Evaluation and optimization of the antimelanogenic activity of 1-(2-cyclohexylmethoxy-6-hydroxy-phenyl)-3-(4-hydroxymethyl-phenyl)-propenone derivatives

Article abstract of DOI:10.3390/molecules24071372

The chemical modification and optimization of biologically active compounds are essential steps in the identification of promising lead compounds for drug development. We previously reported the anti-melanogenic activity of 1-(2-cyclohexylmethoxy-6-hydrox

Full text of DOI:10.3390/molecules24071372

molecules
Article
Evaluation and Optimization of the Anti-Melanogenic
Activity of 1-(2-Cyclohexylmethoxy-6-
hydroxy-phenyl)-3-(4-hydroxymethyl-phenyl)-
propenone Derivatives
Byung-Hak Kim 1,2 , Soo-Nam Hong 3, Sang-Kyu Ye 1,2,4,5,* and Jung-Youl Park 6,
*
1
Department of Pharmacology and Biomedical Sciences, Seoul National University College of Medicine,
103 Daehak-ro, Jongno-gu, Seoul 03080, Korea; protein0826@snu.ac.kr
Biomedical Science Project (BK21PLUS), Seoul National University College of Medicine, 103 Daehak-ro,
Jongno-gu, Seoul 03080, Korea
Department of Beauty Care, Bucheon University, 25 Sinheung-ro 56 beon-gil, Bucheon-si, Gyeonggi-do
14632, Korea; sn1228@naver.com
Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, 103 Daehak-ro,
Jongno-gu, Seoul 03080, Korea
Neuro-Immune Information Storage Network Research Center, Seoul National University College of
Medicine, 103 Daehak-ro, Jongno-gu, Seoul 03080, Korea
2
3
4
5
6
Department of Applied Chemistry, Daejeon University, 62 Daehak-ro, Dong-gu, Daejeon 34520, Korea
Correspondence: sangkyu@snu.ac.kr (S.-K.Y.); jypark0908@dju.kr (J.-Y.P.); Tel.: +82-2-740-8281 (S.-K.Y.);
+82-42-280-4976 (J.-Y.P.); Fax: +82-2-745-7996 (S.-K.Y.)
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Academic Editor: Tiziano Tuccinardi
Received: 18 March 2019; Accepted: 5 April 2019; Published: 8 April 2019
Abstract: The chemical modification and optimization of biologically active compounds are essential
steps in the identification of promising lead compounds for drug development. We previously reported
the anti-melanogenic activity of 1-(2-cyclohexylmethoxy-6-hydroxy-phenyl)-3-(4-hydroxymethyl-
phenyl)-propenone (chalcone 21). In this study, we synthesized 21 derivatives of chalcone 21
and evaluated their anti-melanogenic activity in
(Cyclohexylmethoxy)phenyl)-3-oxoprop-1-en-1-yl)phenyl)acetamide (chalcone 21-21) exhibited the
strongest inhibition of cellular melanin production, with an IC50 value of 0.54 M. It was more potent
than chalcone 21 and the known anti-melanogenic agents kojic acid and arbutin, whose IC50 values
were 4.9, 38.5, and 148.4 M, respectively. Chalcone 21-21 decreased the expression and activity
α-MSH-induced B16F10 cells. (E)-N-(4-(3-(2-
µ
µ
of tyrosinase. It also decreased the expression of TRP1, TRP2 and MITF, the phosphorylation of
CREB and ERK1/2, and the transcriptional activity of MITF and CRE. Our results demonstrate that
chalcone-21-21 is an effective lead compound with anti-melanogenic activity.
Keywords: alpha-melanocyte stimulating hormone (α-MSH); chalcone; melanin biosynthesis;
(E)-N-(4-(3-(2-(cyclohexylmethoxy)phenyl)-3-oxoprop-1-en-1-yl)phenyl)acetamide (chalcone 21-21);
tyrosinase
1. Introduction
Melanin is a group of dark polymer pigments synthesized in melanocytes. It is found in almost all
animals, and is responsible for the various pigmentation of the skin, hair, and eyes. Several beneficial
functions of melanin have been identified, including the protection of skin cancer development
by protecting cells from damage due to sunlight, hormonal factors, cytokines, toxic drugs, and
other chemicals [
1,2]. However, abnormal loss or accumulation of melanin results in dermatological
disorders such as vitiligo, freckles, melasma, chloasma, ephelides, senile lentigines, and melanoderma.
Molecules 2019, 24, 1372; doi:10.3390/molecules24071372
www.mdpi.com/journal/molecules

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In addition, melanin can induce skin inflammatory responses such as eczema, allergic contact
dermatitis, and irritant contact dermatitis [ ]. These disorders can lead to serious and emotionally
3–5
distressing problems, and development of anti-melanogenic agents is necessary to address them.
Melanin biosynthesis is mediated by a series of intricate signaling pathways initiated by many
intrinsic and extrinsic stimuli, including ultraviolet (UV) radiation, nitric oxide (NO), stem cell factor
(SCF), and hormones such as alpha-melanocyte stimulating hormone (α-MSH), epinephrine, and
adrenocorticotropic hormone (ACTH). These stimuli activate many signaling pathways, such as
those involving inositol trisphosphate/diacylglycerol (IP3/DAG)/protein kinase C (PKC), guanylate
cyclase/cyclic guanosine monophosphate (GC/cGMP), c-Kit, and adenylate cyclase/cyclic adenosine
monophosphate (AC/cAMP) [4,6,7].
Chalcones are aromatic ketones consisting of two aromatic rings linked to a three-carbon
α,β-unsaturated carbonyl system as a central core. They are naturally occurring in plants that
have been used in traditional folk medicine. Chalcones possess many biological effects, including
anti-inflammatory, antimicrobial, antifungal, antioxidative, cytotoxic, antiproliferative, antitumor, and
chemopreventive effects. In addition, chalcones are considered to be intermediates in the biosynthesis
of flavonoids and isoflavonoids, abundant substances in edible plants that show a wide spectrum of
biological activities [8–12]. We reported previously that the chalcone derivative 1-(2-cyclohexylmethoxy-
6-hydroxy-phenyl)-3-(4-hydroxymethyl-phenyl)-propenone (chalcone 21) has anti-melanogenic
activity [13]. In an attempt to increase its anti-melanogenic activity, we synthesized 21 derivatives via
the aldol condensation of 2- and/or 6-substituted acetophenones and appropriate benzaldehydes in the
presence of potassium hydroxide as a catalyst in ethanol. The largest difference with the derivatives
previously reported is the presence of an hydroxyl group in the R2 position. Among the derivatives,
we identified (E)-N-(4-(3-(2-(cyclohexylmethoxy)phenyl)-3-oxoprop-1-en-1-yl)phenyl)acetamide
(chalcone 21-21) as exhibiting the strongest anti-melanogenic activity (approximately 10-fold more
effective than chalcone 21).
2. Results and Discussion
2.1. Chemistry
We previously reported the anti-melanogenic activity of chalcone 21 [13]. To obtain more potent
anti-melanogenic agents, we synthesized a series of chalcone derivatives by aldol condensation reactions
(Scheme 1) [8,
14]. Detailed synthetic procedures and the 1H-NMR and 13C-NMR spectroscopic data
are shown in the Materials and Methods.
Scheme 1. Synthetic scheme of the chalcone derivatives 21-1 ~ 21-21. The chalcone derivatives
were synthesized by the aldol condensation of 2- and/or 6-substituted acetophenones and appropriate
benzaldehydes in the presence of KOH as a catalyst in ethanol. R1, 4-OTf-benzyl, benzyl, or cyclohexylmethyl;
R2, hydroxyl; R3, ethyl, chloro, methoxy, carboxylic acid, or acetylamine.
2.2. Biological Evaluation
2.2.1. Cellular Melanin Production
(2E)-1,3-Diphenyl-propene-1-one forms the core structure of chalcones, consisting of two aromatic
rings linked to a three-carbon
activity of the 21 chalcone derivatives (21-1 through 21-21), we measured the inhibition of cellular
melanin production in -MSH-stimulated mouse melanoma B16F10 cells. -MSH is the main intrinsic
stimulator of melanogenesis, via a melanocortin receptor type 1 (MC1R)-mediated cAMP/protein
α,β-unsaturated carbonyl (Figure 1A). To determine the anti-melanogenic
α
α

Molecules 2019, 24, 1372
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kinase A (PKA) pathway [15
,16]. Cells were incubated for 72 h with vehicle alone or with various
concentrations of each compound in the presence of
by measuring the absorbance at 405 nm of the cell-free culture medium. The cellular melanin levels
were 34.4 1.8 g/mL in vehicle-treated resting cells, and were dramatically increased to 135.2
2.5 g/mL by -MSH stimulation. The degree of inhibiton of the melanin production varied among the
α-MSH, and melanin content was determined
±
µ
±
µ
α
tested compound. For each, the percent inhibition of melanin production, the half maximal inhibitory
concentration (IC50), and the cell viability of cultures exposed at 3 µM are shown in Table 1.
We observed that chalcones 21-5, 21-13, and 21-21 exhibited strong anti-melanogenic activity,
with IC50 values of 0.88, 1.6, and 0.54 µM, respectively. Weak cytotoxic activity was associated with
those compounds containing an acetamido or carboxyl group at the R3 position. Chalcones 2-2,
2-7, 2-9, and 2-11 exhibited moderate anti-melanogenic activity, with IC50 values of 1.2, 2.3, 2.6, and
2.0 µM, respectively; weak to moderate cytotoxic activity was observed for these forms containing
a chloride atom at the R3 position. Other chalcone compounds with ethyl or methoxy group at the
R3 position exhibited very weak to weak anti-melanogenic activity, and weak to moderate cytotoxic
activity. The strong, moderate, and weak anti-melanogenic and cytotoxic activities were divided into
over 75%, 50~75%, and less than 50% at 3 µM of each compound. Based on the structure-activity
relationship (SAR), moderate to strong anti-melanogenic activity was observed when benzyloxy,
4-trifluoromethoxybenzyloxy or cyclohexylmethoxy group was located at the R1 position and chloride
or acetamido group was located at the R3 position. However, hydrogen or hydroxyl group at the
R2 position had little affected. In fact, benzyloxy, 4-trifluoromethoxybenzyloxy and chloride groups
exhibited moderate cytotoxic activity, while cyclohexylmethoxy and acetamido groups exhibited weak
cytotoxic activity. Overall, the anti-melanogenic activity of chalcone derivatives is the strongest when
cyclohexylmethoxy and acetamido groups are located at the R1 and R3 positions. These data suggest
the significance of acetamido or carboxyl groups at the R3 position to anti-melanogenic activity in the
derivatives of chalcone 21. In particular, chalcone 21-21 exhibited the strongest inhibition of melanin
production, and was approximately 10-fold more effective than chalcone 21 (Figure 1B and Table 1).
These results are preliminary data at the simple screening step and require further analysis to determine
the action mechanisms against anti-melanogenic activity.
Figure 1.
(A) Core-structure of chalcones. (B) Chemical structure of chalcone 21 (left) and chalcone
21-21 (right).

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Table 1. Inhibitory effect of the derivatives of chalcone 21 on melanin production in α-MSH-stimulated B16F10 cells. B16F10 cells were incubated for 3 days with each
compound in the presence of α-MSH (10 nM) and data are expressed as mean ±SD based on three independent experiments (n = 3).
Substitutions
Compound
Viability (%) at 3 µM
Inhibition (%) at 3 µM
IC50 (µM)
R1
R2
R3
Chalcone 21
21-1
cyclohexylmethoxy
4-trifluoromethoxybenzyloxy
4-trifluoromethoxybenzyloxy
4-trifluoromethoxybenzyloxy
4-trifluoromethoxybenzyloxy
4-trifluoromethoxybenzyloxy
benzyloxy
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
H
H
H
H
H
H
CH2OH
CH2CH3
Cl
41.7 ± 3.3
26.3 ± 6.5
73.9 ± 6.8
47.2 ± 7.8
38.8 ± 6.2
89.4 ± 8.4
42.8 ± 7.1
65.0 ± 4.9
35.7 ± 4.6
55.8 ± 6.2
36.6 ± 5.6
72.0 ± 7.0
35.7 ± 5.8
91.8 ± 6.4
47.1 ± 6.4
23.8 ± 4.9
21.6 ± 6.1
9.4 ± 5.4
95.1 ± 3.9
79.7 ± 3.9
60.6 ± 3.2
78.6 ± 2.7
78.9 ± 2.1
76.5 ± 3.1
85.0 ± 4.6
59.8 ± 3.9
74.4 ± 3.1
72.9 ± 2.2
77.0 ± 2.6
67.8 ± 3.5
81.6 ± 3.5
71.2 ± 2.8
74.5 ± 3.8
78.3 ± 2.9
80.4 ± 3.0
81.8 ± 4.8
87.6 ± 4.2
76.4 ± 3.6
90.2 ± 3.2
86.3 ± 2.5
>3.0
>3.0
1.2
>3.0
>3.0
0.88
>3.0
2.3
>3.0
2.6
>3.0
2.0
21-2
21-3
21-4
21-5
21-6
21-7
21-8
21-9
21-10
21-11
21-12
21-13
21-14
21-15
21-16
21-17
21-18
21-19
21-20
21-21
OCH3
COOH
NHCOCH3
CH2CH3
Cl
benzyloxy
benzyloxy
benzyloxy
benzyloxy
benzyloxy
benzyloxy
benzyloxy
benzyloxy
OCH3
COOH
CH2CH3
Cl
OCH3
>3.0
1.6
COOH
NHCOCH3
CH2CH3
OCH3
NHCOCH3
CH2CH3
Cl
>3.0
>3.0
>3.0
>3.0
>3.0
>3.0
>3.0
0.54
OH
OH
OH
cyclohexylmethoxy
cyclohexylmethoxy
cyclohexylmethoxy
cyclohexylmethoxy
29.1 ± 4.9
49.7 ± 7.7
19.8 ± 8.5
92.0 ± 5.5
H
H
H
COOH
NHCOCH3

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2.2.2. Anti-Melanogenic Activity of Chalcone 21-21
Because chalcone 21-21 exhibited the greatest inhibition of melanin production in
α-MSH-stimulated
B16F10 cells, we investigated the mechanisms underlying its anti-melanogenic effects. First, we determined
the cytotoxic activity of chalcone 21-21. It exhibited weak to no cytotoxic activity in vitro at
concentrations of up to 10
in concentration- and time-dependent manners. Treatment with 10
moderate cytotoxic activity, but the cytotoxic activity of chalcone 21-21 was not significantly different at
concentrations of up to 100 M in the absence or presence of -MSH (Figure 2A,B). Therefore, chalcone
M in subsequent in vitro experiments to exclude the
µM, in cells cultured for 24–72 h; above 10
µM, cytotoxic activity increased
µM for 72 h showed weak to
µ
α
21-21 was used at concentrations of up to 3
µ
effects of cytotoxicity.
To determine the effect of chalcone 21-21 on melanin production, cellular melanin release was
measured in B16F10 cells, followed by incubation for 72 h with various concentrations of chalcone
21-21 in the absence or presence of
vehicle-treated resting cells and 139.8
α
-MSH. The released melanin levels were 31.3
5.5 g/mL in -MSH-stimulated cells. Treatment with chalcone
5.5%
M, and 92.0
M (Figure 2C). Kojic acid and arbutin, well-known
anti-melanogenic agents that act by inhibiting tyrosinase activity [17 18] were used as positive controls.
Both compounds also reduced melanin production in a concentration-dependent manners with IC50
values of 38.5 M and 148.4 M, respectively (Figure 2D). These results indicate that chalcone 21-21
±
2.3 µg/mL in
±
µ
α
21-21 decreased melanin release in a concentration-dependent manner, corresponding to 16.9
inhibition at 0.1 M, 38.4 5.2% inhibition at 0.3 M, 72.2 5.2% inhibition at 1
4.2% inhibition at 3 M, for an IC50 value of 0.54
±
µ
±
µ
±
µ
±
µ
µ
,
µ
µ
has significant potential anti-melanogenic activity.
Figure 2. Chalcone 21-21 inhibits melanin production. (A,B) B16F10 cells were incubated for the
indicated time-intervals with vehicle (0.1% DMSO) alone or various concentrations of chalcone 21-21 in
the absence or presence of -MSH (10 nM). Cell viability was measured using the EZ-CyTox Enhanced
Cell Viability Assay Reagent, and is represented as % control compared to the vehicle alone-treated
group. Results are represented as mean SD from three independent experiments (n = 3). p-values of
* p < 0.05 and ** p < 0.005 compared to the vehicle- or -MSH-treated group were considered statistically
significant. ( ) Cells were incubated for 72 h as mentioned above in Figure 2B. Melanin contents (
and inhibition percentage of melanin production ( ) are shown. Results are represented as mean SD
from three independent experiments (n = 3). p-values of # p < 0.005 compared to the vehicle-treated
group and * p < 0.05 and ** p < 0.005 compared to the -MSH-treated group were considered statistically
significant. Kojic acid and arbutin were used as positive controls.
α
±
α
C
,
D
C)
D
±
α

Molecules 2019, 24, 1372
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2.2.3. Inhibition of Tyrosinase Activity
Next, we evaluated whether chalcone 21-21 could inhibit tyrosinase activity. Tyrosinase is a
copper-dependent enzyme that catalyzes the conversion of l-tyrosine to l-3,4-dihydroxyphenylalanine
(l-DOPA), the rate-limiting step in melanin biosynthesis [19
levels of tyrosinase, B16F10 cells were incubated for 72 h with various concentrations of chalcone
21-21 in the absence or presence of -MSH. Both the mRNA and protein levels of tyrosinase were
just detectable in vehicle-treated resting cells, and the levels were markedly up-regulated by -MSH
,20]. To determine the mRNA and protein
α
α
stimulation. These up-regulated expression levels decreased by treatment with chalcone 21-21 in a
concentration-dependent manner (Figure 3A).
Tyrosinase activity was further determined in cell lysates of B16F10 cells that were incubated for
72 h with various concentrations of chalcone 21-21 in the absence or presence of α-MSH. To perform
zymography, lysate proteins were separated by native SDS-PAGE, and the gels were incubated with
l-DOPA for the colorimetric detection of l-dopaquinone, which is the tyrosinase-catalyzed metabolite
of l-DOPA and a precursor of melanin [21
resting cells, but was dramatically increased by
,
22]. Tyrosinase activity was very weak in vehicle-treated
-MSH stimulation. Treatment with chalcone 21-21
α
decreased tyrosinase activity in a concentration-dependent manner (Figure 3B).
We also evaluated tyrosinase promoter activity to determine whether the expression of tyrosinase
was regulated at the transcriptional level. B16F10 cells were transiently transfected with a tyrosinase
reporter plasmid construct containing the human tyrosinase promoter together with the pRL-TK
Renilla control vector. After stimulation with
cells. Treatment with chalcone 21-21 decreased
concentration-dependent manner, corresponding to 14.5
inhibition at 0.3 M, 70.7 3.3% inhibition at 1 M, and 94.5
These results suggest that chalcone 21-21 has potential for development of skin-lightening agent.
α
-MSH, luciferase activity increased 7.4-fold over resting
-MSH-induced tyrosinase promoter activity in a
5.2% inhibition at 0.1 M, 36.4 4.4%
1.2% inhibition at 3 M (Figure 3C).
α
±
µ
±
µ
±
µ
±
µ
Figure 3. Chalcone 21-21 inhibits tyrosinase activity. (
vehicle (0.1% DMSO) alone or various concentrations of chalcone 21-21 in the absence or presence of
-MSH (10 nM). The level of tyrosinase was determined by Western blot or RT-PCR analyses. GAPDH
served as a loading control. ( ) Total cell lysates were prepared as mentioned above in Figure 3A.
Tyrosinase activity was determined by zymography, and the band densities are represented. ( ) B16F10
A) B16F10 cells were incubated for 72 h with
α
B
C
cells were transiently transfected with a reporter construct of tyrosinase together with the pRL-TK.
The cells were incubated for 24 h with vehicle (0.1% DMSO) alone or with various concentrations
of chalcone 21-21 in the absence or presence of
and firefly luciferase activities were normalized to that of Renilla luciferase. Results are represented
as mean
SD from three independent experiments (n = 3). p-values of # p < 0.005 compared to the
α-MSH (10 nM). Luciferase activity was measured
±
vehicle-treated group and * p < 0.05 and ** p < 0.005 compared to the α-MSH-treated group were
considered statistically significant.
2.2.4. Inhibition of Melanogenesis-Related Proteins
Melanin biosynthesis occurs in melanosomes, membrane-bound organelles within melanocytes.
In mammals, melanin biosynthesis is a tightly regulated complex network consisting of multiple

Molecules 2019, 24, 1372
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important factors including tyrosinase-related protein (TRP)1 and TRP2, which share numerous structural
similarities due to amino acid sequences that are 40% homologous to that of tyrosinase [ 20]. Therefore,
we investigated whether chalcone 21-21 could affect the protein levels of melanogenesis-related
TRP1 and TRP2 by Western blot analysis using whole-cell lysates from -MSH-stimulated B16F10
cells. Both protein levels were hardly detectable in vehicle-treated resting cells, but their expression
levels were dramatically increased by stimulation with -MSH. Chalcone 21-21 treatment resulted in
decreasing the -MSH-induced protein synthesis of TRP1 and TRP2 in a concentration-dependent
1,
α
α
α
manner (Figure 4A).
Microphthalmia-associated transcription factor (MITF) is the master transcription factor in melanin
biosynthesis, initiating the transcription of genes encoding tyrosinase, TRP1, and TRP2 [23]. The mRNA
and protein levels of MITF were detactable in vehicle-treated resting B16F10 cells, and were dramatically
increased by stimulation with α-MSH. Treatment with chalcone 21-21 decreased expression at both
levels in a concentration-dependent manner (Figure 4B). We further determined the transcriptional
regulation of MITF using a reporter system. A luciferase expression plasmid under the control of the
human MITF promoter was transiently transfected together with the pRL-TK Renilla control vector
into B16F10 cells. Stimulation with
vehicle-treated resting cells. Treatment with chalcone 21-21 reduced
activity in a concentration-dependent manner, corresponding to 13.2
3.3% inhibition at 0.3 M, 67.4 3.6% inhibition at 1 M, and 89.5 3.3% inhibition at 3 µ
These results indicate that chalcone 21-21 down-regulates multiple melanogenesis-related proteins
including tyrosinase, TRP1, TRP2, and MITF, specifically via the transcriptional regulation of MITF.
α
-MSH increased luciferase activity 4.8-fold compared to the
-MSH-induced MITF luciferase
3.9% inhibition at 0.1 M, 36.2
M (Figure 4C).
α
±
µ
±
µ
±
µ
±
Figure 4. Chalcone 21-21 inhibits the expression of melanogenesis-related proteins. (
were incubated for 24 h with vehicle (0.1% DMSO) alone or various concentrations of chalcone 21-21 in
the absence or presence of -MSH (10 nM). The levels of TRP1, TRP2, and MITF were determined by
Western blot or RT-PCR analyses. GAPDH served as a loading control. ( ) B16F10 cells were transiently
A,B) B16F10 cells
α
C
transfected with a reporter construct of MITF together with the pRL-TK. The cells were incubated for
24 h with vehicle (0.1% DMSO) alone or with various concentrations of chalcone 21-21 in the absence
or presence of α-MSH (10 nM). Luciferase activity was measured and firefly luciferase activities were
normalized to that of Renilla luciferase. Results are represented as mean SD from three independent
±
experiments (n = 3). p-values of # p < 0.005 compared to the vehicle-treated group and * p < 0.05 and
** p < 0.005 compared to the α-MSH-treated group were considered statistically significant.
2.2.5. Inhibition of ERK and CREB Signaling
Canonical
α-MSH-induced melanogenesis signaling is mediated by MC1R, which activates
cAMP/PKA, resulting in the phosphorylation of the cAMP response element-binding protein (CREB)
and regulation of MITF. CREB can act as a transcription factor to induce several genes including MITF,
and elevated MITF activity is reinforced and regulated by extracellular signal-regulated kinase (ERK)
activation. In addition, ERK2 can activate MITF by serine phosphorylation at residue 73 [1,6,20,23].
This indicates that the signaling pathways for melanin biosynthesis involve cross-talk, and may be
closely linked. Therefore, we determined whether chalcone 21-21 could affect ERK1/2 and/or CREB.
Phosphorylation of ERK1/2 and CREB was barely detectable in vehicle-treated resting B16F10 cells,

Molecules 2019, 24, 1372
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but was dramatically increased by
α
-MSH stimulation. The levels of phosphorylated ERK1/2 and
CREB decreased by treatment with chalcone 21-21 in a concentration-dependent manner (Figure 5A).
We also determined the transcriptional activity of CREB by measuring CRE-luciferase activity.
B16F10 cells were transiently transfected with a CRE reporter plasmid construct containing the human
CRE promoter together with the pRL-TK Renilla control vector.
α
-MSH stimulation increased luciferase
activity by 6.1-fold over that in the vehicle-treated resting cells.
α-MSH-induced CRE-luciferase activity
decreased by chalcone 21-21 treatment in a concentration-dependent manner, corresponding to 14.0
1.9% inhibition at 0.1 M, 36.3 3.3% inhibition at 0.3 M, 69.5 3.4% inhibition at 1 M, and 92.6
0.4% inhibition at 3
is an important target of chalcone 21-21 for anti-melanogenic activity.
±
±
µ
±
µ
±
µ
µ
M (Figure 5B). These results suggest that inhibition of ERK1/2 and CREB activities
Figure 5. Chalcone 21-21 inhibits ERK and CREB signaling. (
vehicle (0.1% DMSO) alone or with various concentrations of chalcone 21-21 in the absence or presence
of -MSH (10 nM). The levels of phospho-ERK1/2, ERK1/2, phospho-CREB, and CREB were determined
by Western blot analysis. GAPDH served as a loading control. ( ) B16F10 cells were transiently
A) B16F10 cells were incubated for 1 h with
α
B
transfected with a reporter construct of CRE together with the pRL-TK. The cells were incubated for
24 h with vehicle (0.1% DMSO) alone or with various concentrations of chalcone 21-21 in the absence
or presence of α-MSH (10 nM). Luciferase activity was measured and firefly luciferase activities were
normalized to that of Renilla luciferase. Results are represented as mean
±
SD from three independent
experiments (n = 3). p-values of # p < 0.005 compared to the vehicle-treated group and * p < 0.05 and
** p < 0.005 compared to the α-MSH-treated group were considered statistically significant.
3. Materials and Methods
3.1. Chemical Synthesis
To synthesize the derivatives of chalcone 21, anhydrous ethanol was added 2- and/or 6-substituted
acetophenones, followed by KOH as a base, together with the appropriate benzaldehydes, at room
temperature. The mixture was refluxed for 2~10 h, and then evaporated to remove the ethanol
solvent. Methylene chloride was added to the concentrated solution, which was neutralized with HCl.
The methylene chloride extract layer was dried with anhydrous MgSO4 and evaporated under reduced
pressure. Reaction progress was monitored using an analytical thin layer chromatography (TLC)
on pre-coated Merck silica-gel Kiesegel 60 F254 plates and the spots were detected under UV light
(254 nm). Flash chromatography was conducted using a silica gel 230–400 mesh. Flash chromatography
was performed, and 21 compounds were obtained from the liquid products (Table 1). Proton nuclear
magnetic resonance (1H-NMR) and carbon-13 NMR (13C-NMR) spectra were recorded at 400 (1H-NMR)
and 100 (13C-NMR) MHz on a Bruker ARX 400 (Bruker, Billerica, MA, USA) spectrometer and mass
spectrometry (MS) data were collected using an AB SCIEX API 3200TM (SCIEX, Ontario, Canada)
LC-MS/MS system. The chemical shifts were reported downfield in ppm (δ) relative to internal
tetramethylsilane (TMS), and coupling constants were reported in Hertz (Hz). All chemicals and
solvents were purchased from Sigma–Aldrich (St. Louis, MO, USA).

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3.1.1. (E)-3-(4-Ethylphenyl)-1-(2-hydroxy-6-((4-(trifluoromethoxy)benzyl)oxy)phenyl)prop-2-
en-1-one (21-1)
1H-NMR (400 MHz, CDCl3):
δ 13.3 (s, 1H), 7.77 (d, J = 2.8 Hz, 1H), 7.50 (d, J = 8.8 Hz, 1H), 7.46 (d,
J = 8.8 Hz, 1H), 7.45–7.35 (m, 3H), 7.20–7.15 (m, 3H), 6.66 (dd, J = 8.4, 24.4 Hz, 1H), 6.47 (dd, J = 8.0,
33.6 Hz, 1H), 5.13 (s, 2H), 2.36-2.29 (m, 3H), 1.24 (t, J = 7.6 Hz, 3H); 13C-NMR (100 MHz, DMSO-d6): d
194.6, 158.8, 157.9, 148.3, 147.2, 144.1, 136.6, 133.0, 132.4, 130.0, 129.0, 128.8, 128.0, 121.8, 121.3, 119.2,
115.9, 110.0, 104.0, 69.4, 28.5, 15.6; LC-MS (ESI) 443.2 (M + H).
3.1.2. (E)-3-(4-Chlorophenyl)-1-(2-hydroxy-6-((4-(trifluoromethoxy)benzyl)oxy)phenyl)prop-2-
en-1-one (21-2)
1H-NMR (400 MHz, CDCl3):
δ 13.3 (s, 1H), 7.82–7.63 (m, 2H), 7.49 (d, J = 8.6 Hz, 2H), 7.40 (t, J = 8.3 Hz,
1H), 7.25–7.15 (m, 4H), 7.10 (d, J = 8.5 Hz, 2H), 6.68 (d, J = 8.4 Hz, 1H), 6.52 (d, J = 8.1 Hz, 1H), 5.13 (s,
2H); 13C-NMR (100 MHz, DMSO-d6): d 193.2. 158.6, 157.7, 146.5, 145.1, 136.9, 133.5, 133.3, 129.7, 129.3,
128.7, 128.0, 128.7, 114.5, 114.0, 110.5, 70.8; LC-MS (ESI) 489.1 (M + H).
3.1.3. (E)-1-(2-Hydroxy-6-((4-(trifluoromethoxy)benzyl)oxy)phenyl)-3-(4-methoxyphenyl)prop-2-
en-1-one (21-3)
1H-NMR (400 MHz, CDCl3):
δ 13.5 (s, 1H), 7.82–7.60 (m, 2H), 7.52 (d, J = 8.4 Hz, 2H), 7.36 (t, J = 8.4 Hz,
1H), 7.26–7.10 (m, 4H), 6.76 (d, J = 8.8 Hz, 2H), 6.66 (d, J = 8.4 Hz, 1H), 6.51 (d, J = 8.4 Hz, 1H), 5.13
(s, 2H), 3.83 (s, 3H); 13C-NMR (100 MHz, DMSO-d6): d 194.4, 158.6, 157.7, 148.1, 146.9, 144.1, 136.4,
132.8, 132.2, 130.0, 129.0, 128.6, 128.0, 121.5, 121.3, 119.1, 115.7, 110.0, 104.0, 69.8, 55.8; LC-MS (ESI) 445.1
(M + H).
3.1.4. (E)-4-(3-(2-Hydroxy-6-((4-(trifluoromethoxy)benzyl)oxy)phenyl)-3-oxoprop-1-en-1-yl)benzoic
acid (21-4)
1H-NMR (400 MHz, CDCl3):
δ 13.2 (s, 1H), 7.96 (d, J = 7.9 Hz, 2H), 7.84 (d, J = 15.8 Hz, 1H), 7.71 (d, J =
15.6 Hz, 1H), 7.54–7.38 (m, 3H), 7.34–7.13 (m, 5H), 6.68 (d, J = 8.6 Hz, 1H), 6.54 (d, J = 8.4 Hz, 1H), 5.14
(s, 2H); 13C-NMR (100 MHz, DMSO-d6): d 194.6, 158.3, 157.6, 148.3, 143.4, 143.3, 136.7, 132.7, 130.1,
129.9, 129.8, 128.5, 121.3, 119.2, 116.2, 110.0, 103.9, 69.3; LC-MS (ESI) 459.1 (M + H).
3.1.5. (E)-N-(4-(3-(2-Hydroxy-6-((4-(trifluoromethoxy)benzyl)oxy)phenyl)-3-oxoprop-1-en-1-yl)
phenyl)acetamide (21-5)
1H-NMR (400 MHz, CDCl3):
δ 13.3 (s, 1H), 7.72 (s, 2H), 7.54–7.35 (m, 5H), 7.34–7.09 (m, 5H), 6.67 (d,
J = 8.1 Hz, 1H), 6.52 (d, J = 8.0 Hz, 1H), 5.14 (s, 3H), 2.21 (s, 3H); 13C-NMR (100 MHz, DMSO-d6): d
193.2, 168.9, 165.3, 160.1, 146.5, 145.1, 137.7, 136.9, 130.8, 129.3, 129.0, 128.8, 121.5, 114.5, 114.0, 110.5,
105.3, 69.8, 24.0; LC-MS (ESI) 472.1 (M + H).
3.1.6. (E)-1-(2-(Benzyloxy)-6-hydroxyphenyl)-3-(4-ethylphenyl)prop-2-en-1-one (21-6)
1H-NMR (400 MHz, CDCl3):
δ 13.6 (s, 1H), 7.76 (dd, J = 15.6, 40.9 Hz, 2H), 7.50–7.27 (m, 6H), 7.01 (s,
4H), 6.61 (d, J = 8.3 Hz, 1H), 6.46 (d, J = 8.2 Hz, 1H), 5.04 (s, 2H), 2.61 (q, J = 7.6 Hz, 2H), 1.21 (t, J =
7.6 Hz, 3H); 13C-NMR (100 MHz, DMSO-d6): d 194.7, 159.2, 158.2, 147.3, 144.3, 137.0, 133.2, 129.0, 128.3,
127.8, 115.5, 110.0, 103.9, 70.5, 28.6, 15.7; LC-MS (ESI) 359.2 (M + H).
3.1.7. (E)-1-(2-(Benzyloxy)-6-hydroxyphenyl)-3-(4-chlorophenyl)prop-2-en-1-one (21-7)
1H-NMR (400 MHz, CDCl3):
δ 13.5 (s, 1H), 7.77 (d, J = 15.6 Hz, 1H), 7.61 (d, J = 15.6 Hz, 1H), 7.54–7.29
(m, 6H), 7.12 (d, J = 8.0 Hz, 2H), 6.91 (d, J = 8.0 Hz, 2H), 6.62 (d, J = 8.2 Hz, 1H), 6.49 (d, J = 8.1 Hz, 1H),
5.05 (s, 2H); 13C-NMR (100 MHz, DMSO-d6): d 194.5, 159.0, 158.2, 142.4, 137.0, 135.4, 133.8, 130.5, 129.4,
128.8, 128.4, 128.3, 109.9, 104.0, 70.5; LC-MS (ESI) 365.1 (M + H).

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3.1.8. (E)-1-(2-(Benzyloxy)-6-hydroxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (21-8)
1H-NMR (400 MHz, CDCl3):
δ 14.00 (s, 1H), 7.77 (dd, J = 15.6, 27.5 Hz, 2H), 7.52–7.45 (m, 2H), 7.44–7.32
(m, 4H), 7.32–7.14 (m, 1H), 7.07 (d, J = 8.7 Hz, 2H), 6.73 (d, J = 8.7 Hz, 2H), 6.45 (d, J = 8.5 Hz, 1H), 5.08
(s, 2H), 3.97 (s, 3H); 13C-NMR (100 MHz, DMSO-d6): d 194.5, 161.7, 159.1, 158.1, 144.3, 137.1, 133.0,
130.8, 128.9, 128.4, 128.3, 127.4, 126.3, 115.6, 114.9, 109.9, 103.9, 70.4, 55.8; LC-MS (ESI) 360.8 (M+).
3.1.9. (E)-4-(3-(2-(Benzyloxy)-6-hydroxyphenyl)-3-oxoprop-1-en-1-yl)benzoic acid (21-9)
1H-NMR (400 MHz, CDCl3):
δ 7.96–7.88 (m, 2H), 7.74–7.67 (m, 1H), 7.54–7.35 (m, 6H), 7.33–7.08 (m,
5H), 6.67 (d, J = 8.0 Hz, 1H), 6.57 (d, J = 8.3 Hz, 1H), 5.14 (s, 2H); 13C-NMR (100 MHz, DMSO-d6): d
194.6, 158.9, 158.0, 143.1, 137.4, 137.1, 133.1, 130.2, 129.9, 128.8, 128.6, 128.5, 128.3, 128.2, 115.7, 110.0,
103.9, 70.4; LC-MS (ESI) 374.9 (M+).
3.1.10. (E)-1-(2-(Benzyloxy)phenyl)-3-(4-ethylphenyl)prop-2-en-1-one (21-10)
1H-NMR (400 MHz, CDCl3):
δ 12.9 (s, 1H), 10.0 (s, 1H), 7.96–7.88 (m, 2H), 7.81 (d, J = 8.1 Hz, 2H),
7.67–7.57 (m, 3H), 7.50 (t, J = 8.5 Hz, 1H), 7.36 (d, J = 8.0 Hz, 2H), 7.30–7.24 (m, 3H), 7.03 (d, J = 8.4
Hz, 1H), 7.00–6.87 (m, 1H), 2.78–2.66 (m, 3H), 1.27 (ddd, J = 2.2, 7.6, 7.6 Hz, 3H); 13C-NMR (100 MHz,
CDCl3) d 192.4, 157.4, 146.9, 143.2, 136.3, 133.0, 132.7, 130.8, 129.6, 128.6, 128.5. 128.3, 127.5, 126.5, 121.2,
113.0, 70.7, 28.9, 15.4; LC-MS (ESI) 343.2 (M + H).
3.1.11. (E)-1-(2-(Benzyloxy)phenyl)-3-(4-chlorophenyl)prop-2-en-1-one (21-11)
1H-NMR (400 MHz, CDCl3):
δ 7.77 (dd, J = 1.6, 7.6 Hz, 1H), 7.59 (d, J = 16 Hz, 1H), 7.53–7.38 (m, 4H),
7.35–7.20 (m, 8H), 7.11–7.02 (m, 2H), 5.17 (s, 2H); 13C-NMR (100 MHz, CDCl3) d 192.8, 161.1, 145.2,
136.7, 135.5, 133.5, 133.2, 131.1, 129.9, 128.9, 128.7, 127.6, 127.1, 126.1, 122.3, 118.7, 117.2, 70.6; LC-MS
(ESI) 349.1 (M + H).
3.1.12. (E)-1-(2-(Benzyloxy)phenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (21-12)
1H-NMR (400 MHz, CDCl3):
δ 7.72 (dd, J = 2.0, 7.7 Hz, 1H), 7.61 (d, J = 15.8 Hz, 1H), 7.48–7.40 (m, 4H),
7.36–7.34 (m, 2H), 7.31–7.26 (m, 3H), 7.08–7.04 (m, 2H), 6.83 (d, J = 8.8 Hz, 2H), 5.17 (s, 2H), 3.84 (s, 3H);
13C-NMR (100 MHz, CDCl3): 192.3, 161.3, 157.4, 136.4, 132.9, 130.8, 130.1, 129.7, 128.6, 128.0, 127.9,
127.5, 125.2, 121.1, 114.2, 113.0, 70.7, 55.4; LC-MS (ESI) 345.2 (M + H).
3.1.13. (E)-4-(3-(2-(Benzyloxy)phenyl)-3-oxoprop-1-en-1-yl)benzoic acid (21-13)
1H-NMR (400 MHz, CDCl3):
δ 8.01 (d, J = 8.4 Hz, 2H), 7.81 (dd, J = 1.9, 7.7 Hz, 1H), 7.63 (d, J = 5.3 Hz,
2H), 7.52 (td, J = 2.0, 8.8 Hz, 1H), 7.45–7.43 (m, 2H), 7.40 (d, J = 8.4 Hz, 2H), 7.35–7.31 (m, 3H), 7.12–7.07
(m, 2H), 5.18 (s, 2H), 2.18 (s, 1H); 13C-NMR (100 MHz, CDCl3): d 192.8, 169.3, 161.1, 145.2, 140.4,
136.7, 135.5. 131.3, 130.2, 129.4, 129.0, 128.7, 127.6, 127.1. 126.1, 121.5, 118.7, 117.1, 70.2; LC-MS (ESI)
358.1 (M+).
3.1.14. (E)-N-(4-(3-(2-(Benzyloxy)phenyl)-3-oxoprop-1-en-1-yl)phenyl)acetamide (21-14)
1H-NMR (400 MHz, CDCl3):
δ 7.75-7.67 (m, 2H), 7.46–7.39 (m, 4H), 7.32–7.25 (m, 4H), 7.12–7.03 (m,
3H), 6.76 (d, J = 8.4 Hz, 1H), 6.59 (d, J = 8.5 Hz, 2H), 5.16 (s, 2H), 4.06-3.94 (m, 3H); 13C-NMR (100 MHz,
CDCl3): d 192.8, 168.9, 162.1, 145.0, 137.7, 136.8, 135.4, 131.3, 130.8, 129.0, 128.7, 127.4, 127.0, 126.1,
121.5. 120.5, 114.7, 113.7, 70.4, 29.1; LC-MS (ESI) 372.2 (M + H).
3.1.15. (E)-3-(4-Ethylphenyl)-1-(2-hydroxyphenyl)prop-2-en-1-one (21-15)
1H-NMR (400 MHz, CDCl3):
δ 12.89 (s, 1H), 7.93–7.88 (m, 3H), 7.63–7.57 (m, 3H), 7.48 (t, J = 8.6 Hz, 1H),
7.25 (s, 1H), 7.02 (d, J = 8.3 Hz, 1H), 6.93 (t, J = 8.0 Hz, 1H), 2.69 (1, J = 5.7 Hz, 2H), 1.26 (t, J = 5.1 Hz
,
3H); 13C-NMR (100 MHz, CDCl3): d 193.8, 163.6, 147.9, 145.6, 136.3, 132.2, 130.7, 129.6. 128.8, 128.6,
119.1, 118.9, 118.8, 118.6, 118.4, 28.9, 15.3; LC-MS (ESI) 253.1 (M + H).

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3.1.16. (E)-1-(2-Hydroxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (21-16)
1H-NMR (400 MHz, CDCl3):
δ 12.98 (s, 1H), 7.89–7.87 (m, 2H), 7.57 (d, J = 8.8 Hz, 2H), 7.50–7.43 (m,
2H), 7.00 (dd, J = 0.8, 8.4 Hz, 1H), 6.92–6.88 (m, 3H), 3.81 (s, 3H); 13C-NMR (100 MHz, CDCl3): d 193.7,
163.6, 163.5, 162.0, 145.3, 136.1, 130.5, 129.5, 127.4, 127.3, 120.1, 118.7, 118.6, 117.6, 114.5, 55.4; LC-MS
(ESI) 255.1 (M + H).
3.1.17. (E)-N-(4-(3-(2-Hydroxyphenyl)-3-oxoprop-1-en-1-yl)phenyl)acetamide (21-17)
1H-NMR (400 MHz, CDCl3):
δ 12.88 (s, 1H), 9.92 (s, 1H), 7.94-7.90 (m, 1H), 7.85 (d, J = 8.9 Hz, 2H),
7.72-7.42 (m, 5H), 7.04 (t, J = 7.9 Hz, 1H), 6.95 (t, J = 7.6 Hz, 1H), 2.23 (s, 3H); 13C-NMR (100 MHz,
CDCl3): d 193.9, 162.9, 146.9, 135.49, 135.47, 131.0, 129.6, 118.7, 117.7, 114.3, 114.2, 114.15, 114.13, 114.1,
29.1; LC-MS (ESI) 282.1 (M + H).
3.1.18. (E)-1-(2-(Cyclohexylmethoxy)phenyl)-3-(4-ethylphenyl)prop-2-en-1-one (21-18)
1H-NMR (400 MHz, CDCl3):
δ 7.68–7.62 (m, 2H), 7.50 (d, J = 8.1 Hz, 2H), 7.48 (s, 1H), 7.45–7.40 (m,
1H), 7.45–7.40 (m, 1H), 7.21 (d, J = 8.1 Hz, 2H), 7.00 (td, J = 0.6, 7.5 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 3.83
(d, J = 5.8 Hz, 2H), 2.66 (q, J = 7.6 Hz, 1H), 1.82–1.57 (m, 6H), 1.24 (t, J = 7.6Hz, 3H), 1.19–1.01 (m, 5H);
13C-NMR (100 MHz, CDCl3): d 192.8, 158.0, 145.1, 143.4, 135.1, 132.4, 192.8, 128.5, 125.7, 120.8, 118.7,
114.9, 74.8, 38.4, 30.0, 28.2, 26.2, 25.8, 14.5; LC-MS (ESI) 349.2 (M + H).
3.1.19. (E)-3-(4-Chlorophenyl)-1-(2-(cyclohexylmethoxy)phenyl)prop-2-en-1-one (21-19)
1H-NMR (400 MHz, CDCl3):
δ 7.78–7.74 (m, 4H), 7.69 (d, J = 8.4Hz, 2H), 7.65–7.61 (m, 2H), 7.20 (d, J =
8.4 Hz, 2H), 3.84 (s, 2H), 1.84–1.58 (m, 6H), 1.20–1.02 (m, 5H); 13C-NMR (100 MHz, CDCl3): d 192.8,
158.0, 143.1, 135.0, 133.5, 133.6, 131.0, 130.0, 128.9, 125.7, 120.8, 118.7, 115.0, 74.8, 38.4, 30.0, 28.2, 26.2,
25.8; LC-MS (ESI) 355.1 (M + H).
3.1.20. (E)-4-(3-(2-(Cyclohexylmethoxy)phenyl)-3-oxoprop-1-en-1-yl)benzoic acid (21-20)
1H-NMR (400 MHz, CDCl3):
δ 8.13 (d, J = 8.3 Hz, 2H), 7.73–7.46 (m, 6H), 7.03 (t, J = 7.5 Hz, 1H), 6.98
(d, J = 8.5 Hz, 1H), 3.87 (d, J = 5.8 Hz, 2H), 1.83–1.58 (m, 6H), 1.17–1.00 (m, 5H); 13C-NMR (100 MHz,
CDCl3): d 193.0, 168.2, 157.9, 145.0, 140.4, 135.1, 131.2, 130.2, 129.3, 129.0, 125.7, 120.8, 118.7, 114.5, 75.0,
38.5, 30.0, 26.0, 25.8; LC-MS (ESI) 365.1 (M + H).
3.1.21. (E)-N-(4-(3-(2-(Cyclohexylmethoxy)phenyl)-3-oxoprop-1-en-1-yl)phenyl)acetamide (21-21)
1H-NMR (400 MHz, CDCl3):
δ 8.42 (s, 1H), 7.65–7.57 (m, 4H), 7.51 (d, J = 8.6 Hz, 2H), 7.46–7.39 (m,
2H), 7.00 (t, J = 7.4 Hz, 1H), 6.95 (d, J = 8.4 Hz, 1H), 3.82 (d, J = 5.8 Hz, 2H), 2.17 (s, 3H), 1.81–1.56 (m,
6H), 1.16–1.01 (m, 5H); 13C-NMR (100 MHz, CDCl3): d 192.9, 167.0, 156.8, 143.2, 137.7, 135.1, 132.2,
130.8, 129.5, 125.4, 121.6. 120.8, 118.5, 114.1, 74.9, 38.4, 30.0, 26.2, 25.9. 24.5; LC-MS (ESI) 378.2 (M + H).
3.2. Biological Assays
3.2.1. Reagents
α
-MSH, kojic acid, arbutin, l-DOPA, and synthetic melanin were obtained from Sigma-Aldrich
(St. Louis, MO, USA). Primary antibodies specific for tyrosinase, TRP1, TRP2, and MITF were obtained
from Novus Biologicals, and phospho-ERK1/2, ERK1/2, phospho-CREB, CREB, and GAPDH were
obtained from Cell Signaling Technology (Danvers, MA, USA). All other chemicals were purchased
from Sigma-Aldrich, unless otherwise noted.
3.2.2. Cell Line and Culture Conditions
The mouse melanoma cell line B16F10 was obtained from the American Type Culture
Collection (ATCC, Manassas, VA, USA) and was maintained in Dulbecco’s modified Eagle’s medium

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(DMEM, Hyclone, Pittsburgh, PA, USA) supplemented with 10% fetal bovine serum (FBS) and 1%
penicillin-streptomycin solution (Gibco BRL, Grand Island, NY, USA) at 37 ◦C in a humidified incubator
suppling 5% CO2.
3.2.3. Cellular Melanin Contents
Cells were seeded at a density of 2,500/well in 96-well culture plates and incubated for 24 h.
The cells were incubated for 72 h with vehicle (0.1% dimethyl sulfoxide, DMSO) alone or with various
concentrations of chalcone 21-21 in the absence or presence of α-MSH (10 nM). Melanin contents were
determined in the cell-free culture medium by measuring the absorbance at 405 nm using a microplate
reader. Results were calculated from a standard curve generated using synthetic melanin. Kojic acid
and arbutin were used as positive controls.
3.2.4. Cell Viability Assay
Cells were seeded at a density of 2500/well in 96-well culture plates. After overnight incubation,
cells were treated with vehicle (0.1% DMSO) alone or with various concentrations of chalcone 21-21 for
appropriate time intervals in the absence or presence of α-MSH (10 nM). Cell viability was measured
by the absorbance at 450 nm in a microplate reader (Molecular Devices) after further incubation for
2–4 h at 37 ◦C, followed by the addition with EZ-CyTox Enhanced Cell Viability Assay Reagent (Daeil
Lab Service) according to the manufacturer’s protocols.
3.2.5. Zymography
Cells were incubated for 72 h with vehicle (0.1% DMSO) alone or various concentrations of
chalcone 21-21 in the absence or presence of α-MSH (10 nM). Whole-cell lysates were prepared on
ice for 1 h using a sodium phosphate lysis buffer containing 0.1 M sodium phosphate buffer (pH 6.8),
1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF) and phosphatase inhibitor cocktail
(Thermo Scientific, Pittsburgh, PA, USA). Soluble protein samples were prepared by centrifugation at
13,000 rpm for 10 min at 4 ◦C and the samples were resolved with Laemmli sample buffer without
β-mercaptoethanol by native SDS-PAGE. The gels were equilibrated and soaked in 100 mM sodium
phosphate buffer (pH 6.8) containing 5 mM l-DOPA until colorimetric detection of tyrosinase.
3.2.6. Western Blot Analysis
Whole-cell lysates were prepared on ice for 30 min using a lysis buffer containing 50 mM Tris-HCl
(pH 7.4), 350 mM NaCl, 1% Triton X-100, 0.5% Nonidet P-40, 10% glycerol, 0.1% SDS, 1 mM EDTA, 1 mM
EGTA, 1 mM Na3VO4, 1 mM PMSF, and phosphatase inhibitor cocktail (Thermo Scientific, Pittsburgh,
PA, USA). The lysates were resolved by SDS-PAGE and transferred onto nitrocellulose membrane
(Sigma-Aldrich, St. Louis, MO, USA). The membrane was blocked in a blocking buffer containing 5%
skim-milk and subsequently incubated with appropriate primary antibodies for overnight at 4 ◦C.
Blots were washed and incubated with horseradish peroxidase-conjugated secondary antibodies at
room temperature for 2 h, and the signals were detected using an ECL reagent (SurModics, Eden
Prairie, MN, USA).
3.2.7. Semi-Quantitative RT-PCR
TotalRNAwasisolatedusingaTRIzolReagent(Invitrogen, Carlsbad, CA, USA)andsemi-quantitative
reverse-transcription PCR (RT-PCR) was performed using an AccuPower RT-PCR PreMix (Bioneer,
Daejeon, Korea) according to the manufacturer’s protocol. Briefly, total RNA was reverse-transcribed
on a thermal cycler programmed at 42 ◦C for 1 h and PCR was performed 30 cycles at 94 ◦C for 30 s,
58 ◦C for 30 s and 72 ◦C for 30 s, and ending with one cycle at 72 ◦C for 5 min. The amplicons were
resolved on agarose gels with DNA SafeStain (Lamda Biotech, Ballwin, MO, USA) and visualized using

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a Gel Doc 2000 imaging system (Bio-Rad Laboratories, Hercules, CA, USA). The primer sequences
were described in Table 2.
Table 2. Primer sequences for the RT-PCR.
Gene
Primer
Forward
Reverse
Forward
Reverse
Forward
Reverse
Sequence
50-GAC GGT CAC TGC AGA CTT TG-30
50-GCC ATG ACC AGG ATG AC-30
50-GAC GGT CAC TGC AGA CTT TG-30
50-GCC ATG ACC AGG ATG AC-30
Tyrosinase
MITF
50-TGA AGG TCG GTG TGA ACG GAT TTC GC-30
50-CAT GTA GGC CAT GAG GTC CAC CAC-30
GAPDH
3.2.8. Transfection and Promoter Assay
Cells were transiently transfected with the pTyr-luciferase, pMITF-luciferase, or pCRE-luciferase
construct together with the pRL-TK vector (Promega, Madison, WI, USA) using a Lipofectamine®
PLUSTM reagent (Life Technologies, Carlsbad, CA, USA) as described previously [13]. After 24 h, cells
were incubated for 24 h with vehicle (0.1% DMSO) alone or with various concentrations of chalcone
21-21 in the absence or presence of α-MSH (10 nM). Luciferase activities were measured using a
Dual-Glo luciferase assay system (Promega) and firefly luciferase activity was normalized to that of
Renilla luciferase.
3.2.9. Statistics
Data are presented as mean
using one-way analysis of variance, followed by Dunnett’s multiple comparison tests. Statistical
±
standard deviation (SD) and statistical differences were determined
significance was considered to be significant at p < 0.05.
4. Conclusions
Melanin is synthesized in the melanosomes of melanocytes, and is transferred to and eventually
distributed in the epidermis [24]. Although it has a skin protective role, abnormal accumulation can
lead to various skin disorders, indicating a need for the development of anti-melanogenic agents. In this
study, we synthesized a series of derivatives of chalcone 21 to optimize and increase its previously
reported anti-melanogenic activity [13]. Activity was evaluated in α-MSH-stimulated B16F10 cells
by measuring cellular melanin production. Based on the SAR, moderate to strong biological activity
with weak cytotoxicity was observed when an acetamido group, carboxyl group, or chloride atom was
located at the R3 position. Among the derivatives, chalcone 21-21 had the strongest inhibitory effect on
cellular melanin production, with an IC50 value of 0.54 µM. This is more potent than chalcone 21 and
the known anti-melanogenic agents kojic acid and arbutin, which had IC50 values of 4.9, 38.5, and
148.4 µM, respectively.
Melanin biosynthesis is tightly regulated by several complex signaling networks. Among the
signaling networks, tyrosinase, TPR1, TPR2, MITF, CREB, and ERK1/2 are well-known key regulators of
melanin synthesis [1,6,19–23]. Chalcone 21-21 inhibited the expression and activity of tyrosinase, which
catalyzes the rate-limiting step in melanin biosynthesis by catalyzing the conversion of L-tyrosine to
L-DOPA by hydroxylation. In addition, chalcone 21-21 inhibited the expression and transcriptional
activity of MITF, a master transcription factor that drives the expression of target genes encoding
tyrosinase, TRP1, and TRP2 [23], this inhibition resulted in reducing the expression of MITF-regulated
target genes. Furthermore, chalcone 21-21 inhibited the phosphorylation of ERK1/2 and CREB, as well
as the transcriptional activity of CREB. ERK/CREB signaling plays an important role in regulating the
transcription and phosphorylation of MITF. Our results indicate that the anti-melanogenic activity

Molecules 2019, 24, 1372
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of chalcone 21-21 is associated with the inhibition of ERK/CREB signaling, thereby targeting the
expression of MITF and MITF-regulated genes including those encoding tyrosinase, TRP1, and TRP2
(Figure 6). Finally, our results demonstrate that chalcone-21-21 is an effective anti-melanogenic agent
and may be beneficial in the treatment of aesthetic problems and hyperpigmentation disorders.
Figure 6. A schematic diagram illustrating the proposed action mechanisms of chalcone 21-21. Chalcone
21-21 specifically targets the ERK/CREB signaling, thereby inhibiting MITF and MITF-regulated
target genes including those encoding tyrosinase, TRP1, and TRP2. AC, adenylate cyclase; α-MSH,
alpha-melanocyte stimulating hormone; ATP, adenosine triphosphate; cAMP, cyclic adenosine
monophosphate; CREB, cAMP response element-binding protein; ERK, extracellular signal-regulated
kinase; MAP kinase, mitogen-activated protein kinase; MC1R, melanocortin receptor type 1; MITF,
microphthalmia-associated transcription factor; PKA, protein kinase A; ROS, reactive oxygen species;
TRP, tyrosinase-related protein; TYR, tyrosinase.
Author Contributions: B.-H.K. conceived the experimental design, performed the biological assays, analyzed the
data and wrote the paper. J.-Y.P. carried out the synthesis of the 21 chalcone derivatives. S.-N.H. provided the
reagents and gave critical comments. S.-K.Y. and J.-Y.P. conceived the experimental design, provided the reagents
and supervised the project. All authors read and approved the final manuscript.
Funding: This work was supported by grants from the National Research Foundation of Korea (NRF) funded
by the Korea government (2014R1A2A1A11053203, 2017R1A2B2006839, and 2018R1A5A2025964 to S.-K.Y.) and
the Cooperative Research Program of Basic Medical Science and Clinical Science from Seoul National University
College of Medicine (800-20160092 to S.-K.Y.).
Conflicts of Interest: The authors declare no conflicts of interest.
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