Design, synthesis and anti-melanogenic effect of cinnamamide derivatives

Article abstract of DOI:10.1016/j.bmc.2018.10.014

Pigmentation disorders are attributed to excessive melanin which can be produced by tyrosinase. Therefore, tyrosinase is supposed to be a vital target for the treatment of disorders associated with overpigmentation. Based on our previous findings that an (E)-β-phenyl-α,β-unsaturated carbonyl scaffold can play a key role in the inhibition of tyrosinase activity, and the fact that cinnamic acid is a safe natural substance with a scaffolded structure, it was speculated that appropriate cinnamic acid derivatives may exhibit potent tyrosinase inhibitory activity. Thus, ten cinnamamides were designed, and synthesized by using a Horner-Emmons olefination as the key step. Cinnamamides 4 (93.72% inhibition), 9 (78.97% inhibition), and 10 (59.09% inhibition) with either a 2,4-dihydroxyphenyl, or 4-hydroxy-3-methoxyphenyl substituent showed much higher mushroom tyrosinase inhibition at 25 μM than kojic acid (18.81% inhibition), used as a positive control. Especially, the two cinnamamides 4 and 9 having a 2,4-dihydroxyphenyl group showed the strongest inhibition. Docking simulation with tyrosinase revealed that these three cinnamamides, 4, 9, and 10, bind to the active site of tyrosinase more strongly than kojic acid. Cell-based experiments carried out using B16F10 murine skin melanoma cells demonstrated that all three cinnamamides effectively inhibited cellular tyrosinase activity and melanin production in the cells without cytotoxicity. There was a close correlation between cellular tyrosinase activity and melanin content, indicating that the inhibitory effect of the three cinnamamides on melanin production is mainly attributed to their capability for cellular tyrosinase inhibition. These results imply that cinnamamides having the (E)-β-phenyl-α,β-unsaturated carbonyl scaffolds are promising candidates for skin-lighting agents.

Full text of DOI:10.1016/j.bmc.2018.10.014

Accepted Manuscript
Design, synthesis and anti-melanogenic effect of cinnamamide derivatives
Sultan Ullah, Yujin Park, Muhammad Ikram, Sanggwon Lee, Chaeun Park,
Dongwan Kang, Jungho Yang, Jinia Akter, Sik Yoon, Pusoon Chun, Hyung
Ryong Moon
PII:
S0968-0896(18)31467-6
https://doi.org/10.1016/j.bmc.2018.10.014
BMC 14573
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
16 August 2018
16 October 2018
17 October 2018
Please cite this article as: Ullah, S., Park, Y., Ikram, M., Lee, S., Park, C., Kang, D., Yang, J., Akter, J., Yoon, S.,
Chun, P., Moon, H.R., Design, synthesis and anti-melanogenic effect of cinnamamide derivatives, Bioorganic &
Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.10.014
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Design, synthesis and anti-melanogenic effect of cinnamamide derivatives
Sultan Ullaha, Yujin Parka, Muhammad Ikramb,c, Sanggwon Leea, Chaeun Parka, Dongwan
Kanga, Jungho Yanga, Jinia Aktera, Sik Yoonc, Pusoon Chund, Hyung Ryong Moona,*
aLaboratory of Medicinal Chemistry, College of Pharmacy, Pusan National University, Busan
46241, South Korea
bDepartment of Pharmacy, COMSATS University Islamabad, Abbottabad Campus,
Abbottabad 22060, Pakistan
cDepartment of Anatomy, Pusan National University School of Medicine, 49 Busandaehak-ro,
Mulgeum-eup, Yangsan-si, Gyeongsangnam-do, 50612, South Korea.
dCollege of Pharmacy and Inje Institute of Pharmaceutical Sciences and Research, Inje
University, Gimhae, Gyeongnam 50834, South Korea
*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 51 510 2815; Fax: +82 51 513 6754.
Abstract
Pigmentation disorders are attributed to excessive melanin which can be produced by
tyrosinase. Therefore, tyrosinase is supposed to be a vital target for the treatment of disorders
associated with overpigmentation. Based on our previous findings that an (E)--phenyl-,-
unsaturated carbonyl scaffold can play a key role in the inhibition of tyrosinase activity, and
the fact that cinnamic acid is a safe natural substance with a scaffolded structure, it was
speculated that appropriate cinnamic acid derivatives may exhibit potent tyrosinase inhibitory
activity. Thus, ten cinnamamides were designed, and synthesized by using a Horner-Emmons
olefination as the key step. Cinnamamides 4 (93.72% inhibition), 9 (78.97% inhibition), and
10 (59.09% inhibition) with either a 2,4-dihydroxyphenyl, or 4-hydroxy-3-methoxyphenyl

substituent showed much higher mushroom tyrosinase inhibition at 25 µM than kojic acid
(18.81% inhibition), used as a positive control. Especially, the two cinnamamides 4 and 9
having a 2,4-dihydroxyphenyl group showed the strongest inhibition. Docking simulation with
tyrosinase revealed that these three cinnamamides, 4, 9, and 10, bind to the active site of
tyrosinase more strongly than kojic acid. Cell-based experiments carried out using B16F10
murine skin melanoma cells demonstrated that all three cinnamamides effectively inhibited
cellular tyrosinase activity and melanin production in the cells without cytotoxicity. There was
a close correlation between cellular tyrosinase activity and melanin content, indicating that the
inhibitory effect of the three cinnamamides on melanin production is mainly attributed to their
capability for cellular tyrosinase inhibition. These results imply that cinnamamides having the
(E)--phenyl-,-unsaturated carbonyl scaffolds are promising candidates for skin-lighting
agents
Key words: cinnamamide, melanin, tyrosinase inhibitor, docking, B10F16 melanoma cells.
1. Introduction
Melanin is a generic term for pigment complexes present in the tissues of plants and animals.
In humans, melanin is biosynthesized in the melanosome of melanocytes present in the
epidermal basal layer and melanin production is generally initiated by exposure to UV light.1
In plants, melanin is produced in excess when the mixture of tyrosinase and tyrosinase
substrates is contacted with oxygen by destruction of the cell structure, such as exfoliation of
the agricultural product peels such as vegetables and fruits, or external physical impacts.
Melanin is a major factor in determining the colour of the skin, pupil and hair.2 Their colour is
mainly determined by the amounts and ratios of eumelanin and pheomelanin. Eumelanin and
pheomelanin are synthesized through different synthetic pathways from dopaquinone, a
common intermediate in the melanin biosynthesis process.2,3
Melanin can block UV rays and protect skin cells from harmful UV radiation. However, local

melanin deposits on the skin, such as senile spots, cause cosmetic problems.4-6 In addition,
melanin causes browning of agricultural products,7 causing damage to flavour and tissue,
which ultimately lowers the marketability. Thus, compounds that effectively inhibit
melanogenesis can be used as therapeutic agents for skin diseases such as hyperpigmentation,
cosmetics, and antibrowning agents for agricultural products in the food industry.
Melanin is synthesized through chemical and enzymatic reactions starting from L-tyrosine, and
several enzymes are involved in melanin biosynthesis, including tyrosinase, which is widely
distributed in nature, from bacteria to plants and humans.8 Tyrosinase is involved in three
chemical reactions in the melanin biosynthesis process, and in the rate-limiting step which
oxidizes L-tyrosine to dopaquinone.9 Thus, tyrosinase has been widely recognized as an
important target enzyme for the development of whitening agents. This enzyme is a
metalloenzyme with two copper (II) ions at the active site and it acts as an oxidase that oxidizes
the phenolic ring.10 Therefore, some researchers have attempted to develop tyrosinase
inhibitors by synthesizing a copper chelator.11,12
For the past several years, our laboratory has synthesized a variety of phenolic compounds
mimicking the chemical structure of natural substrates, L-dopa and L-tyrosine, for tyrosinase
and has evaluated their tyrosinase inhibitory activity to find competitive tyrosinase inhibitors
that bind to the active site of tyrosinase more strongly than L-tyrosine and L-dopa.13-18 From
these studies, we concluded that compounds with an (E)--phenyl-,-unsaturated carbonyl
scaffold exhibit profound tyrosinase-inhibitory activity. Cinnamic acid, a natural compound
isolated from cinnamon or blossom,19 has been reported to have extensive physiological
activities20-27 and also to show mild tyrosinase inhibitory activity.28
Cinnamic acid has the (E)--phenyl-,-unsaturated carbonyl scaffold in view of the molecular
structure and is not cytotoxic.29 Additionally, it showed potential towards to the inhibition of
tyrosinase although the potency was mild. Amide bonds are widely present in many medicines

available in the market and are used as important functional groups because they are
structurally stable and can act as hydrogen bond donors and/or receptors. Therefore, we
targeted a variety of cinnamamide derivatives to find potent tyrosinase inhibitors.
In this study, as part of our ongoing efforts to find novel tyrosinase inhibitors derived from
natural products and featuring the (E)-scaffold, we synthesized a series of cinnamamide
analogues and examined their anti-tyrosinase activity using mushroom tyrosinase and their
anti-melanogenic effect through cell-based assays using a murine cell line.
2. Results and Discussion
2.1. Chemistry
Our strategy for cinnamic acid derivatization was to synthesize stable cinnamamides with
various substituents on the –phenyl ring, which could be easily prepared from Horner-
Emmons olefination of benzaldehydes having various substituents in appropriate phenyl
positions with triethyl phosphonoacetate followed by saponification and coupling reaction with
secondary cyclic amines such as pyrrolidine and piperidine. According to the structure-activity
relationship (SAR) data we have collected, compounds having at least one hydroxyl group on
the –phenyl ring of the (E)-motif, that is, 4-hydroxyphenyl, 4-hydroxy-3-methoxyphenyl, 2,4-
dihydroxyphenyl, 3-hydroxy-4-methoxyphenyl and 3,4-dihydroxyphenyl, generally showed
potent inhibition towards tyrosinase. Therefore, cinnamamide derivatives with these five
phenyl rings were synthesized. First, the hydroxyl groups of five benzaldehydes (3-hydroxy-
4-methoxybenzaldehyde (11), 4-hydroxybenzaldehyde (12), 3,4-dihydroxybenzaldehyde (13),
2,4-dihydroxybenzaldehyde (14), and 4-hydroxy-3-methoxybenzaldehyde (15)) were
protected as benzyl ethers, giving compounds 16 – 20, as shown in Scheme 1. Horner-Emmons
olefination between aldehydes 16 – 20 and triethyl phosphonoacetate in the presence of bases
(K2CO3, and a catalytic amount of DBU (1,8-diazabicyclo[5.4.0]undec-7-ene)) afforded ethyl

cinnamates 21 – 25, which were hydrolysed to produce the corresponding cinnamic acids 26 –
30. After the activation of the carboxylic acid functionality of 26 – 30 to the corresponding
mixed anhydrides using isobutyl chloroformate in the presence of N-methylmorpholine,
treatment of two secondary cyclic amines, pyrrolidine and piperidine, afforded the
corresponding cinnamamide derivatives 31 – 35 and 36 – 40, respectively. Finally,
debenzylation was conducted under reflux and acidic conditions using concentrated HCl and
acetic acid to produce the corresponding cinnamamide analogues 1 – 3, 5 – 8, and 10. However,
these acidic conditions did not afford cinnamamides 4 and 9 with the 2,4-dihydroxyphenyl
group, but rather produced a mixture of compounds with high polarity. Use of BBr3 as an
alternative Lewis acid for removal of the benzyl groups of 34 and 39, interestingly, produced
the desired cinnamamides 4 and 9 with the 2,4-dihydroxyphenyl moiety in a moderate yields.
The structures of the synthesized cinnamamides 1 – 10 were confirmed by 1H and 13C nuclear
magnetic resonance (NMR) and mass spectroscopy. The geometry of the double bond of the
final compounds was assigned as an (E)-form on the basis of J values (15.0 – 15.6 Hz) of the
vinylic peaks in the 1H NMR spectra.

R1
O
R1
O
R1
O
R2
R3
b
R2
R3
a
R2
R3
O
H
H
21
22
23
24
25
: R1 = H, R2 = OBn, R3 = OMe
: R1 = H, R2 = H, R3 = OBn
: R1 = H, R2 = OBn, R3 = OBn
: R1 = OBn, R2 = H, R3 = OBn
: R1 = H, R2 = OMe, R3 = OBn
11
12
13
14
15
: R1 = H, R2 = OH, R3 = OMe
: R1 = H, R2 = H, R3 = OH
: R1 = H, R2 = OH, R3 = OH
: R1 = OH, R2 = H, R3 = OH
: R1 = H, R2 = OMe, R3 = OH
16
17
18
19
20
: R1 = H, R2 = OBn, R3 = OMe
: R1 = H, R2 = H, R3 = OBn
: R1 = H, R2 = OBn, R3 = OBn
: R1 = OBn, R2 = H, R3 = OBn
: R1 = H, R2 = OMe, R3 = OBn
R1
O
R1
O
R2
e
R2
d
c
N
OH
n
R3
R3
31
32
33
34
35
36
37
38
39
40
: n = 1, R1 = H, R2 = OBn, R3 = OMe
: n = 1, R1 = H, R2 = H, R3 = OBn
: n = 1, R1 = H, R2 = OBn, R3 = OBn
: n = 1, R1 = OBn, R2 = H, R3 = OBn
: n = 1, R1 = H, R2 = OMe, R3 = OBn
: n = 2, R1 = H, R2 = OBn, R3 = OMe
: n = 2, R1 = H, R2 = H, R3 = OBn
: n = 2, R1 = H, R2 = OBn, R3 = OBn
: n = 2, R1 = OBn, R2 = H, R3 = OBn
: n = 2, R1 = H, R2 = OMe, R3 = OBn
26
27
28
29
30
: R1 = H, R2 = OBn, R3 = OMe
: R1 = H, R2 = H, R3 = OBn
: R1 = H, R2 = OBn, R3 = OBn
: R1 = OBn, R2 = H, R3 = OBn
: R1 = H, R2 = OMe, R3 = OBn
R1
O
R2
R3
N
n
1
2
3
4
5
6
7
8
9
: n = 1, R1 = H, R2 = OH, R3 = OMe
: n = 1, R1 = H, R2 = H, R3 = OH
: n = 1, R1 = H, R2 = OH, R3 = OH
: n = 1, R1 = OH, R2 = H, R3 = OH
: n = 1, R1 = H, R2 = OMe, R3 = OH
: n = 2, R1 = H, R2 = OH, R3 = OMe
: n = 2, R1 = H, R2 = H, R3 = OH
: n = 2, R1 = H, R2 = OH, R3 = OH
: n = 2, R1 = OH, R2 = H, R3 = OH
10
: n = 2, R1 = H, R2 = OMe, R3 = OH
Scheme 1. Synthetic scheme of cinnamamide derivatives 1 – 10. Reagents and conditions:
a) BnBr, K2CO3, CH3CN, reflux, 24 h; b) (EtO)2P(=O)CH2CO2Et, K2CO3, DBU (cat. amount),
DCM, DMF, rt for 11, 13, and 15, or 70 °C for 12, and 14, 24 – 36 h; c) 1N-NaOH aqueous
solution, 1,4-dioxane, rt, 48 h; d) i-BuO2CCl, N-methylmorpholine, THF, rt, 30 min, and then,
pyrrolidine, or piperidine, rt, 24 h; and e) c-HCl, AcOH, reflux, 40 min, or DCM, 4 – 8 equiv.
BBr3, -40 0C, 30 min.

2.2. Mushroom tyrosinase inhibitory effect of cinnamamides 1 – 10.
The inhibitory effects of the ten synthesized cinnamamide derivatives on mushroom tyrosinase
were examined at 25 M using kojic acid as a positive control, as depicted in Table 1. Overall,
cinnamamide derivatives 6 – 10 with piperidine exhibited slightly higher inhibition than
cinnamamide derivatives 1 – 5 with pyrrolidine, except for compounds 4 and 9 with a 2,4-
dihydroxy group. Compounds, 1 and 6, with a 3-hydroxy-4-methoxy group, showed slightly
greater inhibitory activity with 26.42±3.49 and 36.70±6.52% inhibition, respectively, than
kojic acid (18.81±1.25% inhibition). According to our cumulative SAR data, compounds with
a 3-hydroxy-4-methoxyphenyl group generally showed stronger inhibition than compounds
with a 4-hydroxy-3-methoxy group.16,18,30,31. Interestingly, therefore, compound 10
(59.09±1.40% inhibition) with a 4-hydroxy-3-methoxy group showed more potent inhibitory
activity than compound 6 (36.70±6.52% inhibition) with a 3-hydroxy-4-methoxy group.
Compounds 2 and 7, with a 4-hydroxy group, exhibited similar or slightly less inhibition
(13.65±3.78 and 19.07±3.74% inhibitions) than kojic acid. Introduction of an additional
hydroxyl group at position 3 of the –phenyl group of compounds 2 and 7 reduced the
tyrosinase inhibitory activities, such that compounds 3 and 8 with a catechol (3,4-
dihydroxyphenyl) group revealed weak to no inhibition against mushroom tyrosinase. On the
other hand, introduction of an additional hydroxyl group at position 2 of the –phenyl group
of compounds 2 and 7 dramatically increased their tyrosinase inhibitory activities, such that
compounds 4 and 9 with a 2,4-dihydroxy group exhibited the highest inhibitory activity with
93.72±0.25 and 78.97±0.16% inhibitions, respectively. This result is consistent with our
cumulative SAR data showing that compounds having a 2,4-dihydroxyphenyl group generally
show potent tyrosinase inhibitory activities.32-36 These results imply that fine changes in the
chemical structure can have a very large effect on the tyrosinase activity. Because

cinnamamides 4, 9, and 10 revealed the most potent inhibitions against mushroom tyrosinase,
these three compounds were used in the following studies involving cell-based assays.
Table 1. Substitution patterns, and tyrosinase-inhibitory activities of the synthesized
cinnamamide derivatives 1 – 10 and kojic acid
R1
O
R1
O
R2
R3
R2
R3
N
N
1 5
-
6 10
-
Compounds
Compounds
Compound R1
R2
R3
Tyrosinase inhibition (%)a
1
H
H
H
OH
H
OMe
OH
OH
OH
OH
OMe
OH
OH
OH
OH
26.42±3.49
2
13.65±3.78
3
OH
H
3.41±2.25
4
OH
H
93.72±0.25
5
OMe
OH
H
24.00±4.90
6
H
36.70±6.52
7
H
19.07±3.74
8
H
OH
H
12.43±4.29
9
OH
H
78.97±0.16
10
OMe
59.09±1.40
Kojic acid
18.81±1.25
aTyrosinase inhibition was assayed at 25 M using L-tyrosine as a substrate. Results are
presented as mean ± SEMs.
2.3. Docking simulation of cinnamamides 4, 9, 10 and kojic acid with tyrosinase.
Docking simulation was performed using AutoDock Vina 1.1.2 software (The Scripps
Research Institute) to investigate whether cinnamamides 4, 9, and 10 directly inhibit tyrosinase
by binding to its active site. For tyrosinase ligand preparation, the energy minimization tool of
Chem3D Pro 12.0 software (CambridgeSoft Corporation) was used to convert the 2D structures
of the three cinnamamides into the corresponding 3D structures. The 3D structure of Agaricus
bisporus tyrosinase from the Protein Data Bank (ID: 2Y9X) was used for docking simulation
of the cinnamamides. All three cinnamamide derivatives showed stronger binding affinities
with -6.8 ~ -6.9 kcal/mol than kojic acid (-5.7 kcal/mole), used as a positive control, as depicted

in Figure 1d. Interactions involved in docking simulation between tyrosinase and ligands, 4, 9,
and 10 were identified using LigandScout 4.1.0 software. Kojic acid forms - stacking with
His263 and two hydrogen bonds between the hydroxyl group of kojic acid and His259 and
His263 of tyrosinase (Figure 1b). On the other hand, cinnamamide 4 creates three hydrogen
bonds (His85, His 263, and Met280), and cinnamamides 9 and 10 form only one hydrogen
bond with Met280 (Figure 1a). Especially, the 4-hydroxyl group on the phenyl ring of 4
interacts with two histidine residues (His85, His263), like the hydroxyl group of kojic acid. All
three cinnamamide derivatives interact with Val283 in a hydrophobic manner, cinnamamides
4 and 9 commonly exhibit additional hydrophobic interactions with Ala286, and cinnamamide
10 exhibits additional hydrophobic interactions with Phe264. These results indicate that the
three cinnamamide derivatives can bind to the active site of tyrosinase effectively and more
highly inhibit the tyrosinase activity than kojic acid.
Figure 1. Docking simulation of 4, 9, 10 and of kojic acid with tyrosinase and pharmacophore
analysis: (a, and b) Pharmacophore results for 4, 9, 10 and kojic acid, indicating possible
hydrogen-bonding (green arrow), - stacking (violet arrow) and hydrophobic (yellow)
interactions, (c) docking simulation results between 4, 9, or 10 and mushroom tyrosinase, and

(d) docking scores between tyrosinase and 4, 9, 10 or kojic acid.
2.4. Cell viability of cinnamamides 4, 9 and 10 in B10F16 melanoma cells
The cell viabilities of cinnamamides 4, 9 and 10 were determined in B10F16 melanoma cells
by performing WST-8 assay. The three test cinnamamides 4, 9 and 10 were treated at four
different concentrations (0, 5, 10, and 25 µM) with melanoma cells and incubated for 24 h. Up
to 25 µM, no prominent changes occurred in melanoma cell viability, as shown in Figure 2.
Thus, these concentrations were chosen for use in subsequent cell-based assays to evaluate
tyrosinase inhibition and anti-melanogenesis.
Figure 2. Cell viability assay of cinnamamides 4, 9 and 10 in B16F10 melanoma cells.
Cinnamamides were treated at concentrations of 5, 10, and 25 μM. Viabilities are expressed in
% to control, and bars represent standard errors.
2.5. Tyrosinase inhibitory effect of cinnamamides 4, 9 and 10 in α-MSH-stimulated
B16F10 melanoma cells
α-MSH-stimulated B10F16 melanoma cells were treated with four different concentrations (0,
5, 10, and 25 µM) of the three cinnamamides 4, 9 and 10. The cells were incubated for 24 h.
The results of tyrosinase inhibition are indicated in Figure 3.
According to the results, cinnamamides 4 and 10 exhibited the highest tyrosinase inhibitory

effect in α-MSH-stimulated B10F16 melanoma cells at all three concentrations and each
cinnamamide inhibited tyrosinase activity in a concentration-dependent manner. Cinnamamide
9 also suppressed the activity of tyrosinase greater than kojic acid and arbutin (400 µM), used
as positive controls, at 25 µM. The tyrosinase activity inhibitions at concentrations of 10 and
25 µM were 38.33%, and 76.67% for cinnamamide 4, 23.33%, and 64.67% for cinnamamide
9, and 44.00%, and 73.33 % for cinnamamide 10, respectively. The tyrosinase inhibition of the
three cinnamamides 4, 9 and 10 at a concentration of 25 µM was much higher than that of kojic
acid (36.67%) at 25 µM and arbutin (51.67%) at 400 µM. These results demonstrate that our
previous findings showing that compounds with 2,4-dihydroxyphenyl groups exhibit generally
strong tyrosinase inhibition against mushroom tyrosinase can be applied to tyrosinase
inhibition in B16F10 cells.
Tyrosinase activity
4
9
10
100
50
0
**
***
***
***
***
***
***
***
***
***
l
d
i
n
M
M
M
M
M
M
M
M
M
o
i
t
r
µ
µ
µ
µ
µ
µ
µ
µ
µ
c
t
u
A
n
5
0
5
5
0
5
5
0
5
b
r
1
2
1
2
1
2
o
c
i
C
j
A
o
K
Concentration
Figure 3. Tyrosinase inhibitory activity of cinnamamides 4, 9 and 10 in α-MSH-stimulated
B16F10 melanoma cells that were co-treated with cinnamamides 4, 9, and 10, kojic acid (25
µM) or arbutin (400 µM). The asterisks represent the significance difference between the

columns: **, p<0.01; and ***, p<0.001. The bars represent standard errors.
2.6. Anti-melanogenic effect of cinnamamides 4, 9 and 10 in α-MSH-stimulated B16F10
melanoma cells
B10F16 melanoma cells were stimulated by α-MSH to determine the effect of the three
cinnamamides 4, 9 and 10 on the melanin content. The α-MSH-stimulated B16F10 melanoma
cells were co-treated with the three cinnamamides or kojic acid. After incubation for 24 h,
optical densities were measured to evaluate the effect of the three cinnamamides on the melanin
content in α-MSH-stimulated melanoma cells.
According to the results shown in Figure 4, all three cinnamamides exhibited strong decreases
in the melanin content compared to kojic acid at 25 µM in α-MSH-stimulated B16F10
melanoma cells. Of the three cinnamamides, cinnamamide 4 showed the highest melanin
content reduction (78.30%) at 25 µM, followed by cinnamamides 10 (72.67%) and 9 (61.80%);
all three reductions were superior to that of kojic acid (37.63% at 25 µM). All three
cinnamamides decreased melanin content in a dose-dependent manner and showed a decrease
in melanin content at 10 µM equal to or greater than that of kojic acid (25 µM). Considering
the very large similarity between the residual tyrosinase activity depicted in Figure 3 and the
melanin content shown in Figure 4, the anti-melanogenic effect of the three cinnamamides
appears to be mainly due to the tyrosinase inhibitory activity.

Melanin content
9
4
10
100
50
**
***
***
***
***
***
***
***
***
***
0
l
d
i
M
M
µ
M
M
M
M
M
M
M
o
r
µ
µ
µ
µ
µ
µ
µ
µ
c
t
A
n
5
0
5
5
0
5
5
0
5
1
2
1
2
1
2
o
c
i
j
C
o
K
Concentration
Figure 4. Anti-melanogenic effect of cinnamamides 4, 9 and 10 in α-MSH-stimulated B16F10
melanoma cells that were co-treated with cinnamamides 4, 9, and 10 or kojic acid (25 µM).
The asterisks represent the significant difference between the columns: **, p<0.01; and ***,
p<0.001. The bars represent standard errors.
2.7. DPPH radical scavenging activity of cinnamamides 1 – 10
The radical scavenging activity of the cinnamamides was determined by performing DPPH
assay. Cinnamamides 1 – 10 and L-ascorbic acid were added at a final concentration of 1 mM
to a DPPH methanol solution and radical scavenging activities were measured after 30 min.
The results are listed in Table 2.
According to our previous studies, compounds having a 3,4-dihydroxyl or 4-hydroxy-3-
methoxyl substituent on the –phenyl ring of the (E)-scaffold generally exhibit a high DPPH
radical scavenging activity, whereas compounds that lose the substituent at position 3 exhibit
dramatically decreased DPPH radical scavenging activity.15,16 Like our previous findings,
cinnamamides 3, 5, 8, and 10 with 3,4-dihydroxyphenyl and 4-hydroxy-3-methoxyphenyl

groups exhibited high DPPH radical scavenging activity (83.16%, 82.09%, 82.85%, and
80.69%, respectively) similar to that of L-ascorbic acid (84.64%), whereas cinnamamides 2,
and 7 with no substituent at the 3-position on the phenyl ring showed very low scavenging
activity.
Reactive oxygen species (ROS) is known to induce melanogenesis through tyrosinase
activation.37 Therefore, the inhibitory effect of cinnamamide 10 on the melanin production
shown in Figure 4 seems to be due to both the indirect inhibition of tyrosinase by antioxidant
activity and the direct inhibition of tyrosinase.
Table 2. DPPH radical scavenging activities of the synthesized cinnamamide derivatives 1 –
10, and kojic acid
R1
O
R1
O
R2
R3
R2
R3
N
N
1 5
-
6 10
-
Compounds
Compounds
Compound
DPPH radical
Compound
DPPH radical
scavenging activity (%)
scavenging activity (%)
1
2
3
4
5
34.19±0.24
6
37.59±0.09
3.51±1.09
7
0.91±0.48
83.16±0.05
8
82.85±0.49
22.33±1.11
9
30.67±1.71
82.09±0.21
10
80.69±0.28
L-ascorbic acid
84.64±0.32
The radical scavenging activities were measured at 30 min after the addition of cinnamamides
with a final concentration of 1.0 mM to the DPPH methanol solution. The presented results are
the means±SDs of three independent experiments.
3. Conclusion
Cinnamamides with an (E)--phenyl-,-unsaturated carbonyl scaffold were designed

as potential tyrosinase inhibitors. Cinnamamides 1 – 10 were prepared using Horner-Emmons
olefination as a key step. Of the 10 cinnamamides, three, namely 4, 9 and 10, exhibited greatly
superior tyrosinase inhibition against mushroom tyrosinase than that of kojic acid. In docking
simulations using tyrosinase, all three cinnamamides showed stronger binding affinities than
kojic acid and pharmacophore analysis revealed that these cinnamamides bind to the same
binding site as the natural substrates of tyrosinase. The three cinnamamides effectively
suppressed melanin content and inhibited tyrosinase activity to extents far superior to that
achieved by kojic acid in B16F10 murine melanoma cells without cytotoxicity. These results
support the promising potential of cinnamamides for use as anti-melanogenic agents in
pigmentation disorders.
4. Materials and Methods
4.1. General methods
1H NMR and 13C NMR data were obtained by a Varian Unity INOVA 400 spectrometer
or a Varian Unity AS500 spectrometer (Agilent Technologies, Santa Clara, CA, USA) using
DMSO-d6 and CDCl3 as solvents. All chemical shifts were measured in parts per million (ppm)
versus residual solvent or deuterated peaks (H 7.24 and C 77.0 for CDCl3, H 2.50 and C 39.7
for DMSO-d6). Coupling constants are represented 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)
and Agilent Accurate Mass Q-TOF liquid-chromatography mass spectrometer (Agilent, Santa
Clara, CA, USA), respectively. All reactions were conducted under a nitrogen atmosphere and
monitored by thin layer chromatography (TLC; Merck precoated 60F245 plates). All anhydrous
solvents were distilled over Na/benzophenone or CaH2 before use.
4.1.1. General procedure for the preparation of compounds 16 – 20.

Benzaldehydes 11 – 15 (5.00 g), benzyl bromide (1.0 equiv.), potassium carbonate (1.0
equiv.) and acetonitrile (50 mL) were added to a 250 mL round-bottom flask and the reaction
mixture was refluxed for 24 h. Acetonitrile was evaporated on completion of the reaction and
the resulting residues were partitioned between dichloromethane and water. The
dichloromethane layer was dried with MgSO4, filtered, and evaporated under reduced pressure
to give the products 16 - 20 as a white or grey solid in yields of 95 – 97%.
4.1.1.1 3-(Benzyloxy)-4-methoxybenzaldehyde (16). White solid, 97% yield. 1H NMR (500
MHz, CDCl3) δ 9.82 (s, 1 H, CHO), 7.47 (dd, 1 H, J = 8.0, 2.0 Hz, 6-H), 7.47 – 7.44 (m, 3 H,
2-H, 2′-H, 6′-H), 7.38 (t, 2 H, J = 7.5 Hz, 3′-H, 5′-H), 7.32 (t, 1 H, J = 7.5 Hz, 4′-H), 7.00 (d, 1
13
H, J = 8.0 Hz, 5-H), 5.19 (s, 2 H, benzylic H), 3.96 (s, 3 H, OCH3); C NMR (100 MHz,
CDCl3) δ 191.0, 155.2, 148.9, 136.5, 130.2, 128.8, 128.3, 127.7, 127.1, 111.6, 111.0, 71.1,
56.4.
1
4.1.1.2. 4-(Benzyloxy)benzaldehyde (17). White solid, 97% yield. H NMR (500 MHz,
CDCl3) δ 9.89 (s, 1 H, CHO), 7.84 (d, 2 H, J = 8.5 Hz, 2-H, 6-H), 7.44 (d, 2 H, J = 8.0 Hz, 2′-
H, 6′-H), 7.41 (t, 2 H, J = 8.0 Hz, 3′-H, 5′-H), 7.35 (t, 1 H, J = 7.5 Hz, 4′-H), 7.08 (d, 2 H, J =
13
8.5 Hz, 3-H, 5-H), 5.15 (s, 2 H, benzylic H); C NMR (100 MHz, CDCl3) δ 191.0, 163.9,
136.1, 132.2, 130.3, 128.9, 128.5, 127.7, 115.3, 70.5.
4.1.1.3. 3,4-Bis(benzyloxy)benzaldehyde (18). White solid, 96% yield. 1H NMR (500 MHz,
CDCl3) δ 9.81 (s, 1 H, CHO), 7.49 (d, 1 H, J = 2.0 Hz, 2-H), 7.48 – 7.36 (m, 9 H, 6-H, 2′-H,
3′-H, 5′-H, 6′-H, 2′′-H, 3′′-H, 5′′-H, 6′′-H), 7.34 – 7.30 (m, 2 H, 4′-H, 4′′-H), 7.02 (d, 1 H, J =
8.0 Hz, 5-H), 5.26 (s, 2 H, benzylic H), 5.22 (s, 2 H, benzylic H); 13C NMR (100 MHz, CDCl3)
δ 191.0, 154.4, 149.4, 136.7, 136.4, 130.5, 128.9, 128.8, 128.3, 128.2, 127.5, 127.2, 126.9,
113.3, 112.5, 71.2, 71.0.
1
4.1.1.4. 2,4-Bis(benzyloxy)benzaldehyde (19). Gray solid, 95 % yield. H NMR (500 MHz,
CDCl3) δ 10.39 (s, 1 H, CHO), 7.84 (d, 1 H, J = 9.0 Hz, 6-H), 7.45 – 7.38 (m, 8 H, 2′-H, 3′-H,

5′-H, 6′-H, 2′′-H, 3′′-H, 5′′-H, 6′′-H), 7.37 – 7.34 (m, 2 H, 4′-H, 4′′-H), 6.64 (dd, 1 H, J = 9.0,
2.0 Hz, 5-H), 6.60 (d, 1 H, J = 2.0 Hz, 3-H), 5.14 (s, 2 H, benzylic H), 5.11 (s, 2 H, benzylic
13
H); C NMR (100 MHz, CDCl3) δ 188.5, 165.4, 162.9, 136.1, 136.1, 130.7, 129.0, 128.9,
128.6, 128.5, 127.8, 127.5, 119.7, 107.2, 100.3, 70.6, 70.4.
4.1.1.5. 4-(Benzyloxy)-3-methoxybenzaldehyde (20). White solid, 97% yield. 1H NMR (500
MHz, CDCl3) δ 9.84 (s, 1 H, CHO), 7.45 – 7.42 (m, 3 H, 2-H, 2′-H, 6′-H), 7.40 – 7.37 (m, 3 H,
6-H, 3′-H, 5′-H), 7.33 (t, 1 H, J = 7.5 Hz, 4′-H), 6.99 (d, 1 H, J = 8.0 Hz, 5-H), 5.25 (s, 2 H,
benzylic H), 3.95 (s, 3 H, OCH3); 13C NMR (100 MHz, CDCl3) δ 191.1, 153.8, 150.3, 136.2,
130.5, 128.9, 128.4, 127.4, 126.8, 112.6, 109.5, 71.1, 56.3.
4.1.2. General procedure for the synthesis of compounds 26 – 30 via compounds 21 -25.
To a stirred solution of compounds 16 – 20 (2.00 g), triethyl phosphonoacetate (1.1
equiv.), and potassium carbonate (2.0 equiv.) in dichloromethane/N,N-dimethylformamide (2:1,
15 mL) was added a catalytic amount of DBU (0.03 equiv.). The reaction mixture was stirred
0
at room temperature (compounds 11, 13, and 15) or 70 C (12, and 14) for 24 – 36 h. After
evaporation of dichloromethane, ice water was added to the reaction mixture which was then
stirred for 30 min to give precipitates. After filtration, the filter cake was washed with a plenty
of water, and dried to produce ethyl cinnamates 21 – 25 as a white or gray solid in yields of 95
– 98%. The ethyl cinnamates were used directly in the next reaction without characterization.
Compounds 21 – 25 (2.57 to 4.11 g) were added to a 100 mL round-bottom flask and
1,4-dioxane (20 mL) and 1N-NaOH aqueous solution (8.0 – 16.0 equiv.) were added
subsequently. The reaction mixture was stirred at room temperature for 48 h. After completion
of the reaction, the reaction mixture was acidified until pH 2 by 2N-HCl aqueous solution. The
reaction mixture was stirred at room temperature for 30 min and then the resultant solid was
filtered. The filter cake was washed with a plenty of water, and dried to give the cinnamic acid

derivatives 26 – 30 as a white or grey solid in yields of 100%.
4.1.2.1. (E)-3-(3-(Benzyloxy)-4-methoxyphenyl)acrylic acid (26). White solid, 100% yield.
1H NMR (500 MHz, DMSO-d6) δ 12.19 (brs, 1 H, COOH), 7.49 (d, 1 H, J = 16.0 Hz, 3-vinylic
H), 7.45 (d, 2 H, J = 7.5 Hz, 2′-H, 6′-H), 7.41 (d, 1 H, J = 2.0 Hz, 2´-H), 7.38 (t, 2 H, J = 8.0
Hz, 3′-H, 5′-H), 7.32 (t, 1 H, J = 7.5 Hz, 4′-H), 7.20 (dd, 1 H, J = 8.0, 2.0 Hz, 6´-H), 6.98 (d, 1
H, J = 8.0 Hz, 5´-H), 6.40 (d, 1 H, J = 16.0 Hz, 2-vinylic H), 5.12 (s, 2 H, benzylic H), 3.78 (s,
3 H, OCH3); 13C NMR (100 MHz, DMSO-d6) δ 168.5, 151.7, 148.6, 144.7, 137.6, 129.1, 128.6,
128.5, 127.6, 123.7, 117.4, 112.5, 112.4, 70.5, 56.3.
1
4.1.2.2. (E)-3-(4-(Benzyloxy)phenyl)acrylic acid (27). White solid, 100% yield. H NMR
(500 MHz, DMSO-d6) δ 12.25 (brs, 1 H, COOH), 7.61 (d, 2 H, J = 9.0 Hz, 2-H, 6-H), 7.52 (d,
1 H, J = 16.0 Hz, 3-vinylic H), 7.43 (d, 2 H, J = 7.5 Hz, 2′-H, 6′-H), 7.38 (t, 2 H, J = 7.5 Hz,
3′-H, 5′-H), 7.31 (t, 1 H, J = 7.5 Hz, 4′-H), 7.02 (d, 2 H, J = 8.0 Hz, 3-H, 5-H), 6.36 (d, 1 H, J
= 16.0 Hz, 2-vinylic H), 5.14 (s, 2 H, benzylic H); 13C NMR (100 MHz, DMSO-d6) δ 168.5,
160.6, 144.3, 137.4, 130.6, 129.1, 128.6, 128.4, 127.7, 117.4, 115.8, 70.0.
1
4.1.2.3. (E)-3-(3,4-Bis(benzyloxy)phenyl)acrylic acid (28). White solid, 100% yield. H
NMR (500 MHz, DMSO-d6) δ 12.22 (brs, 1 H, COOH), 7.47 (d, 1 H, J = 16.0 Hz, 3-vinylic
H), 7.46 -7.41 (m, 5 H, 2-H, 2′-H, 6′-H, 2′′-H, 6′′-H), 7.37 (t, 2 H, J = 7.5 Hz, 3′-H, 5′-H), 7.36
(t, 2 H, J = 7.5 Hz, 3′′-H, 5′′-H), 7.30 (t, 2 H, J = 7.5 Hz, 4′-H, 4′′-H), 7.17 (dd, 1 H, J = 8.5,
2.0 Hz, 6-H), 7.05 (d, 1 H, J = 8.5 Hz, 5-H), 6.40 (d, 1 H, J = 16.0 Hz, 2-vinylic H), 5.18 (s,
2 H, benzylic H), 5.16 (s, 2 H, benzylic H); 13C NMR (100 MHz, DMSO-d6) δ 168.5, 150.7,
148.9, 144.6, 137.8, 137.6, 129.1, 129.1, 128.5, 128.5, 128.3, 128.1, 123.5, 117.7, 117.5,
114.5, 113.5, 70.6, 70.5.
4.1.2.4. (E)-3-(2,4-Bis(benzyloxy)phenyl)acrylic acid (29). Grey solid, 100% yield. 1H NMR
(500 MHz, DMSO-d6) δ 12.10 (s, 1 H, COOH), 7.77 (d, 1 H, J = 16.0 Hz, 3-vinylic H), 7.62
(d, 1 H, J = 9.0 Hz, 6-H), 7.45 – 7.31 (m, 10 H, 2ⅹPh), 6.81 (d, 1 H, J = 2.0 Hz, 3-H), 6.65

(dd, 1 H, J = 9.0, 2.0 Hz, 5-H), 7.38 (d, 1 H, J = 16.0 Hz, 2-vinylic H), 5.19 (s, 2 H, benzylic
H), 5.14 (s, 2 H, benzylic H); 13C NMR (100 MHz, DMSO-d6) δ 168.8, 162.1, 158.8, 139.3,
137.3, 137.3, 130.6, 129.2, 129.1, 128.7, 128.6, 128.5, 128.4, 117.3, 116.6, 107.8, 101.2, 70.5,
70.2.
4.1.2.5. (E)-3-(4-(Benzyloxy)-3-methoxyphenyl)acrylic acid (30). White solid, 100% yield.
1H NMR (500 MHz, DMSO-d6) δ 12.17 (brs, 1 H, COOH), 7.50 (d, 1 H, J = 16.0 Hz, 3-vinylic
H), 7.42 (d, 2 H, J = 7.5 Hz, 2′-H, 6′-H), 7.38 (t, 2 H, J = 7.5 Hz, 3′-H, 5′-H), 7.32 (t, 1 H, J =
7.5 Hz, 4′-H), 7.32 (d, 1 H, J = 2.0 Hz, 2-H), 7.16 (dd, 1 H, J = 8.0, 2.0 Hz, 6-H), 7.04 (d, 1 H,
J = 8.0 Hz, 5-H), 6.43 (d, 1 H, J = 16.0 Hz, 2-vinylic H), 5.11 (s, 2 H, benzylic H), 3.80 (s, 3
H, OCH3); 13C NMR (100 MHz, DMSO-d6) δ 168.5, 150.3, 149.9, 144.7, 137.4, 129.1, 128.6,
128.5, 128.0, 123.1, 117.5, 113.7, 111.2, 70.4, 56.3.
4.1.3. General procedure for the preparation of compounds 31 – 40.
A solution of compounds 26 – 30 (100 mg), isobutyl chloroformate (2.0 equiv.), and
N-methylmorpholine (2.5 equiv.) in anhydrous THF (5 mL) was stirred at room temperature
for 30 min. Then cyclic amine (pyrrolidine and piperidine) (2.0 equiv.) was added to the
reaction mixture which was then stirred at room temperature for 24 h. After completion of the
reaction, the reaction mixture was partitioned between ethyl acetate and water. The organic
layer was washed with brine, dried with MgSO4 and filtered. The filtrate was evaporated in
reduced pressure. The resultant residue was purified by column chromatography using
methylene chloride and methanol (30 – 70:1) as the eluent. The products 31 – 40 were obtained
as a white or yellow solid in yields of 80 – 91%.
4.1.3.1 (E)-3-(3-(Benzyloxy)-4-methoxyphenyl)-1-(pyrrolidin-1-yl)prop-2-en-1-one (31).
Yellowish solid, 80% yield. 1H NMR (400 MHz, CDCl3) δ 7.57 (d, 1 H, J = 15.2 Hz, 3-vinylic
H), 7.42 (d, 2 H, J = 8.0 Hz, 2′-H, 6′-H), 7.34 (t, 2 H, J = 8.0 Hz, 3′-H, 5′-H), 7.26 (t, 1 H, J =
8.0 Hz, 4′-H), 7.09 (d, 1 H, J = 8.0, 2.0 Hz, 6-H), 7.03 (d, 1 H, J = 2.0 Hz, 2-H), 6.84 (d, 1 H,

J = 8.0 Hz, 5-H), 6.45 (d, 1 H, J = 15.2, 2-vinylic H), 5.18 (s, 2 H, benzylic H), 3.87 (s, 3 H,
OCH3), 3.59 – 3.53 (m, 4 H, 2′′-H, 5′′-H), 1.95 – 1.88 (m, 4 H, 3′′-H, 4′′-H); 13C NMR (100
MHz, CDCl3) δ 165.1, 151.3, 148.4, 141.6, 137.1, 128.8, 128.4, 128.2, 127.6, 122.4, 116.9,
113.3, 111.7, 71.5, 56.2, 46.7, 46.2, 26.4, 24.6.
4.1.3.2. (E)-3-(4-(Benzyloxy)phenyl)-1-(pyrrolidin-1-yl)prop-2-en-1-one (32). Yellowish
white solid, 91% yield. 1H NMR (500 MHz, CDCl3) δ 7.66 (d, 1 H, J = 15.5 Hz, 3-vinylic H),
7.48 (d, 2 H, J = 8.5 Hz, 2-H, 6-H), 7.43 (d, 2 H, J = 7.5 Hz, 2′-H, 6′-H), 7.39 (t, 2 H, J = 7.5
Hz, 3′-H, 5′-H), 7.33 (t, 1 H, J = 7.5 Hz, 4′-H), 6.96 (d, 2 H, J = 8.5 Hz, 3-H, 5-H), 6.60 (d, 1
H, J = 15.5 Hz, 2-vinylic H), 5.08 (s, 2 H, benzylic H), 3.60 (brt, 4 H, J = 6.0 Hz, 2′′-H, 5′′-H),
1.94 – 1.91 (m, 4 H, 3′′-H, 4′′-H); 13C NMR (100 MHz, CDCl3) δ 165.3, 160.2, 141.9, 136.7,
129.7, 128.8, 128.4, 128.3, 127.7, 116.4, 115.3 70.2, 46.6, 25.8.
4.1.3.3. (E)-3-(3,4-bis(Benzyloxy)phenyl)-1-(pyrrolidin-1-yl)prop-2-en-1-one (33). White
solid, 80% yield. 1H NMR (400 MHz, CDCl3) δ 7.62 (d, 1 H, J = 15.6 Hz, 3-vinylic H), 7.43 –
7.25 (m, 10 H, 2ⅹPh), 7.07 (d, 1 H, d = 2.0 Hz, 2-H), 7.04 (dd, 1 H, J = 8.8, 2.0 Hz, 6-H), 6.86
(d, 1 H, J = 8.8 Hz, 5-H), 6.46 (d, 1 H, J = 15.6 Hz, 2-vinylic H), 5.15 (s, 2 H, benzylic H),
5.15 (s, 2 H, benzylic H), 3.55 (brt, 4 H, J = 6.4 Hz, 2′′-H, 5′′-H), 1.90 – 1.85 (m, 4 H, 3′′-H,
4′′-H); 13C NMR (100 MHz, CDCl3) δ 166.0, 161.0, 137.7, 136.7, 136.5, 132.6, 128.9, 128.8,
128.4, 128.3, 127.7, 118.7, 118.1, 106.3, 100.7, 70.7, 70.4, 46.4, 46.0, 26.2, 24.6.
4.1.3.4. (E)-3-(2,4-bis(Benzyloxy)phenyl)-1-(pyrrolidin-1-yl)prop-2-en-1-one (34). White
solid, 84% yield. 1H NMR (400 MHz, CDCl3) δ 7.73 (d, 1 H, J = 16.0 Hz, 3-vinylic H), 7.45 –
7.28 (m, 11 H, 6-H, 2ⅹPh), 6.81 (d, 1 H, J = 16.0 Hz, 2-vinylic H), 6.60 (d, 1 H, J = 2.0 Hz,
3-H), 6.55 (dd, 1 H, J = 8.0, 2.0 Hz, 5-H), 5.04 (s, 2 H, benzylic H), 5.03 (s, 2 H, benzylic H),
3.49 (brm, 2 H, 2′′-H, 5′′-H), 3.17 (brm, 2 H, 2′′-H, 5′′-H), 1.80 (brm, 4 H, 3′′-H, 4′′-H); 13C
NMR (100 MHz, CDCl3) δ 166.1, 161.0, 159.3, 137.9, 136.7, 136.5, 132.7, 128.9, 128.8, 128.4,
128.4, 128.4, 127.4, 118.6, 118.1, 106.3, 100.7, 70.7, 70.4, 46.3, 46.1, 26.1, 24.5.

4.1.3.5. (E)-3-(4-(Benzyloxy)-3-methoxyphenyl)-1-(pyrrolidin-1-yl)prop-2-en-1-one (35).
Yellowish white solid, 89% yield. 1H NMR (500 MHz, CDCl3) δ 7.63 (d, 1 H, J = 15.5 Hz, 3-
vinylic H), 7.42 (d, 2 H, J = 7.5 Hz, 2′-H, 6′-H), 7.36 (t, 2 H, J = 7.5 Hz, 3′-H, 5′-H), 7.30 (t, 1
H, J = 7.5 Hz, 4′-H), 7.06 – 7.03 (m, 2 H, 2-H, 6-H), 6.85 (d, 1 H, J = 9.0 Hz, 5-H), 6.57 (d, 1
H, J = 15.5, 2-vinylic H), 5.17 (s, 2 H, benzylic H), 3.91 (s, 3 H, OCH3), 3.60 (brt, 4 H, J = 6.0
Hz, 2′′-H, 5′′-H), 1.85 (brm, 4 H, 3′′-H, 4′′ H); 13C NMR (100 MHz, CDCl3) δ 165.2, 155.5,
149.9, 142.3, 136.9, 128.8, 128.2, 127.4, 121.8, 116.6, 113.7, 110.9, 71.1, 56.3, 46.1, 25.5.
4.1.3.6. (E)-3-(3-(Benzyloxy)-4-methoxyphenyl)-1-(piperidin-1-yl)prop-2-en-1-one (36).
Yellow solid, 87% yield. 1H NMR (500 MHz, CDCl3) δ 7.54 (d, 1 H, J = 15.5 Hz, 3-vinylic
H), 7.45 (d, 2 H, J = 7.5 Hz, 2′-H, 6′-H), 7.37 (t, 2 H, J = 7.5 Hz, 3′-H, 5′-H), 7.30 (t, 1 H, J =
7.5 Hz, 4′-H), 7.10 (dd, 1 H, J = 8.5, 1.5 Hz, 6-H), 7.05 (d, 1 H, J = 1.5 Hz, 2-H), 6.86 (d, 1 H,
J = 8.5 Hz, 5-H), 6.65 (d, 1 H, J = 15.5 Hz, 2-vinylic H), 5.17 (s, 2 H, benzylic H), 3.90 (s, 3
H, OCH3), 3.59 (brt, 4 H, J = 5.0 Hz, 2′′-H, 6′′-H), 1.69 – 1.65 (m, 2 H, 3′′-Ha, 5′′-Ha), 1.62 –
1.58 (m, 4 H, 3′′-Hb, 4′′-H, 5′′-Hb); 13C NMR (100 MHz, CDCl3) δ 165.7, 151.3, 148.4, 142.4,
137.1, 128.8, 128.5, 128.2, 127.6, 122.3, 115.5, 113.2, 111.7, 71.5, 56.2, 45.1, 26.4, 24.8.
4.1.3.7. (E)-3-(4-(Benzyloxy)phenyl)-1-(piperidin-1-yl)prop-2-en-1-one (37). Yellowish
white solid, 89% yield. 1H NMR (500 MHz, CDCl3) δ 7.63 (d, 1 H, J = 15.5 Hz, 3-vinylic H),
7.45 (d, 2 H, J = 9.0 Hz, 2-H, 6-H), 7.42 (d, 2 H, J = 7.5 Hz, 2′-H, 6′-H), 7.39 (t, 2 H, J = 7.5
Hz, 3′-H, 5′-H), 7.33 (t, 1 H, J = 7.5 Hz, 4′-H), 6.96 (d, 2 H, J = 9.0 Hz, 3-H, 5-H), 6.77 (d, 1
H, J = 15.5 Hz, 2-vinylic H), 5.09 (s, 2 H, benzylic H), 3.62 (brt, 4 H, J = 5.5 Hz, 2′′-H, 6′′-H),
1.69 – 1.66 (m, 2 H, 3′′-Ha, 5′′-Ha), 1.62 – 1.58 (m, 4 H, 3′′-Hb, 4′′-H, 5′′-Hb); 13C NMR (100
MHz, CDCl3) δ 165.9, 160.1, 142.5, 136.8, 129.5, 128.8, 128.6, 128.3, 127.7, 115.3, 115.1,
70.3, 45.6, 26.4, 24.8.
4.1.3.8. (E)-3-(3,4-Bis(benzyloxy)phenyl)-1-(piperidin-1-yl)prop-2-en-1-one (38). White
solid, 85% yield. 1H NMR (500 MHz, CDCl3) δ 7.54 (d, 1 H, J = 15.5 Hz, 3-vinylic H), 7.46 –

7.29 (m, 10 H, 2ⅹPh), 7.09 (d, 1 H, J = 1.5 Hz, 2-H), 7.06 (dd, 1 H, J = 8.5, 1.5 Hz, 6-H), 6.90
(d, 1 H, J = 8.5 Hz, 5-H), 6.66 (d, 1 H, J = 15.5 Hz, 2-vinylic H), 5.18 (s, 4 H, 2ⅹbenzylic H),
3.60 (brt, 4 H, J = 5.0 Hz, 2′′-H, 6′′-H), 1.69 – 1.66 (m, 2 H, 3′′-Ha, 5′′-Ha), 1.62 – 1.58 (m, 4
13
H, 3′′-Hb, 4′′-H, 5′′-Hb); C NMR (100 MHz, CDCl3) δ 165.8, 150.6, 149.0, 142.8, 137.3,
137.0, 129.1, 128.8, 128.7, 128.1, 127.6, 127.4, 122.4, 115.4, 114.6, 114.4, 71.8, 71.2, 45.5,
26.4, 24.8.
4.1.3.9. (E)-3-(2,4-Bis(benzyloxy)phenyl)-1-(piperidin-1-yl)prop-2-en-1-one (39). White
solid, 81% yield. 1H NMR (500 MHz, CDCl3) δ 7.76 (d, 1 H, J = 15.5 Hz, 3-vinylic H), 7.47 –
7.33 (m, 11 H, 6-H, 2ⅹPh), 7.02 (d, 1 H, J = 15.5 Hz, 2-vinylic H), 6.64 (d, 1 H, J = 2.0 Hz,
3-H), 6.59 (dd, 1 H, J = 8.5, 2.0 Hz, 5-H), 5.07 (s, 2 H, benzylic H), 5.05 (s, 2 H, benzylic H),
3.43 (brm, 4 H, 2′′-H, 6′′-H), 1.63 – 1.58 (m, 2 H, 3′′-Ha, 5′′-Ha), 1.49 - 146 (m, 4 H, 3′′-Hb,
4′′-H, 5′′-Hb); 13C NMR (100 MHz, CDCl3) δ 166.7, 161.0, 159.2, 139.1, 136.7, 136.5, 132.8,
128.9, 128.9, 128.5, 128.4, 128.3, 127.8, 118.1, 116.7, 106.3, 100.7, 70.7, 70.4, 45.4, 26.3, 24.9.
4.1.3.10. (E)-3-(4-(Benzyloxy)-3-methoxyphenyl)-1-(piperidin-1-yl)prop-2-en-1-one (40).
Yellow solid, 88% yield. 1H NMR (500 MHz, CDCl3) δ 7.58 (d, 1 H, J = 15.5 Hz, 3-vinylic
H), 7.43 (d, 2 H, J = 7.5 Hz, 2′-H, 6′-H), 7.36 (t, 2 H, J = 7.5 Hz, 3′-H, 5′-H), 7.30 (t, 1 H, J =
7.5 Hz, 4′-H), 7.04 (d, 1 H, J = 1.5 Hz, 6-H), 7.03 (dd, 1 H, J = 9.0, 1.5 Hz, 2-H), 6.85 (d, 1 H,
J = 9.0 Hz, 5-H), 6.74 (d, 1 H, J = 15.5 Hz, 2-vinylic H), 5.18 (s, 2 H, benzylic H), 3.92 (s, 3
H, OCH3), 3.62 (brt, 4 H, J = 5.5 Hz, 2′′-H, 6′′-H), 1.69 – 1.65 (m, 2 H, 3′′-Ha, 5′′-Ha), 1.62 –
1.58 (m, 4 H, 3′′-Hb, 4′′-H, 5′′-Hb); 13C NMR (100 MHz, CDCl3) δ 165.8, 149.8, 149.8, 142.9,
136.9, 129.0, 128.8, 128.2, 127.4, 121.7, 115.4, 113.7, 110.8, 71.1, 56.3, 45.5, 26.4, 24.8.
4.1.4. General procedure for the preparation of cinnamamides 1 – 3, 5 – 8 and 10.
Compounds 31 – 33, 35 – 38 and 40 (80 mg) were added to a 25 mL round-bottom
flask, along with concentrated HCl and acetic acid (1:1, 2 mL), and the reaction mixture was

refluxed for 40 min. After completion of the reaction, the mixture was neutralised by 2 N NaOH
aqueous solution and partitioned between methylene chloride and water. The organic layer was
dried with anhydrous MgSO4, and filtered and the filtrate was evaporated in reduced pressure.
The resulting residue was purified by column chromatography using methylene chloride and
methanol (10 – 30:1) as the eluent. Cinnamamides 1 – 3, 5 – 8 and 10 were produced as a
yellow or white solid in yields of 67-74 %.
4.1.4.1. (E)-3-(3-Hydroxy-4-methoxyphenyl)-1-(pyrrolidin-1-yl)prop-2-en-1-one (1).
Yellow solid, 70% yield. 1H NMR (400 MHz, CDCl3) δ 7.60 (d, 1 H, J = 15.6 Hz, 3-H), 7.17
(d, 1 H, J = 1.2 Hz, 2′-H), 6.98 (dd, 1 H, J = 8.0, 1.2 Hz, 6′-H), 6.79 (d, 1 H, J = 8.0 Hz, 5′-H),
6.54 (d, 1 H, J = 15.6 Hz, 2-H), 3.87 (s, 3 H, OCH3), 3.56 (t, 4 H, J = 6.4 Hz, 2′′-H, 5′′-H), 1.90
(brm, 4 H, 3′′-H, 4′′-H); 13C NMR (100 MHz, CDCl3) δ 165.4, 148.5, 146.1, 142.1, 129.0, 121.7,
116.8, 113.1, 110.8, 56.2, 46.4, 26.4, 24.6; LRMAS (ESI-) m/z 246 (M-H)-.
4.1.4.2. (E)-3-(4-Hydroxyphenyl)-1-(pyrrolidin-1-yl)prop-2-en-1-one (2). Yellowish white
solid, 72% yield. 1H NMR (500 MHz, CDCl3) δ 7.56 (d, 1 H, J = 15.0 Hz, 3-H), 7.34 (d, 2 H,
J = 7.5 Hz, 2′-H, 6′-H), 6.81 (d, 2 H, J = 7.5 Hz, 3′-H, 5′-H), 6.49 (d, 1 H, J = 15.0 Hz, 2-H),
13
3.55 (brm, 4 H, 2′′-H, 5′′-H), 1.96 (brm, 2 H, 3′′-H), 1.87 (brm, 2 H, 4′′-H); C NMR (100
MHz, CDCl3) δ 166.1, 159.1, 142.8, 129.9, 126.8, 116.0, 115.0, 47.0, 46.4, 26.2, 24.5; LRMAS
(ESI-) m/z 216 (M-H)-.
4.1.4.3. (E)-3-(3,4-Dihydroxyphenyl)-1-(pyrrolidin-1-yl)prop-2-en-1-one (3).38 White solid,
68% yield. 1H NMR (500 MHz, DMSO-d6) δ 9.37 (brs, 1 H, OH), 9.00 (brs, 1 H, OH), 7.28
(d, 1 H, J = 15.5 Hz, 3-H), 7.03 (s, 1 H, 2′-H), 6.93 (d, 1 H, J = 8.0 Hz, 6′-H), 6.72 (d, 1 H, J =
8.0 Hz, 5′-H), 6.62 (d, 1 H, J = 15.5 Hz, 2-H), 3.57 (t, 2 H, J = 6.5 Hz, 2′′-H), 3.35 (t, 2 H, J =
6.5 Hz, 5′′-H) 1.88 (quint, 2 H, J = 6.5 Hz, 3′′-H), 1.77 (quint, 2 H, J = 6.5 Hz, 4′′-H); 13C NMR
(100 MHz, DMSO-d6) δ 164.6, 148.0, 146.1, 141.4, 127.2, 121.3, 116.8, 116.3, 115.2, 46.5,
46.2, 26.3, 24.6; LRMAS (ESI-) m/z 232 (M-H)-.

4.1.4.4. (E)-3-(4-Hydroxy-3-methoxyphenyl)-1-(pyrrolidin-1-yl)prop-2-en-1-one (5).
Yellow solid, 71% yield. 1H NMR (500 MHz, CDCl3) δ 7.64 (d, 1 H, J = 15.5 Hz, 3-H), 7.10
(d, 1 H, J = 8.0 Hz, 6′-H), 7.00 (s, 1 H, 2′-H), 6.91 (d, 1 H, J = 8.0 Hz, 5′-H), 6.57 (d, 1 H, J =
15.5 Hz, 2-H), 3.91 (s, 3 H, OCH3), 3.61 (brm, 4 H, 2′′-H, 5′′-H), 1.95 (brm, 4 H, 3′′-H, 4′′-H);
13C NMR (100 MHz, CDCl3) δ 165.3, 147.6, 146.9, 142.2, 128.0, 122.1, 116.3, 115.0, 110.3,
56.2, 46.6, 26.2, 24.6; LRMAS (ESI-) m/z 246 (M-H)-.
4.1.4.5. (E)-3-(3-Hydroxy-4-methoxyphenyl)-1-(piperidin-1-yl)prop-2-en-1-one (6).
Yellowish white solid, 72% yield. 1H NMR (400 MHz, CDCl3) δ 7.56 (d, 1 H, J = 15.2 Hz,
3-H), 7.14 (s, 1 H, 2′-H), 6.97 (d, 1 H, J = 8.4 Hz, 6′-H), 6.79 (d, 1 H, J = 8.4 Hz, 5′-H), 6.72
(d, 1 H, J = 15.2 Hz, 2-H), 3.88 (s, 3 H, OCH3), 3.60 – 3.58 (m, 4 H, 2′′-H, 6′′-H) 1.63 - 1.58
(m, 6 H, 3′′-H, 4′′-H, 5′′-H); 13C NMR (100 MHz, CDCl3) δ 165.9, 148.2, 146.0, 142.6, 129.2,
121.7, 115.7, 112.8, 110.8, 56.2, 45.8, 26.4, 24.8; LRMAS (ESI-) m/z 260 (M-H)-.
4.1.4.6. (E)-3-(4-Hydroxyphenyl)-1-(piperidin-1-yl)prop-2-en-1-one (7). Yellowish white
solid, 74% yield. 1H NMR (500 MHz, CDCl3) δ 7.59 (d, 1 H, J = 15.0 Hz, 3-H), 7.34 (d, 2 H,
J = 8.5 Hz, 2′-H, 6′-H), 6.87 (d, 2 H, J = 8.5 Hz, 3′-H, 5′-H), 6.72 (d, 1 H, J = 15.0 Hz, 2-H),
3.65 – 3.60 (m, 4 H, 2′′-H, 6′′-H), 1.69 - 1.58 (m, 6 H, 3′′-H, 4′′-H, 5′′-H); 13C NMR (100 MHz,
CDCl3) δ 166.6, 158.9, 143.6, 129.8, 127.1, 116.2, 113.8, 47.5, 43.9, 26.8, 25.9, 24.7; LRMAS
(ESI-) m/z 230 (M-H)-.
4.1.4.7. (E)-3-(3,4-Dihydroxyphenyl)-1-(piperidin-1-yl)prop-2-en-1-one (8).39 White solid,
67% yield. 1H NMR (500 MHz, CDCl3 + a few drops of CD3OD) δ 7.39 (d, 1 H, J = 15.0 Hz,
3-H), 7.02 (s, 1 H, 2′-H), 6.88 (d, 1 H, J = 8.0 Hz, 6′-H), 6.76 (d, 1 H, J = 8.0 Hz, 5′-H), 6.64
(d, 1 H, J = 15.0 Hz, 2-H), 3.56 – 3.54 (m, 4 H, 2′′-H, 6′′-H), 1.63 – 1.53 (m, 6 H, 3′′-H, 4′′-H,
13
5′′-H); C NMR (100 MHz, CDCl3 + a few drops of CD3OD) δ 166.9, 147.2, 144.9, 144.1,
127.4, 121.3, 115.4, 114.5, 113.4, 47.5, 43.9, 26.4, 25.9, 24.5; LRMAS (ESI-) m/z 246 (M-H)-.
4.1.4.8. (E)-3-(4-Hydroxy-3-methoxyphenyl)-1-(piperidin-1-yl)prop-2-en-1-one (10).

Yellowish white solid, 73% yield. 1H NMR (400 MHz, CDCl3) δ 7.59 (d, 1 H, J = 15.2 Hz,
3-H), 7.07 (dd, 1 H, J = 8.0, 1.6 Hz, 6′-H), 6.97 (d, 1 H, J = 1.6 Hz, 2′-H), 6.88 (d, 1 H, J = 8.0
Hz, 5-H), 6.71 (d, 1 H, J = 15.2 Hz, 2-H), 3.90 (s, 3 H, OCH3), 3.61 – 3.59 (m, 4 H, 2′′-H, 6′′-
H) 1.64 – 1.55 (m, 6 H, 3′′-H, 4′′-H, 5′′-H); 13C NMR (100 MHz, CDCl3) δ 165.9, 147.5, 146.9,
143.0, 128.1, 122.0, 115.0, 114.9, 110.1, 56.2, 45.4, 43.9, 26.4, 24.8; LRMAS (ESI-) m/z 275
(M-H)-; HRMS (ESI-) m/z C15H18NO3 (M-H)- calcd 260.1292, obsd 260.1299.
4.1.5. General procedure for the preparation of cinnamamides 4 and 9.
To a 25 mL round-bottom flask, a solution of compounds 34 and 39 (80 mg) in
anhydrous methylene chloride (3 mL) was added under a nitrogen atmosphere. BBr3 (4.0 equiv.)
was added to the reaction mixture at -40 0C which was then stirred for 30 min. After completion
of the reaction, the mixture was neutralised by the addition of an equal amount of pyridine to
the used BBr3, and then an equal amount of methanol was added to the mixture. The reaction
mixture was evaporated under reduced pressure. The resultant residue was purified by column
chromatography using methylene chloride and methanol (10 – 30:1) as the eluent.
Cinnamamides 4 and 9 were obtained as a white solid in yields of 65 – 67%.
4.1.5.1. (E)-3-(2,4-Dihydroxyphenyl)-1-(pyrrolidin-1-yl)prop-2-en-1-one (4). White solid,
65% yield. 1H NMR (500 MHz, DMSO-d6) δ 9.86 (s, 1 H, OH), 9.64 (s, 1 H, OH), 7.62 (d, 1
H, J = 15.5 Hz, 3-H), 7.39 (d, 1 H, J = 8.0 Hz, 6′-H), 6.69 (d, 1 H, J = 15.5 Hz, 2-H), 6.32 (d,
1 H, J = 2.5 Hz, 3′-H), 6.23 (dd, 1 H, J = 8.0, 2.5, 2 Hz, 5′-H), 3.53 (t, 2 H, J = 7.0 Hz, 2′′-H),
3.35 (t, 2 H, J = 7.0 Hz, 5′′-H), 1.89 (quint, 2 H, J = 6.5 Hz, 3′′-H), 1.77 (quint, 2 H, J = 6.5
Hz, 5′′-H); 13C NMR (100 MHz, DMSO-d6) δ 165.2, 160.6, 158.4, 136.8, 130.2, 115.7, 114.2,
108.1, 103.1, 46.5, 46.2, 26.3, 24.6; LRMAS (ESI-) m/z 232 (M-H)-; HRMS (ESI-) m/z
C13H14NO3 (M-H)- calcd 232.0979, obsd 232.0987.
4.1.5.2. (E)-3-(2,4-Dihydroxyphenyl)-1-(piperidin-1-yl)prop-2-en-1-one (9). White solid,
67% yield. 1H NMR (500 MHz, DMSO-d6) δ 9.81 (s, 1 H, OH), 9.64 (s, 1 H, OH), 7.64 (d, 1

H, J = 15.5 Hz, 3-H), 7.44 (d, 1 H, J = 8.5 Hz, 6′-H), 6.92 (d, 1 H, J = 15.5 Hz, 2-H), 6.31 (d,
1 H, J = 2.0 Hz, 3′-H), 6.22 (dd, 1 H, J = 8.5, 2.0 Hz, 5′-H), 3.51 (brm, 4 H, 2′′-H, 6′′-H), 1.60
13
– 1.43 (m, 6 H, 3′′-H, 4′′-H, 5′′-H); C NMR (100 MHz, DMSO-d6) δ 165.8, 160.5, 158.3,
137.7, 130.1, 114.3, 113.8, 108.0, 103.1, 46.6, 43.2, 27.2, 26.2, 24.9; LRMAS (ESI-) m/z 246
(M-H)-; HRMS (ESI-) m/z C14H16NO3 (M-H)- calcd 246.1136, obsd 246.1146.
4.2. Biological experiments
4.2.1. Mushroom tyrosinase inhibition assay
Cinnamamide derivatives 1 – 10 were assessed for mushroom tyrosinase inhibition
with slight modification of a known method.40 A 200 µL mixture of 20 µL of tyrosinase
solution (1,000 U/mL), 170 µL of substrate solution (14.7 mM phosphate buffer, 293 µL L-
tyrosine solution) and 10 µL of the test compounds (final concentration: 25 µM) was added to
a 96-well plate. The plates were incubated at 37 0C for 30 min and the optical densities were
calculated using a microplate reader (VersaMaxTM, Molecular Devices, Sunnyvale, CA, USA)
at 450 nm. Kojic acid (25 µM) and arbutin (400 µM) were used as positive controls. The
experiments were replicated three times. The following formula was used to calculate the
tyrosinase inhibition:
%Inhibition = [1-(A/B) X 100)]
where A represents the absorbance of the test compounds and B the absorbance of the non-
treated control.
4.2.2. In silico docking simulation of cinnamamides 4, 9, and 10 with tyrosinase
Docking simulation of the three cinnamamides 4, 9, and 10, or kojic acid with tyrosinase
enzyme was performed according to the procedure used in previous work41,42. 3D structures of
the cinnamamides and kojic acid were created on Chem3D Pro 12.0 software. AutoDock Vina
1.1.2, and Chimera softwares were used for the docking score calculation between the test

compounds and tyrosinase enzyme. The 3D structure of tyrosinase (Agaricus bisporus) was
obtained from Protein Data Bank (PDB ID: 2Y9X). Pharmacophore models were created on a
LigandScout 4.1.0, indicating possible interactions between ligands and the amino acid
residues of tyrosinase.
4.2.3. Cell culture
Murine B16F10 melanoma cells were purchased from the American Type Culture
Collection (ATCC, Manassas, VA, USA). Cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) containing 10% fetal bovine serum (FBS), obtained from Gibco/Thermo
Fisher Scientific (Carlsbad, CA, USA), with 100 IU/mL penicillin, and 100 μg/mL
streptomycin in a humidified atmosphere with 5% CO2 at 37 ºC. Cell viability, melanin content
and tyrosinase activity assays were performed on cells cultured in either a 96-well plate or 24-
well dish. All experiments were performed in triplicate.
4.2.4. Cell viability assay in B16F10 melanoma cells
Cell viabilities were determined using WST-8 assay43. B16F10 melanoma cells were
seeded in a 96-well plate at a density of 1x104 cells per well and incubated in a humidified
condition with 5% CO2 at 37 °C for 24 h. The three test cinnamamides 4, 9 and 10 at four
different concentrations (0, 5, 10, and 25 µM) were added to the cells and further incubated for
24 h under the above mentioned conditions. The following day the cultured cells were treated
with WST-8 reagents and incubated for 30 min to 2 h at 37 °C and the cell viability was
assessed using the EZ-Cytox assay (EZ-3000, Daeil Lab Service, Seoul, Korea) in 96-well
microtiter plates by reading at an OD of 450 nm. The experiments were replicated three times.
4.2.5. Tyrosinase activity assay in B16F10 melanoma cells
The tyrosinase activity assay was performed with minor modification of the standard
procedure44. Briefly, B16F10 cells at 5x104 cells per well were cultured in a 96-well plate
followed by incubation in a humidified environment containing 5% CO2 at 37°C. After 24 h,

the cells were treated either with α-MSH (1 µM) and the three test cinnamamides 4, 9 and 10
(0, 5, 10, 25 µM), or with α-MSH (1 µM) and kojic acid (25 µM) and further incubated for 24
h under the above mentioned conditions. The following day the cells were washed with PBS
2-3 times and then broken with lysis buffer (100 μL containing 50 mM PBS (90 μL, pH 6.8),
0.1 mM PMSF (5 μL), and 1% Triton X-100 (5 μL)). The lysates of the cells were then frozen
at -80 °C for 30 min followed by centrifugation at 12,000 rpm for 30 min at 4 °C. Supernatant
of the lysates was transferred to a 96-well plate and 80 µL of supernatant with 20 µL of 10 mM
L-dopa were incubated at 37 °C for 30 min. Optical densities were measured at 500 nm by
using a microplate reader (Tecan, Männedorf, Switzerland). The experiments were performed
in triplicate.
4.2.6. Anti-melanogenesis assay in B16F10 melanoma cells
To assess the inhibitory effect of the three test cinnamamides 4, 9 and 10 on melanin
production, melanin content assay was performed using slight modification of the standard
method previously reported.45 Briefly, B16F10 cells were seeded at a density of 5x104
cells/well in a 24-well plate and the cells were cultured in 5% CO2 incubator at 37 °C. After 24
h, the cells were treated with α-MSH (1 µM) plus either four different concentrations of the
three test cinnamamides (0, 5, 10, and 25 µM), or kojic acid (25 µM) and further incubated for
24 h under the above mentioned conditions. The cells were washed with PBS buffer 2-3 times
to remove the media content and incubated with 1 N NaOH solution (200 µL) to dissolve
melanin. After transferring the solution to 96-well plates, the absorbances of the dissolved
melanin solution were measured at 405 nm using a microplate reader. All experiments were
performed in triplicate.
4.2.7. DPPH radical scavenging activity assay.
A previously reported method with slight modification was adopted to determine the
DPPH radical scavenging abilities of the test cinnamamides 1 – 10.46 Briefly, 180 µL of a

DPPH methanol solution (0.2 mM) was mixed with 20 µL of each cinnamamide 1 – 10 (10
mM in DMSO) in 96-well plates. L-Ascorbic acid was used as a positive standard reference
material. Next, the mixed solution in 96-wells plates was incubated in the dark for 30 min and
the absorbances were measured at 517 nm using a VersaMaxTM microplate reader. All
experiments were performed in triplicate. The following formula was used to calculate the
DPPH scavenging activities of 1 – 10:
scavenging activity (%) = [(Ac–As)/Ac × 100]
where Ac is the absorbance of the non-treated control and As is the absorbances of the
cinnamamides.
4.2.8. Statistical analysis
GraphPad Prism 5 software (La Jolla, CA, USA) was used to determine the statistical
analysis. All experiments were performed in triplicate. Calculated results are shown as means
± SEMs. Intergroup significance differences were calculated by using one-way ANOVA and
Tukey’s test. Statistical significance was considered at < 0.05 of two-sided P-values.
Acknowledgments
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).
References
1. Prota G. Regulatory mechanisms of melanogenesis: beyond the tyrosinase concept. J Invest
Dermatol. 1993;100(2): S156-S161.
2. Plant N. Strategies for using in vitro screens in drug metabolism. Drug Discovery Today.
2004;9(7): 328-336.
3. Stein B, Sutherland MSK. IL-6 as a drug discovery target. Drug Discovery Today.
1998;3(5): 202-213.
4. McEvily AJ, Iyengar R, Otwell WS. Inhibition of enzymatic browning in foods and
beverages. Crit Rev Food Sci Nutr. 1992;32(3): 253-273.
5. Maeda K, Fukuda M. In vitro effectiveness of several whitening cosmetic components in
human melanocytes. J Soc Cosmet Chem. 1991;42(2): 361-368.