Synthesized quercetin derivatives stimulate melanogenesis in B16 melanoma cells by influencing the expression of melanin biosynthesis proteins MITF and p38 MAPK

Article abstract of DOI:10.1016/j.bmc.2014.04.053

In order to understand the effect of structure-activity relationships on melanogenesis using B16 melanoma cells, 19 quercetin derivatives were synthesized. Among the synthesized compounds, 3-O-methylquercetin (11) and 3′,4′,7-O-trimethylquercetin (14) increased melanin content more potently than the positive control theophylline, while exhibiting low cytotoxicity. Compound 11 exhibited less melanogenesis-stimulating activity than compound 14. However, 11 increased the expression of tyrosinase and tyrosinase-related protein 1 (TRP-1) to a greater extent than 14, thereby suggesting that melanogenesis in melanoma cells does not depend solely on the expression of the enzymes catalyzing melanin biosynthesis. Furthermore, 14 also stimulated the expression of the microphthalmia-associated transcription factor (MITF) and p-p38 mitogen activated protein kinase (MAPK), while they were not increased by 11. These results suggest that 11 may enhance the expression of tyrosinase and TRP-1 by regulating the proteasomal degradation of melanogenic enzymes and/or by activating other transcriptional factors regulating enzyme expression.

Full text of DOI:10.1016/j.bmc.2014.04.053

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesized quercetin derivatives stimulate melanogenesis in B16
melanoma cells by influencing the expression of melanin
biosynthesis proteins MITF and p38 MAPK
⇑
Kosei Yamauchi, Tohru Mitsunaga , Mizuho Inagaki, Tohru Suzuki
The United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, 501-1193 Gifu, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to understand the effect of structure–activity relationships on melanogenesis using B16 mela-
noma cells, 19 quercetin derivatives were synthesized. Among the synthesized compounds, 3-O-meth-
ylquercetin (11) and 30,40,7-O-trimethylquercetin (14) increased melanin content more potently than
the positive control theophylline, while exhibiting low cytotoxicity. Compound 11 exhibited less melano-
genesis-stimulating activity than compound 14. However, 11 increased the expression of tyrosinase and
tyrosinase-related protein 1 (TRP-1) to a greater extent than 14, thereby suggesting that melanogenesis
in melanoma cells does not depend solely on the expression of the enzymes catalyzing melanin biosyn-
thesis. Furthermore, 14 also stimulated the expression of the microphthalmia-associated transcription
factor (MITF) and p-p38 mitogen activated protein kinase (MAPK), while they were not increased by
11. These results suggest that 11 may enhance the expression of tyrosinase and TRP-1 by regulating
the proteasomal degradation of melanogenic enzymes and/or by activating other transcriptional factors
regulating enzyme expression.
Received 26 March 2014
Revised 23 April 2014
Accepted 25 April 2014
Available online xxxx
Keywords:
B16 melanoma cells
Tyrosinase
TRP-1
Quercetin
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
of the skin. Therefore, controlling melanogenesis is important for
maintaining the good health and cosmetic appearance of the
Melanin, a pigment present in several tissues in the human
body, plays an important role in preventing skin cancer caused
by ultraviolet (UV) rays.1,2 The overall level of melanogenesis is
changed by aging, stress, and damage caused by the UV rays,
resulting in the appearance of gray hairs, sunburn, and mottling
human body. A number of studies have investigated novel pharma-
cological and herbal agents aimed at controlling melanogenesis.3–7
Tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2 are key
enzymes involved in melanin biosynthesis. Tyrosinase, an enzyme
containing copper ions, catalyzes two separate reactions within the
melanin biosynthetic pathway.8 Tyrosinase catalyzes the first step
in melanin biosynthesis that is, the hydroxylation of
-3,4-dihydroxyphenylalanine ( -DOPA), as well as the subsequent
oxidation of -DOPA to -DOPA quinone. Two types of melanin are
L-tyrosine to
Abbreviations:
albumin; cAMP, cyclic adenosine monophosphate; DMEM, Dulbecco’s modified
Eagle medium; DMF, dimethylformamide; -DOPA, -3,4-dihydroxyphenylalanine;
a-MSH, a-melanocyte-stimulating hormone; BSA, bovine serum
L
L
L
L
L
L
ECL, enhanced chemiluminescence; EtOAc, ethyl acetate; EtOH, ethanol; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; HMBC, heteronuclear multiple-bond
correlations; HRP, horseradish peroxidase; IR, infrared; JNK, c-Jun N-terminal
kinase; MAPK, mitogen-activated protein kinase; MALDI, matrix-assisted laser
desorption/ionization; MeOH, methanol; MS, mass spectroscopy; MITF, microph-
thalmia-associated transcription factor; MTT, microculture tetrazolium technique;
NMR, nuclear magnetic resonance; PBS, phosphate-buffered saline; Pd/C, palladium
on carbon; p-HPLC, preparative high-performance liquid chromatography; PVDF,
polyvinylidene difluoride; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel
electrophoresis; RIPA, radioimmunoprecipitation assay; TBST, tris-buffered saline
Tween20; THF, tetrahydrofuran; TRP, tyrosinase-related protein; UPLC–TOFMS,
ultra performance liquid chromatography time-of-flight mass spectrometry; UV,
ultraviolet.
ultimately biosynthesized, reddish-orange and blackish-brown
pigments called pheomelanin and eumelanin, respectively, with
the enzymes TRP-1 and TRP-2 playing a key role in the biosynthe-
sis of eumelanin.9
Melanin is biosynthesized in the melanosome in the perinuclear
region of the melanocyte and transported in the mature melano-
some to the periphery of the cell.10–12 The melanosome is further
transported to the hair matrix or the keratinocyte present above
the melanocyte. The keratinocyte that receives the melanin under-
goes cornification, resulting in skin pigmentation. Similarly, hair
pigmentation occurs due to melanin released on the outside of
the melanocyte. Despite the importance of the extracellular
⇑
Corresponding author. Tel./fax: +81 58 293 2920.
E-mail address: mitunaga@gifu-u.ac.jp (T. Mitsunaga).
http://dx.doi.org/10.1016/j.bmc.2014.04.053
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Yamauchi, K.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.053

2
K. Yamauchi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
pigment stores, an evaluation of melanogenesis activity using mel-
anin-controlling agents has only been performed on intracellular
melanin in B16 melanoma cells thus far.13–15 The present study
focused on evaluating extracellular melanin released from B16
melanoma cells and investigated the levels of melanin released
from the B16 melanoma cells in addition to measuring the intracel-
lular melanin content.
Quercetin is a flavonoid present as a glycoside in various fruits
and vegetables.16–18 A number of studies have demonstrated that
quercetin exhibits a variety of pharmacological effects, including
antioxidant and anti-cancer activities,19,20 while some reports relate
to effectiveness in controlling melanogenesis. Quercetin is recog-
nized as a potent inhibitor of tyrosinase activity and melanogenesis,
as evidenced by the studies performed in the mouse B16 melanoma
cells.21 However, quercetin has been reported to elicit the opposite
effect and accelerate melanogenesis in human melanoma cells.22
Furthermore, it was reported that the direction of its melanogene-
sis-regulating activity depends on the concentration of quercetin
used.23 A small number of studies have shown that quercetin deriv-
derivatives for understanding the mechanisms underlying the
observed activity.
2. Materials and methods
2.1. General experimental procedures
1H and 13C NMR spectra were recorded in methanol-d4, acetone-
d6 or dimethylsulfoxide-d6 using JEOL EC600 M Hz NMR (JEOL,
Tokyo, Japan). Coupling constants were expressed in Hz, and the
chemical shifts were expressed on a d (ppm) scale. Ultra perfor-
mance liquid chromatography time-of-flight mass spectrometry
(UPLC–TOFMS; WatersÒXevo™ QT for MS) was performed using
a C18 column (2.1 mm
u
ꢀ 100 mm length; Waters, Milford, MA,
USA). UPLC–TOFMS data were collected in the negative ionization
mode. The capillary voltage was 3.0 kV. Cone and desolvation gas
flow rates were set at 50 L/h and 1000 L/h, respectively, and the
source and desolvation temperatures were 150 °C and 500 °C,
respectively. Matrix-assisted laser desorption/ionization TOFMS
(MALDI-TOFMS) spectra were measured on a Shimadzu AXIMA-
Resonance spectrometer (Kyoto, Japan) equipped with a nitrogen
laser (k = 337 nm). Samples were mixed with the matrix (2,3-dihy-
droxybenzoic acid in 30% acetonitrile, 10 mg/mL) and loaded onto
a 384-well MALDI sample plate. Preparative HPLC (SHIMAZU
LC-6AD) was performed using an Inertsil ODS-3 column (20 mm
atives can control melanogenesis. Quercetin-3-O-b-D-glucoside
enhances melanogenesis by stimulating the expression of TRP-1
and TRP-2.24 In our previous study, we evaluated the effect on mela-
nogenesis of two quercetin glycosides, 40-O-b-
D
-glucopyranosyl-
-glucopyranoside (3)
-glucopyranosyl-querce-
D-glucopyranoside isolated
quercetin-3-O-b-
and 40-O-b-
-glucopyranosyl-(1?2)-b-
tin-3-O-b- -glucopyranosyl-(1?4)-b-
D-glucopyranosyl-(1?4)-b-D
D
D
D
u
ꢀ 250 mm; GL Sciences, Tokyo, Japan) with gradient elution.
from Helminthostachys zeylanica root extract.25 While compound 3
was found to stimulate melanogenesis, the latter compound had
no effect, suggesting that the structure and positions of the sugars
in the quercetin glycosides may affect their melanogenesis-modu-
lating activity in B16 melanoma cells. Furthermore, in order to
investigate the structure–activity relationship of quercetin glyco-
sides, ten structurally distinct quercetin glycosides (Fig. 1) were
synthesized, and their effects on intracellular melanogenesis were
Microculture tetrazolium technique (MTT) assay kit and bovine
serum albumin (BSA) was purchased from Sigma–Aldrich (St.
Louis, MO, USA). Antibodies against tyrosinase (H-109), TRP-1
(H-90), TRP-2 (H-150), p38 MAPK (H-147), and p-p38 MAPK
(D-8) were purchased from Santa Cruz Bio technology (Santa Cruz,
CA, USA). Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG
donkey antibody (NA934) and HRP-conjugated anti-mouse IgG
sheep antibody (NA931) were purchased from GE Healthcare (Pis-
catawawy, NJ, USA), while MITF-specific antibody (EPR9731) and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific
antibody (GTX100118) were purchased from Abcam (Cambridge,
MA, USA) and GeneTex (Irvine, CA, USA) respectively. Radioimmu-
noprecipitation assay (RIPA) buffer (ab156034) and protease inhib-
itors cocktail (539134) were purchased from Abcam (Cambridge,
MA, USA) and Merck Millipore (Billerica, MA, USA). Other commer-
cially available products were purchased from Wako Chemicals
(Richmond, VA, USA). IR spectra were recorded on a Perkin-Elmer
Spectrum 100 FT-IR system (Waltham, MA, USA). UV spectra were
recorded on a Shimadzu SPD-M20A diode array detector. Optical
determined. Quercetin-3-O-b-
D
-glucopyranoside (1), quercetin-3-
-glucopyranoside (2), and 3
O-b- -glucopyranosyl-(1?4)-b-
D
D
stimulated intracellular melanogenesis in our previous report,26
suggesting the potency of the quercetin derivatives as stimulators
of melanogenesis. In this study, 9 quercetin derivatives were newly
synthesized, and their melanogenesis-stimulating activities deter-
mined by measuring both intra- and extracellular melanin levels
in order to clarify the structure–activity relationships of the querce-
tin derivatives. Furthermore, the expression of proteins related to
melanin biosynthesis were determined by western blot analysis in
B16 melanoma cells treated with the synthesized quercetin
OH
3'
R2
2'
1'
quercetin
R1=R2=R3=OH
Compound
4'
5'
8
1
4
R3
O
O
7
8a
2
6'
R1=glucose, R2=R3=OH
1
2
3
3
R1=OCH3, R2=R3=OH
11
6
4a
R1
5
R1=cellobiose, R2=R3=OH
R1=cellobiose, R2=glucose, R3=OH
12 R1=R2=OCH3, R3=OH
13 R1=R3=OCH3, R2=OH
14 R1=R2=R3=OCH3
OH
4
5
6
R1=cellobiose, R2=OH, R3=glucose
R1=cellobiose, R2=cellobiose, R3=OH
R1=cellobiose, R2=OH, R3=cellobiose
R1=glucose, R2=glucose, R3=OH
R1=glucose, R2=OH, R3=glucose
15 R2=OCH3, R1=R3=OH
16 R3=OCH3, R1=R2=OH
17 R1=OH, R2=R3=OCH3
7
8
18 R1=OAc, R2=R3=OH
O
9
R1=glucose, R2=cellobiose, R3=OH
R1=glucose, R2=OH, R3=cellobiose
19 R1=
, R2=R3=OH
OAc
A1
A2
O
A3
A5
A4
10
OAc
AcO
A6
OAc
Figure 1. Structures of synthesized quercetin derivatives.
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K. Yamauchi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
rotations were measured using a JASCO P-2300 system (Easton,
MD, USA).
H-60); 13C NMR (CD3OD,150 MHz) dC 55.0 (40-OCH3), 59.2 (3-
OCH3), 93.4 (C-8), 98.4 (C-6), 104.6 (C-4a), 114.8 (C-20, 50), 120.8
(C-60), 122.8 (C-10), 138.5 (C-3), 146.3 (C-30), 150.3 (C-40), 156.2
(C-2), 157.1 (C-8a), 161.8 (C-5), 164.6 (C-7), 178.7 (C-4); UPLC–
2.2. Synthesis of quercetin derivatives
TOFMS m/z 329.0645 [MꢁH]ꢁ.
25
2.2.1. Synthesis of compounds 1–10
Compound 13: UV kmax 209, 256, 357 nm; [
a
]
D ꢁ19.6° (c 0.10,
Compounds 1–10 were synthesized using methods presented in
THF); IR (KBr) mmax 3415, 1657, 1595, 1498, 1441, 1350 cmꢁ1 1H
;
our previous publication.26
NMR (Acetone-d6, 600 MHz) dH 3.84 (3H, s, 3-OCH3), 3.89 (3H, s,
7-OCH3), 6.28 (1H, dr s, H-6), 6.62 (1H, br s, H-8), 6.97 (1H, d,
J = 8.22 Hz, H-50), 7.57 (1H, br d, J = 8.22 Hz, H-60), 7.70 (1H, br s,
H-20); 13C NMR (Acetone-d6,150 MHz) dC 55.6 (7-OCH3), 59.3
(3-OCH3), 91.9 (C-8), 97.6 (C-6), 105.7 (C-4a), 115.4 (C-20), 115.6
(C-50), 121.3 (C-60), 122.1 (C-10), 138.6 (C-3), 145.0 (C-30), 148.3
(C-40), 156.1 (C-2), 156.9 (C-8a), 162.0 (C-5), 165.7 (C-7), 178.8
2.2.2. Synthesis of 3-O-methylquercetin (11)
Rutin (5.00 g, 8.19 mmol), K2CO3 (9.04 g, 65.52 mmol), and BnBr
(7.79 mL, 65.52 mmol) were added to 60 mL of dimethylformam-
ide (DMF), and the mixture was stirred for 10 h under argon at
room temperature. The resulting mixture was diluted with
150 mL of ethyl acetate (EtOAc) and washed with water
(2 ꢀ 150 mL). The residue obtained after evaporation of the solvent
was dissolved in 100 mL of 1 N HCl, and the mixture was refluxed
at 80 °C for 2 h.
The precipitate was allowed to cool and filtered. Next, 1.0 g of
the precipitate, K2CO3 (22.5 g, 163 mmol), and dimethylsulfate
(17.5 mL, 184 mmol) were added to 26 mL of DMF, and the mixture
was stirred for 6 h at 65 °C. The reaction mixture was diluted with
150 mL of EtOAc and washed with water (2 ꢀ 150 mL). After the
EtOAc phase was dried using Na2SO4, the reactant was obtained
by evaporating the solvent.
(C-4); UPLC–TOFMS m/z 329.0653 [MꢁH]ꢁ.
25
Compound 14: UV kmax 209, 255, 354 nm; [
a
]
D ꢁ26.1° (c 0.52,
1H
THF); IR (KBr) mmax 3420, 1656, 1595, 1501, 1441, 1333 cmꢁ1
;
NMR (dimethylsulfoxide-d6, 600 MHz) dH 3.76 (3H, s, 3-OCH3),
3.83 (6H, s, 40-OCH3, 7-OCH3), 6.33 (1H, dr s, H-6), 6.69 (1H, br s,
H-8), 7.07 (1H, d, J = 8.22 Hz, H-50), 7.54 (2H, br s, H-60, 20); 13C
NMR (dimethylsulfoxide-d6,150 MHz) dC 56.2 (40-OCH3), 56.6 (7-
OCH3), 60.2 (3-OCH3), 92.8 (C-8), 98.3 (C-6), 105.7 (C-4a), 115.6
(C-20, 50), 121.0 (C-60), 122.7 (C-10), 138.7 (C-3), 146.9 (C-30),
150.8 (C-40), 156.1 (C-2), 156.8 (C-8a), 161.4 (C-5), 165.7 (C-7),
178.6 (C-4); UPLC–TOFMS m/z 343.0825 [MꢁH]ꢁ.
The reactant was dissolved in 40 mL of ethanol (EtOH)/tetrahy-
drofuran (THF) (1:1, v/v), and 200 mg of 10% Pd/C was added. The
mixture was stirred for 1 h at room temperature under 0.05 MPa
hydrogen. The Pd/C was filtered off. After the solvent was evapo-
rated, 11 was isolated by preparative HPLC with an ODS-3 column
2.2.4. Synthesis of 40-O-methylquercetin (15), 7-O-
methylquercetin (16) and 40, 7-O-dimethylquercetin (17)
Rutin (200 mg, 0.328 mmol), K2CO3 (90.6 mg, 0.656 mmol), and
dimethylsulfate (62.2 lL, 0.656 mmol) were added to 10 mL of
(20 mm
u
ꢀ 250 mm). Elution was performed with a linear gradi-
DMF, and the mixture was stirred for 6 h at 65 °C. The resulting
mixture was added to 50 mL of 2.5 N HCl and refluxed at 80 °C
for 2 h. The reaction mixture was diluted with 150 mL of EtOAc
and washed with water (2 ꢀ 150 mL). The reaction mixture was
obtained by drying the EtOAc phase using Na2SO4. After evaporat-
ing the solvent, compounds 15, 16, and 17 were obtained by pre-
ent of MeOH/0.05% aqueous solution TFA (0 min, 50/50; 50 min,
100/0; 60 min, 100/0) to obtain 11 as a yellowish powder with
41.1% yield. The structure of the synthesized methylquercetin 11
was confirmed using NMR, UPLC–TOFMS, UV, and IR spectra, and
by measuring the specific optical rotation: UV kmax 204, 255,
25
357 nm; [
a
]
ꢁ3.75° (c 0.75, MeOH); IR (KBr) mmax 3402, 1655,
parative HPLC with an ODS-3 column (20 mm
u
ꢀ 250 mm).
D
1607, 1505, 1440, 1361 cmꢁ1
;
1H NMR (CD3OD, 600 MHz) dH 3.75
Compounds were eluted with the linear gradient MeOH/0.05%
aqueous solution of TFA (0 min, 60/40; 50 min, 100/0; 60 min,
100/0) to obtain 15, 16 and 17 as yellowish powders with yields
of 4.1%, 10.0%, and 7.7%, respectively. The structures of the synthe-
sized compounds 15, 16, and 17 were confirmed using NMR,
MALDI-TOFMS, UV and IR spectra, and by the measurements of
(3H, s, 3-OCH3), 6.16 (1H, d, J = 2.04 Hz, H-6), 6.36 (1H, d,
J = 2.04 Hz, H-8), 6.88 (1H, d, J = 8.28 Hz, H-50), 7.50 (1H, dd,
J = 8.22, 2.04 Hz, H-60), 7.59 (1H, d, J = 2.04 Hz, H-20); 13C NMR
(CD3OD,150 MHz) dC 59.2 (3-OCH3), 93.4 (C-8), 98.4 (C-6), 104.5
(C-4a), 115.1 (C-20, 50), 121.0 (C-60), 121.6 (C-10), 138.2 (C-3),
145.1 (C-30), 148.6 (C-40), 156.6 (C-2), 157.1 (C-8a), 161.7 (C-5),
164.5 (C-7), 178.7 (C-4); UPLC–TOFMS m/z 315.049 [M–H]ꢁ.
specific optical rotation.
25
Compound 15: UV kmax 207, 254, 368 nm; [
a
]
D ꢁ26.9° (c 0.20,
MeOH); IR (KBr)
m
max 3430, 1655, 1617, 1499, 1456 cmꢁ1; 1H NMR
2.2.3. Synthesis of 3,40-O-dimethylquercetin (12), 3,7-O-
dimethylquercetin (13) and 3,40,7-O-trimethylquercetin (14)
Methylquercetin 11 (100 mg, 0.316 mmol), K2CO3 (43.7 mg,
(dimethylsulfoxide-d6, 600 MHz) dH 3.81 (3H, s, 40-OCH3), 6.15 (1H,
d, J = 2.04 Hz, H-6), 6.39 (1H, d, J = 2.04 Hz, H-8), 7.05 (1H, d,
J = 8.22 Hz, H-50), 7.62 (1H, dd, J = 8.22, 2.76 Hz, H-60), 7.63 (1H,
d, J = 2.10 Hz, H-20); 13C NMR (dimethylsulfoxide-d6,150 MHz) dC
56.1 (40-OCH3), 93.9 (C-8), 98.7 (C-6), 103.6 (C-4a), 112.4 (C-50),
115.1 (C-20), 120.3 (C-60), 123.9 (C-10), 136.7 (C-3), 146.7 (C-30),
146.8 (C-2), 149.9 (C-40), 156.7 (C-8a), 161.3 (C-5), 164.5 (C-7),
0.316 mmol), and dimethylsulfate (35.1 lL, 0.316 mmol) were
added to 10 mL of DMF. The mixture was stirred for 6 h at 65 °C.
The resulting mixture was diluted with 30 mL of EtOAc and
washed with water (2 ꢀ 30 mL). After evaporating the solvent,
compounds 12, 13, and 14 were obtained by preparative HPLC with
176.5 (C-4); MALDI-TOFMS m/z 317.0494 [M+H]+.
25
an ODS-3 column (20 mm
u
ꢀ 250 mm). Compounds were eluted
Compound 16: UV kmax 206, 255, 371 nm; [
a
]
D ꢁ17.3° (c 0.20,
with the linear gradient MeOH/0.05% aqueous solution TFA
(0 min, 50/50; 50 min, 100/0; 60 min, 100/0) to obtain 12, 13 and
14 as yellowish powders with yields of 11.9%, 9.1%, and 9.1%,
respectively. The structures of synthesized methylquercetins were
confirmed using NMR, UPLC–TOFMS, UV, and IR spectra, and by
THF); IR (KBr) mmax 3402, 1656, 1593, 1502, 1442, 1324 cmꢁ1 1H
;
NMR (dimethylsulfoxide-d6, 600 MHz) dH 3.82 (3H, s, 7-OCH3),
6.31 (1H, d, J = 2.04 Hz, H-6), 6.60 (1H, d, J = 1.38 Hz, H-8), 6.86
(1H, d, J = 8.22 Hz, H-50), 7.56 (1H, dd, J = 8.22, 2.04 Hz, H-60), 7.69
(1H, d, J = 2.04 Hz, H-20); 13C NMR (dimethylsulfoxide-
d6,150 MHz) dC 56.5 (7-OCH3), 92.4 (C-8), 98.0 (C-6), 104.5 (C-4a),
115.6 (C-20), 116.1 (C-50), 120.5 (C-60), 122.4 (C-10), 136.6 (C-3),
145.6 (C-30), 147.8 (C-2), 148.4 (C-40), 156.6 (C-8a), 160.9 (C-5),
measuring the specific optical rotation.
Compound 12: UV kmax 208, 254, 355 nm; [
MeOH); IR (KBr)
25
a]
D ꢁ0.51° (c 0.90,
m
max 3402, 1654, 1609, 1507, 1441, 1364 cmꢁ1; 1H
NMR (CD3OD, 600 MHz) dH 3.76 (3H, s, 3-OCH3), 3.91 (3H, s, 40-
OCH3), 6.16 (1H, dr s, H-6), 6.36 (1H, br s, H-8), 7.02 (1H, d,
J = 8.22 Hz, H-50), 7.57 (1H, br s, H-20), 7.60 (1H, br d, J = 8.28 Hz,
165.4 (C-7), 176.5 (C-4); MALDI-TOFMS m/z 317.0274 [M+H]+.
25
Compound 17: UV kmax 206, 254, 368 nm; [
a
]
D ꢁ3.72° (c 0.23,
THF); IR (KBr) mmax 3420, 1656, 1594, 1501, 1441, 1332 cmꢁ1
;
1H
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4
K. Yamauchi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
NMR (dimethylsulfoxide-d6, 600 MHz) dH 3.82 (3H, s, 7-OCH3),
3.83 (3H, s, 40-OCH3), 6.32 (1H, d, J = 2.10 Hz, H-6), 6.68 (1H, d,
J = 2.04 Hz, H-8), 7.05 (1H, d, J = 8.94 Hz, H-50), 7.65 (1H, dd,
J = 8.94, 2.04 Hz, H-60), 7.69 (1H, d, J = 2.04 Hz, H-20); 13C NMR
(dimethylsulfoxide-d6,150 MHz) dC 56.2 (40-OCH3), 56.6 (7-OCH3),
92.5 (C-8), 98.0 (C-6), 104.6 (C-4a), 115.3 (C-20), 112.3 (C-50),
120.3 (C-60), 123.8 (C-10), 137.0 (C-3), 146.7 (C-30), 147.3 (C-2),
150.0 (C-40), 156.7 (C-8a), 160.9 (C-5), 165.5 (C-7), 176.6 (C-4);
MALDI-TOFMS m/z 331.0608 [M+H]+.
obtained by preparative HPLC with an ODS-3 column (20 mm
u
ꢀ 250 mm). Fractions were eluted with the linear gradient
MeOH/0.05% TFA aqueous solution (0 min, 50/50; 50 min, 100/0;
60 min, 100/0) to obtain 19 as a yellowish powders with a yield
of 25.2%. The structure of the synthesized 19 was confirmed using
NMR, UPLC–TOFMS, UV, and IR spectra, and the measurement of
25
specific optical rotation: UV kmax 205, 255, 354 nm; [
a]
ꢁ68.1°
D
(c 0.86, MeOH); IR (KBr) mmax 3436, 1747, 1656, 1609, 1498,
1442, 1368 cmꢁ1 1H NMR (CD3OD, 600 MHz) dH 1.89 (3H, s, A2-
;
OAc), 1.93 (3H, s, A4-OAc), 1.96 (3H, s, A3-OAc), 2.14 (3H, s, A6-
OAc), 3.92 (1H, m, H-A5), 3.94 (1H, m, H-A6), 4.03 (1H, m, H-A6),
5.01 (1H, m, H-A4), 5.21 (1H, dd, J = 9.66, 8.28 Hz, H-A2), 5.35
(1H, t, J = 9.66 Hz, H-A3), 5.56 (1H, d, J = 8.28 Hz, H-A1), 6.02 (1H,
d, J = 2.04 Hz, H-6), 6.24 (1H, d, J = 2.10 Hz, H-8), 6.85 (1H, d,
J = 8.22 Hz, H-50), 7.48 (1H, dd, J = 8.28, 2.10 Hz, H-60), 7.56 (1H,
d, J = 2.04 Hz, H-20); 13C NMR (CD3OD,150 MHz) dC 19.2 (A2-OAc,
A4-OAc), 19.3 (A3-OAc), 19.7 (A6-OAc), 61.3 (C-A6), 68.6 (C-A4),
71.4 (C-A5), 71.7 (C-A2), 72.8 (C-A3), 93.5 (C-8), 98.4 (C-6), 99.5
(C-A1), 104.4 (C-4a), 114.6 (C-20), 116.2 (C-50), 121.9 (C-10), 122.0
(C-60), 133.3 (C-3), 144.5 (C-30), 148.3 (C-40), 157.0 (C-2), 158.3
(C-8a), 161.5 (C-5), 164.4 (C-7), 169.9 (A4-OAc), 170.3 (A6-OAc),
170.6 (A3-OAc), 171.1 (A2-OAc), 177.4 (C-4); UPLC–TOFMS m/z
631.126 [MꢁH]ꢁ.
2.2.5. Synthesis of 3-O-acetylquercetin (18)
Rutin (5.00 g, 8.19 mmol), K2CO3 (9.04 g, 65.52 mmol), and BnBr
(7.79 mL, 65.52 mmol) were added to 60 mL of DMF, and the mix-
ture was stirred for 10 h under argon at room temperature. The
resulting mixture was diluted with 150 mL of EtOAc and washed
with water (2 ꢀ 150 mL). The residue obtained by evaporating
the solvent was dissolved in 100 mL of 1 N HCl and refluxed at
80 °C for 2 h. The obtained precipitate was filtered after cooling,
and 100 lg was acetylated overnight using acetic anhydride
(1.0 mL, 10.6 mmol) in 1.0 mL of pyridine at room temperature.
The reaction mixture was diluted with 50 mL of EtOAc and washed
with water (2 ꢀ 50 mL). After the EtOAc phase was dried using Na2-
SO4, the reactant was obtained by evaporating the solvent.
The reaction mixture was dissolved in 40 mL of EtOH / THF (1:1,
v/v), and 100 mg of 10% Pd/C was added. The mixture was stirred
for 1 h at room temperature under 0.05 MPa hydrogen. After the
Pd/C was removed by filtration, the solvent was evaporated to
obtain 18 as a yellowish powder with 39.0% yield from original
rutin. It was purified using preparative HPLC with an ODS-3 col-
2.3. Tyrosinase activity assay
Measurements of tyrosinase activity were performed using a
technique modified from previous reports.27,28 Briefly, 60
l
L ali-
quots of the sample were added to a 96-well plate. Following addi-
tion of 30 L of mushroom tyrosinase (333 U/mL in phosphate
buffer, 50 mM, pH 6.5), and 110 L of substrates (2 mM -tyrosine
or 2 mM -DOPA), samples were incubated at 37 °C for 30 min.
umn (20 mm
u
ꢀ 250 mm L). Compound 18 was eluted using a lin-
l
ear gradient of MeOH/0.05% aqueous solution of TFA (0 min, 50/50;
50 min, 100/0; 60 min, 100/0). The structure of the synthesized 18
was confirmed using NMR, UPLC–TOFMS, UV, and IR spectra, and
l
L
L
Absorbance was measured at 510 nm using a microplate reader.
Each experiment was repeated twice. Tyrosinase activity was
expressed as a percentage of the activity measured in control cells
incubated with vehicle (solvent DMSO/water) without sample
materials.
the measurement of specific optical rotation: UV kmax 204, 255,
25
350 nm; [
a]
ꢁ2.67° (c 0.55, MeOH); IR (KBr) mmax 3402, 1656,
D
1604, 1507, 1444, 1367 cmꢁ1 1H NMR (CD3OD, 600 MHz) dH 2.30
;
(3H, s, 3-OAc), 6.20 (1H, d, J = 2.10 Hz, H-6), 6.40 (1H, d,
J = 2.04 Hz, H-8), 6.88 (1H, d, J = 8.94 Hz, H-50), 7.27 (1H, dd,
J = 8.94, 2.04 Hz, H-60), 7.32 (1H, d, J = 2.10 Hz, H-20); 13C NMR
(CD3OD,150 MHz) dC 19.1 (3-OAc), 93.7 (C-8), 98.8 (C-6), 103.9
(C-4a), 114.8 (C-20), 115.2 (C-50), 120.8 (C-60), 120.7 (C-10), 130.1
(C-3), 145.3 (C-30), 149.1 (C-40), 157.0 (C-2), 157.3 (C-8a), 161.7
(C-5), 164.9 (C-7), 168.5 (3-OAc), 175.9 (C-4); UPLC–TOFMS m/z
343.043 [MꢁH]ꢁ.
2.4. Cell culture
Murine melanoma B16-F0 cells (DS Pharma Biomedical, Osaka,
Japan) were grown in Dulbecco’s modified Eagle medium (DMEM)
supplemented with 10% fetal bovine serum, 100,000 unit/L penicil-
lin, and 100 mg/L streptomycin. Cells were cultured at 37 °C in
humidified atmosphere of 5% CO2.
2.2.6. Synthesis of quercetin-3-O-tetra-O-acetyl-b-D-
glucopyranoside (19)
2.5. Measurement of cellular melanin content
Rutin (5.00 g, 8.19 mmol), K2CO3 (9.04 g, 65.52 mmol), and BnBr
(7.79 mL, 65.52 mmol) were added to 60 mL of DMF, and the mix-
ture was stirred for 10 h under argon at room temperature. The
resulting mixture was diluted with 150 mL of EtOAc and washed
with water (2 ꢀ 150 mL). The residue obtained after evaporation
of the solvent was dissolved in 100 mL of 1 N HCl and refluxed at
80 °C for 2 h.
After the mixture was cooled and the precipitate filtered,
200 mg of the precipitate, K2CO3 (83.4 mg, 0.604 mmol), and acet-
obromoglucose (250 mg, 0.604 mmol) were added to 2.0 mL of
DMF, and the mixture was stirred for 6 h at room temperature
under argon. The reaction mixture was diluted with 30 mL of
EtOAc and washed with water (2 ꢀ 30 mL). After the EtOAc phase
was dried using Na2SO4, the reactant was obtained by evaporating
the solvent.
Briefly, confluent cultures of B16 melanoma cells were rinsed in
phosphate-buffered saline (PBS) and removed using 0.25% trypsin/
EDTA. The cells were loaded into a 24-well plate (5.0 ꢀ 104 cells/
well) and allowed to adhere at 37 °C for 24 h. Sample compounds
prepared at 200–10
added and the cells incubated for 72 h. Following incubation, cell
medium was collected and 200 L aliquots were loaded into a
lM for 1–10 and 50–6.25 lM for 11–19 were
l
96-well plate. The absorbance of the medium was measured at
510 nm by using a microplate reader and used as a measure of
extracellular melanin contents. The cells were washed with PBS
following lysis in 600
30 min to solubilize the melanin. A portion of the resulting lysate
(250 L) was loaded into a 96-well microplate, and the absorbance
lL of 1 M NaOH by heating at 100 °C for
l
was measured at 405 nm using a microplate reader. Measured
absorbance was used as an index of intracellular melanin contents.
Each experiment was repeated twice. The melanin-producing
activities were expressed as a percentage of the activity measured
in the control cells treated with DMSO without sample materials.
The reaction mixture was dissolved in 40 mL of EtOH/THF (1:1,
v/v). Following addition of 100 mg of 10% Pd/C, the mixture was
stirred for 2 h at room temperature under 0.05 MPa hydrogen.
Pd/C was filtered, and, after the solvent was evaporated, 19 was
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K. Yamauchi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
2.6. Cell viability
ther methylation of 11 was performed to obtain compounds 12–
14.
Measurement of cell viability was performed according to a pre-
viously described method,29 using the microculture tetrazolium
technique (MTT). Cultures were initiated in 24-well plates at
5.0 ꢀ 104 cells per well. Following incubation with compounds
prepared the same concentrations as described in measurement
Compounds 15–17 were synthesized according to the route
shown in Scheme 2. Following methylation of rutin by dimethyl-
sulfate, rutinose was removed by acid hydrolysis to obtain 15–17.
Compounds 18 and 19 were synthesized according to the route
shown in Scheme 3. In order to specifically append the acetyl group
or acetoglucose at the C-3 hydroxyl group, the phenolic hydroxyl
group of rutin was protected by benzylation using the method
described above. Following acid hydrolysis, the C-3 hydroxyl group
was acetylated or glucosylated by acetic acid anhydride or acetob-
romoglucose to yield 18 or 19, respectively.
of cellular melanin content for 72 h, 50 lL of MTT reagent (5 mg/
mL of 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium
bromide in PBS) was added to each well. The plates were incubated
in a humidified atmosphere of 5% CO2 at 37 °C for 4 h. After the
medium was removed, 1.0 mL of isopropyl alcohol (containing
0.04 N HCl) was added to each well, and a 150
l
L sample were
All compounds (1–19) were purified using preparative high-
performance liquid chromatography (p-HPLC). Structural analysis
of compounds 11–19 was performed by nuclear magnetic reso-
nance (NMR), mass spectrometry (MS), infrared (IR) spectroscopy,
UV spectroscopy, and the measurement of specific optical rotation.
The protons existing in the methoxyl group of compound 11 were
correlated with C-3, as shown in Figure 2, indicating that the meth-
oxyl group is bonded to the C-3 position. The key heteronuclear
multiple-bond correlations (HMBC) within compound 14 are also
presented in Figure 2, showing the binding position of the meth-
oxyl group. The anomeric proton of acetoglucose of 19 was corre-
lated with C-3, as shown in Figure 2, indicating that acetoglucose
binds to the C-3 position as a glycoside. Similarly, HMBC correla-
tions indicated the exact position of the substituent groups in the
other synthesized quercetin derivatives (not shown). Comparing
the data with the spectrum of quercetin itself,30,31 the position of
the acetyl group in 18 is likely to be at C-3, as suggested by the
observed chemical shift of the C-3 carbon (130.1 ppm).
withdrawn and transferred to a 96-well plate. Absorbance was
measured at 590 nm by using a microplate reader. Each experi-
ment was repeated twice. Cell viability was expressed as a percent-
age of the viability measured in control cells treated with solvent
DMSO without sample materials.
2.7. Western blot analysis
B16 melanoma cells treated with the samples at 12.5–0 lM for
72 h were lysed with radioimmunoprecipitation assay (RIPA)
buffer containing protease inhibitor cocktail at 0 °C for 30 min. Pro-
tein concentrations were determined using a Bradford protein
assay kit (Thermo) and a BSA solution as a standard. Cell lysates
were loaded at 10 lg of protein per lane and separated by sodium
dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) on
10% polyacrylamide gel. Proteins were subsequently transferred
onto a polyvinylidene difluoride (PVDF) membrane (Millipore,
Billerica, MA, USA) using a semi-dry transfer system (Atto) run at
150 mA for 30 min. The membrane was blocked with 2% BSA in
tris-buffered saline Tween20 (TBST) at 4 °C overnight. After wash-
ing, the membranes were incubated with dilutions of rabbit mono-
clonal anti-MITF (1:1000), rabbit polyclonal anti-tyrosinase
(1:200), rabbit polyclonal anti-TRP-1 (1:200), rabbit polyclonal
anti-TRP-2 (1:100), rabbit polyclonal anti-p38 MAPK (1:100), or
mouse monoclonal anti-p-p38 MAPK (1:200) antibodies. Following
incubation for 2 h, the membranes were washed and incubated
with 1:5000 diluted HRP-conjugated secondary antibody for 2 h.
Following addition of the Luminata™ Forte (Millipore), protein
density was visualized using enhanced chemiluminescence (ECL)
detection system (LAS-3000, Fujifilm, Tokyo, Japan) and quantified
using the Multi Gauge V3.0 quantification system (Fujifilm).
3.2. Melanogenesis-stimulating activities of quercetin
derivatives
The melanogenesis-stimulating activities of synthesized quer-
cetin glycosides were determined by measuring both intra- and
extracellular melanin content in B16 melanoma cells. The effects
of compounds 1–3 and 11–19 on cell viability and melanogenesis
are shown in Table 1. In our previous study, we evaluated the mod-
ulation of intracellular melanin levels by 1–10 with theophylline as
a positive control.26 The current experiment investigated the
effects of quercetin derivatives on extracellular melanin release
and melanogenesis. On measuring the melanogenesis activity
assay for each compounds, we adopted a concentration which
was not shown strong cytotoxicity of the B16 melanoma cells. As
shown in Table 1, quercetin glycosides 1, 2, and 3 stimulated intra-
cellular melanogenesis in a dose-dependent manner. However,
none of the synthesized quercetin glycosides increased the extra-
cellular levels of melanin. On the other hand, quercetin methyle-
thers 11–14 increased both intra- and extracellular melanin
content (Table 1), demonstrating higher melanogenesis-stimula-
tion than theophylline, a positive control. Significant effects were
observed on the extracellular melanin levels. Comparing the
activities of compounds 11–14, medium of cells incubated with
2.8. Statistical analysis
All data were expressed as means SD values. Statistical signif-
icance of differences were evaluated using the Student’s t-test.
3. Results and discussion
3.1. Synthesis of quercetin derivatives
Quercetin derivatives 1–19 were synthesized using commer-
cially available rutin as the starting material. The syntheses of
quercetin glycosides 1–10 were performed according to the
previously described methods.26 The methylquercetin derivatives
11–14 were synthesized according to the synthesis route shown
in Scheme 1. The first step in this synthetic approach involved
append the methyl group at the C-3 hydroxyl group of quercetin.
All phenolic hydroxyl groups of rutin were protected by benzyla-
tion. After removing rutinose by acid hydrolysis, the free C-3
hydroxyl group was methylated using dimethylsulfate. Debenzyla-
tion was subsequently performed by hydrogenation using
palladium on carbon (Pd/C), yielding 3-O-methylquercetin 11. Fur-
50 lM of compound 11 showed 224.9% higher extracellular mela-
nin levels compared to controls. The increases of melanin levels
following incubation with compounds 12–14 were higher than
220%, even at 6.25 lM, indicating the most potent stimulation of
extracellular melanin levels in this study. Furthermore, the
3-hydroxyl quercetin methylethers such as 15–17, 3-O-acethylqu-
ercetin (18), and quercetin-3-O-b-D-2,3,4,6-tetra-O-acetoglucopy-
lanoside (19) showed no stimulatory effect on the extracellular
melanin levels, suggesting that the 3-methoxyl group of com-
pounds 11–14 is an essential moiety for stimulation activity. Addi-
tionally the 40 and/or 7-methoxyl group may further increase the
melanogenesis-stimulating activity. Compounds 12–14 showed
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6
K. Yamauchi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
OH
OH
OH
OBn
HO
O
O
1) BnBr
K2CO3 / DMF
OH
OH
OBn
O
OH
H2/Pd/C
HO
O
O
BnO
O
OH
O
OH
O
O CH3
O CH3
2) MeOH / HCl
O
rutin
OH
OBn O
HO
OH
3) Dimethyl sulfate
K2CO3 / DMF
OH
11
OH
O
OH
OH
Dimethyl sulfate
K2CO3 / DMF
CH3
H3C
OH
O
CH3
HO
O
O
O
H3C
O
O
O
O CH3
O CH3
O CH3
OH
O
OH
O
OH
13
14
12
Scheme 1. Synthesis route of methylquercetins 11–14.
OH
OH
OH
CH3
O
OH
OH
OH
CH3
OH
O
H3C
O
O
O
HO
OH
O
O
H3C
OH
O
O
OH
OH
HO
O
O
OH
Dimethyl sulfate
K2CO3 / DMF
O
O
OH
O
OH
OH
O
OH
O
O
OH
O
OH
O
OH
O
OH
O
O
OH
O
OH
O
O
O
O
O
rutin
HO
OH
HO
OH
HO
OH
OH
OH
HO
OH
OH
OH
OH
OH
OH
O
CH3
H3C
OH
H3C
O
CH3
MeOH / HCl
HO
O
O
O
O
O
OH
OH
OH
OH
O
OH
O
OH
O
16
17
15
Scheme 2. Synthesis route of methylquercetins 15–17.
more potent melanogenesis-stimulating activity compared to 11.
Importantly, 12 and 13 were associated with high cell cytotoxicity
in the cell viability studies, while 14 exhibited high cell viability.
These differences in cell cytotoxicity between the quercetin meth-
ylethers may depend on the presence of both 40 and 7-methoxyl
groups.
In order to understand the involvement of the tyrosinase
enzyme in the stimulation of melanogenesis, the activity of mush-
room tyrosinase was measured following incubation with querce-
tin derivatives. However, no effect on tyrosinase activity was
observed with any of the quercetin derivatives synthesized in this
study (data not shown). Therefore, the quercetin methylethers may
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K. Yamauchi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
OH
OBn
OBn
OH
OH
HO
O
BnO
O
OH
OAc
HO
O
O
OAc
OH
O
1) BnBr
OH
OBn O
H2/Pd/C
K2CO3 / DMF
O
OH
18
OH
OBn
OH
OH
O
OBn
OH
O
2) MeOH / HCl
rutin
HO
O
BnO
O
O
3) Acetic acid anhydride
Pyridine
O
OAc
OAc
OAc
O
O
OAc
OAc
OAc
HO
OH
OH
O
OBn O
AcO
or
OH
O
Acetobromoglucose
K2CO3 / DMF
AcO
19
Scheme 3. Synthesis route of methylquercetins 18, 19.
OH
H
OH
CH3
OH
H
OH
H
O
HO
H
O
O
H
OH
H
H
H
H
O
H
O
O
H
H
HO
H
O
O
H
O
H3C
H
OH
OAc
H
O
O
O
CH3
OH
AcO
CH3
OH
OAc
OAc
Figure 2. Key HMBC correlations of compounds 11, 14, and 19.
contribute to the expression of tyrosinase or related genes in B16
melanoma cells.
cells.34,35 Similarly, the compounds showing melanogenesis-
stimulating activity in this report may also control the levels of
tyrosinase and the proteins that modulate its expression, as sug-
gested by the observation that the stimulatory activity does not
depend on the tyrosinase activity.
3.3. Effect of compounds 11 and 14 on the expression of
proteins involved in melanin biosynthesis
As presented in Table 1, compound 14 showed significant intra-
and extracellular melanogenesis-stimulating activity, with low
cytotoxicity. The effects of compound 14 on the expressions of pro-
teins related to melanin biosynthesis, such as tyrosinase, TRP-1,
TRP-2, MITF, p-p38 MAPK, and p38 MAPK were investigated in
order to identify the specific biosynthetic step associated with its
activity. As shown in Figure 3C and D, 14 increased the expression
of tyrosinase, TRP-1, TRP-2, MITF, and p-p38 MAPK in a dose
dependent manner in B16 melanoma cells. Conversely, the expres-
sion of p38 MAPK was not increased by 14, indicating that it
stimulates melanin biosynthesis by stimulating the p38 MAPK
phosphorylation. Furthermore, the melanogenesis-stimulating
effects of 11 were determined in order to compare the activity
on the expression of the proteins. Comparing the activities of 11
and 14 on the expression of proteins related to melanin biosynthe-
sis, 11 was found to increase the expression ratio of the tyrosinase
and TRP-1 to a greater extent than 14 (Fig. 3B and D). Nevertheless,
extracellular melanogenesis-stimulating activity of 11 was lower
than that of 14 (Table 1), suggesting that melanogenesis in mela-
noma cells is not solely dependent on the expression of tyrosinase
and TRP-1. Furthermore, 14 significantly stimulated the expression
of MITF and p-p38 MAPK, which enhance the expression of tyros-
inase, TRP-1, and TRP-2. Conversely, 11 did not alter the expression
In general, the signaling pathway modulating the expression of
melanogenic enzymes in melanoma cells comprises the following
steps. First, extracellular messengers, such as
a-melanocyte-stimu-
lating hormone ( -MSH) and histamine, interact with receptors on
a
the melanocyte. Receptor stimulation increases intracellular cyclic
adenosine monophosphate (cAMP) levels in the melanocyte, stim-
ulating a number of intracellular kinase pathways, such as the p38
mitogen activated protein kinase (p38 MAPK), extracellular signal-
regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). These
kinase signaling cascades regulate the expression of microphthal-
mia-associated transcription factor (MITF),32 which acts as a tran-
scriptional factor regulating the expression of tyrosinase, TRP-1,
and TRP-2.
Some studies have reported that melanogenesis-modulating
agents regulate the expression of tyrosinase, TRP-1, and TRP-2 by
regulating the expression of p38 MAPK, ERK, JNK, and MITF. For
example, the mechanism of melanogenesis-stimulating activity of
a lignan cubebin was found to involve the enhancement of p38
MAPK activity in B16 melanoma cells.33 Additionally, the compo-
nents isolated from Nardostachys chinensis and Rhodiola rosea crude
extracts were reported to inhibit melanin biosynthesis by sup-
pressing the expression of MITF and tyrosinase in B16 melanoma
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8
K. Yamauchi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Table 1
Intra and extracellular melanogenesis activity and cell viability in B16 melanoma cells by the synthesized quercetin derivatives 1–3 and 11–19
Cell viability and melanogenesis activity (%)
200
lM
100
l
M
10 lM
1
Intercellular melanogenesis activity
Extracellular melanogenesis activity
Cell viability
206.9 2.3⁄⁄
86.1 2.7
157.1 8.6⁄
85.1 1.7
80.9 5.5
116.3 1.3
91.0 4.1
105 3.8
68.4 6.9⁄
2
Intercellular melanogenesis activity
Extracellular melanogenesis activity
Cell viability
176.0 9.1⁄
85.2 4.9
81.1 3.0
126.8 9.1⁄
91.0 6.3
93.2 0.6
102.6 2.9
83.3 5.8
99.6 1.5
3
Intercellular melanogenesis activity
Extracellular melanogenesis activity
Cell viability
190.5 10.2⁄
125.5 9.3
74.8 2.1
151.5 8.1⁄
116.0 12.6
86.8 2.0
137.0 0.8⁄
99.7 21.7
90.0 6.9
50
lM
25
lM
12.5
lM
6.25 lM
11
Intercellular melanogenesis activity
Extracellular melanogenesis activity
Cell viability
157.3 8.4⁄
224.9 18.2⁄
74.4 3.8
146.5 15.3
130.8 5.8⁄
88.1 4.1
137.0 23.6
124.1 1.4
98.2 6.7
—
—
—
12
Intercellular melanogenesis activity
Extracellular melanogenesis activity
Cell viability
—
—
—
166.6 0.0⁄⁄
346.7 2.9⁄⁄
63.2 2.1⁄⁄
178.8 9.6⁄⁄
309.5 14.5⁄⁄
50.0 0.5⁄⁄
132.3 8.1⁄
229.5 17.6⁄
75.6 2.9⁄⁄
13
Intercellular melanogenesis activity
Extracellular melanogenesis activity
Cell viability
—
—
—
187.6 2.5⁄⁄
265.5 5.9⁄⁄
54.1 0.55⁄⁄⁄
171.2 0.0⁄⁄
304.3 4.0⁄⁄
60.4 4.2⁄⁄
134.0 4.3⁄
222.8 12.8⁄⁄
80.4 0.5⁄⁄
14
Intercellular melanogenesis activity
Extracellular melanogenesis activity
Cell viability
—
—
—
203.4 3.4⁄⁄
298.7 3.7⁄⁄
90.2 4.3
181.4 9.0⁄⁄
228.0 7.0⁄⁄
101.9 2.2
127.7 4.3⁄
225.5 10.8⁄⁄
95.3 1.8
15
Intercellular melanogenesis activity
Extracellular melanogenesis activity
Cell viability
—
—
—
106.9 7.1
97.9 0.0
100.9 1.3
133.5 4.4
95.1 0.2
103.0 4.3
125.6 5.7
97.5 2.2
103.6 3.6
16
Intercellular melanogenesis activity
Extracellular melanogenesis activity
Cell viability
—
—
—
75.8 12.9
114.3 1.1
91.6 4.5
87.6 2.6
105.2 1.3
99.4 5.1
102.4 12.0
105.5 1.4
92.7 0.5
17
Intercellular melanogenesis activity
Extracellular melanogenesis activity
Cell viability
—
—
—
100.5 10.0
100.1 0.3
101.3 3.7
106.1 8.1
98.0 0.0
101.4 1.9
110.2 2.5
100.5 0.5
99.7 1.2
18
Intercellular melanogenesis activity
Extracellular melanogenesis activity
Cell viability
104.6 3.1
126.6 2.3
110.0 7.9
114.3 3.6
102.6 6.2
120.4 0.3
99.0 8.3
87.6 5.1
110.0 3.8
—
—
—
19
Intercellular melanogenesis activity
Extracellular melanogenesis activity
Cell viability
148.4 1.4⁄⁄
96.1 1.8
103.7 1.1
121.4 17.1
101.5 1.4
101.8 0.2
122.4 2.1
89.3 0.4
113.5 8.1
—
—
—
Theophylline
500
lM
250
l
M
125 lM
Intercellular melanogenesis activity
Extracellular melanogenesis activity
Cell viability
166.8 31.7⁄
204 1.6⁄⁄
91.4 1.3
131.7 1.9
183.2 3.2⁄⁄
94.4 4.1
127.6 6.6
170.7 0.7⁄⁄
95.5 11.0
Data are expressed as means SD. (n = 2). ⁄p 6 0.05 and ⁄⁄p 6 0.01 compared with respective control values.
—: not done.
of MITF and p-p38 MAPK, despite enhancing the expression of
tyrosinase, TRP-1, and TRP-2. Except for MITF, no transcriptional
factors controlling tyrosinase expression have been reported thus
far. These results may therefore indicate that 11 enhances the
expression of tyrosinase, TRP-1, and TRP-2 by stimulating tran-
scriptional factors that are yet to be identified. The levels of mela-
nogenic enzymes in the melanocyte are also regulated by protein
degradation by proteasome through ubiquitination.36 Fatty acids,
a major component of the cell membranes, were previously
reported to regulate melanin biosynthesis by controlling the degra-
dation of tyrosinase, TRP-1, and TRP-2.37 Therefore, in addition to
the possible effect on a transcriptional factor, an alternative expla-
nation for the enhancing activity of 11 on the levels of melanogenic
enzymes may involve the inhibition of the degradation of melano-
genic enzymes through ubiquitination. Compared to 14, 11
increased the expression of melanogenic enzymes, but showed less
melanogenesis-stimulating activity, as described above. Addition-
ally, the levels of p-p38 MAPK and MITF were not increased by
the addition of 11. If the two phenomena were related to each
other, MITF and/or p-p38 MAPK may play an important role in
the regulation of melanogenesis not only by enhancing the expres-
sions of the enzymes, but also through affecting other factors, such
as the transportation and/or degradation of melanogenic enzymes.
Tyrosinase, TRP-1, and TRP-2 are expressed through a MITF-reg-
ulated process and transported to the melanosome. Melanin is bio-
synthesized in the melanosome by the action of the melanogenic
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K. Yamauchi et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
9
A
B
600
OH
OH
500
400
300
HO
O
O
O
OH
CH3
Control
3.1µM
6.3µM
12.5µM
11
12.5µM
Control 3.1µM 6.3µM
Tyrosinase
TRP1
200
100
0
TRP2
MITF
p-p38MAPK
p38MAPK
GAPDH
Tyrosinase
TRP1
TRP2
MITF
p-p38MAPK p38MAPK
C
D
OH
600
500
400
300
200
100
0
O
CH3
O
O
H3
C
O
OH
O
CH3
14
Control
3.1µM
6.3µM
12.5µM
12.5µ
Control 3.1µ
6.3µ
Tyrosinase
TRP1
TRP2
MITF
p-p38MAPK
p38MAPK
GAPDH
Tyrosinase
TRP1
TRP2
MITF p-p38MAPK p38MAPK
Figure 3. Effect of 11 and 14 on the expression of tyrosinase, TRP1, TRP2, MITF, p-p38MAPK, and p38MAPK in B16 melanoma cells. (A) Representative blots of 11. (B)
Quantification of the ratio of protein expressions in melanoma cells treated by 11. The data show the means SD from three independent experiments. (C) Representative
blots of 14. (D) Quantification of the ratio of protein expressions in melanoma cells treated by 14. The data show the means SD from three independent experiments.
⁄p 6 0.05 and ⁄⁄p 6 0.01 compared with control values.
enzymes. The mature melanosome is specifically transported to
the periphery of the cell from the perinuclear region of the melano-
cytes through a process that involves a wide variety of cellular
transport proteins, including actin, myosin Va, Rab27A, and
Slac2-a.10–12 Compound 14 may accelerate the transportation of
melanogenic enzymes to the melanosome and/or transportation
of melanosome to the outside of the cells by regulating the expres-
sion of proteins related to cellular transport. However, 11 may
elicit less potent stimulation of the transportation of melanogenic
enzymes to the melanosome and/or the transportation of melano-
some as compared to 14.
tyrosinase, TRP-1, and TRP-2 by stimulating currently unidentified
transcriptional factors and/or by regulating the proteasomal degra-
dation of melanogenic enzymes. Further investigations are
required in order to elucidate the exact mechanism of action that
underlies the observed activities of 11. The discovery of the novel
melanogenesis-modulating activity of quercetin methylethers in
B16 melanoma cells supports further research into its potential
anti-graying applications or into possible uses for such compounds
as cosmetic products that regulate skin pigmentation.
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