenyl-α,β-unsaturated carbonyl scaffold of an endomethylene type on
tyrosinase inhibition, 2-phenyl-1,4-naphthoquinone derivatives were synthesized by Michael addition of substituted
benzenes to 1,4-naphthoquinone and subsequently an auto-oxidation. Most of the derivatives potently inhibited mushroom
tyrosinase and four derivatives, including 1c (IC50 = 22.00 1.63 μM), more potently inhibited mushroom tyrosinase than
kojic acid (IC50 = 37.86 2.21 μM). Cell-based assays using B16F10 cells (a melanoma cell line) showed 1c dose-
dependently suppressed melanin production and more potently inhibited tyrosinase activity than kojic acid. Docking
simulation results between 1c and tyrosinase and a kinetic study suggest that 1c is a competitive inhibitor that binds to the
active site of tyrosinase. These results support that anti-melanogenic effect of naphthoquinone derivatives results from their
tyrosinase inhibiting activity and the (E)-β-phenyl-α,β-unsaturated carbonyl scaffold of an endomethylene type can confer
tyrosinase-inhibitory activity.
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Keywords Tyrosinase inhibitor (E)-β-phenyl-α,β-unsaturated carbonyl 2-phenyl-1 4-naphthoquinone HMBC Docking
simulation
Introduction
1979). Tyrosinase is a metalloenzyme that contains two
copper(II) ions at its active site, and is involved in the
consecutive oxidation reactions responsible for converting
L-tyrosine into dopaquinone via L-dopa (Hearing and
Tsukamoto 1991). Dopaquinone is an unstable entity but is
transformed in vivo to eumelanin (a type of melanin
responsible for brown and black colours) by reactions
involving mercapto-containing substances, such as, cysteine
and glutathione or to phenomelanin, which is responsible
for yellow and red colours. Furthermore, eumelanin to
pheomelanin ratio in skin is primary determinant of skin
colour. Melanins protect skin cells from ultraviolet radia-
tion, but excessive accumulations of melanin can cause
hyperpigmentation, such as senile lentigines, melasma and
freckles and aesthetic problems.
Tyrosinase has attracted attention because it plays an
important role in the biosynthesis of melanin (Solano 2014),
a core determinant of the colours of hair, pupils and skin
and the browning of vegetables and fruits (Mayer and Harel
Electronic supplementary material Supplementary information:
The online version of this article (https://doi.org/10.1007/s00044-018-
2267-9) contains supplementary material, which is available to
authorized users.
* Pusoon Chun
pusoon@inje.ac.kr
* Hyung R. Moon
Over past several decades, a large number of naturally
occurring substances and synthetic compounds have been
tested with respect to their abilities to inhibit tyrosinase for
the purpose of discovering novel whitening agents.
Although many compounds have been found to inhibit
tyrosinase activity, relatively few are used for clinical pur-
poses due to a lack potency and side effects, which include
mhr108@pusan.ac.kr
1
College of Pharmacy, Pusan National University, Busan 609-735,
South Korea
2
College of Pharmacy and Inje Institute of Pharmaceutical Sciences
and Research, Inje University, Gimhae, Gyeongnam 50834, South
Korea

Medicinal Chemistry Research
Therefore, in this study, we investigated whether com-
pounds with an endomethylene type of the scaffold still
exhibited large tyrosinase inhibiting activity. For this pur-
pose, a series of 2-(substituted phenyl)-1,4-naphthoquinone
derivatives containing the endomethylene type of the (E)-β-
phenyl-α,β-unsaturated carbonyl scaffold were designed
and synthesized by a Michael reaction followed by an auto-
oxidation. The synthesized naphthoquinones were assessed
for tyrosinase-inhibitory activity using mushroom tyr-
osinase and their abilities to inhibit melanin biosynthesis,
and tyrosinase activity was examined using B16F10 cells (a
murine melanoma cell line).
Fig. 1 Exomethylene and endomethylene types of the (E)-β-phenyl-α,
β-unsaturated carbonyl scaffold
cytotoxicity and mutagenicity. The skin lightning agents
containing salicylhydroxamic acid, kojic acid (Gonçalez
et al. 2013, Ki et al. 2013, Kumar et al. 2013), magnesium
L-ascorbyl-2-phosphate, phenols (Jimbow 1991), hydro-
xyanisole, azelaic acid (Breathnach et al. 1989, Garcia-
Lopez 1989), tretinoin, mercury salts, corticosteroids
(Neering 1975), retinoid (Griffiths et al. 1993, Kimbrough-
Green et al. 1994), N-acetyl-4-S-cysteaminylphenol, arbutin
(hydroquinone-β-D-glucopyranoside), hydroquinone (HQ)
(Arndt and Fitzpatrick 1965, Fitzpatrick et al. 1966, Heil-
gemeir and Balda 1981, Kligman and Willis 1975) and
monobenzyl hydroquinone are generally used in cosmetics.
They are accepted globally, although having certain draw-
backs and side effects. The application of kojic acid has
been limited due to its carcinogenic effect and issue of
instability during storage. HQ is considered a toxic agent to
mammalian cells and revealed a series of unwanted effects
such as irritation, hypochromia, contact dermatitis, ochro-
nosis, burning and chestnut spots on the nails (Curto et al.
1999, Engasser 1984, Fisher 1983, Romaguera and Grimalt
1985). Due to side effects, the EU Cosmetic Regulation
completely bans the application of corticosteroids, HQ,
monobenzyl hydroquinone, mercury salts and tretinoin as
whitening agents. Although many tyrosinase inhibitors have
proved effective in in vitro studies, only a few of them
showed good results in clinical trials. Therefore, there is a
need to identify novel, safe tyrosinase inhibitors with
greater anti-melanogenic efficacies and better selectivities.
For the past several years, our laboratory has been
searching for potent tyrosinase inhibitors capable of being
used as skin-lightening agents. Based on the chemical
structures of L-tyrosine and L-dopa, which are both natural
substrates of tyrosinase, we have designed and synthesized
several compounds including MHY498 (Kim et al. 2013),
5-(substituted benzylidene)hydantoins (Ha et al. 2011),
benzylidene-linked thiohydantoins (Kim et al. 2014) and
MHY2081 (Kang et al. 2015) as competitive tyrosinase
inhibitors, which inhibit tyrosinase-inhibitory activity more
potently than kojic acid, a well-known strong tyrosinase
inhibitor. An analysis of the chemical structures of these
active compounds revealed they all possessed the (E)-β-
phenyl-α,β-unsaturated carbonyl scaffold, which indicated
that this scaffold importantly confers tyrosinase-inhibitory
activity. The scaffold of the above-mentioned tyrosinase
inhibitors is an exomethylene type, which shown in Fig. 1.
Materials and methods
Chemistry
All chemical reagents were commercially available and
used without further purification. Low-resolution mass
spectrometry (LRMS) and high-resolution mass spectro-
metry (HRMS) data were obtained on an Expression CMS
(Advion, Ithaca, NY, USA) and a 6530 Accurate Mass
quadrupole time-of-flight liquid-chromatography mass
spectrometer (Agilent), respectively. Nuclear Magnetic
Resonance (NMR) data were recorded on a Varian Unity
INOVA
400
spectrometer
and
Varian
Unity
AS500 spectrometer (Agilent Technologies, Santa Clara,
CA, USA), using DMSO-d6 and chemical shifts were
reported in parts per million (ppm) with reference to the
respective residual solvent or deuterated peaks (δH 7.24 and
δC 77.0 for CDCl3, δH 2.50 and δC 39.7 for DMSO-d6).
Coupling constants are reported in hertz. The abbreviations
used are as follows: s (singlet), brs (broad singlet), d
(doublet), t (triplets), q (quartet), td (triplet of doublets), dd
(doublet of doublets) or m (multiplet). All the reactions
described below were performed under nitrogen atmosphere
and monitored by thin-layer chromatography (TLC). TLC
was performed on Merck precoated 60F254 plates. All
anhydrous solvents were distilled over CaH2 or Na/benzo-
phenone prior to use. Identity of all final compounds was
confirmed by 1D (1H and 13C) and 2D (NOESY) NMR and
mass spectrometry.
The general procedure for the preparation of 2-
(substituted phenyl)naphthalene-1,4-dione
derivatives (1a–1f)
Appropriate phenols (50 mg) in acetic acid (1.0 mL) were
added to a stirred solution of 1,4-naphthoquinone (2.0 eq.)
in acetic acid (2.0 mL) in a 25 mL round bottom flask and
then 2M-H2SO4 (0.5 mL) was added. The reaction mixture
was stirred at room temperature for 2–10 h under a nitrogen

Medicinal Chemistry Research
atmosphere. After addition of water, the reaction mixture
was neutralized with saturated NaHCO3 solution and par-
titioned between ethyl acetate and water. The organic layer
was dried over anhydrous MgSO4, filtered and evaporated
under reduced pressure. The resulting residue was purified
by column chromatography (hexane:ethyl acetate = 7:1–
1:1) to give solids at yields of 17–86%.
2-(3,4-Dihydroxyphenyl)naphthalene-1,4-dione (1e)
1
Yield: 42%; H NMR (500 MHz, DMSO-d6) δ 9.33 (brs,
2H, OH), 8.06–8.04 (m, 1H, 6-H, or 7-H), 7.99–7.97 (m,
1H, 6-H, or 7-H), 7.88–7.84 (m, 2H, 5-, 8-H), 7.08 (d, 1H,
J = 1.5 Hz, 2′-H), 6.98 (dd, 1H, J = 8.0, 2.0 Hz, 6′-H), 6.97
(s, 1H, 3-H), 6.81 (d, 1H, J = 8.0 Hz, 5′-H); 13C NMR (100
MHz, CD3OD+CDCl3) δ 185.7, 185.2, 147.8, 147.4, 144.5,
133.9, 133.4, 132.7, 132.1, 127.1, 125.9, 125.3, 122.7,
120.6, 116.5, 115.3; LRMS (ESI-) m/z 265 (M-H)−
(Janeczko et al. 2016).
2-(2,4-Dihydroxyphenyl)naphthalene-1,4-dione (1a)
1
(Redaelli et al. 2015). Yield: 86%; H NMR (500 MHz,
DMSO-d6) δ 9.67 (s, 1H, OH), 9.66 (s, 1H, OH), 8.02–7.97
(m, 2H, 6-, 7-H), 7.86–7.84 (m, 2H, 5-, 8-H), 7.05 (d, 1H, J
= 8.5 Hz, 6′-H), 7.00 (s, 1H, 3-H), 6.37 (d, 1H, J = 1.5 Hz,
3′-H), 6.30 (dd, 1H, J = 8.5, 1.5 Hz, 5′-H); 13C NMR (100
MHz, DMSO-d6) δ 185.4, 184.5, 160.6, 157.5, 147.9,
135.4, 134.7, 134.6, 133.0, 132.9, 132.2, 127.1, 126.0,
112.7, 107.3, 103.3 (Redaelli et al., 2015).
2-(2,4-Dimethoxyphenyl)naphthalene-1,4-dione (1f)
1
Yield: 84%; H NMR (500 MHz, CDCl3) δ 8.15–8.10 (m,
2H, 6-, 7-H), 7.76–7.74 (m, 2H, 5-, 8-H), 7.21 (d, 1H, J =
8.5 Hz, 6′-H), 7.03 (s, 1H, 3-H), 6.58 (dd, 1H, J = 8.5, 2.0
Hz, 5′-H), 6.43 (d, 1H, J = 2.0 Hz, 3′-H), 3.86 (s, 3H, CH3),
3.78 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) δ
185.6, 184.2, 162.5, 158.8, 147.6, 136.5, 133.8, 133.7,
132.9, 132.4, 131.8, 127.1, 126.1, 116.0, 104.9, 99.2, 55.9,
55.7.
2-(4-Hydroxy-3-methoxyphenyl)naphthalene-1,4-dione (1b)
1
Yield: 37%; H NMR (500 MHz, CDCl3) δ 8.17–8.07 (m,
2H, 6-, 7-H), 7.76–7.74 (m, 2H, 5-, 8-H), 7.16 (m, 2H, 2′-,
6′-H), 7.03 (s, 1H, 3-H), 6.98 (d, 1H, J = 8.0 Hz, 5′-H),
3.94 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3) δ 185.5,
185.1, 148.1, 147.7, 146.6, 133.9 (×2), 132.8, 132.3, 127.2,
126.1, 125.5, 123.6, 114.9, 112.4, 56.3; LRMS (ESI-) m/z
279 (M-H)−; HRMS (ESI-) m/z C17H12O4 (M-H)− calcd
279.0663, obsd 279.0660.
Biological evaluation
Mushroom tyrosinase inhibition assay
The mushroom tyrosinase-inhibitory assay for the synthe-
sized compounds 1a–1f was carried out according to the
known method with slight modifications (Hyun et al. 2008).
To a 96-well microplate, a total of 200 µL mixture was
added, containing 20 µL mushroom tyrosinase solution
(1000 U/mL), 10 µL of each test compounds (final con-
centration 30 µM) and 170 µL of substrate solution (293 µM
L-tyrosine solution and 14.7 mM potassium phosphate
buffer (pH 6.5) solution (1:1, v/v)). The microplate was
incubated for 30 min at 37 °C. A VersaMaxTM microplate
reader (Molecular Devices, Sunnylvale, CA, USA) was
used to measure the absorbance of dopachrome contents
produced during incubation at 450 nm. A volume of 50 µM
of kojic acid and 500 µM of arbutin were used as positive
controls, respectively. All experiments were repeated for
three times. Formula [Inhibition (%) = [1−(A/B)] × 100(A
= the absorbance of test compounds and B = the absor-
bance of the blank control with no test compound)] was
used for the calculation of tyrosinase inhibition. The IC50
value of compound 1c was determined in three different
concentrations.
2-(4-Hydroxy-2-methoxyphenyl)naphthalene-1,4-dione (1c)
Yield: 37%; 1H NMR (500 MHz, DMSO-d6) δ 9.88 (s, 1H,
OH), 8.02–7.98 (m, 2H, 6-, 7-H), 7.87–7.85 (m, 2H, 5-, 8-
H), 7.10 (d, 1H, J = 8.0 Hz, 6′-H), 6.95 (s, 1H, 3-H), 6.49
(s, 1H, 3′-H), 6.43 (d, 1H, J = 8.0 Hz, 5′-H), 3.66 (s, 3H,
CH3); 13C NMR (100 MHz, DMSO-d6) δ 185.3, 184.1,
161.0, 159.1, 148.2, 135.7, 134.6, 134.5, 132.8, 132.2,
132.1, 127.0, 126.0, 114.4, 107.8, 99.9, 55.9; LRMS (ESI-)
m/z 279 (M-H)−; HRMS (ESI-) m/z C17H11O4 (M-H)− calcd
279.0663, obsd 279.0665.
2-(2-Hydroxy-4-methoxyphenyl)naphthalene-1,4-dione (1d)
Yield: 17%; 1H NMR (500 MHz, DMSO-d6) δ 9.84 (s, 1H,
OH), 8.03–7.98 (m, 2H, 6-, 7-H), 7.87–7.85 (m, 2H, 5-, 8-
H), 7.16 (d, 1H, J = 8.0 Hz, 6′-H), 7.02 (s, 1H, 3-H), 6.48
(dd, 1H, J = 8.0, 2.0 Hz, 5′-H), 6.43 (d, 1H, J = 2.0 Hz, 3′-
H), 3.74 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) δ
185.4, 184.3, 162.1, 157.3, 147.7, 136.0, 134.7, 134.6,
133.0, 132.7, 132.2, 127.1, 126.1, 114.4, 105.5, 102.0,
55.8; LRMS (ESI-) m/z 279 (M-H)−; HRMS (ESI-) m/z
C17H11O4 (M-H)− calcd 279.0663, obsd 279.0664.
Kinetic analysis of tyrosinase inhibition
Kinetic analysis study was performed to determine the
nature of inhibition of compound 1c using mushroom

Medicinal Chemistry Research
tyrosinase. Different concentration of a substrate, L- tyr-
osine (0.5 mM, 1 mM, 2 mM or 4 mM), a tyrosinase
enzyme (20 µL or 1000 U/mL) and test samples (0 µL, 5 µL,
10 µL or 25 µL) were mixed together and added to a 96-well
microplate in total of 200 µL final solution. The 96-well
microplates were incubated at 37 °C for 30 min. An ELISA
reader (Tecan, Austria) was used to measure the absorbance
of dopachrome produced during incubation at 450 nm. Km
(Michaelis constant) and Vmax (maximum velocity) were
calculated using a Lineweaver–Burk plot and a nature of
enzyme inhibition of the test compound 1c was determined.
The experiments were repeated for three times.
removed, formazan crystals were dissolved in DMSO/EtOH
(1:1, 200 µL) and moved to a 96-well ELISA plate. At 570
nm, well absorbances were detected by an Elisa reader.
Experiments were replicated for three times.
Tyrosinase activity of compound 1c in B16F10
melanoma cells
The standard procedure with slight modifications was fol-
lowed for the measuring of tyrosinase activity in B16F10
cells (Chen et al. 2009). The cells were cultured in a 24-well
plate in the amount of 5 × 104 cells per well. The 24-well
plates were stored in a humidified environment containing
5% CO2 at 37 °C for 24 h. Test compound 1c (0, 5, 10 or 25
µM), α-MSH (1 µM) and kojic acid (25 µM) were added to
the cells and further incubated for 24 h under the same
conditions. The cells were washed with PBS twice and
lysed with lysis buffer containing 50 mM PBS (90 μL, pH
6.8), 0.1 mM PMSF (5 μL) and 1% Triton X-100 (5 μL).
The lysed cells were frozen at −80 °C for 30 min. Frozen
lysates were centrifuged at 12,000 rpm for 30 min at 4 °C to
give a supernatant. A volume of 20 µL of L-dopa (10 mM)
was mixed with 80 µL of the supernatant in 96-well ELISA
plates and incubated at 37 °C for 30 min. Optical densities
were obtained at 500 nm. All experiments were repeated for
three times.
In silico docking simulation of compound 1c and
tyrosinase
Docking studies were carried out between tyrosinase, and
kojic acid, or compound 1c. First of all, 3D structure of
compound 1c was created with the help of Chem3D pro
12.0. From PDB (Protein Data Bank), the 3D structure of
Agaricus bisporus tyrosinase (PDB ID: 2Y9X) was
obtained. The docking score of compound 1c was deter-
mined by AutoDock Vina and Chimera software according
to the known procedure (Morris et al. 1998, Moustakas
et al. 2006). A pharmacophore between 1c and tyrosinase
were created by LigandScout software, which showed the
binding interactions between the tyrosinase and compound
1c.
Melanin production in B16F10 melanoma cells
Cell culture
For the determination of inhibitory effect of compound 1c
on melanin biosynthesis, a melanin content assay was car-
ried out according to the known method with slight mod-
ifications (Chen et al. 2009). B16F10 cells were distributed
in a 24-well plate (5 × 104 cells/well). Then the cells were
stored in 5% CO2 incubator for an overnight at 37 °C. Test
compound 1c (0, 5, 10 or 25 µM), α-MSH (1 µM) and kojic
acid (25 µM) were added to the cells and further incubated
for 24 h under the same conditions. After cleaning of the
cells with PBS buffer, 1 N NaOH solution (200 µL) was
added to the cells. Lysates were transferred to 96-well plates
to measure the dissolved melanin amounts and the absor-
bances were checked at 405 nm using an enzyme-linked
immunosorbent assay reader. All experiments were repeated
for three times.
Mouse melanoma cells B16F10 were obtained from a
Korean Cell Line Bank. In a humidified atmosphere (5%
CO2) and at 37 °C temperature, B16F10 cells were cultured
in DMEM (Dulbecco’s modified Eagle’s medium) having
FBS (10% heat-inactivated fetal bovine serum) and strep-
tomycin/penicillin (100 IU/50 µg/mL). To determine cell
viability, melanin content and tyrosinase activity, cells were
cultured in 24-well plates. All experiments were repeated
for three times.
Viability assay of compound 1c in B16F10
melanoma cells
MTT assay was performed for the determination of cell
viabilities (Shin et al. 2008). The cells were cultured in a
24-well plate in the amount of 5 × 104 cells per well. The
24-well plates were stored in a humidified environment
containing 5% CO2 at 37 °C for 24 h. Different concentra-
tions (0, 5, 10, 25 µM) of test compound 1c were added to
the cells and further incubated for 24 h under the same
conditions. Then the cells were treated with MTT solution
and incubated for 2 h at 37 °C. After supernatant were
DPPH radical scavenging activity assay
A known method with slight modifications was followed to
determine the DPPH radical scavenging abilities of the test
compounds 1a–1f (Matos et al. 2015). A volume of 20 μL
of the test compounds 1a–1f (10 mM in DMSO) was added
to a 96-well plate, and then 180 μL of a DPPH methanol
solution (0.2 mM) was added. L-Ascorbic acid was used as

Medicinal Chemistry Research
Scheme 1 Reagents and
conditions: 4N-H2SO4 aqueous
solution/acetic acid (v/v = 1:4)
a standard. The 96-well plate were incubated in the dark for
30 min and absorbances were measured at 517 nm using
VersaMaxTM microplate reader. All experiments were per-
formed in triplicates. The following formula was used for
the calculation of radical scavenging activities of 1a–1f.
Scavenging activityð%Þ¼½ðAc À AsÞ=AcŠÂ100
Fig. 2 Key HMBC correlations of compounds 1c and 1d
where Ac is the optical density of the non-treated control
and As is the optical density of the test sample.
prepared from 1,4-naphthoquinone and hydroxyl-
substituted benzenes using the Michael reaction and sub-
sequent spontaneous oxidation, as depicted in Scheme 1.
Reactions between 1,4-naphthoquinone and resorcinol (1,3-
dihydroxybenzene), 2-methoxyphenol, 3-methoxyphenol,
catechol (1,2-dihydroxybenzene) or 1,3-dimethoxybenzene
in an acidic co-solvent (4N-H2SO4 plus acetic acid; v/v =
1:4) afforded the desired 2-(hydroxyl-/methoxy-substituted
phenyl)naphthoquinone derivatives, 1a–1f. It was note-
worthy that reaction with 3-methoxyphenol produced two
regioisomers, 2-(4-hydroxy-2-methoxyphenyl)naphthalene-
1,4-dione (1c) and 2-(2-hydroxy-4-methoxyphenyl)naph-
thalene-1,4-dione (1d), respectively, in a ratio of 4:1, pre-
sumably due to the ortho- and para-orientations of the
hydroxyl substituent on the β-phenyl ring. We used 1D (1H
and 13C) and 2D NMR spectra (COSY, HSQC and HMBC)
to assign these two regioisomers.
According to the HMBC data of 1c (Supporting infor-
mation), the proton (9.88 ppm) of 4′-hydroxyl group
showed correlations with three carbons (C′-3, 99.9 ppm; C
′-4, 161.0 ppm; C′-5 and 107.8 ppm) (Fig. 2), indicating
that compound 1c was 2-(4-hydroxy-2-methoxyphenyl)
naphthalene-1,4-dione. The structure of compound 1d was
determined to be 2-(2-hydroxy-4-methoxyphenyl)naphtha-
lene-1,4-dione based on HMBC data (Supporting informa-
tion). In particular, the proton (9.84 ppm) of 2′-hydroxyl
group showed correlations with three carbons (C′-1, 114.4
ppm; C′-2, 157.3 ppm and C′-3, 102.0 ppm). The reactions
between 1,4-naphthoquinone and 2-methoxyphenol and
1,2-dihydroxybenzene each produced only one detectable
regioisomer, that is, compounds 1b and 1e, respectively, the
structures of which were easily determined by analysing the
splitting patterns of protons on the 2-phenyl ring of the
Statistical analysis
GraphPad software (La Jolla, CA, USA) was used for the
statistical analysis. All experiments were performed three
times. Results were calculated as means SEMs. The sig-
nificance of intergroup differences was measured using one-
way INOVA and Tukey’s test. Two-sided P-values of <0.05
were considered statistically significant.
Results and discussion
Chemistry
According to our previous results, the presence of a
hydroxyl group on the β-phenyl ring of the (E)-β-phenyl-α,β
unsaturated carbonyl scaffold generally enhances
tyrosinase-inhibitory activity. Therefore, the synthesis of 2-
phenyl-1,4-naphthoquinone derivatives 1a–1e with at least
one hydroxyl substituent on the 2-phenyl ring was carried
out (Scheme 1). The synthetic method based on a Pd-
catalyzed coupling reaction (the Suzuki–Miyaura reaction)
(Maluenda and Navarro 2015), between 2-halo-1,4-naph-
thoquinone and substituted phenylboronic acid was exclu-
ded due to cost considerations. Instead, we focused on the
chemical properties of 1,4-naphthoquinone, because it can
serve as a good Michael acceptor and hydroxy-substituted
benzenes can act as good nucleophiles due to the strong
electron-donating property of the hydroxyl substituent.
Thus, we speculated that 2-(hydroxy-/methoxy-substituted
phenyl)naphthoquinone derivatives 1a–1f could be

Medicinal Chemistry Research
Table 1 Substitution patterns, reaction time and tyrosinase-inhibitory
activities by the prepared 2-(substituted phenyl)-1,4-naphthoquinone
derivatives, 1a–1f, arbutin and kojic acid
regioisomers. On the other hand, reaction between 1,4-
naphthoquinone and phloroglucinol (1,3,5-trihydrox-
ybenzene) gave a compound consisting of two 1,4-naph-
thoquinones and one phloroglucinol under the same
reaction conditions (data not shown), instead of the desired
2-(1,3,5-trihydroxyphenyl)naphthoquinone; although 1H
and 13C NMR and mass data of the compound were
obtained, its chemical structure could not be determined. Of
the six 2-phenylnaphthoquinone derivatives produced, 2-(4-
hydroxy-3-methoxyphenyl)naphthalene-1,4-dione (1b), 2-
(4-hydroxy-2-methoxyphenyl)naphthalene-1,4-dione (1c),
and 2-(2-hydroxy-4-methoxyphenyl)naphthalene-1,4-dione
(1d) were unknown compounds. In all products, the pre-
sence of only one vinylic proton peak (3-H) of the 1,4-
naphthoquinone moiety indicated that only one phenyl
groups were attached to the 2-position of 1,4-naphthoqui-
none and that auto-oxidation had occurred after Michael
addition in each case. The vinylic proton peaks (H-3) of all
six 2-phenyl-1,4-naphthoquinone derivatives (1a–1f)
revealed in the 6.90–7.03 ppm range.
Compound R1
R2
H
R3
Reaction
timea (h)
Tyrosinase
inhibitionb (%)
1a
OH
OH 12
51.39 6.99
51.48 5.30
54.28 5.30
NId
1b
H
OMe OH 12
1c
OMe
OH
H
H
H
OH 10
OMe 10
1d
1e
OH OH 11
OMe 12
NI
1f
OMe
H
49.40 3.28
32.65 1.77
47.49 3.32
Arbutinc
Kojic acid
NI no inhibition
aMichael addition and autoxidation reaction time
Biological evaluation
bTyrosinase inhibition was performed using L-tyrosine at 30 μM of
each inhibitor or 30 μM of kojic acid
Mushroom tyrosinase assay
cThe assays were carried out at 500 μM of arbutin
dResults are indicated as mean standard error of three experiments’
The tyrosinase-inhibitory effects of the synthesized 2-phe-
nyl-1,4-naphthoquinone derivatives on commercially
available mushroom tyrosinase were assayed at 30 μM and
arbutin (Cui et al. 2005) (500 μM) and kojic acid (30 μM)
were used as positive controls. As shown in Table 1,
compounds 1a–1c and 1f were found to more potently
inhibit mushroom tyrosinase activity than the positive
controls. Compound 1e, which contained a 2-catechol group
failed to inhibit tyrosinase. Notably, 1f with no hydroxyl
group on its β-phenyl ring, inhibited mushroom tyrosinase
as potently as kojic acid (49.40 3.28% vs. 47.49 3.32%
inhibition, respectively). Although 1a exhibited potent
tyrosinase-inhibitory activity at 30 μM, it also showed
appreciable toxicity in B16F10 cells at a concentration of ≥
10 μM (data not shown). Compound 1d having a 2-
mean
Cell viability assay
120
100
Control
2.5 uM
5 uM
80
60
40
20
0
10 uM
5 uM
10 uM
2.5 uM
Control
Concentration of 1c
Fig. 3 Effect of 1c on B16F10 cell viability. The viabilities of cells
treated with 1c (2.5 μM, 5 μM or 10 μM) are indicated as percentage
viabilities vs. untreated controls
hydroxy-4-methyoxyphenyl group did not exhibit
a
tyrosinase-inhibitory activity, but compound 1c in which
the 4-methoxy group of 1d was substituted with a 4-
hydroxy group showed a large inhibition. On the basis of
IC50 values, compound 1c (IC50 = 22.00 1.63 μM) inhib-
ited greater tyrosinase inhibitory than 1b (IC50 = 24.91
2.12 μM) or kojic acid (IC50 = 37.86 2.21 μM), and nei-
ther showed appreciable cytotoxicity at a concentration of
≤10 μM. To the best of our knowledge, these compounds
are the first naphthoquinone derivatives with tyrosinase-
inhibitory activity. Because of its greater tyrosinase-
inhibitory activity than kojic acid based on IC50 values,
compound 1c was used in following studies.
Cell studies
The effect of compound 1c on B16F10 cell viability was
examined using an MTT assay. The obtained results
showed that exposure to compound 1c at concentrations
of ≤ 10 µM for 24 h had no appreciable effect (Fig. 3).
The inhibitory effect of compound 1c on melanogenesis
in B16F10 melanoma cells was evaluated by measuring
intracellular melanin contents in cells co-treated with 1c and

Medicinal Chemistry Research
Melanin contents
Table 2 The effect of 2-(substituted phenyl)-1,4-naphthoquinone
derivatives, 1a–1f, on the DPPH radical scavenging ability
120
100
80
60
40
20
0
***
Compound
DPPH radical scavenging activity (%)a
Control
2.5 uM
5 uM
10 uM
10 uM
***
***
***
1a
87.27 0.44
78.19 0.81
87.10 0.70
83.58 1.70
87.62 0.29
25.02 0.58
87.51 0.18
***
1b
1c
1d
1e
5 uM
10 uM
10 uM
1f
2.5 uM
Control
1c kojic acid
L-Ascorbic acid
aAt 30 min after treatment of a DPPH methanol solution, the radical
scavenging activity of each compound at a concentration of 1.0 mM
was examined. All experiments were independently carried out in
triplicate. Results are shown as mean standard error of the mean of
three experiments
Fig. 4 Effects of 1c on melanogenesis. B16F10 cells were treated with
α-MSH and 1c or kojic acid for 24 h. The asterisk indicates significant
differences between cells treated with 1c or kojic acid and non-treated
control cells: ***p < 0.001
Tyrosinase activity
120
100
Control
2.5 uM
5 uM
10 uM
10 uM
***
80
***
***
60
40
20
0
***
***
5 uM
10 uM
10 uM
2.5 uM
Control
1c kojic acid
Fig. 5 Effects of compound 1c on cellular tyrosinase activity. B16F10
cells were treated with α-MSH and 1c or kojic acid for 24 h. The
asterisk denotes significant differences between cells treated with 1c or
kojic acid and non-treated control cells: ***p < 0.001
Fig. 6 Mode of mushroom tyrosinase inhibition by compound 1c. The
type of inhibition caused by 1c was investigated using Lineweaver–
Burk plots. The results are mean of 1/V values, where V is the increase
in O.D. per minute at different L-tyrosine concentrations. The mod-
ified Michaelis–Menten equation was used: 1/Vmax = 1/Km(1+[S]
/Ki), where Ki is the inhibition constant, V the reaction velocity, Km
Michaelis constant and S the concentration of L-tyrosine
α-MSH (α-melanocyte-stimulating hormone) and cells
treated with α-MSH only. Melanin contents were found to
dose-dependently decrease as the concentration of 1c
increased (Fig. 4). Furthermore, compound 1c inhibited
melanogenesis more effectively than kojic acid at 10 μM.
When we examined the inhibitory effect of compound 1c
on tyrosinase activity in B16F10 cells treated with α-MSH,
it was found 1c dose-dependently inhibited tyrosinase
activity (Fig. 5). In particular, at 10 μM compound 1c
suppressed tyrosinase activity much more strongly than
kojic acid. The similar patterns of tyrosinase activity inhi-
bition and melanin content reduction by 1c, suggest its anti-
melanogenic effect was probably due to the inhibition of
tyrosinase activity.
scavenging assay. As shown in Table 2, most of the deri-
vatives, including 1c, exhibited radical scavenging activity
similar to that of L-ascorbic acid (positive control), sug-
gesting the inhibitory effect of 1c on melanin production
might be partially due to its radical scavenging activity.
Mechanism of action
A Lineweaver–Burk plot was used to investigate the mode
of mushroom tyrosinase inhibition by 1c. As shown in Fig.
6, four straight lines with different slopes that intersected
the y-axis at the same point were obtained, which indicated
Vmax values were not affected by the concentration of 1c.
As the concentration of 1c increased, Km values also
increased gradually. These observations showed compound
1c competitively inhibits mushroom tyrosinase activity in a
dose-dependent manner similar to other competitive
DPPH radical scavenging activity
It has been reported that ROS (reactive oxygen species)
induce melanogenesis by activating tyrosinase (Gillbro and
Olsson 2011, Valverde et al. 1996) and thus, we investi-
gated the scavenging effects of the synthesized 2-(hydroxyl/
methoxy-substituted phenyl)naphthoquinone derivatives
using a DPPH (2,2-diphenyl-1-picrylhydrazyl) radical

Medicinal Chemistry Research
Fig. 7 Docking simulation of
interactions between tyrosinase
and 1c or kojic acid and
pharmacophore analysis. a, b
Pharmacophore results for 1c
and kojic acid. c Docking result
for the interaction between 1c
and tyrosinase. d Tyrosinase
docking scores for 1c and kojic
acid. Green arrow: hydrogen-
bonding, violet arrow: π–π
stacking, yellow: hydrophobic
interaction
tyrosinase inhibitors (Yi et al. 2010, Xie et al. 2017, Pintus
et al. 2017).
methoxy-substituted phenyl)naphthoquinone derivatives
1a–1f were synthesized by Michael reaction and subsequent
spontaneous oxidation. Of the six compounds prepared,
four showed either matched or bettered the mushroom
tyrosinase-inhibitory effect of kojic acid (IC50 = 37.86
2.21 μM) and 1c exhibited the strongest inhibitory effect
(IC50 = 22.00 1.63 μM). Furthermore, this observation
was supported by the results of our kinetic study and in
silico docking simulation, which showed compound 1c
competitively inhibits tyrosinase. In addition, cell-based
assays using B16F10 cells showed that 1c exerted its anti-
melanogenic effect by inhibiting cellular tyrosinase without
appreciable cytotoxicity. These results support that the (E)-
β-phenyl-α,β-unsaturated carbonyl scaffold of the endo-
methylene type has an intrinsic ability to facilitate the
inhibition of tyrosinase.
Docking studies
To determine whether 1c binds directly to the active site of
tyrosinase, docking simulation was carried out with Auto-
Dock Vina (Di Muzio et al. 2017) using the 3D structure of
mushroom tyrosinase (PDB: 2Y9X), 1c as a ligand, and kojic
acid as a positive reference ligand. Compound 1c was found
to have a stronger binding affinity (−6.6 kcal/mol) for the
active site than kojic acid (−5.7 kcal/mol) (Fig. 7d).
LigandScout 3.1.2 was used to identify the binding residues
of 1c and kojic acid responsible for their binding with tyr-
osinase. The results obtained showed that 1c directly hydro-
gen bonds to the active site of tyrosinase using its 4′-hydroxyl
group at two amino acid residues (His61 and His259) (Fig.
7a,c). In addition, the results showed the benzene ring of the
1,4-naphthoquinone of 1c interacts hydrophobically with
Phe264 of tyrosinase and that its 2-phenyl ring interacts
hydrophobically with the two amino acid residues Val283 and
Val286. However, LigandScout results indicated kojic acid
interacts with tyrosinase by forming one hydrogen bond with
Asn260 and by π–π stacking with His263 (Fig. 7b). Thus, the
greater binding energy of 1c to tyrosinase than of kojic acid to
tyrosinase appears to explain the greater interaction between
1c and tyrosinase.
Acknowledgements This research was supported by the Basic Science
Research Program through the National Research Foundation of Korea
(NRF) funded by the Korean Ministry of Education (NRF-
2017R1D1A1B03027888).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
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