In vitro anti-melanogenic effects of chimeric compounds, 2-(substituted benzylidene)-1,3-indanedione derivatives with a β-phenyl-α, β -unsaturated dicarbonyl scaffold

Article abstract of DOI:10.1016/j.bioorg.2021.104688

Tyrosinase is considered a key contributor to melanogenesis, and safe, potent tyrosinase inhibitors are needed for medical and cosmetic purposes to treat skin hyperpigmentation and prevent fruit and vegetable browning. According to our accumulated SAR dat

Full text of DOI:10.1016/j.bioorg.2021.104688

Bioorganic Chemistry 109 (2021) 104688
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
In vitro anti-melanogenic effects of chimeric compounds, 2-(substituted
benzylidene)-1,3-indanedione derivatives with a β-phenyl-α, β -unsaturated
dicarbonyl scaffold
,
,
,
,
Il Young Ryua 1, Inkyu Choia 1, Hee Jin Junga 1, Sultan Ullahb 1, Heejeong Choia,
Md. Al-Amina, Pusoon Chunc, Hyung Ryong Moona
,
*
a College of Pharmacy, Pusan National University, Busan 46241, South Korea
b Department of Molecular Medicine, The Scripps Research Institute, FL 33458, USA
c College of Pharmacy and Inje Institute of Pharmaceutical Sciences and Research, Inje University, Gimhae, Gyeongnam 50834, South Korea
A R T I C L E I N F O
A B S T R A C T
Keywords:
Tyrosinase is considered a key contributor to melanogenesis, and safe, potent tyrosinase inhibitors are needed for
medical and cosmetic purposes to treat skin hyperpigmentation and prevent fruit and vegetable browning. Ac-
Tyrosinase inhibitor
Indanedione
cording to our accumulated SAR data on tyrosinase inhibitors, the β-phenyl-α,β-unsaturated carbonyl scaffold in
β-phenyl-
α
either E or Z configurations, can confer potent tyrosinase inhibitory activity. In this study, twelve indanedione
derivatives were synthesized as chimeric compounds with a β-phenyl- ,β-unsaturated dicarbonyl scaffold. Two of
these derivatives, that is, compounds 2 and 3 (85% and 96% inhibition, respectively), at 50 M inhibited
β-unsaturated dicarbonyl
Anti-melanogenic effect
Docking simulation
Competitive inhibitor
Chimeric compound
Kojic acid
α
μ
mushroom tyrosinase markedly more potently than kojic acid (49% inhibition). Docking studies predicted that
compounds 2 and 3 both inhibited tyrosinase competitively, and these findings were supported by Lineweaver-
Burk plots. In addition, both compounds inhibited tyrosinase activity and reduced melanin contents in B16F10
cells more than kojic acid without perceptible cytotoxicity. These results support the notion that chimeric
compounds with the β-phenyl-
α,β-unsaturated dicarbonyl scaffold represent promising starting points for the
development of potent tyrosinase inhibitors.
1. Introduction
Melanin is the pigment largely responsible for the color of skin and
functionally acts as a barrier against ultraviolet radiation [7]. However,
elevated levels of melanin in skin create aesthetic problems such as
melasma, freckles, and age spots, and are also associated with the
pathogenesis of melanoma [8–15], especially in the middle-aged and
elderly [16]. Thus, research studies have focused on the development of
tyrosinase inhibitors that prevent the production of excess melanin, and
to date, have identified a large number of potent natural and synthetic
tyrosinase inhibitors [9,17–27]. Some of these inhibitors such as arbu-
tin, kojic acid and hydroquinone are being used as topical whitening or
anti-hyperpigmentation agents [28,29]. However, although hydroqui-
none and kojic acid are used as whitening agents, they are also associ-
ated with an elevated risk of thyroid cancer [30] and with nephrotoxic
[31], genotoxic [13], and cytotoxic (to melanocytes) effects [32].
Arbutin, another tyrosinase inhibitor, has fewer side effects but is hy-
drolyzed to D-glucose and hydroquinone by primary skin microflora
Tyrosinases (also known as polyphenol oxidases) play key roles in
mammalian melanogenesis and in the enzymatic browning of fruit or
fungi [1]. The active site of tyrosinase contains two central copper(II)
ions, which interact with histidine residues in the common mushroom
(Agaricus bisporus) and human malignant melanoma tyrosinase [2,3].
Tyrosinases exist in various forms such as immature, mature and active
forms in animals, plants and fungi [4]. Melanogenesis can be defined as
the process that results in the formation of the dark macromolecular
pigment melanin via a series of enzymatic and chemical reactions.
Tyrosinase catalyzes different reactions in the melanin biosynthetic
pathway in melanocytes such as the hydroxylation of L-tyrosine to L-
DOPA and the oxidation of the L-DOPA to dopaquinone, which in man, is
converted by a series of complex reactions involving cyclization and
oxidative polymerizations to melanin [5,6].
* Corresponding author at: Laboratory of Medicinal Chemistry, College of Pharmacy, Pusan National University, Busan 46241, South Korea.
E-mail address: mhr108@pusan.ac.kr (H.R. Moon).
1
These authors (I.Y. Ryu, I. Choi, H.J. Jung, and S. Ullah) contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2021.104688
Received 3 September 2020; Received in revised form 15 January 2021; Accepted 22 January 2021
Available online 2 February 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

I.Y. Ryu et al.
Bioorganic Chemistry 109 (2021) 104688
(Staphylococcus epidermidis and Staphylococcus aureus) [33].
2. Results and discussions
Natural tyrosinase inhibitors are generally considered to be free of
harmful side effects, but they have low potencies, poor stabilities, and
are expensive, due to the lack of rich natural sources. Accordingly, sci-
entists have focused on the development of low cost, potent synthetic
tyrosinase inhibitors [34]. We have synthesized many tyrosinase in-
hibitors over the last decade [35–47], and as shown by Fig. 1, our studies
have demonstrated that compounds containing the β-phenyl-
2.1. Chemistry
Indanedione derivatives have been synthesized previously by many
research groups [49–51]. As depicted in Scheme 2, 2-(substituted
benzylidene)-1,3-indanedione derivatives with the β-phenyl-
α
,β-unsaturated dicarbonyl scaffold were synthesized. The derivatives 1
α
,β-unsaturated carbonyl scaffold often exhibit potent tyrosinase
– 12 were easily prepared by condensation between 1H and indene-1,3
(2H)-dione and an appropriate benzaldehyde in 1 M HCl acetic acid
solution. Twelve benzaldehydes including 2,4- and 3,4-dihydroxyben-
zaldehyde were used and condensation yields ranged from 43.1 to
99.8% (Table 1). The structures of the final compounds were confirmed
by 1H and 13C NMR spectroscopy and high- and low-resolution mass
spectroscopy.
inhibitory activity. Initially, we synthesized compounds with the (E)
scaffold geometry because they could be synthesized more easily and
because they tend to be more stable than corresponding compounds
with the (Z)-scaffold geometry.
In a previous study, to determine whether compounds with the (Z)
geometry also exhibit potent tyrosinase inhibitory activities, we pre-
pared a series of these compounds by condensing an appropriate benz-
aldehyde with 3-phenylisoxazol-5(4H)-one, which was easily prepared
by reacting ethyl benzoylacetate with hydroxylamine·HCl in the pres-
ence of DABCO (1,4-diazabicyclo[2.2.2]octane) (Scheme 1) [48]. Due to
steric hindrance by the phenyl group of 3-phenylisoxazol-5(4H)-one,
2.2. Biology
2.2.1. Mushroom tyrosinase inhibition assay
The inhibitory effects of the synthesized 2-(substituted benzylidene)-
1,3-indanedione derivatives 1 – 12 on mushroom tyrosinase were
investigated. Kojic acid, a well-known tyrosinase inhibitor, was used as
the positive reference control. As depicted in Table 2, residual tyrosinase
activities were determined after treating mushroom tyrosinase with
compounds with a (Z)-β-phenyl-α,β-unsaturated carbonyl scaffold were
predominately obtained. A (Z)-derivative with a 2,4-dihydroxyl sub-
stituent on the β-phenyl ring of the scaffold more potently inhibited
mushroom and B16F10 (a melanoma derived cell-line) tyrosinase than
kojic acid, which suggested the β-phenyl-α,β-unsaturated carbonyl
each test compound at 50 μM. Residual tyrosinase activity after treat-
scaffold can confer potent tyrosinase inhibitory activity, regardless of
scaffold geometry.
ment with kojic acid was 50%. Of 12 test compounds, compounds 2, 3,
and 8 more potently inhibited tyrosinase than kojic acid. Compound 8
with a 2,4-dimethoxy substituent on the β-phenyl ring of the scaffold
achieved a residual tyrosinase activity of 44%. According to our cu-
mulative structure–activity relationship (SAR) data for tyrosinase inhi-
bition, compounds with at least one hydroxyl group on the β-phenyl ring
of the scaffold, such as 4-hydroxyl, 2,4-dihydroxyl, 3,4-dihydroxyl, 4-
hydroxy-3-methoxyl, and 3-hydroxy-4-methoxyl, generally exhibit
potent tyrosinase inhibitory activity. Therefore, it is notable that com-
pound 8 showed stronger tyrosinase inhibition than kojic acid.
Furthermore, our SAR data indicate that a 2,4-dihydroxyl substituent on
the β-phenyl ring of the scaffold also markedly enhances tyrosinase in-
hibition. As was expected, compound 2 with the 2,4-dihydroxyl sub-
stituent reduced tyrosinase activity more than compound 8 or kojic acid
(residual activity 14%). However, compound 3 with a 3,4-dihydroxyl
Based on our previous findings, we wondered whether chimeric
compounds with a β-phenyl-α,β-unsaturated dicarbonyl scaffold (Fig. 2)
also exhibit high tyrosinase inhibitory activity. In this study, we report
the synthesis of 2-(substituted benzylidene)-1,3-indanedione derivatives
and their abilities to inhibit mushroom tyrosinase and tyrosinase in
B16F10 cells (a murine melanoma cell-line). In addition, we investi-
gated their abilities to inhibit melanogenesis in B16F10 cells and their
cytotoxic effects on this cell-line.
Fig. 1. Compounds containing the β-phenyl-
α
,β-unsaturated carbonyl scaffold that exhibit potent tyrosinase inhibitory activity.
2

I.Y. Ryu et al.
Bioorganic Chemistry 109 (2021) 104688
Scheme 1. Schematic of the 3-phenylisoxazol-5(4H)-one based synthesis of compounds containing the (Z)-β-phenyl-
α
,β-unsaturated carbonyl scaffold.
we calculated binding energies of these ligands with mushroom tyrosi-
nase and calculated the RMSD (root mean square deviation) values of
hydrogen bonding and of interactions with the copper ions of tyrosinase.
Binding interactions are shown in Fig. 3 in 2D and 3D format and in
Fig. 5. As shown in Fig. 3b, compounds 2 and 3 occupied the same
binding pocket as kojic acid. However, a detailed study of binding in-
teractions (Fig. 3a) showed that compound 2 interacted more strongly
with tyrosinase than compound 3 or kojic acid. Summarizing, kojic acid
formed a hydrogen bond with Gly281 using its hydroxymethyl group
and π-π stacking interactions between the pyranone ring of kojic acid
and the imidazole rings of tyrosinase His259 and His263. In addition,
one non-classical hydrogen bond interacting with aromatic hydrogen
(https://www.schrodinger.com/training/videos/docking-receptor-grid
-generation/hydrogen-bonds-aromatic-hydrogens-and-halogens) was
observed between the oxygen of the 3-hydroxyl pyranone group of kojic
acid and the imidazolyl hydrogen of His259.
Compound 2 formed two hydrogen bonds and π-π stacking, π-cation
interactions, and hydrophilic interactions with various polar amino acid
residues in the active site of tyrosinase. The hydroxyl groups of com-
pound 2 at positions 2 and 4 on its phenyl ring formed two hydrogen
bonds with Asn260 and Met280 of tyrosinase, respectively. In addition,
Fig. 2. Chimeric compounds, 2-benzylidene-indanediones with the β-phenyl-
α
,β-unsaturated dicarbonyl scaffold.
the phenyl and the benzene rings of indanedione formed π-π stacking
and π-cation interactions with the His263 and Arg268 amino acid resi-
substituent on the β-phenyl ring had the greatest inhibitory effect and
suppressed tyrosinase activity to only 4%. Log P values were obtained by
ChemDraw Ultra 12.0 and the log P values of all synthesized compounds
were better than the standard kojic acid.
dues of tyrosinase, respectively. As shown in Fig. 5a, one non-classical
hydrogen bond was also observed between the carbonyl group of
Met280 and the hydrogen at position 5 of the phenyl ring.
Compound 3 interacted with tyrosinase less than compound 2, and
notably, it did not form a classical hydrogen bond. Rather, it formed two
Because compounds 2 and 3 inhibited tyrosinase more than kojic
acid, we measured the IC50 values of these derivatives and of kojic acid
using different concentrations. The IC50 value of kojic acid was 50.10 ±
π-π stacking interactions between the indanedione benzene ring and the
Hie244 (protonated His244) imidazole ring and between the phenyl ring
of indanedione and the His259 imidazole ring. However, compound 3
did interact with a Cu ion, that is, the 4-hydroxyl group on the phenyl
ring of 3 coordinated with this ion (Fig. 3a). One non-classical hydrogen
bond was also observed between the indanedione carbonyl oxygen and
the aromatic hydrogen of Phe264 (Fig. 5a). The docking simulations
showed that compounds 2 and 3 bound to the active site of tyrosinase.
According to the docking simulation results in Fig. 3, compound 2
did not interact with copper ions, whereas compound 3 interacted with
copper ions. To prove these results experimentally [52], the UV–visible
spectra for compounds 2 and 3 were measured in the presence of
mushroom tyrosinase with or without CuSO4 (0.125 mM) and the
spectra are shown in Fig. 4. Regardless of the presence or absence of
CuSO4, the spectra of compound 2 were almost consistent (λmax = 481
2.63
2.07
μM, whereas those of 2 and 3 were 32.15 ± 0.63 μM and 17.98 ±
μ
M, respectively, that is, the 2 compounds inhibited tyrosinase 1.5-
and 2.8-fold more than kojic acid (Table 2). Further studies are needed
to determine whether compounds containing β-phenyl-α,β-unsaturated
dicarbonyl have superior tyrosinase inhibitory effects compared to
compounds containing β-phenyl-α,β-unsaturated carbonyl.
2.2.2. In silico studies
2.2.2.1. Binding pockets and enzyme-ligand interactions. In silico docking
simulation with mushroom tyrosinase was performed using Schrodinger
Suite (release 2020–2) to predict the mode of action responsible for
tyrosinase inhibition by compounds 2 and 3. During the docking study,
3

I.Y. Ryu et al.
Bioorganic Chemistry 109 (2021) 104688
Scheme 2. Synthesis of 2-(substituted benzylidene)-1,3-indanedione derivatives 1 – 12. Reagents and conditions: (a) 1 M HCl in acetic acid, rt, 9 – 24 h, 43.1
– 99.8%.
Table 1
Table 2
Synthetic yields and substitution patterns of 2-(substituted benzylidene)-1,3-
Residual tyrosinase activities after treatment with 2-(substituted benzylidene)-
1,3-indanedione derivatives 1 – 12, and the IC50 and Log P values.
Compound
Residual tyrosinase activity (%)
IC50
(μM)
aLog P
Control
100
–
1
82.71 ± 5.84
14.35 ± 1.52
3.74 ± 1.68
99.81 ± 14.08
99.26 ± 1.47
95.09 ± 2.28
67.50 ± 4.06
44.32 ± 2.75
66.53 ± 4.41
99.95 ± 3.76
82.16 ± 5.31
72.47 ± 2.47
50.01 ± 1.98
> 100
2.23
1.84
1.84
2.11
2.45
2.11
2.50
2.37
2.37
1.98
2.24
3.06
ꢀ 2.45
indanedione derivatives 1 – 12.
2
32.15 ± 0.63
17.98 ± 2.07
> 100
3
4
5
> 100
6
> 100
Compound
R1
R2
R3
R4
Synthetic yield (%)
7
90.24 ± 2.91
47.26 ± 1.52
87.08 ± 2.67
> 100
8
1
H
H
OH
H
86.8
43.1
75.7
75.1
99.8
80.8
65.4
93.4
83.0
79.2
57.7
75.1
9
2
OH
H
H
OH
H
10
3
OH
OMe
OEt
OH
H
OH
H
11
> 100
4
H
OH
H
12
> 100
5
H
OH
H
Kojic acid
50.10 ± 2.63
6
H
OMe
OMe
OMe
OMe
OH
H
7
H
H
Tyrosinase inhibitory activity experiments were conducted at a concentration of
8
OMe
H
H
H
50
μ
M of derivatives and kojic acid. aLog P values were obtained by ChemDraw
9
OMe
OMe
OMe
Br
H
Ultra 12.0.
10
11
12
H
OMe
OMe
H
H
OMe
OH
H
distances between copper 400 and 401 and the carbonyl oxygen of kojic
acid were 3.16 Å and 2.68 Å, respectively. Based on these interactions,
kojic acid achieved a docking score of ꢀ 4.56 kcal/mole. Compound 2
achieved a higher docking score (ꢀ 5.96 kcal/mol) than kojic acid or
compound 3, probably because it interacted with tyrosinase in more
ways. Compound 2 formed two hydrogen bonds with lengths of 2.03 Å
and 2.07 Å and one aromatic hydrogen bond of length 2.62 Å (Fig. 5). It
nm). This result means that compound 2 does not interact with Cu2+ in
the active site of tyrosinase. However, in the case of compound 3, in the
absence of CuSO4, the spectrum showed a value of λmax at 502 nm, and
the addition of excess CuSO4 induced hypsochromic shift (λmax = 465
nm), implying that compound 3 interacts with Cu2+ in the active site of
tyrosinase.
also formed two π-π stacking interactions and interacted with Cu400 and
Cu401 at distances of 4.56 and 3.46 Å, respectively. On the other hand,
compound 3 did not form a hydrogen bond, but its 4-hydroxy on the
phenyl ring interacted closely with Cu400 and Cu401 with bond dis-
tances of 2.58 and 2.30 Å, respectively. These results indicate that
compound 3 bound deeper within the catalytic site of tyrosinase than
2.2.2.2. Docking scores and binding analysis. Docking scores of com-
pound 2, compound 3, and kojic acid with mushroom tyrosinase were
determined using glide XP (Fig. 5b). Binding interactions were assessed
by measuring atomic distances associated with binding interactions. In
the case of kojic acid, the length of the hydrogen bond formed was 2.04
Å, whereas the aromatic hydrogen bond length was 3.33 Å (Fig. 5a).
Kojic acid was also found to be located close to both copper ions. The
compound 2 or kojic acid. Compound 3 also formed two π-π stacking
interactions and an aromatic hydrogen bond of length 2.69 Å. The col-
lective effects of these interactions led to stronger binding affinity
4

I.Y. Ryu et al.
Bioorganic Chemistry 109 (2021) 104688
Fig. 3. Binding interactions between tyrosinase (PDB ID = 2Y9X) and compound 2, compound 3, and kojic acid. The binding interactions with tyrosinase of all three
compounds were obtained using Maestro 12.4. Fig. 3a and 3b are 2D and 3D representations of tyrosinase-ligand interactions.
Fig. 4. UV–visible spectra of compounds 2 and 3 (0.05 mM). The red line represents the absorbance in the absence of CuSO4 and the blue line represents in the
presence of CuSO4 (0.125 mM). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
between tyrosinase and compound 3 (ꢀ 5.32 kcal/mol) than with kojic
acid (ꢀ 4.56 kcal/mol). These in silico docking simulation results suggest
compounds 2 and 3 strongly bind to the active site of tyrosinase, and
thus, compete with the natural tyrosinase substrates L-tyrosine and L-
dopa.
can be found in the Supporting Information (Fig. S49 – S54).
2.2.3. Kinetic analysis study
Kinetic analyses were conducted on compound 2 at 0, 10, 20, or 40
μ
M and compound 3 at 0, 7.5, 15, or 30 μM (Fig. 6). Tyrosinase inhib-
In addition to compounds 2 and 3, a docking study of the remaining
compounds was performed, and the binding activity was determined in
terms of the docking scores. Compounds 6, 9, 10, and 11 were found to
be inactive, resulting in no docking score. None of the other compounds
showed better binding affinity to mushroom tyrosinase than compounds
2 and 3. The docking scores calculated for these compounds were ꢀ 4.49,
ꢀ 4.62, ꢀ 4.60, ꢀ 4.14, ꢀ 4.57 and ꢀ 4.75 kcal/mol for compounds 1, 4, 5,
7, 8, and 12, respectively. The binding interactions for these compounds
itory activities were investigated in the presence of L-tyrosine (0, 1, 2, or
4 mM for 2, and 0, 2, 4, 8, or 16 mM for 3). To determine inhibition
modes, Lineweaver-Burk plots for each compound were plotted using
kinetic data. Double-reciprocal plots of tyrosinase inhibition by com-
pounds 2 and 3 are shown in Fig. 6. 1/V vs. 1/[L-tyrosine] plots pro-
duced four different lines with different slopes on each graph, and for
each compound, the four different lines intersected at the same point on
the y-axis, indicating that Vmax values remained constant as compound
5

I.Y. Ryu et al.
Bioorganic Chemistry 109 (2021) 104688
Fig. 5. Docking scores and binding analysis findings for interactions between mushroom tyrosinase (PDB ID = 2Y9X) and compound 2, compound 3, and kojic acid.
Docking scores with tyrosinase were obtained using Maestro 12.4. Fig. 5a and 5b show bond lengths between ligands and amino acid residues, and docking scores,
respectively.
concentrations increased and that KM values gradually increased in a
concentration-dependent manner. These results suggest that compounds
2 and 3 competitively inhibit tyrosinase by using the same binding
pocket as natural tyrosinase substrates, as was predicted by docking
simulation. The inhibitory kinetics (Table 3) showed the following: for
2 and 3 did not show perceptible cytotoxicity up to a concentration of
50 M. Accordingly, further studies on the effects of 2 and 3 on tyrosi-
nase inhibition and melanogenesis in vitro were conducted at concen-
trations of ≤50 M.
μ
μ
2; KM = 4.00, 8.95, and 22.10 mM at 7.5, 15, and 30
μ
M, respectively;
M,
M were 2.82
2.2.4.2. Intracellular tyrosinase inhibition. Initially, B16F10 cells were
treated with 5 M of -melanocyte-stimulating hormone ( -MSH) and
200 M of 3-isobutyl-1-methylxanthine (IBMX) for 60 h to enhance
tyrosinase activity and then treated with compounds 2 or 3 (5, 10, or 20
M), or kojic acid (20 M) for 60 h. Results are shown in Fig. 8. Treat-
ment of cells with
and for 3; KM = 2.00, 3.07, and 8.50 mM, at 7.5, 15, and 30
μ
μ
α
α
respectively. Ki values at concentrations of 7.5, 15, and 30
μ
μ
× 10ꢀ 6, 2.09 × 10ꢀ 6, and 2.09 × 10ꢀ 6 M for 2, and 4.70 × 10ꢀ 5, 1.91 ×
10ꢀ 5, and 7.63 × 10ꢀ 6 M for 3, respectively. According to our previous
data [53], kojic acid had 3.16 mM of KM, 2.32 × 10ꢀ 4 M of Ki, and 3.4 ×
μ
μ
α
-MSH and IBMX enhanced intracellular tyrosinase
10ꢀ 2 mM/min of Vmax at 20
μM, respectively. Compounds 2 and 3
activity by 5.8-fold, and compounds 2 and 3 concentration-dependently
reduced this increase (to 2.7- and 2.2-fold, respectively, at 20 µM). On
showed much lower Ki values than kojic acid.
the other hand, kojic acid at 20 µM did not reduce α-MSH and IBMX-
2.2.4. Cell study
induced increases in intracellular tyrosinase activity.
2.2.4.1. Cell viabilities. Before performing in vitro experiments on mu-
rine B16F10 melanoma cells, we examined the effects of compounds 2
and 3 on B16F10 viability using an EZ-Cytox assay (Fig. 7). Compounds
2.2.4.3. Cellular melanin inhibition. The effects of compounds 2 and 3
on melanin production were examined in B16F10 cells. As was per-
formed for cellular tyrosinase activity experiments, cells were treated
6

I.Y. Ryu et al.
Bioorganic Chemistry 109 (2021) 104688
Fig. 6. Lineweaver-Burk plots of mushroom tyrosinase inhibition by compounds 2 and 3. For compound 2, concentrations of 0, 10, 20, or 40
μ
M were used, and for
compound 3, concentrations of 0, 7.5, 15, or 30
16 mM of L-tyrosine for 3.
μM were used. Each experiment was conducted in the presence of 0, 1, 2, or 4 mM of L-tyrosine for 2, and 0, 2, 4, 8, or
intracellular melanin contents and showed much stronger anti-
Table 3
melanogeic effect than kojic acid (Fig. 9a and b). At 20
did not reduce extracellular melanin levels.
μM, kojic acid
Kinetic analysis of compounds 2 and 3.
Concentration (
μ
M)
Vmax (mM/min)
KM (mM)
Ki (M)
These results suggest the inhibitory effects of compounds 2 and 3 on
melanin production were due to tyrosinase inhibition. Numerous reports
have determined only intracellular melanin levels to confirm the anti-
melanogenic effects of the compounds, but we have examined both
intracellular and extracellular melanin levels. Our results show that
because the melanin synthesized by the B16F10 cells was released to the
outside of the cells, the degree of inhibition of intracellular melanin
levels influenced the extracellular melanin levels, and tyrosinase in-
hibitors had a greater effect on intracellular than extracellular melanin
levels.
Compound 2 7.5
3.3 × 10ꢀ 2
3.3 × 10ꢀ 2
3.3 × 10ꢀ 2
4.0 × 10ꢀ 2
4.0 × 10ꢀ 2
4.0 × 10ꢀ 2
4.00
8.95
22.10
2.00
3.07
8.50
2.82 × 10ꢀ 6
2.09 × 10ꢀ 6
1.56 × 10ꢀ 6
4.70 × 10ꢀ 5
1.91 × 10ꢀ 5
7.63 × 10ꢀ 6
15
30
Compound 3 7.5
15
30
Data are mean values of 1/V (inverse of the increase in absorbance at a wave-
length of 475 nm per min (ΔOD475/min)), of three independent experiments
conducted using different L-tyrosine concentrations. The Lineweaver-Burk plot
equation is: 1/V = 1/Vmax + KM/ Vmax × 1/[S] and the modified Michaelis-
Menten equation is 1/Vmax = (1 + [I]/Ki) × 1/KM, where V is the reaction
rate, Vmax is the maximum reaction rate, KM is the Michaelis-Menten constant,
[S] is substrate concentration, [I] is inhibitor concentration, and Ki is the inhi-
bition constant.
3. Conclusions
To examine whether chimeric compounds with the β-phenyl-
α
,β-unsaturated dicarbonyl scaffold plays an essential role in the inhi-
bition of tyrosinase, twelve 2-(substituted benzylidene)-1,3-indanedione
derivatives with the β-phenyl- ,β-unsaturated dicarbonyl scaffold were
with α-MSH (5 μM) and IBMX (200 μM) before being treated with 2, 3, or
α
kojic acid. The effects of 2 and 3 on melanin contents are shown in Fig. 9.
Treatment with compound 2 did not reduce extracellular melanin con-
synthesized by Knoevenagel condensation. Mushroom tyrosinase
inhibitory assays showed two 1,3-indanedione derivatives (compounds
2 and 3) with a 2,4- and 3,4- hydroxyls on the phenyl ring of the scaffold,
respectively, inhibited tyrosinase significantly more than kojic acid.
Docking studies on compounds 2 and 3 predicted that both bind more
strongly to the active site of tyrosinase than kojic acid. Our kinetic study
tents in the concentration range 5–20 μM (Fig. 9a), whereas it dose-
dependently reduced intracellular melanin contents (Fig. 9b). At 20
M, compound 2 decreased intracellular melanin contents to the same
extent as 20 M kojic acid. On the other hand, compound 3 dose-
dependently and significantly reduced both extracellular and
μ
μ
Fig. 7. Effects of compounds 2 and 3 on B16F10 cell viability. Cell viabilities were determined using EZ-Cytox solution at compound concentrations of 1 – 50 μM.
7

I.Y. Ryu et al.
Bioorganic Chemistry 109 (2021) 104688
Fig. 8. Effects of compounds 2 and 3 on intracellular tyrosinase inhibitory activity in B16F10 melanoma cells. Each experiment was carried out in triplicate and
results are presented as means ± SEMs. ###P < 0.001, compared with the untreated controls; ***p < 0.001, compared with
α–MSH and IBMX-treated cells. α–MSH,
α
-melanocyte-stimulating hormone; IBMX, 3-isobutyl-1-methylxanthine; KA, kojic acid.
Fig. 9. Effects of compounds 2 and 3 on melanin pro-
duction in B16F10 melanoma cells. Experiments were
carried out in triplicate and results are presented as
means ± SEMs. (a) and (b) Extracellular and intracel-
lular melanin contents of compound 2, respectively. (c)
and (d) Extracellular and intracellular melanin contents
of compound 3, respectively. ###P < 0.001, compared
with untreated controls; **p < 0.01, and ***p < 0.001,
compared with
-melanocyte-stimulating hormone; IBMX, 3-isobutyl-1-
methylxanthine; KA, kojic acid.
α–MSH and IBMX-treated cells. α–MSH,
α
8

I.Y. Ryu et al.
Bioorganic Chemistry 109 (2021) 104688
indicated that compounds 2 and 3 are a competitive tyrosinase inhibitor
(Vmax remained constant regardless of concentration). In vitro experi-
ments in B16F10 cells showed that compounds 2 and 3 dose-
dependently inhibited intracellular melanin production and intracel-
lular tyrosinase activity with no perceptible cytotoxicity. These results
CDCl3) δ 9.58 (brs, 1H, OH), 8.42 (s, 1H, 2′-H), 7.55 – 7.51 (m, 2H, 5-H,
6-H), 7.41 – 7.38 (m, 2H, 4-H, 7-H), 7.35 (s, 1H, vinylic H), 7.25 (d, 1H,
J = 8.4 Hz, 6′-H), 6.57 (d, 1H, J = 8.4 Hz, 5′-H), 3.64 (s, 3H, CH3); 13C
NMR (100 MHz, DMSO‑d6 + CDCl3) δ 190.6, 189.6, 153.2, 147.6, 147.6,
142.1, 139.6, 135.0, 134.8, 132.0, 125.6, 125.2, 122.7, 122.7, 116.2,
115.7, 55.9; LRMS (ESI-) m/z 279 (Mꢀ H)ꢀ , 264 (Mꢀ Hꢀ CH3)ꢀ ; HRMS
(ESI + ) m/z C17H13O4 (M + H)+ calcd. 281.0808, obsd. 281.0803.
4.1.2.5. 2-(3-Ethoxy-4-hydroxybenzylidene)-1H-indene-1,3(2H)-dione
(5). Yellow solid, 99.8% yield. 1H NMR (500 MHz, DMSO‑d6) δ 10.51
(brs, 1H, OH), 8.68 (s, 1H, 2′-H), 7.93 – 7.84 (m, 5H, 4-H, 5-H, 6-H, 7-H,
6′-H), 7.71 (s, 1H, vinylic H), 6.93 (d, 1H, J = 8.0 Hz, 5′-H), 4.17 (q, 2H,
J = 6.5 Hz, CH2CH3), 1.41 (t, 3H, J = 6.5 Hz, CH2CH3); 13C NMR (100
MHz, DMSO‑d6) δ 190.7, 189.9, 154.1, 147.6, 147.3, 142.3, 139.8,
136.2, 136.0, 132.4, 125.8, 125.6, 123.5, 123.3, 118.3, 116.4, 64.5,
15.3; LRMS (ESI-) m/z 293 (Mꢀ H)ꢀ , 264 (Mꢀ Hꢀ C2H5)ꢀ ; HRMS (ESI + )
m/z C18H15O4 (M + H)+ calcd. 295.0965, obsd. 295.0966.
suggest that chimeric compounds with the β-phenyl-α,β-unsaturated
dicarbonyl scaffold provide a basis for the design of potent tyrosinase
inhibitors.
4. Experimental
4.1. Chemistry
4.1.1. General methods
1H NMR and 13C NMR data were obtained using a Varian Unity
INOVA 400 spectrometer or a Varian Unity AS500 spectrometer (Agilent
Technologies, Santa Clara, CA, USA); DMSO‑d6 or CDCl3 were used as
solvents. All chemical shifts were measured in parts per million (ppm)
versus 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 pre-
sented in hertz. The following abbreviations are used for 1H NMR:
singlet (s), broad singlet (brs), doublet (d), doublet of doublets (dd),
broad doublet (brd), triplet (t), broad triplet (brt), quartet (q) or
multiplet (m). Low-resolution and high-resolution mass data were
recorded on an Expression CMS (Advion Ithaca, NY, USA) in ESI positive
and/or negative mode or an Agilent Accurate Mass Q-TOF liquid-
chromatograph/mass spectrometer (Agilent, Santa Clara, CA, USA) in
ESI positive mode, respectively. All reactions were conducted under
nitrogen and monitored by thin-layer chromatography (TLC; Merck
precoated 60F245 plates). Solvents were distilled over Na/benzophenone
or CaH2 before use.
4.1.2.6.
2-(3-Hydroxy-4-methoxybenzylidene)-1H-indene-1,3(2H)-
dione (6). Yellow solid, 80.8% yield. 1H NMR (400 MHz, CDCl3) δ 8.21
(s, 1H, 2′-H), 8.05 (d, 1H, J = 8.8 Hz, 6′-H), 7.98 – 7.95 (m, 2H, 5-H, 6-
H), 7.78 (s, 1H, vinylic H), 7.78 – 7.76 (m, 2H, 4-H, 7-H), 6.95 (d, 1H, J
= 8.8 Hz, 5′-H), 5.72 (s, 1H, OH), 3.98 (s, 3H, CH3); 13C NMR (100 MHz,
CDCl3) δ 191.0, 189.6, 151.5, 147.4, 147.4, 145.6, 142.7, 135.3, 135.1,
129.7, 127.4, 127.3, 123.4, 123.3, 120.0, 110.6, 56.4; LRMS (ESI-) m/z
279 (Mꢀ H)ꢀ , 264 (Mꢀ Hꢀ CH3)ꢀ ; HRMS (ESI + ) m/z C17H13O4 (M +
H)+ calcd. 281.0808, obsd. 281.0809.
4.1.2.7. 2-(4-Methoxybenzylidene)-1H-indene-1,3(2H)-dione (7). Yel-
low solid, 65.4% yield. 1H NMR (500 MHz, DMSO‑d6) δ 8.57 (d, 2H, J =
9.0 Hz, 2′-H, 6′-H), 7.93 – 7.88 (m, 4H, 4-H, 5-H, 6-H, 7-H), 7.76 (s, 1H,
vinylic H), 7.09 (d, 2H, J = 9.0 Hz, 3′-H, 5′-H), 3.87 (s, 3H, CH3); 13C
NMR (100 MHz, DMSO‑d6) δ 190.5, 189.6, 164.4, 146.4, 142.4, 139.9,
137.7, 136.3, 136.2, 126.9, 126.6, 123.5, 123.5, 115.1, 56.4; LRMS (ESI
+ ) m/z 265 (M + H)+; LRMS (ESI-) m/z 295 (M + MeOH-H)ꢀ , 248
(Mꢀ Hꢀ CH3)ꢀ ; HRMS (ESI + ) m/z C17H13O4 (M + H)+ calcd. 265.0859,
obsd. 265.0858.
4.1.2. General procedure for the synthesis of 2-(substituted benzylidene)-
1,3-indanedione derivatives 1 – 12: A solution of 1,3-indanedione (100
mg, 0.68 mmol) and an appropriate benzaldehyde (1.0 – 1.1 equiv.) in 1
M HCl acetic acid (0.4 mL) was stirred at room temperature for 9 – 24 h.
After adding water to the reaction mixture, the precipitate generated
was filtered and washed with water and dichloromethane or hexane/
dichloromethane (5:1 – 1:1) to give pure 2-(substituted benzylidene)-
1,3-indanedione derivatives 1 – 12 in yields of 43.1 – 99.8% (Scheme 2).
4.1.2.1. 2-(4-Hydroxybenzylidene)-1H-indene-1,3(2H)-dione (1).
Light brown solid, 86.8% yield. 1H NMR (400 MHz, DMSO‑d6) δ 10.84
(brs, 1H, OH), 8.48 (d, 2H, J = 7.2 Hz, 2′-H, 6′-H), 7.89 – 7.83 (m, 4H, 4-
H, 5-H, 6-H, 7-H), 7.69 (s, 1H, vinylic H), 6.89 (d, 2H, J = 7.2 Hz, 3′-H,
5′-H); 13C NMR (100 MHz, DMSO‑d6) δ 190.6, 189.7, 164.0, 146.9,
142.3, 139.9, 138.3, 136.2, 136.0, 125.8, 125.3, 123.4, 123.4, 116.7;
LRMS (ESI-) m/z 249 (Mꢀ H)ꢀ ; HRMS (ESI + ) m/z C16H11O3 (M + H)+
calcd. 251.0703, obsd. 251.0703.
4.1.2.8. 2-(2,4-Dimethoxybenzylidene)-1H-indene-1,3(2H)-dione (8).
Yellow solid, 93.4% yield. 1H NMR (500 MHz, DMSO‑d6) δ 9.14 (d, 1H,
J = 8.5 Hz, 6′-H), 8.23 (s, 1H, vinylic H), 7.94 – 7.89 (m, 4H), 6.73 (d,
1H, J = 8.5 Hz, 5′-H), 6.69 (s, 1H, 3′-H), 3.96 (s, 3H, CH3), 3.91 (s, 3H,
CH3); 13C NMR (100 MHz, CDCl3) δ 191.4, 190.0, 166.6, 163.2, 142.5,
141.2, 140.2, 136.8, 135.0, 134.8, 125.7, 123.1, 123.1, 116.3, 105.8,
97.9, 56.0, 55.9; LRMS (ESI + ) m/z 349 (M + MeOH + Na)+, 317 (M +
Na)+, 295 (M + H)+; HRMS (ESI + ) m/z C18H15O4 (M + H)+ calcd.
295.0965, obsd. 295.0965.
4.1.2.9. 2-(3,4-Dimethoxybenzylidene)-1H-indene-1,3(2H)-dione (9).
Yellow solid, 83.0% yield. 1H NMR (500 MHz, DMSO‑d6) δ 8.67 (s, 1H,
2′-H), 8.01 (d, 1H, J = 8.0 Hz, 6′-H), 7.96 – 7.90 (m, 4H, 4-H, 5-H, 6-H, 7-
H), 7.78 (s, 1H, vinylic H), 7.14 (d, 1H, J = 8.0 Hz, 5′-H), 3.90 (s, 3H,
CH3), 3.89 (s, 3H, CH3); 13C NMR (150 MHz, DMSO‑d6) δ 190.1, 189.4,
154.2, 148.8, 147.0, 142.2, 139.7, 135.7, 135.5, 131.5, 126.6, 126.5,
123.2, 123.0, 116.0, 111.5, 56.2, 56.0; LRMS (ESI-) m/z 325 (M +
MeOH-H)ꢀ , 293 (Mꢀ H)ꢀ ; HRMS (ESI + ) m/z C18H15O4 (M + H)+ calcd.
295.0965, obsd. 295.0969.
4.1.2.2. 2-(2,4-Dihydroxybenzylidene)-1H-indene-1,3(2H)-dione (2).
Brown solid, 43.1% yield. 1H NMR (400 MHz, DMSO‑d6) δ 10.95 (s, 1H,
OH), 10.81 (s, 1H, OH), 9.08 (d, 1H, J = 8.8 Hz, 6′-H), 8.25 (s, 1H,
vinylic H), 7.87 – 7.84 (m, 4H, 4-H, 5-H, 6-H, 7-H), 6.40 (d, 1H, J = 2.4
Hz, 3′-H), 6.39 (dd, 1H, J = 8.8, 2.4 Hz, 5′-H); 13C NMR (100 MHz,
DMSO‑d6) δ 191.3, 190.0, 166.7, 164.1, 142.1, 140.9, 139.7, 136.6,
135.9, 135.7, 123.1, 123.1, 113.8, 109.3, 102.5; LRMS (ESI-) m/z 265
(Mꢀ H)ꢀ ; HRMS (ESI + ) m/z C16H11O4 (M + H)+ calcd. 267.0652, obsd.
267.0649.
4.1.2.10.
2-(4-Hydroxy-3,5-dimethoxybenzylidene)-1H-indene-1,3
(2H)-dione (10). Orange solid, 79.2% yield. 1H NMR (400 MHz, CDCl3
+ 2 drops of DMSO‑d6) δ 8.68 (brs, 1H, OH), 7.72 (s, 2H, 2′-H, 6′-H),
7.65 – 7.62 (m, 2H, 5-H, 6-H), 7.50 – 7.48 (m, 2H, 4-H, 7-H), 7.45 (s, 1H,
vinylic H), 3.70 (s, 6H, 2 × CH3); 13C NMR (100 MHz, CDCl3 + 2 drops of
DMSO‑d6) δ 190.7, 189.7, 148.0, 147.6, 142.4, 142.3, 139.7, 135.1,
134.9, 125.8, 124.4, 122.9, 122.9, 112.6, 56.4; LRMS (ESI-) m/z 309
(Mꢀ H)ꢀ , 294 (Mꢀ Hꢀ CH3)ꢀ ; HRMS (ESI + ) m/z C18H15O5 (M + H)+
calcd. 311.0914, obsd. 311.0914.
4.1.2.3. 2-(3,4-Dihydroxybenzylidene)-1H-indene-1,3(2H)-dione (3).
Yellow solid, 75.7% yield. 1H NMR (400 MHz, DMSO‑d6) δ 10.47 (s, 1H,
OH), 9.54 (s, 1H, OH), 8.30 (s, 1H, 2′-H), 7.89 – 7.83 (m, 4H, 4-H, 5-H, 6-
H, 7-H), 7.80 (d, 1H, J = 8.0 Hz, 6′-H), 7.60 (s, 1H, vinylic H), 6.87 (d,
1H, J = 8.0 Hz, 5′-H); 13C NMR (100 MHz, DMSO‑d6) δ 190.7, 189.6,
153.3, 147.6, 146.0, 142.3, 139.8, 136.1, 135.9, 131.0, 125.8, 125.5,
123.3, 123.3, 121.3, 116.4; LRMS (ESI-) m/z 265 (Mꢀ H)ꢀ ; HRMS (ESI +
) m/z C16H11O4 (M + H)+ calcd. 267.0652, obsd. 267.0650.
4.1.2.11. 2-(3,4,5-Trimethoxybenzylidene)-1H-indene-1,3(2H)-dione
(11). Orange solid, 57.7% yield. 1H NMR (500 MHz, DMSO‑d6) δ 8.07 (s,
2H, 2′-H, 6′-H), 7.96 – 7.89 (m, 4H, 4-H, 5-H, 6-H, 7-H), 7.75 (s, 1H,
vinylic H), 3.87 (s, 6H, 2 × CH3), 3.79 (s, 3H, CH3); 13C NMR (100 MHz,
DMSO‑d6) δ 190.1, 189.5, 153.1, 147.2, 143.1, 142.6, 140.0, 136.1,
4.1.2.4.
2-(4-Hydroxy-3-methoxybenzylidene)-1H-indene-1,3(2H)-
1
dione (4). Yellow solid, 75.1% yield. H NMR (400 MHz, DMSO‑d6
+
9

I.Y. Ryu et al.
Bioorganic Chemistry 109 (2021) 104688
136.0, 128.8, 128.2, 123.6, 123.4, 112.6, 60.9, 56.6; LRMS (ESI + ) m/z
379 (M + MeOH + Na)+, 347 (M + Na)+, 325 (M + H)+; HRMS (ESI + )
m/z C19H17O5 (M + H)+ calcd. 325.1071, obsd. 325.1075.
methods [56,57] using compound 2 and 3 concentrations of 0, 10, 20, or
40 μM and 0, 7.5, 15, and 30 μM, respectively. L-Tyrosine was used at
concentrations of 0, 1, 2, or 4 mM for compound 2 and 0, 2, 4, 8, or 16
mM for compound 3, respectively. Initial rates of dopachrome formation
were determined from the initial linear portion of absorbance versus
time plots at 10 min intervals up to 50 min after enzyme addition. In-
hibition types were determined using Lineweaver-Burk plots of dop-
achrome formation rateꢀ 1 (1/V) versus substrate concentrationꢀ 1 (1/[L-
tyrosine] mMꢀ 1).
4.1.2.12.
2-(3-Bromo-4-hydroxybenzylidene)-1H-indene-1,3(2H)-
dione (12). Orange solid, 75.1% yield. 1H NMR (400 MHz, DMSO‑d6) δ
11.68 (s, 1H, OH), 9.06 (d, 1H, J = 2.0 Hz, 2′-H), 8.26 (dd, 1H, J = 8.8,
2.0 Hz, 6′-H), 7.94 – 7.85 (m, 4H, 4-H, 5-H, 6-H, 7-H), 7.68 (s, 1H,
vinylic H), 7.04 (d, 1H, J = 8.8 Hz, 5′-H); 13C NMR (100 MHz, DMSO‑d6)
δ 190.3, 189.8, 159.9, 145.3, 142.4, 140.0, 139.6, 137.4, 136.4, 136.3,
127.2, 126.6, 123.6, 123.5, 117.0, 110.5; LRMS (ESI-) m/z 329 (Mꢀ H)ꢀ ,
327 (Mꢀ 2ꢀ H)ꢀ .
4.2.5. Cell culture
Murine B16F10 melanoma cells were obtained from the American
Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured
in DMEM (Dulbecco’s Modified Eagle Medium) containing 10% FBS
4.2. Biology
(fetal bovine serum), 100 IU/mL penicillin, and 100 μg/mL strepto-
4.2.1. Mushroom tyrosinase inhibition assay of compounds 1–12 and kojic
acid
mycin (Gibco/Thermo Fisher Scientific; Carlsbad, CA, USA) in 5% CO2
humidified atmosphere at 37 0C. The cultured cells were used for sub-
sequent experiments.
A standard procedure was used to determine the mushroom tyrosi-
nase inhibitory activities of the 12 test compounds, with some modifi-
cation [54]. Two hundred
tyrosinase solution, 170
buffer, and 293 L L-tyrosine solution), and 10
μL of a mixture consisting of 20 μL of
4.2.6. Viabilities of B16F10 melanoma cells treated with compounds 2 or 3
The EZ-Cytox (EZ-3000, Daeil Lab Service, Seoul, Korea) assay was
used to measure cell viabilities after treatment with various concentra-
tions (1, 2, 5, 10, 20, or 50 µM) of compounds 2 or 3 [60]. B16F10 cells
were seeded (1x104 cells/well) in 96 well plates and incubated for 24 h
μ
L of substrate solution (14.7 mM phosphate
μ
μ
L of a test compound (1 –
12, final concentration: 50 μM) was placed in the wells of a 96 well plate.
The plates were incubated at 37◦ C for 30 min and optical densities were
measured using a microplate reader (VersaMaxTM) at 450 nm. Kojic acid
◦
in humidified 5% CO2 atmosphere at 37 C. After treatment with test
(50 μM) was used as the positive control. Experiments were repeated
compounds, cells were incubated for an additional 24 h. The next day,
the EZ-Cytox reagent was added to cultured cells in a ratio of 1:10 and
incubated at 37 ◦C for 2 h. Cell viabilities were calculated by measuring
optical densities at 450 nm. Experiments were conducted independently
three times.
independently three times. The following formula was used to calculate
tyrosinase inhibition.
%Inhibition = [1-(A/B)] × 100
A represents test compound absorbance and B the absorbance of the
untreated control.
4.2.7. Cellular tyrosinase activities of B16F10 melanoma cells after
treatments of compounds 2 or 3
Cellular tyrosinase inhibitory activities were evaluated by deter-
mining the rate of L-DOPA oxidation as previously described with slight
modification [61]. B16F10 cells were cultured at a density of 5x104 cells
per well, allowed to attach to the bottom of wells in a 6-well plate, and
then incubated overnight in a humidified 5% CO2 atmosphere at 37 ◦C.
4.2.2. In silico docking simulations of compound 2, compound 3, and kojic
acid with tyrosinase
Schrodinger Suite (2020–2) was used for the in silico docking simu-
lation studies. The three-dimensional (3D) structure of mushroom
tyrosinase (Agaricus bisporus) (PDB ID: 2Y9X) was imported from the
Protein Data Bank (PDB), and the protein structure of tyrosinase was
prepared using the protein preparation wizard in Maestro 12.4 to
improve docking results prior to molecular docking experiments. Un-
wanted protein chains were removed. The protein structure of tyrosi-
nase was optimized by adding hydrogen atoms, then water molecules
were removed and the structure was minimized. The active site of
tyrosinase was defined using the co-crystal ligand binding site of the
PDB and literature data [55–57]. Compounds 2, 3, and kojic acid were
imported to the entry list of Maestro 12.4 in CDXML format and pre-
pared for docking using LigPrep. Molecular docking experiments were
performed for compounds 2 and 3 and kojic acid using the Glide docking
protocol [58]. Predicted binding energies (docking scores) and binding
interactions of ligands within the active region of tyrosinase were ob-
tained using Glide extra precision (XP) [59].
The control and treatment groups were activated with 5 µM α-MSH plus
200 µM IBMX. Cells were treated with test compounds 2 or 3 (final
concentrations: 0, 5, 10, or 20 µM) or kojic acid (final concentration: 20
µM) and held for an additional 60 h under standard conditions. Cells
were washed 2–3 times with PBS, lysed with lysis buffer (100 μL) (50
mM phosphate buffer (pH 6.5), 0.1 mM phenylmethylsulfonyl fluoride
(PMSF), and 1% Triton X-100) and frozen at ꢀ 80 ◦C for 30 min. Lysed
cells were centrifuged at 12,000 rpm for 30 min at 4 ◦C, and lysates (80
µL) were incubated with 20 µL of L-dopa (2 mg/mL) at 37 ◦C for 30 min.
Optical densities were measured at 492 nm using a microplate reader
¨
(Tecan, Mannedorf, Switzerland). Experiments were conducted inde-
pendently three times.
4.2.8. Melanin inhibition by compounds 2 and 3 in B16F10 melanoma cells
Melanin inhibition assays of compounds 2 and 3 in B16F10 cells
were performed as previously described with slight modification [62].
B16F10 cells were cultured at a density of 5x104 cells per well in 6-well
plates, allowed to attach, and then incubated overnight in a humidified
4.2.3. Assay of copper-interacting ability [52]
The mixture consisting of 5 μL of a sample compound (2 or 3, 0.05
mM), 5
50
μL of the aqueous solution of mushroom tyrosinase (139 units),
5% CO2 atmosphere at 37 ◦C. They were then treated with 5 µM
α-MSH
μ
L of distilled water, and 90 μL of 50 mM phosphate buffer (pH 6.5)
with or without CuSO4 (0.125 mM) was incubated at 37 ◦C for 30 min,
and then the UV–visible spectra (350 – 600 nm) were measured using
microplate spectrophotometer (Multiskan GO, Thermo Scientific, CA,
USA).
plus 200 µM IBMX, and compounds 2 and 3 (final concentrations: 0, 5,
10, and 20 µM) or kojic acid (final concentration: 20 µM) were added.
Cells were then left for 60 h under standard conditions. For extracellular
melanin content, the culture media was directly measured at 405 nm
using a microplate reader. For intracellular melanin content, the cell
pellets were washed 2–3 times with PBS buffer, treated with 200 μL of 1
4.2.4. Kinetic analysis
N NaOH, and incubated at 60 ◦C for 1 h to extract the melanin. Melanin
absorbances were measured at 405 nm. All results were normalized to
the total protein concentration of the cell pellet using a Bicinchoninic
Compounds 2 and 3 most potently inhibited mushroom tyrosinase
and were selected for the kinetic analysis. The inhibition kinetics of
compounds 2 and 3 were investigated using previously reported
10

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Bioorganic Chemistry 109 (2021) 104688
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Acknowledgments
This work was supported by a National Research Foundation of
Korea (NRF) grant funded by the Korea government (MSIT) (No. NRF-
2020R1A2C1004198).
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