Inhibitory effects of N-(acryloyl)benzamide derivatives on tyrosinase and melanogenesis

Article abstract of DOI:10.1016/j.bmc.2019.07.034

Targeting of tyrosinase has proven to be the best means of identifying safe, efficacious, and potent tyrosinase inhibitors for whitening skin. We designed and synthesized ten NAB (N-(acryloyl)benzamide) derivatives (1a–1j) using the Horner-Wadsworth-Emmon

Full text of DOI:10.1016/j.bmc.2019.07.034

Accepted Manuscript
Inhibitory effects of N-(acryloyl)benzamide derivatives on tyrosinase and mel-
anogenesis
Sanggwon Lee, Sultan Ullah, Chaeun Park, Hee Won Lee, Dongwan Kang,
Jungho Yang, Jinia Akter, Yujin Park, Pusoon Chun, Hyung Ryong Moon
PII:
S0968-0896(19)30959-9
https://doi.org/10.1016/j.bmc.2019.07.034
BMC 15018
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
11 June 2019
9 July 2019
19 July 2019
Please cite this article as: Lee, S., Ullah, S., Park, C., Won Lee, H., Kang, D., Yang, J., Akter, J., Park, Y., Chun,
P., Ryong Moon, H., Inhibitory effects of N-(acryloyl)benzamide derivatives on tyrosinase and melanogenesis,
Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.07.034
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Inhibitory effects of N-(acryloyl)benzamide derivatives on tyrosinase and
melanogenesis
a,§
a,§
a,§
a
a
Sanggwon Lee , Sultan Ullah , Chaeun Park , Hee Won Lee , Dongwan Kang , Jungho
a
a
a
b
a,
Yang , Jinia Akter , Yujin Park , Pusoon Chun , Hyung Ryong Moon *
aLaboratory of Medicinal Chemistry, College of Pharmacy, Pusan National University, Busan
4
6241, South Korea
bCollege of Pharmacy and Inje Institute of Pharmaceutical Sciences and Research, Inje
University, Gimhae, Gyeongnam 50834, South Korea
§These authors (S. Lee, and S. Ullah, and C. Park) equally contributed to this work.
*
Author to whom correspondence should be addressed.
Hyung Ryong Moon, Laboratory of Medicinal Chemistry, College of Pharmacy, Pusan National
University, Busan 46241, Republic of Korea. E-mail address: mhr108@pusan.ac.kr; Tel.: +82
5
1 510 2815; Fax: +82 51 513 6754.

Abstract
Targeting of tyrosinase has proven to be the best means of identifying safe, efficacious, and
potent tyrosinase inhibitors for whitening skin. We designed and synthesized ten NAB (N-
(acryloyl)benzamide) derivatives (1a – 1j) using the Horner-Wadsworth-Emmons olefination of
diethyl (2-benzamido-2-oxoethyl)phosphonate and appropriate benzaldehydes. A mushroom
tyrosinase inhibitory assay showed compounds 1a (36.71±2.14% inhibition) and 1j
(
25.99±2.77% inhibition) inhibited tyrosinase more than the other eight NAB derivatives and
kojic acid (21.56±2.93% inhibition), and docking studies indicated 1a (-6.9 kcal/mole) and 1j (-
.5 kcal/mole) had stronger binding affinities for tyrosinase than kojic acid (-5.7 kcal/mole). At a
7
concentration of 25 μM, 1a and 1j were nontoxic in B16F10 melanoma cells and exhibited
stronger tyrosinase inhibition (59.70% and 76.77%, respectively) than kojic acid (50.30%
inhibition) or arbutin (41.78% inhibition at 400 μM). Similarly, in B16F10 melanoma cells,
compounds 1a and 1j at 25 μM decreased total melanin content by 47.97% and 61.77%,
respectively (kojic acid; 38.98%). Similarities between inhibitions of tyrosinase activity and
melanin contents suggested the anti-melanogenic effects of 1a and 1j were due to tyrosinase
inhibition. The excellent DPPH scavenging activity of 1j suggests it might enhance in vivo effect
on melanin contents. The study suggests compound 1j offers a potential starting point for the
development of safe, potent tyrosinase inhibitors.
Key words: (N-(acryloyl)benzamide) derivatives, Horner-Wadsworth-Emmons olefination,
melanogenesis, tyrosinase, B16F10 melanoma cells, inhibitor.

1
. Introduction
Tyrosinase (EC 1.14.18.1), is a type 3, di-copper containing enzyme that produces melanin and
is widely distributed in plants and animals. The tyrosinase is composed of glycoproteins and its
levels are regulated by two degradation systems, that is, lysosomal/endosomal and proteasomal
1
systems. The structure of tyrosinase from Streptomyces Castaneoglobisporus was first
determined in 2006, but other crystal structures have been reported in different species since.2
Structurally, tyrosinase is composed of a central domain, a C-terminal domain and an N-terminal
3
domain. The central domain consists of two copper(II) ions and six histidine residues.
According to the crystal structures of tyrosinases from Streptomyces castaneoblobisporus,
Aspergillus oryzae, Agaricus bisporus, and Bacillus megaterium, its central domain is
responsible for its catalytic effects. The formation of a thioether bond between a cysteine residue
and a histidine residue in the active site of tyrosinase stabilizes the histidine residue. Histidine is
also coordinated with one of the copper ions for the catalytic mechanism. The N-terminal
4
domain of tyrosinase determines its final shape in the presence of a transit peptide, which is
responsible for melanosome transfer in mushrooms and in humans. The catalytic activity of
tyrosinase is highly dependent on its two copper ions (CuA and CuB) and six histidine residues.5
Tyrosinase can exist in oxy, met, or deoxy forms.6-9 In a resting state, tyrosinase mainly exists in
the met form and cannot oxidize phenols. To enable it to act as a catalyst, it must first be
transformed into the deoxy form, which is readily oxidized to the oxy form. For this reason, the
met form is viewed as the best target for tyrosinase inhibitors.10
Due to the ability of tyrosinase to catalyze the rate-limiting step of melanin production, it is
considered an important target for the treatment of abnormal melanogenesis. Researchers have
tried many approaches based on the modulation of tyrosinase expression,11 maturation,12,13

1
4
15
degradation, and direct catalytic activity, to control abnormal melanin production. However,
although a large number of tyrosinase inhibitors have been identified, relatively few are used as
skin lightening agents because they lack potency and have poor safety profiles.15 The skin
lightening agents commonly used in the cosmetic market are hydroquinone, monobenzyl
hydroquinone, arbutin,16-20 kojic acid,21 salicylhydroxamic acid,22 azelaic acid,23 and
corticosteroids.22,23 However, the use of hydroquinone is restricted because it is cytotoxic to
mammalian cells and its use is associated with serious side effects, which include skin irritation,
burning, contact dermatitis, hypochromia, and chestnut spots over nails.24-27 Similarly, the use of
kojic acid is restricted due to its instability and carcinogenicity.16,21 Furthermore, EU cosmetic
regulations ban the use of hydroquinone and of corticosteroids as skin whitening agents. Thus,
there is a need for safe, effective tyrosinase inhibitor for skin whitening agents.
As part of our continued efforts to identify safe melanogenic materials, we synthesized a series
of tyrosinase inhibitors and found that compounds possessing the (E)-β-phenyl-α,β-unsaturated
carbonyl motif showed excellent tyrosinase inhibitory activity. Analogues containing the (E)-β-
phenyl-α,β-unsaturated carbonyl motif have also been reported to have many different biological
2
8
29
30
activities including anticancer agents, anti-convalescent agents, anti-oxidant agents, anti-
3
1
32,33
viral agents, and anti-malarial agents.
The natural substrates of tyrosinase are L-tyrosine
and L-dopa, which have hydroxyl groups on their phenyl ring. The hydroxyl groups may play an
important role in binding to the tyrosinase enzyme. Therefore, we want to introduce different
substituents such as hydroxyl, methoxyl, and bromo groups to the β-phenyl ring of the carbonyl
motif and to compare the effects of the substituents on tyrosinase inhibition. For this purpose, N-
(
acryloyl)benzamide (NAB) derivatives,34-37 which also contain the (E)-β-phenyl-α,β-unsaturated
carbonyl motif, were synthesized as potential tyrosinase inhibitors, and evaluated with respect to

their abilities to inhibit mushroom tyrosinase, and tyrosinase activity and melanin production in
B16F10 melanoma cells.
2
. Results and discussions
2
.1. Chemistry
A strategy for the preparation of NAB derivatives is depicted in Scheme 1. It was envisioned that
NAB derivatives could be produced by condensation between diethyl (2-benzamido-2-
oxoethyl)phosphonate (3) and appropriate benzaldehydes. Reaction between benzamide and
3
8
chloroacetyl chloride gave N-acyl benzamide 2 in 58% yield, and subsequent reaction between
3
8
2
and triethyl phosphite afforded phosphonate 3 a key intermediate for the synthesis of NAB
derivatives by Michaelis-Arbuzov rearrangement. To synthesize NAB derivatives 1i and 1j,
which contain a 4-hydroxyphenyl or a 3,4-dihydroxyphenyl, respectively, the hydroxyl groups of
4
-hydroxybenzaldehyde and 3,4-dihydroxybenzaldehyde were first protected with a
tetrahydropyranyl (THP) ether using 3,4-dihydro-2H-pyran in the presence of pyridinium p-
toluenesulfonate to afford compounds 4 (90.9%) and 5 (79.7%), respectively. Horner-
Wadsworth-Emmons olefination39 of phosphonate 3 with different substituted benzaldehydes
produced the desired NAB derivatives 1a – 1h and the THP-protected NAB derivatives 6 and 7.
These two derivatives were deprotected under acidic conditions using 2M-HCl in 1,4-dioxane to
give the NAB derivatives 1i and 1j. The structures of NAB derivatives 1a – 1j were confirmed
1
13
by H and C NMR and high resolution mass spectrometry.

O
O
O
O
O
O
O
P
O
O
O
O
R2
R3
Cl
P
e
N
H
+
b
N
N
O
H
R2
H
O
3
2
6 (R = H, R3 = OTHP)
2
R1
THPO
2
3
R2
R3
4 (R2 = H)
(R2 = OTHP)
f
f
7 (R , R = OTHP)
a
O
c
1i
(R2 = H, R3 = OH)
2
5
1j (R , R3 = OH)
O
R4
O
d
NH2
R1
O
O
R2
R3
+
O
R2
N
H
O
Cl
HO
Cl
R4
R2 = H or OH
1
4
1
1
1
1
1
1
1
1
a (R , R = H, R2 = OH, R3 = OMe)
1
2
4
3
b (R , R , R = H, R = OMe)
1
4
2
3
c (R , R = H, R , R = OMe)
d (R , R4 = H, R , R = OMe)
2
1
3
e (R1 = H, R , R , R = OMe)
2
3
4
f (R1 = H, R , R = OMe, R3 = OH)
g (R , R4 = H, R2 = Br, R3 = OH)
h (R1 = H, R , R4 = Br, R3 = OH)
2
4
1
2
Scheme 1. Synthetic scheme for NAB derivatives 1a – 1j. Reagents and conditions: a) reflux, 50
o
min, 58%; b) 80 C, 24 h, 51%; c) NaH, THF, rt, 1 – 12 h, 26 – 86%; d) pyridinium p-
toluenesulfonate, DCM, rt, 2 – 50 h, 91% for 4, and 80% for 5; e) NaH, THF, rt, 3 h, 69% for 6,
and 88% for 7; f) 2M-HCl, 1,4-dioxane, rt, 30 min, 63% for 1i, 6 h, 49% for 1j.
2
.2. Mushroom tyrosinase inhibition assay for NAB derivatives 1a – 1j
Mushroom tyrosinase inhibitions by NAB derivatives 1a – 1j were determined at a concentration
of 25 µM, and kojic acid (25 µM; a well-known tyrosinase inhibitor) was used as the control.
Results are summarized in Table 1. Although all NAB derivatives harboured the β-phenyl-α,β-
unsaturated carbonyl motif, the types and positions of substituents on the β-phenyl ring of NAB
derivatives were found to greatly influence their activities. Among the four NAB derivatives
with a β-3,4-disubstituted-phenyl ring, compound 1a with a 3-hydroxy-4-methoxyphenyl ring
and compound 1j with a 3,4-dihydroxylphenyl ring showed greater tyrosinase inhibitory activity
(
36.71±2.14 and 25.99±2.77%, respectively) than kojic acid (21.55±2.30%). On the other hand,
NAB derivatives (1c – 1e) either failed (1e) or only weakly inhibited tyrosinase (5.49±2.21% for
c, 8.05±2.84% for 1d). Compound 1b did not have a hydroxyl group like 1c – 1e but
1

moderately inhibited tyrosinase activity by 17.14±3.94%. Derivative 1i, which possessed a 4-
hydroxyphenyl ring, also potently inhibited tyrosinase (by 19.18±1.14%). Notably insertion of a
hydroxyl group into position 3 of the -phenyl ring of 1i increased inhibitory activity (1i:
1
9.18±1.14% vs. 1j: 25.99±2.77%), whereas the insertions of methoxyl groups into positions 3
and 5 greatly diminished inhibitory activity (1i: 19.18±1.14% vs. 1f: 5.54±2.35%). The
additional introduction of bromo groups into positions 3 and/or 5 also reduced inhibitory activity
(1i: 19.18±1.14% vs. 1g: 11.32±2.90% and 1h: no inhibition). These results suggest the β-
phenyl-α,β-unsaturated carbonyl motif plays a key role in the inhibition of tyrosinase and that
substituent type and location on the β-phenyl ring sensitively affect tyrosinase inhibition.

Table 1. Inhibitory effects of NAB derivatives 1a – 1j and of kojic acid on mushroom tyrosinase
R1
O
O
R2
R3
N
H
R4
Compound
R1
H
R2
R3
R4
H
Tyrosinase inhibition (%)
36.71±2.14
17.14±3.94
5.49±2.21
8.05±2.84
aNI
1
a
OH
H
OMe
OMe
OMe
OMe
OMe
OH
1
b
H
H
1
c
H
OMe
H
H
1
d
OMe
H
H
1
e
OMe
OMe
Br
OMe
OMe
H
1
f
H
5.54±2.35
11.32±2.90
NI
1
g
H
OH
1
h
H
Br
OH
Br
H
1
i
H
H
OH
19.18±1.14
25.99±2.77
21.55±2.30
1
j
H
OH
OH
H
Kojic acid
Tyrosinase inhibitions were measured at a derivative concentration of 25 µM. L-Tyrosine was
a
used as a substrate. Results are expressed as means ± SEMs. NI: no inhibition
2
.3. Docking simulation study of derivatives 1a and 1j
To investigate interactions with tyrosinase at the molecular level, docking simulations were
performed using NAB derivatives 1a and 1j, which most inhibited mushroom tyrosinase. We
used AutoDock Vina 1.1.2 docking software developed by the Scripps Research Institute for the

simulations. Using the energy minimization tool of Chem3D Pro 12.0 software from
CambrigeSoft Corporation, we created stable 3D structures of ligands 1a and 1j. The structure of
mushroom tyrosinase was obtained from the Protein Data Bank (ID: 2Y9X). As shown in Figure
1
d, ligands 1a and 1j showed stronger binding affinities for tyrosinase (-6.4 and -7.5 kcal/mole,
respectively) than kojic acid (-5.7 kcal/mole). To investigate the natures of binding interactions,
we used a LigandScout 4.3.0 software to produce pharmacophore models. As shown in Figure
1
b, kojic acid interacted with amino acid His263 by π-π stacking and formed two hydrogen
bonds with the amino acid residues of His259 and His263. The ligand 1a interacted
hydrophobically with the tyrosinase amino acids Val248, Met257, Phe264, and Val283. On the
other hand, compound 1j interacted hydrophobically with Val248, Phe264, and Val283. These
results indicate the 1a and 1j bind strongly to tyrosinase at its active site.
Figure 1. Binding scores and interactions of NAB derivatives 1a and 1j and kojic acid with
tyrosinase. (a, and b) Pharmacophore results for 1a and 1j and kojic acid obtained using
LigandScount 4.3.0 showed probable hydrogen-bonding (green arrows), π-π stacking (violet

arrow), and hydrophobic interactions (yellow) between 1a, 1c, and kojic acid and amino acid
residues of tyrosinase, (c) 3D pharmacophore models of ligands 1a and 1j and tyrosinase, and (d)
binding scores for interactions between ligands 1a, 1j, and kojic acid and tyrosinase.
2
.4. Cytotoxicities of NAB derivatives 1a and 1j
Derivatives 1a and 1j were chosen for the cytotoxicity study, which was performed using a
WST-8 assay. NAB derivatives 1a and 1j were prepared at concentrations of 0, 5, 10, and 25 µM
and treated with B16F10 melanoma cells in an incubator for 24 h. As shown in Figure 2, neither
derivative exhibited a cytotoxic effect at concentrations up to 25 µM. Thus, further studies on the
anti-tyrosinase and anti-melanin production activities of 1a and 1j were conducted at
concentrations ≤ 25 µM in B16F10 melanoma cells.

Figure 2. Cytotoxic effects of NAB derivatives 1a and 1j in B16F10 melanoma cells at
concentrations of 5, 10, or 25 µM. Cytotoxic effects are expressed as percentage viabilities
versus non-treated controls. Standard errors are indicated by bars.
2
.5. Mammalian tyrosinase inhibition by NAB derivatives 1a and 1j in B16F10 cells
B16F10 melanoma cells were stimulated with α-MSH hormone and treated with 1a or 1j at
concentrations of 0, 5, 10, or 25 μM, kojic acid (25 μM), or arbutin (400 μM; positive control)

and cellular tyrosinase activities were screened. Cells were incubated in a 5% CO environment
2
for 24 h. Results were obtained by measuring optical densities.
As shown in Figure 3, NAB derivatives 1a and 1j inhibited tyrosinase more potently than kojic
acid or arbutin. At a concentration of 25 µM, 1a (59.70% inhibition) more strongly inhibited
tyrosinase than kojic acid (50.30% inhibition) or arbutin (41.78% inhibition). 1j (76.77%
inhibition) with a 3,4-dihydroxyphenyl ring at the –position of the α,β-unsaturated carbonyl
motif inhibited tyrosinase more at this concentration than 1a, which conflicted with our
mushroom tyrosinase results. We attribute this to structural differences between mushroom
tyrosinase and the mammalian tyrosinase of B16F10 melanoma cells. Both 1a and 1j induced
significant decreases in tyrosinase activities at all concentrations tested versus non-treated
controls, and inhibited tyrosinase activity in a concentration-dependent manner. These results
suggest that the (E)-β-phenyl-α,β-unsaturated carbonyl motif plays an important role in the
inhibitions of mushroom and mammalian tyrosinase.
Tyrosinase activity
Control
1
a
1j
100
*
**
*
**
*
**
***
*
**
***
50
*
**
*
**
0
l
d
n
i
t
o
M
M
M
M
M
M
i
r
µ
µ
µ
µ
µ
µ
c
t
u
n
5
0
5
5
0
5
A
b
r
o
1
2
1
2
c
i
C
j
A
o
K
Concentration

Figure 3. Mammalian tyrosinase inhibition by NAB derivatives 1a and 1j in α-MSH stimulated
B16F10 melanoma cells. The cells were treated with NAB derivatives 1a or 1j (5, 10 or 25 μM),
kojic acid (25 μM), or arbutin (400 μM) in the presence of α-MSH. Asterisks indicate significant
differences between columns: ***, p<0.001. Standard errors are represented by bars.
2
.6. Melanin content assays of B16F10 melanoma cells treated with NAB derivatives 1a or 1j
B16F10 melanoma cells were activated with α-MSH and then treated with 1a, 1j (5, 10, or 25
μM), or kojic acid (25 μM) and incubated for 24 h. Melanin levels were determined by
measuring optical densities.
As indicated in Figure 4, NAB derivatives 1a and 1j significantly reduced melanin production
versus kojic acid. When treated at concentrations of 5, 10, or 25 μM, melanin levels reduced by
9
2
.06%, 31.24%, and 47.97%, respectively, for 1a, and 21.58%, 37.57%, and 61.77% for 1j. At
5 μM, kojic acid reduced melanin content by only 38.98%. Somewhat surprisingly, 1j at 10 μM
reduced melanin levels to the same level as kojic acid at 25 μM. Because tyrosinase controls the
rate-limiting step during melanogenesis, the decrease in melanin content will be closely
associated with the inhibition of tyrosinase. As was expected, the melanin contents shown in
Figure 4 are similar to the tyrosinase activities depicted in Figure 3.

Melanin content
1
a
1j
1
00
**
***
***
***
***
***
5
0
0
***
l
d
o
M
M
M
M
M
M
i
r
c
t
µ
µ
µ
µ
µ
µ
a
n
5
0
5
5
0
5
o
c
i
j
1
2
1
2
C
o
K
Concentration
Figure 4. Effects of NAB derivatives 1a and 1j on melanin levels in α-MSH stimulated B16F10
melanoma cells. B16F10 melanoma cells were treated with 1a, 1j (5, 10 or 25 μM) or kojic acid
(25 μM). Asterisks indicate significance differences between columns: ***, p<0.001. Standard
errors are marked as bars.
2
.7. DPPH radical scavenging assay of NAB derivatives 1a – 1j
NAB derivatives 1a – 1j (1 mM) and L-ascorbic acid were added to a DPPH solution in
methanol, and radical scavenging effects were determined 30 min later by measuring percentage
absorbances (Table 2).
According to our previously reported structure-activity data on DPPH radical scavenging
activity, 40,41 compounds with a 3,4-dihydroxyl substituent on the β-phenyl ring of the β-phenyl-
α,β-unsaturated carbonyl motif usually exhibit high levels of DPPH radical scavenging activity.
This trend was also observed for NAB derivatives. NAB derivative 1j, which possessed a 3,4-
dihydroxyphenyl substituent, showed greatest DPPH radical scavenging activity 82.89±0.32%,

which was similar to that of L-ascorbic acid (84.64±0.32%; positive control) (Table 2). The other
NAB derivatives did not exhibit prominent radical scavenging effects. ROS (reactive oxygen
species) are well known to participate in melanogenesis by mediating the activation of
tyrosinase.
Therefore, it is suggested that NAB derivative 1j may reduce melanin production in vivo by the
dual mechanism of direct tyrosinase activity inhibition and radical scavenging effect.
Table 2. DPPH radical scavenging activity of NAB derivatives 1a – 1j and kojic acid
Compound
DPPH radical scavenging activity (%)
1
a
7.02±1.32
5.61±1.18
4.82±0.51
5.76±0.65
6.90±0.76
3.76±0.20
17.26±1.11
NAa
1
b
1
c
1
d
1
e
1
f
1
g
1
h
1
i
4.89±1.15
82.89±0.32
84.64±0.32
1
j
L-Ascorbic acid

R1
O
O
R2
R3
N
H
R4
Radical scavenging was measured at an NAB derivative and an L-ascorbic acid concentration of
1
mM. Three independent experiments were performed and results are expressed as means ±
a
standard deviations. NA: no activity.
3
. Conclusion
In this study, ten NAB derivatives designated 1a – 1j were synthesized by the Horner-
Wadsworth-Emmons olefination of diethyl (2-benzamido-2-oxoethyl)phosphonate using
appropriate benzaldehydes and screened for mushroom tyrosinase inhibition. Of these 10
compounds, 1a (36.71±2.14% inhibition), and 1j (25.99±2.77% inhibition) inhibited tyrosinase
activities to the greatest extents and had also greater inhibitory effects than kojic acid
(21.56±2.93% inhibition). The mushroom tyrosinase inhibitory results obtained were supported
by our docking study results, which showed 1a (-6.9 kcal/mole) and 1j (-7.5 kcal/mole) bound to
tyrosinase stronger than kojic acid (-5.7 kcal/mole). B16F10 melanoma cells were used to
investigate the safety profiles of NAB derivatives 1a and 1j, and also to determine their effects
on cellular tyrosinase activity and cellular melanin contents. According to the results obtained,
neither 1a nor 1j had a cytotoxic effect on B16F10 melanoma cells at concentrations up to 25
μM. Furthermore, 1a and 1j inhibited tyrosinase more (59.70% and 76.77%, respectively) at 25
μM than kojic acid (50.30% inhibition at 25 µM) or arbutin (41.78% inhibition at 400 μM).
Similarly, at 25 µM 1a and 1j reduced melanin contents by 47.97% and 61.77%, respectively,
whereas kojic acid only reduced it by 38.98%. The similarity between our tyrosinase and
melanin content inhibition results suggests that the anti-melanogenic effects of 1a and 1j are
mainly due to their tyrosinase inhibitory abilities. Compound 1j also exhibited potent DPPH

radical scavenging activity (82.89±0.32%). Collectively, these results imply that 1j is a
promising candidate for the purpose of prevention and treatment of skin hyperpigmentation
diseases.
4
. Materials and Methods
4
.1. Chemistry
Masses of NAB derivatives were obtained by mass spectroscopy. High-resolution mass spectra
were recorded on an Agilent Accurate-Mass Q-TOF liquid–chromatography-mass spectrometer
(Agilent, Santa Clara, CA, USA. Structure elucidation was performed using a Varian Unity
1
13
INOVA H NMR and a C NMR spectrophotometer (Agilent Technologies, Santa Clara, CA,
USA) using DMSO-d and CDCl as solvents. Chemical shifts were measured in ppm (parts per
6
3
million) against residual solvents or deuterated peaks (for DMSO-d ,  39.7 and  2.50, and
6
C
H
for CDCl ,  77.0 and  7.24). Coupling constants were measured in hertz. The following
3
C
H
abbreviations are used to present NMR data: s (singlet), brs (broad singlet), d (doublet), dd
doublet of doublets), t (triplet), td (triplet of doublets), q (quartet), qd (quartet of doublets) and
(
m (multiplet). Reaction were monitored by thin layer chromatography (TLC), which was
performed using 60 F254 glass silica gel plates from Merck. All intermediates and final products
were purified by extraction, filtration, and/or MP silica 40 – 63 (60 Å) column chromatography.

4
.1.1. Synthesis of N-(2-chloroacetyl)benzamide (2). Benzamide (12.10 g, 99.88 mmol) and
chloroacetyl chloride (8.20 mL, 103.01 mmol) were added to a 250 mL round-bottomed flask at
00
C, and then refluxed for 50 min. After cooling to room temperature, diethyl ether (30 mL) was
added and volatiles were removed under reduced pressure. The precipitate that formed was
filtered to obtain 2 as yellowish white solid at a yield of 58%.
1
H NMR (500 MHz, DMSO-d ) δ 11.42 (s, 1H, NH), 7.92 (d, 2H, J = 8.0 Hz, 2′-H, 6′-H), 7.63
6
1
3
(
t, 1H, J = 8.0 Hz, 4′-H), 7.51 (t, 2H, J = 8.0 Hz, 3′-H, 5′-H), 4.76 (s, 2H, CH ); C NMR (100
2
MHz, DMSO-d ) δ 169.1, 167.1, 133.7, 133.2, 129.2, 129.1, 46.5.
6
4
.1.2. Synthesis of diethyl (2-benzamido-2-oxoethyl)phosphonate (3). Compound 2 (3.00 g,
1
5.18 mmol) and triethyl phosphite (6.6 mL, 37.93 mmol) were added to a 100 mL round-
o
bottomed flask and heated at 80 C for 24 h. After cooling, hexane (6.0 mL) was added and the
supernatant was removed; this procedure was repeated 5 times. The resulting residue was
recrystallized from toluene and hexane (3:1) and the solid generated was filtered to give
compound 3. The filtrate was then evaporated and the residue obtained was purified by silica gel
column chromatography using hexane and ethyl acetate (5:1) as eluent to afford additional
compound 3. After recrystallization and column chromatography compound 3 was obtained as a
1
white solid at a yield of 51% (2.294 g). H NMR (500 MHz, CDCl ) δ 9.85 (s, 1H, NH), 7.93 (d,
3
2
4
H, J = 7.5 Hz, 2′-H, 6′-H), 7.58 (t, 1H, J = 7.5 Hz, 4′-H), 7.48 (t, 2H, J = 7.5 Hz, 3′-H, 5′-H),
.17 (qd, 4H, J = 7.0, 6.5 Hz, OCH CH ), 3.47 (d, 2H, J = 21.5 Hz, PCH ), 1.32 (t, 6H, J = 7.0
2
3
2
1
3
Hz, OCH CH ); C NMR (100 MHz, CDCl ) δ 165.5, 165.3, 133.5, 132.7, 129.2, 128.1, 63.3 (d,
2
3
3
J = 6.8 Hz), 36.7 (d, J = 129.8 Hz), 16.5 (d, J = 6.1 Hz).
.1.3. General procedure for the syntheses of compounds 4 and 5. 4-Hydroxybenzaldehyde
2.00 g, 16.38 mmol) or 3,4-dihydroxybenzaldehyde (3.00 g, 21.72 mmol), pyridinium p-
4
(

toluenesulfonate (0.02 equiv.), and DCM (20 mL/g of benzaldehyde) were added to a 250 mL
round-bottomed flask. A solution of 3,4-dihydro-2H-pyran (4.5 mL, 49.32 mmol for 4-
hydroxybenzaldehyde and 9.9 mL, 108.5 mmol of 3,4-dihydroxybenzaldehyde) in DCM (25 mL)
was then added and the reaction mixture was stirred at room temperature for 2 – 50 h. After
reaction completion, the reaction mixture was partitioned between DCM and saturated aqueous
NaHCO solution, and the organic layer was dried over MgSO , filtered, and evaporated under
3
4
reduced pressure. The resultant residue was purified by silica gel column chromatography using
hexane and ethyl acetate (9:1 for 4, and 4:1 for 5) to afford compound 4 (3.069 g, 90.9%) and
compound 5 (5.32 g, 79.7%), respectively. Compound 5 was used immediately in the next
reaction without the structural analysis.
1
4
-((Tetrahydro-2H-pyran-2-yl)oxy)benzaldehyde (4). Yellowish white solid, 91% yield. H
NMR (400 MHz, CDCl ) δ 9.87 (s, 1H, CHO), 7.80 (d, 2H, J = 8.8 Hz, 2-H, 6-H), 7.13 (d, 2H, J
3
=
8.8 Hz, 3-H, 5-H), 5.52 (t, 1H, J = 2.8 Hz, 2′-H), 3.82 (td, 1H, J = 11.2, 2.8 Hz, 6′-Ha), 3.63 –
3
.58 (m, 1H, 6′-Hb), 2.01 – 1.94 (m, 1H), 1.88 – 1.85 (m, 2H), 1.71 – 1.65 (m, 2H), 1.61 – 1.57
1
3
(
m, 1H); C NMR (100 MHz, CDCl ) δ 191.2, 162.3, 132.1, 130.6, 116.7, 96.3, 62.3, 30.2, 25.2,
3
1
8.6.
4
0
0
.1.4. General procedure for the syntheses of compounds 6 and 7. A solution of 3 (118 mg,
.39 mmol for 6, or 181 mg, 0.60 mmol for 7) in THF (2.5 mL) and a solution of 4 (81.4 mg,
.39 mmol) or 5 (185 mg, 0.60 mmol) in THF (2.5 mL) were added to a stirred suspension of
NaH (60% in mineral oil, 39.4 mg, 0.99 mmol for 6, or 72.5 mg, 1.81 mmol for 7) in THF (2.5
mL). The reaction mixture was then stirred at room temperature for 3 h, and saturated NH Cl
4
aqueous solution was added. For the synthesis of 6, water was added and the precipitate
generated was filtered to give 6 (95.3 mg, 68.8%) as a yellow solid. For the synthesis of 7, the

reaction mixture was partitioned between DCM and water, and the filtrate was dried over
MgSO , filtered, and evaporated under reduced pressure to give crude compound 7 (240.3 mg,
4
8
8.1%), which was immediately used in the next step without the structural analysis.
1
(
E)-N-(3-(4-((Tetrahydro-2H-pyran-2-yl)oxy)phenyl)acryloyl)benzamide (6). H NMR (400
MHz, CDCl ) δ 8.70 (s, 1H, NH), 7.88 (d, 2H, J = 8.8 Hz, 2′-H, 6′-H), 7.87 (d, 1H, J = 15.6 Hz,
3

-vinylic H), 7.71 (d, 1H, J = 15.6 Hz, -vinylic H), 7.61 – 7.56 (m, 3H, 2-H, 4-H, 6-H), 7.49 (t,
2
3
1
1
H, J = 8.0 Hz, 3-H, 5-H), 7.04 (d, 2H, J = 8.8 Hz, 3′-H, 5′-H), 5.46 (t, 1H, J = 2.8 Hz, 2′-H),
.85 (td, 1H, J = 11.2, 2.8 Hz, 6′-Ha), 3.63 – 3.57 (m, 1H, 6′-Hb), 2.03 – 1.94 (m, 1H), 1.88 –
1
3
.84 (m, 2H), 1.70 – 1.64 (m, 2H), 1.61 – 1.57 (m, 1H); C NMR (100 MHz, CDCl ) δ 168.0,
3
66.0, 159.5, 146.9, 133.4, 130.6, 129.2, 128.3, 127.9, 117.1, 116.9, 96.3, 62.2, 30.4, 25.3, 18.8.
4
.1.5. General procedure for the syntheses of NAB derivatives 1a – 1h. Anhydrous THF (3
mL) and sodium hydride (2.5 equiv., 60% in mineral oil) were added to a 25 mL round-bottomed
flask, and then a solution of compound 3 (100 mg, 0.33 mmol) in THF (1.0 mL), and a solution
of appropriate benzaldehyde (1.0 equiv.) in THF (1.0 mL) were added. After stirring at room
0
temperature for 1 – 12 h, aqueous ammonium chloride solution was added at 0 C. The
precipitates generated were filtered and the filter cake so obtained was washed with water and
dried to obtain the NAB derivatives 1a – 1h as solids in yields of 26 – 86%.
(E)-N-(3-(3-Hydroxy-4-methoxyphenyl)acryloyl)benzamide (1a). Yellowish white solid, 49%
1
yield. H NMR (400 MHz, DMSO-d ) δ 11.00 (s, 1H, NH), 9.29 (s, 1H, OH), 7.89 (d, 2H, J =
6
8
2
1
.4 Hz, 2-H, 6-H), 7.60 (t, 1H, J = 8.0 Hz, 4-H), 7.55 (d, 1H, J = 15.2 Hz, -vinylic H), 7.50 (t,
H, J = 8.0 Hz, 3-H, 5-H), 7.10 (d, 1H, J = 15.2 Hz, -vinylic H), 7.07 (s, 1H, 2′′-H), 7.06 (d,
1
3
H, J = 8.0 Hz, 6′′-H), 6.94 (d, 1H, J = 8.0 Hz, 5′′-H), 3.78 (s, 3H, OCH ); C NMR (100 MHz,
3

DMSO-d ) δ 167.3, 166.7, 150.8, 147.4, 144.6, 134.2, 133.4, 129.2, 129.0, 127.9, 122.3, 118.9,
6
+
1
14.2, 112.7, 56.3; HRMS (ESI+) m/z C H NO (M+H) calcd 298.1074, obsd 298.1072,
1
7
16
4
+
C H NNaO (M+Na) calcd 320.0893, obsd 320.0894.
1
7
15
4
1
(
E)-N-(3-(4-Methoxyphenyl)acryloyl)benzamide (1b). Yellow solid, 73% yield. H NMR (400
MHz, DMSO-d ) δ 11.03 (s, 1H, NH), 7.90 (d, 2H, J = 8.0 Hz, 2-H, 6-H), 7.65 (d, 1H, J = 15.6
6
Hz, -vinylic H), 7.60 (d, 2H, J = 8.8 Hz, 2′-H, 6′-H), 7.59 (t, 1H, J = 8.0 Hz, 4-H), 7.50 (t, 2H, J
=
8.0 Hz, 3-H, 5-H), 7.17 (d, 1H, J = 15.6 Hz, -vinylic H), 6.99 (d, 2H, J = 8.8 Hz, 3′-H, 5′-H),
1
3
3
1
2
.77 (s, 3H, OCH ); C NMR (100 MHz, DMSO-d ) δ 167.3, 166.8, 161.8, 144.1, 134.2, 133.4,
3 6
+
30.6, 129.2, 129.0, 127.7, 119.2, 115.2, 56.0; HRMS (ESI+) m/z C H NO (M+H) calcd
1
7
16
3
+
82.1125, obsd 282.1127, C H NNaO (M+Na) calcd 304.0944, obsd 304.0946.
1
7
15
3
(E)-N-(3-(3,4-Dimethoxyphenyl)acryloyl)benzamide (1c). Yellowish white solid, 77% yield.
1
H NMR (400 MHz, CDCl ) δ 8.73 (s, 1H, NH), 7.88 (d, 1H, J = 7.6 Hz, 2-H, 6-H), 7.86 (d, 1H,
3
J = 15.6 Hz, -vinylic H), 7.70 (d, 1H, J = 15.6 Hz, -vinylic H), 7.60 (t, 1H, J = 7.6 Hz, 4-H),
7
.45 (t, 2H, J = 7.6 Hz, 3-H, 5-H), 7.19 (dd, 2H, J = 8.4, 1.6 Hz, 6′-H), 7.15 (d, 1H, J = 1.6 Hz,
1
3
2
′-H), 6.86 (d, 1H, J = 8.4 Hz, 5′-H), 3.92 (s, 3H, OCH ), 3.90 (s, 3H, OCH ); C NMR (100
3 3
MHz, CDCl ) δ 167.9, 166.2, 151.8, 149.4, 147.2, 133.4, 133.3, 129.2, 127.9, 127.9, 123.9,
3
+
1
3
17.0, 111.1, 110.1, 56.2, 56.2; HRMS (ESI+) m/z C H NO (M+H) calcd 312.1230, obsd
1
8
18
4
+
12.1225, C H NNaO (M+Na) calcd 334.1050, obsd 334.1052.
1
8
17
4
1
(
E)-N-(3-(2,4-Dimethoxyphenyl)acryloyl)benzamide (1d). White solid, 86% yield. H NMR
(
500 MHz, CDCl ) δ 8.68 (s, 1H, NH), 8.22 (d, 1H, J = 16.0 Hz, -vinylic H), 7.90 (d, 2H, J =
3
7
1
.5 Hz, 2-H, 6-H) 7.80 (d, 1H, J = 16.0 Hz, -vinylic H), 7.62 (d, 1H, J = 8.5 Hz, 6′-H), 7.61 (t,
H, J = 7.5 Hz, 4-H), 7.51 (t, 2H, J = 7.5 Hz, 3-H, 5-H), 6.52 (dd, 1H, J = 8.5, 2.5 Hz, 5′-H),

1
3
6
.46 (d, 1H, J = 2.5 Hz, 3′-H), 3.90 (s, 3H, OCH ), 3.86 (s, 3H, OCH ); C NMR (100 MHz,
3 3
CDCl ) δ 168.4, 166.1, 163.5, 160.5, 142.5, 133.6, 133.2, 131.1, 129.2, 127.9, 117.1, 116.9,
3
+
1
05.6, 98.5, 55.8, 55.7; HRMS (ESI+) m/z C H NO (M+H) calcd 312.1230, obsd 312.1235,
1
8
18
4
+
C H NNaO (M+Na) calcd 334.1050, obsd 334.1053.
1
8
17
4
(E)-N-(3-(3,4,5-Trimethoxyphenyl)acryloyl)benzamide (1e). Yellowish White solid, 73%
1
yield. H NMR (400 MHz, CDCl ) δ 8.68 (s, 1H, NH), 7.88 (d, 2H, J = 7.6 Hz, 2-H, 6-H), 7.83
3
(d, 1H, J = 15.6 Hz, -vinylic H), 7.72 (d, 1H, J = 15.6 Hz, -vinylic H), 7.60 (t, 1H, J = 7.2 Hz,
4
-H), 7.50 (t, 2H, J = 7.6 Hz, 3-H, 5-H), 6.84 (s, 2H, 2′-H, 6′-H), 3.89 (s, 6H, 2ⅹOCH ), 3.88 (s,
3
1
3
3
1
3
H, OCH ); C NMR (100 MHz, CDCl ) δ 167.7, 166.1, 153.6, 147.2, 140.8, 133.5, 133.2,
3 3
+
30.3, 129.3, 127.9, 118.6, 106.0, 61.2, 56.4; HRMS (ESI+) m/z C H NO (M+H) calcd
1
9
20
5
+
42.1336, obsd 342.1332, C H NNaO (M+Na) calcd 364.1155, obsd 364.1149.
1
9
19
5
(E)-N-(3-(4-Hydroxy-3,5-dimethoxyphenyl)acryloyl)benzamide (1f). Yellowish White solid,
1
2
1
6% yield. H NMR (400 MHz, DMSO-d ) δ 7.90 (d, 2H, J = 7.6 Hz, 2-H, 6-H), 7.60 (d, 1H, J =
6
4.8 Hz, -vinylic H), 7.60 (t, 1H, J = 7.6 Hz, 4-H), 7.50 (t, 2H, J = 7.6 Hz, 3-H, 5-H), 7.08 (d,
1
3
1
H, J = 14.8 Hz, -vinylic H), 6.93 (s, 2H, 2′-H, 6′-H), 3.77 (s, 6H, 2ⅹOCH ); C NMR (100
3
MHz, DMSO-d ) δ 167.2, 166.7, 148.8, 145.2, 139.4, 134.3, 133.3, 129.1, 129.0, 125.1, 118.4,
6
+
1
06.7, 56.6; HRMS (ESI+) m/z C H NO (M+H) calcd 328.1179, obsd 328.1174,
1
8
18
5
+
C H NNaO (M+Na) calcd 350.0999, obsd 350.0994.
1
8
17
5
1
(
E)-N-(3-(3-Bromo-4-hydroxyphenyl)acryloyl)benzamide (1g). Yellow solid, 32% yield. H
NMR (400 MHz, DMSO-d ) δ 10.98 (s, 1H), 10.85 (s, 1H), 7.88 (d, 2H, J = 8.0 Hz, 2-H, 6-H),
6
7
.78 (d, 1H, J = 2.0 Hz, 2′-H), 7.58 (dd, 1H, J = 8.4, 2.0 Hz, 6′-H), 7.55 (d, 1H, J = 16.0 Hz, -

vinylic H), 7.50 – 7.45 (m, 3H, 3-H, 4-H, 5-H), 7.11 (d, 1H, J = 16.0 Hz, -vinylic H), 6.96 (d,
1
3
1
1
H, J = 8.4 Hz, 5′-H); C NMR (100 MHz, DMSO-d ) δ 167.3, 166.5, 156.8, 142.7, 133.4,
6
33.3, 129.6, 129.1, 129.0, 128.1, 128.0, 119.8, 117.3, 115.3; HRMS (ESI+) m/z
+
+
C H BrNNaO (M+Na) calcd 367.9893, obsd 367.9889, C H BrNNaO (M+2+Na) calcd
1
6
12
3
16 12
3
3
69.9874, obsd 369.9871.
(E)-N-(3-(3,5-Dibromo-4-hydroxyphenyl)acryloyl)benzamide (1h). Yellowish White solid,
1
3
7
1
2% yield. H NMR (400 MHz, DMSO-d ) δ 10.82 (brs, 1H), 7.88 (d, 2H, J = 7.6 Hz, 2-H, 6-H),
6
.67 (s, 2H, 2′-H, 6′-H), 7.59 (t, 1H, J = 7.6 Hz, 4-H), 7.49 (t, 2H, J = 7.6 Hz, 3-H, 5-H), 7.46 (d,
1
3
H, J = 16.0 Hz, -vinylic H), 6.94 (d, 1H, J = 16.0 Hz, -vinylic H); C NMR (100 MHz,
DMSO-d ) δ 167.1, 166.6, 159.4, 142.9, 134.3, 133.3, 132.7, 129.1, 128.9, 122.8, 116.2, 114.5;
6
+
+
HRMS (ESI+) m/z C H Br NO (M+H) calcd 423.9178, obsd 423.9174, (M+2+H) calcd
1
6
12
2
3
+
4
25.9159, obsd 425.9150, (M+4+H) calcd 427.9141, obsd 427.9136, C H Br NNaO
16 11 2 3
+
+
(
M+Na) calcd 445.8998, obsd 445.8994, (M+2+Na) calcd 447.8978, obsd 447.8978,
+
(
M+4+Na) calcd 449.8960, obsd 44.8955.
4
.1.6. General procedure for the syntheses of compounds 1i and 1j. 2M-HCl solution (2.0
mL) was added to a solution of 6 (64 mg, 0.18 mmol) or 7 (342 mg, 0.76 mmol) in 1,4-dioxane
2.0 mL). The reaction mixture was stirred at room temperature for 30 min for 1i and for 6 h for
j. For 1i, the reaction mixture was partitioned between DCM and water, and the aqueous layer
(
1
was filtered to give 1i. In addition, the organic layer was evaporated under reduced pressure,
hexane was added to the residue, and the resulting precipitate was filtered to give 1i (30.6 mg,
yield 62.9%) as a yellow solid. For 1j, after adding water, the precipitate was filtered and washed
with water to afford 1j (105.9 mg, 49.3%) as a yellowish white solid.

1
(
E)-N-(3-(4-Hydroxyphenyl)acryloyl)benzamide (1i). H NMR (400 MHz, DMSO-d ) δ 10.98
6
(s, 1H, NH), 10.05 (s, 1H, OH), 7.89 (d, 2H, J = 7.6 Hz, 2-H, 6-H), 7.61 (d, 1H, J = 16.0 Hz, -
vinylic H), 7.60 (t, 1H, J = 7.6 Hz, 4-H), 7.50 (t, 2H, J = 7.6 Hz, 3-H, 5-H), 7.48 (d, 2H, J = 8.4
1
3
Hz, 2′-H, 6′-H), 7.10 (d, 1H, J = 16.0 Hz, -vinylic H), 6.80 (d, 2H, J = 8.4 Hz, 3′-H, 5′-H); C
NMR (100 MHz, DMSO-d ) δ 167.3, 166.8, 160.5, 144.6, 134.2, 133.3, 130.9, 129.1, 129.0,
6
+
1
26.2, 118.0, 116.6; HRMS (ESI+) m/z C H NO (M+H) calcd 268.0968, obsd 268.0969,
1
6
14
3
+
C H NNaO (M+Na) calcd 290.0788, obsd 290.0787.
1
6
13
3
1
(
E)-N-(3-(3,4-Dihydroxyphenyl)acryloyl)benzamide (1j). H NMR (400 MHz, DMSO-d ) δ
6
1
7
0.96 (s, 1H, NH), 9.58 (brs, 1H, OH), 9.24 (brs, 1H, OH), 7.89 (d, 2H, J = 7.6 Hz, 2-H, 6-H),
.60 (t, 1H, J = 7.6 Hz, 4-H), 7.53 (d, 1H, J = 15.4 Hz, -vinylic H), 7.50 (t, 2H, J = 7.6 Hz, 3-H,
-H), 7.07 (d, 1H, J = 15.6 Hz, -vinylic H), 7.04 (d, 1H, J = 2.0 Hz, 2′-H), 6.94 (dd, 1H, J =
5
8
1
1
3
.0, 2.0 Hz, 6′-H), 6.75 (d, 1H, J = 8.0 Hz, 5′-H); C NMR (100 MHz, DMSO-d ) δ 167.3,
6
66.9, 149.2, 146.3, 145.1, 134.3, 133.3, 129.1, 129.0, 126.6, 122.5, 117.8, 116.5, 114.8; HRMS
+
+
(
ESI+) m/z C H NO (M+H) calcd 284.0917, obsd 284.0909, C H NNaO (M+Na) calcd
16 14 4 16 13 4
3
06.0737, obsd 306.0718.
4
.2. Docking simulation for NAB derivatives 1a and 1j
Docking simulations for 1a, 1j, and kojic acid were carried out as previously described.42-44
Chem3D Pro 12.0 software was used to generate the 3D structures of 1a and 1j. AutoDock Vina
and Chimera software were used to calculate binding scores between tyrosinase and the two
derivatives and kojic acid. The three-dimensional structure of tyrosinase (Agaricus Bisporus)
was obtained from the Protein Data Bank (PDB ID: 2Y9X). LigandScout software 4.3.0 was

used to generate pharmacophore models showing possible interactions between the three ligands
and the amino acid residues of tyrosinase.
4
.3. Biological Evaluation
4
.3.1. Mushroom tyrosinase inhibition assays of NAB derivatives 1a – 1j
To determine the mushroom tyrosinase inhibition activities of NAB derivatives, we used a slight
4
5
modification of a previously described procedure. NAB derivatives (1a – 1j, 10 µL, 25 µM)
and 20 µL of tyrosinase solution (one thousand units per mL) were added to the 170 µL solution
of 14.7 mM phosphate buffer containing 293 µM of L-tyrosine in the wells of a 96-well plate and
0
incubated for 30 min at 37 C. Percentage tyrosinase inhibition was determined by measuring
optical densities at 450 nm using a shaking microplate reader (VersaMax). Kojic acid (25 µM)
was used as the control. The experiments were repeated three times. The formula used to
calculate % inhibition was;
%Inhibition = 100ⅹ[1-(A/B)]
where is A sample absorbance and B is the absorbance of blank control (non-treated).
.3.2. Cell culture
4
The B16F10 murine melanoma cells used for cell viability, tyrosinase inhibition, and melanin
content assays were obtained from the American Type Culture Collection (ATCC, VS, USA)
and cultured in DMEM (Dulbecco’s medium) containing 10% foetal bovine serum (FBS), 100
0
µg/mL streptomycin, and 100 IU/ml penicillin in a humidified 5% CO atmosphere at 37 C.
2
4
.3.3. Cell viability assay

The cell viabilities of NAB derivatives 1a and 1j were determined using a WST-8 assay, as
4
6
4
previously described. B16F10 murine melanoma cells (5x10 cells per well) were incubated at
0
3
7 C for 24 h in a humidified 5% CO atmosphere in 96-well plates. Compounds 1a and 1j at
2
concentrations of 0, 5, 10, or 25 were added, and cells were re-incubated for 24 h under the same
conditions. On the following day, cells were treated with WST-8 reagents and incubated for 30
0
min at 37 C for 2 h 30 min later, and viabilities were determined using an EZ-Cytox assay (EZ-
3
000, Daeil Lab Service, Seoul) by reading optical densities at 450 nm. Experiments were
repeated three times.
.3.4. Tyrosinase inhibition assay
Tyrosinase inhibition assays of NAB derivatives 1a and 1j were performed as previously
4
4
7
4
described with slight changes. In brief, B16F10 cells were inoculated at 5x10 cells per well in
0
9
6-well plates and incubated for 24 h in a humidified 5% CO atmosphere at 37 C. The cells
2
were then treated with α-MSH (1 µM) and kojic acid (25 µM) or α-MSH (1 µM) and 1a or 1j (0,
, 10, or 25 µM) and re-cultured for 24 h under the same conditions. Cells were then washed
5
with PBS buffer, lysed with lysis buffer (100 µL containing 1% Triton X-100 (5 µL), 0.1 mM
0
PMSF (5 µL), and 50 mM PBS (pH 6.8, 90 µL), frozen at -80 C for 30 min, centrifuged at
0
1
1
2,000 rpm for 30 min at 4 C, transferred to 96-well plates, treated with L-dopa (total volume
0
00 µL; 80 µL of supernatant and 20 µL of 10 mM L-dopa), and incubated for 30 min at 37 C.
Tyrosinase inhibitions were determined by measuring optical densities at 500 nm in a Tecan,
Mannedorf microplate reader. The experiment was performed in triplicate.
4
.3.5. Anti-melanogenic assay

The anti-melanogenic effects of NAB derivatives 1a and 1j were determined by measuring
melanin contents, as previously described with slight changes.48 Briefly, B16F10 cells were
4
inoculated at 5x10 cells per well in 96-well plates and incubated for 24 h in a humidified 5%
0
CO atmosphere at 37 C. Cells were then treated with α-MSH (1 µM) and kojic acid (25 µM) or
2
with α-MSH (1 µM) and NAB derivatives 1a or 1j (0, 5, 10, or 25 µM) and re-cultured for 24 h
as described above. Cells were then washed twice with PBS buffer and treated with 1M-NaOH
aqueous solution (200 µL) for 1 h to dissolve melanin. Solutions were transferred to 96-well
plates and melanin contents were determined by measuring optical densities at 405 nm in a
Tecan, Mannedorf microplate reader. The experiment was performed in triplicate.
4
.3.6. DPPH radical scavenging assay
To determine the antioxidant activities of NAB derivatives 1a – 1j, a DPPH radical scavenging
4
9
assay was performed as previously described with a few changes. In summary, 20 µL of a
DMSO solution of each NAB derivative (10 mM) was mixed with 180 µL of DPPH solution (0.2
mM) in methanol in 96-well plates. L-Ascorbic acid was used as a positive control. Mixtures
were then incubated for 30 min in the dark, and antioxidant activities were determined by
measuring absorbances at 517 nm using a VersaMax microplate reader. The experiments were
performed in triplicate. The formula used to determine DPPH radical scavenging activity was as
follows:
Radical scavenging activity (%) = 100ⅹ[(Ac-As)/Ac]
Where Ac is the absorbance of the non-treated control, and As is the absorbance of a sample or a
L-ascorbic acid treated solution.
4
.3.7. Statistical analysis

Statistical analysis was performed using GraphPad Prism 5 software (La Jolla, CA, USA). All
experiments were performed in triplicate. Results are expressed as means ± SEMs. One-way
ANOVA and Tukey's test were used to determine the significances of intergroup differences. P-
values were considered to be statistically significant.
Acknowledgment
This research was supported by the Basic Science Research Program through the National
Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-
2
017R1D1A1B03027888).
References
1
.
Bonaventure J, Domingues MJ, Larue L. Pigment cell & Melanoma Res. 2013;26:316-
25.
3
2
3
4
5
6
7
.
.
.
.
.
.
Decker H, Schweikardt T, Tuczek F. Ang Chemie Intl Ed. 2006;45:4546-4550.
Ahn SJ, Koketsu M, Ishihara H, et al. Chem Pharm Bull. 2006;54:281-286.
Artés F, Castaner M, Gil M. Food Sci Tech Intl. 1998;4:377-389.
Bae SJ, Ha YM, Park YJ, et al. Eur J Med Chem. 2012;57:383-390.
Ramsden CA, Riley PA. Arkivoc. 2010;1:260-274.
Ramsden CA, Stratford MR, Riley PA. Organic & Biomolecular Chem. 2009;7:3388-
3
390.
Sánchez-Ferrer A, Rodríguez-López JN, García-Cánovas F, García-Carmona F. BBA.
995;1247:1-11.
8
.
1
9
1
.
0.
Muñoz-Muñoz J, Acosta-Motos J, Garcia-Molina F, et al. BBA. 2010;1804:1467-1475.
Agarwal KC. Mechanism-based biochemical standardization of resveratrol products and
their uses thereof. US6414037B1; 2011.
1
1
1.
2.
Kidson S, De Haan J. Exp Cell Res. 1990;188:36-41.
Imokawa G. The J Invest dermatol. 1989;93:100-107.

1
3.
Petrescu SM, Branza-Nichita N, Negroiu G, Petrescu AJ, Dwek RA. Biochemistry.
000;39:5229-5237.
2
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
4.
5.
6.
7.
8.
9.
0.
1.
2.
3.
4.
5.
6.
7.
8.
Saeki H, Oikawa A. J Cellular physiol. 1980;104:171-175.
Ando H, Kondoh H, Ichihashi M, Hearing VJ. The J Invest dermatol. 2007;127:751-761.
Kumar KS, Vani MG, Wang SY, et al. Biofactors. 2013;39:259-270.
Arndt KA, Fitzpatrick TB. JAMA. 1965;194:965-967.
Fitzpatrick T, Arndt K, El Mofty A, Pathak M. Archi Dermatol. 1966;93:589-600.
Heilgemeir G, Balda B. MMW, Munch Med Woch. 1981;123:47.
Kligman AM, Willis I. Archi Dermatol. 1975;111:40-48.
Gonçalez M, Correa MA, Chorilli M. BioMed Res Intl. 2013;2013.
Breathnach AC, Nazzaro-Porro M, Passi S, Zina G. Clinics in Dermatol. 1989;7:106-119.
Garcia-Lopez M. Acta Derm Venereol (Stockh). 1989;143:58-61.
Curto EV, Kwong C, Hermersdörfer H, et al. Biochem Pharmacol. 1999;57:663-672.
Engasser PG. J A A Dermatol. 1984;10:1072-1073.
Fisher A. Cutis. 1983;31:240-244, 250.
Romaguera C, Grimalt F. Contact Dermatitis. 1985;12:183-183.
Barcelos RC, Pastre JC, Vendramini‐Costa DB, et al. ChemMedChem. 2014;9:2725-
2
743.
2
3
9.
0.
Gunia-Krzyżak A, Żelaszczyk D, Rapacz A, et al. Bioorg Med Chem. 2017;25:471-482.
Spasova M, Kortenska‐Kancheva V, Totseva I, Ivanova G, Georgiev L, Milkova T. J
Peptide Sci: an official publication of the European Peptide Society. 2006;12:369-375.
Spasova M, Philipov S, Nikolaeva-Glomb L, Galabov A, Milkova T. Bioorg Med Chem.
3
1.
2
008;16:7457-7461.
3
3
3
3
2.
3.
4.
5.
Georgiev L, Chochkova M, Totseva I, et al. Med Chem Res. 2013;22:4173-4182.
Sova M. Mini Reviews in Med Chem. 2012;12:749-767.
Bandgar BP, Adsul LK, Chavan HV, et al. Bioorg Med Chem. 2012;20:5649-5657.
Kavala V, Wang C-C, Barange DK, Kuo C-W, Lei P-M, Yao C-F. JOC. 2012;77:5022-
5
029.
3
3
3
3
4
6.
7.
8.
9.
0.
Romagnoli R, Prencipe F, Lopez-Cara LC, et al. J Enz Inhi Med Chem. 2018;33:727-742.
Fiorino F, Eiden M, Giese A, et al. Bioorg Med Chem. 2012;20:5001-5011.
Goodman SN, Jacobsen EN. Adv Syn Cat. 2002;344:953-956.
Vanderwal CD, Jacobsen EN. JACS. 2004;126:14724-14725.
Do Hyun Kim SJK, Ullah S, Yun HY, Chun P, Moon HR. Drug Des Devel Ther.
2
017;11:827.
4
4
4
4
1.
2.
3.
4.
Kim SJ, Yang J, Lee S, et al. Biorg Med Chem. 2018.
Morris GM, Goodsell DS, Halliday RS, et al. J Comp Chem. 1998;19:1639-1662.
Moustakas DT, Lang PT, Pegg S, et al. J Comput Aided Mol Des. 2006;20:601-619.
Bagherzadeh K, Shirgahi Talari F, Sharifi A, Ganjali MR, Saboury AA, Amanlou M. J
Biomol Struct Dyn. 2015;33:487-501.
4
4
5.
6.
Hyun SK, Lee W-H, Jeong DM, Kim Y, Choi JS. Biol Pharm Bull. 2008;31:154-158.
Ishiyama M, Tominaga H, Shiga M, Sasamoto K, Ohkura Y, Ueno K. Biol Pharm Bull.
1
996;19:1518-1520.
4
7.
Bae SJ, Ha YM, Kim J-A, et al. BBA. 2013;77:65-72.