Synthesis of dihydroresveratrol glycosides and evaluation of their activity against melanogenesis in B16F0 melanoma cells

Article abstract of DOI:10.1016/j.ejmech.2014.09.092

Dihydroresveratrol glucoside 1 isolated from Camellia oleifera and its xyloside derivative 2 were synthesized for the first time in 5 steps from TBS-protected aldehyde 4. Natural product 1 is a potent melanogenesis inhibitor in B16F0 melanoma cells (appro

Full text of DOI:10.1016/j.ejmech.2014.09.092

European Journal of Medicinal Chemistry 87 (2014) 862e867
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis of dihydroresveratrol glycosides and evaluation of their
activity against melanogenesis in B16F0 melanoma cells
a
b
,
c
c
c
b
Chisato Oode , Wataru Shimada , Yukiko Izutsu , Mariko Yokota , Takehiro Iwadate ,
a, b, *
Ken-ichi Nihei
a
Department of Applied Biological Chemistry, Faculty of Agriculture, Utsunomiya University, Tochigi 321-0943, Japan
Department of Applied Life Science, United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Tokyo 183-8509,
b
Japan
c
Nikkol Group Cosmos Technical Center Co., Ltd., Tokyo 174-0046, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 August 2014
Received in revised form
Dihydroresveratrol glucoside 1 isolated from Camellia oleifera and its xyloside derivative 2 were syn-
thesized for the first time in 5 steps from TBS-protected aldehyde 4. Natural product 1 is a potent
melanogenesis inhibitor in B16F0 melanoma cells (approximately 40 fold more potent than kojic acid). In
contrast, the synthetic product 2 stimulates melanogenesis, suggesting that a single hydroxymethyl
group in the glycoside substituent of dihydroresveratrols is responsible for inhibition or activation of
melanogenesis.
24 September 2014
Accepted 29 September 2014
Available online 2 October 2014
©
2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Dihydroresveratrol glycoside
Camellia oleifera
Glycosylation
Melanogenesis inhibitor
Melanogenesis activator
1
. Introduction
and cardol [7,11]. Despite containing the pharmacologically active
resorcinol motif, there has been no report on the total synthesis of 1
or its development towards the identification of novel anti-
melanogenesis agent. This study describes the chemical synthesis
of 1e3, along with the evaluation of their activity against melano-
genesis observed in B16F0 melanoma cell culture (Fig. 1).
The abnormal formation of the melanin pigment causes serious
dermal problems, including melasma, solar lentigines, and ephe-
lides [1e3]. Initial steps in melanin biosynthesis include two re-
actions, which are catalyzed by the cupper-containing
oxidoreductase tyrosinase (EC 1.14.18.1): hydroxylation of
L-tyro-
sine and oxidation of -DOPA [4]. Thus, a number of tyrosinase in-
L
2
. Results and discussion
hibitors have been used for the regulation of melanogenesis [5e7].
In addition, melanin accumulation and related phenomena can be
correlated with Parkinson's disease and melanoma development
2.1. Synthesis of 1 and 2
[
8,9]. Accordingly, melanogenesis regulating agents possessing
Steps for the chemical synthesis of natural product 1 are illus-
novel scaffolds are required for chemical-based functional research
for brain and malignant cancer.
trated in Scheme 1. TBS-protected aldehyde 4 [12] was reacted with
the ylide generated from phosphonium salt 5 [13] in the presence of
lithium hexamethyldisilazide (LiHMDS) to obtain stilbene 6 in
In our continuing efforts towards the identification of tyrosinase
inhibitors, we have now focused our attention on dihydroresvera-
trol glucoside 1 isolated from Camellia oleifera (Theaceae) [10]. This
glucoside has the resorcinol moiety that is frequently found in the
structure of potent tyrosinase inhibitors such as 4-hexylresorcinol
1
excellent yield [14]. Analysis by H NMR indicates that the ratio of
cis and trans stilbenes is 3:2. Hydrogenation of 6 using Pearlman's
catalyst [15] furnished phenol 7 in 80% yield. Although this syn-
thetic scheme is similar to that reported previously [16,17], TBS
protection of the starting material led to an improvement in the
synthetic route and afforded high yields of the product. Glycosyl-
ation of 7 using the glucosyl imidate 8 as the glucosyl donor and
trifluoromethanesulfonate (TMSOTf) as the Lewis acid at low
*
Corresponding author. Department of Applied Biochemistry, Faculty of Agri-
culture, Utsunomiya University, Utsunomiya, Tochigi 321-0943, Japan.
E-mail address: nihei98@cc.utsunomiya-u.ac.jp (K.-i. Nihei).
http://dx.doi.org/10.1016/j.ejmech.2014.09.092
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

C. Oode et al. / European Journal of Medicinal Chemistry 87 (2014) 862e867
863
temperatures afforded glycoside 9 in 82% yield. Use of tetrabuty-
lammonium fluoride (TBAF) converted 9 to resorcinol 10 in 51%
yield. Transesterification of 10 using sodium methoxide, followed
by the treatment with Amberlite IR-120H (to remove sodium ions)
provided the natural product 1 in 71% yield. Although analytical
data for 1 are nearly consistent with those reported previously [10],
NMR assignments have been corrected by careful inspections of
NMR data resulting from HMQC and HMBC experiments. Conse-
quently, the natural product 1 was synthesized for the first time via
5
steps from 4 in 24% overall yield.
A previous report describes the synthesis of the bibenzyl
glucoside [17], an analogue of 1, which has been designed on the
basis of the structure of the natural bibenzyl xyloside. As a result,
tyrosinase inhibitory activity of the bibenzyl glucoside was 1.8 fold
weaker than that of the xyloside. Thus, xyloside 2 was prepared in
an effort to furnish a potent and simple melanogenesis inhibitor.
Steps for the chemical synthesis of 2 are illustrated in Scheme 2.
Glycosylation of 7 with the xylosyl donor 11 in the presence of the
ꢁ
catalytic amount of TMSOTf at ꢀ20 C afforded xyloside 12 in 50%
yield. Treatment with TBAF transformed 12 to resorcinol 13 quan-
titatively. The removal of acetyl groups in 12 using sodium
Fig. 1. Structure of dihydroresveratrol derivatives 1e3.
Scheme 1. Synthesis of dihydroresveratrol glucoside 1.
Scheme 2. Synthesis of dihydroresveratrol xyloside 2.

864
C. Oode et al. / European Journal of Medicinal Chemistry 87 (2014) 862e867
methoxide afforded the target compound 2 in excellent yield.
Consequently, 2 was synthesized via 5 steps from 4 in 43% overall
yield.
Supplementary data). However, the protein content reduced (29%
of control), suggesting that 3 possesses an adverse effect on the
protein biosynthesis in B16F0 melanoma cells.
Activity of the synthesized compound 2 was evaluated in B16F0
melanoma cells by an assay similar to the one used for evaluating
the activity of 1 (Fig. 3). With an increase in the concentration of 2
in the medium, the melanin content in the cells increased. On
2
.2. Biological evaluation of 1e3 against melanogenesis
Anti-melanogenesis activity of 1 was evaluated in B16F0
melanoma cells (Fig. 2) [18,19]. Kojic acid, a typical melanogen-
esis inhibitor, was used as the positive control. Melanin content
of the cells decreased with the use of increasing concentrations of
treatment of 27.6 mM (10 mg/mL) of 2, melanin production was
stimulated to provide 130% of negative control. Furthermore, pro-
tein content reduced insignificantly (94% of control), indicating that
2 does not suppress the biosynthesis of the cellular proteins
1. When the cultured cells were treated with 25.5 mM (10 mg/mL)
of 1, the melanin amount reduced significantly (74% of negative
significantly. Concentrations of 2 up to 27.6 mM did not have any
control). Similar reduction in the melanin content was observed
influence on the cell viability (102% cells are viable in the group
treated with 2 when compared with the negative control group),
which was determined by the MTT assay.
Thus, synthetic derivative 2 acts as a melanogenesis activator.
While this result is in contrast to our stated objective of seeking to
identify potent inhibitors, the discovery of a melanogenesis acti-
vator is a remarkable result. Melanogenesis activators, such as the
derivatives of coumarin and stilbene isolated from several plants,
(
79% of negative control) when the B16F0 melanoma cells were
treated with 1 mM of kojic acid. Therefore, anti-melanogenesis
activity of 1 is approximately 40 fold higher than that of kojic
acid. The total protein content was estimated by bicinchoninic
acid (BCA) protein assay [20]. The protein content after treatment
of 25.5 mM of 1 represented 98% of negative control, suggesting
that the natural glucoside 1 has an insignificant effect on the
protein biosynthesis in B16F0 melanoma cells. In addition, the
measurement of cell viability in a 3-(4,5-di-methylthiazol-2-yl)-
have been used to treat hypopigmentation [22,23]. In particular, the
0
stilbene derivative, 2,3,5,4 -tetrahydroxystilbene-2-O-
b
-D-gluco-
2
,5-diphenyltetrazolium bromide (MTT) assay [21] revealed no
significant decrease in cell viability even at concentrations as
high as 25.5 M (102% cells are viable in the group treated with 1
side, isolated from the extracts of Polygonum multiflorum (Polygo-
naceae) stimulated melanogenesis by activating mitogen-activated
protein (MAP) kinase and inducing microphthalmia-associated
transcription factor (MITF) of tyrosinase in B16 melanoma cells
[24]. Although additional investigations are required to gain insight
into the mode of action of 2, structural comparison between this
stilbene and 2 indicated that the activity of 2 can emerge from
similar pathways.
m
when compared to negative control group). These results sug-
gested that compound 1 does not have any adverse effects on cell
viability.
Likewise, alycone 3 acts as an effective melanogenesis inhibitor
because the melanin amount reduced significantly to treat 43.4
mM
(
10 g/mL) of 3 in the cell culture experiment (for details, see the
m
150.0
140.0
130.0
120.0
110.0
100.0
150.0
140.0
130.0
120.0
110.0
100.0
*
*
9
0.0
80.0
0.0
9
8
7
6
5
4
3
2
1
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
*
7
60.0
50.0
40.0
30.0
20.0
10.0
0.0
0
.0
0
3.2
6.4
12.8
25.5
0
3.5
6.9
13.8
27.6
1
(μM)
2 (μM)
Fig. 2. Melanin content in B16F0 melanoma cells following treatment with 1 for 72 h.
The data are expressed as melanin content (% of control) and each column represents
the mean ± SD of 3 determinations. Differences are considered significant if P < 0.05
Fig. 3. Melanin content in B16F0 melanoma cells following treatment with 2 for 72 h.
The data are expressed as melanin content (% of control) and each column represents
the mean ± SD of 3 determinations. Differences are considered significant if P < 0.05
(indicated by asterisk).
(
indicated by asterisk).

C. Oode et al. / European Journal of Medicinal Chemistry 87 (2014) 862e867
865
0
0
3
. Conclusions
J ¼ 16.0, 1H, H-7), 6.94 (d, J ¼ 8.6, 2H, H-3 , H-5 ), 6.83 (d, J ¼ 16.0,
0
1
H, H-7 ), 6.59 (d, J ¼ 2.2, 2H, H-2, H-6), 6.23 (t, J ¼ 2.2, 1H, H-4),
The primary difference in the structure of the two dihydror-
5.07 (s, 2H, Bn), 0.99 (s, 18H, TBS), 0.09 (s, 12H, TBS). ESIHRMS m/z
esveratrol glycosides is the presence or the absence of the
hydroxymethyl group in the sugar moiety. This subtle modification
drastically changes the molecular property from an inhibitor to an
activator of melanogenesis. In addition, the glycosylation of dihy-
droresveratrol suppresses their effect on the protein biosynthesis in
B16F0 melanoma cells.
Since C. oleifera has been widely and traditionally used as an
ingredient in food, cosmetics, and medicine [25e27], the natural
product 1 is likely to be safe for use against hyperpigmentation.
Othereffective agents commonly used include kojic acid and arbutin
547.3069 [MþH]þ (calcd for C33
47 3 2
H O Si , 547.3064).
0
4.3. 3,5-Di(tert-butyldimethylsilyloxy)-4 -hydroxybibenzyl (7)
To a solution of 6 (0.71 g, 1.3 mmol) in Et
10% Pd(OH) on carbon (71 mg). The reaction mixture was stirred
for 4 h under a balloon of H at room temperature and filtered
through a Celite pad. The filtrate was concentrated, and the res-
idue was purified by silica gel chromatography (5e10% EtOAc in
hexane) to give the title compound 7 (0.48 g, 80%) as a white solid.
2
O (6 mL) was added
2
2
ꢀ1 1
[28,29]. In contrast, anti-hypopigmentation agents as the synthetic
IR (film) nmax 3417, 1160, 836, 781 cm . H NMR (400 MHz, CDCl )
3
0
0
0
0
derivative 2 can be used to remedy dermal disorders [30,31], which
can be caused in part due to the use of inappropriate cosmetic in-
gredients. Moreover, molecules that increase or decrease melanin
biosynthesis are useful tools for examining the cellular mechanisms
underlying melanogenesis and for potentially revealing previously
unknown therapeutic targets for melanin-related diseases [32].
Further investigation for clarifying the molecular logic of the dihy-
droresveratrol glycosides against melanogenesis is underway.
d
6.99 (d, J ¼ 8.6, 2H, H-2 , H-6 ), 6.71 (d, J ¼ 8.6, 2H, H-3 , H-5 ),
6.25 (d, J ¼ 2.2, 2H, H-2, H-6), 6.16 (t, J ¼ 2.2, 1H, H-4), 4.79 (bs, 1H,
0
OH), 2.75 (m, 4H, H-7, H-7 ), 0.95 (s, 18H, TBS), 0.15 (s, 12H, TBS).
13
0
C NMR (100 MHz, CDCl
3
)
d
156.3 (C-3, C-5), 153.6 (C-4 ), 143.7 (C-
1), 133.9 (C-1 ), 129.5 (C-2 , C-6 ), 115.1 (C-3 , C-5 ), 113.8 (C-2, C-6),
0
0
0
0
0
0
109.7 (C-4), 38.0 (C-7), 36.7 (C-7 ), 25.7 (TBS), 18.2 (TBS), ꢀ4.4
þ
(TBS). ESIHRMS m/z 459.2747 [MþH] (calcd for C26
43 3 2
H O Si ,
459.2751).
0
00 00 00 00
4
. Experimental
4.4. 4 -(2 ,3 ,4 ,6 -Tetra-O-acetyl-b-D-glucopyranosyl)-3,5-di(tert-
butyldimethylsilyloxy)bibenzyl (9)
4
.1. General experimental procedures
ꢁ
To a cold (ꢀ20 C) and stirred solution of 7 (0.20 g, 0.44 mmol) and
NMR spectra were recorded in CD
3
OD or CDCl
3
on a JEOL EX-400
2,3,4,6-tetra-O-acetyl-
(0.40 g, 0.81 mmol) in CH
lution (0.11 M in CH
a-D
-glucopyranosyl trichloroacetimidate (8)
Cl (6 mL) was added 73 L of TMSOTf so-
, 8.0 mol). After being stirred for 80 min
L) and
1
13
spectrometer ( H at 400 MHz and C at 100 MHz). Chemical shifts
were recorded as ppm relative to the solvent signal for CD OD
3.30 ppm for H NMR, 49.0 ppm for C NMR) or for CDCl
2
2
m
3
Cl
2 2
m
1
13
ꢁ
(
(
3
at ꢀ20 C, the resultant solution was quenched with TEA (15
m
1
13
7.24 ppm for H NMR, 77.0 ppm for C NMR). HRMS spectra were
allowed to warm to room temperature. Concentration in vacuo, fol-
lowed by silica gel chromatography (20e40% EtOAc in hexane) gave
the title compound 9 (0.28 g, 82% from 7) as a colorless solid.
measured on an AB SCIEX TripleTOF 5600 mass spectrometer fitted
with an electrospray ion source in positive ionization mode. IR
spectra were measured with a PerkineElmer Paragon 1000 PC FT-IR
spectrometer. Optical rotations were recorded on a Jasco P-1020
polarimeter. Preparative RP-HPLC was performed on a Hitachi
LaChrome Elite instrument equipped with an Inertsil ODS-3 (5
4
2
2
3
ꢀ1 1
nmax 1759, 1163, 832, 781 cm . H
[
a
]
D
ꢀ2.81 (c 1.13, CHCl
3
). IR (film)
7.05 (d, J ¼ 8.6, 2H, H-2 , H-6 ), 6.86(d, J ¼ 8.6,
2H, H-3 , H-5 ), 6.24 (d, J ¼ 2.2, 2H, H-2, H-6), 6.17 (t, J ¼ 2.2,1H, H-4),
0
0
NMR (400 MHz, CDCl ) d
3
0
0
0
0
00
m
m,
.6 ꢂ 250 mm) with a flow rate of 1.0 mL/min and detected at
54 nm. Compound 3 was prepared from commercially available
5.26 (t, J¼ 9.4,1H, H-3 ), 5.23 (dd, J¼ 7.6,9.4,1H, H-4 ), 5.14 (dd, J¼ 7.6,
0
0
00
00
9.4,1H, H-2 ), 5.01 (d, J¼ 7.6,1H, H-1 ), 4.27 (dd, J¼ 5.3,12.2,1H, H-6 ),
0
0
00
4.14 (dd, J ¼ 2.4, 12.2, 1H, H-6 ), 3.82 (ddd, J ¼ 2.4, 5.3, 7.6, 1H, H-5 ),
0
resveratrol by catalytic hydrogenation using 5% Pd/C and the
spectral data are consisted with those in the previous report [33].
2.77 (m, 4H, H-7, H-7 ), 2.05 (s, 3H, Ac), 2.04 (s, 3H, Ac), 2.02 (s, 3H, Ac),
13
2.01 (s, 3H, Ac), 0.94 (s,18H, TBS), 0.13 (s,12H, TBS). C NMR(100 MHz,
CDCl 170.7 (Ac), 170.3 (Ac), 169.5 (Ac), 169.4 (Ac), 156.3 (C-3, C-5),
155.1 (C-4 ), 143.4 (C-1), 136.8 (C-1 ), 129.5 (C-2 , C-6 ), 116.9 (C-3 , C-
),113.7(C-2, C-6),109.8 (C-4), 99.3 (C-1 ), 72.7 (C-5 ),71.9(C-3 ),71.1
(C-2 ), 68.3 (C-4 ), 61.9 (C-6 ), 37.8 (C-7), 36.7 (C-7 ), 25.7 (TBS), 20.7
3
) d
0
0
0
0
0
0
4.2. 4 -Benzyloxy-3,5-di(tert-butyldimethylsilyloxy)stilbene (6)
0
00 00 00
5
0
0
00
00
0
To a suspension of 5 [13] (1.3 g, 2.6 mmol) in THF (10 mL), 1.5 mL
ꢁ
of LiHMDS (1.3 M in THF, 2.0 mmol) was added at 0 C. The resultant
solution was stirred at room temperature for 0.5 h. The solution was
then cooled to 0 C, and a THF (2 mL) solution of 4 [12] (0.47 g,
(Ac), 20.63 (Ac), 20.61 (Ac), 20.59 (Ac),18.2 (TBS), ꢀ4.4 (TBS). ESIHRMS
m/z 789.3691 [MþH]þ (calcd for C40
61 2
H O12Si , 789.3702).
ꢁ
0
00 00 00 00
1.3 mmol) was slowly added. After being stirred for 1 h at room
4.5. 4 -(2 ,3 ,4 ,6 -Tetra-O-acetyl-b-D-glucopyranosyl)-3,5-
temperature, saturated aqueous NH Cl solution (30 mL) was
4
dihydroxybibenzyl (10)
poured into the reaction mixture. The resultant mixture was
extracted with EtOAc (100 mL). The organic layer was washed with
ꢁ
To a cold (0 C) and stirred solution of 9 (0.10 g, 0.13 mmol) in THF
saturated aqueous NH
(
(
4
Cl solution (30 mL
30 mL ꢂ 2). The aqueous layers were extracted with EtOAc
30 mL ꢂ 3), and the combined organic layers were dried over
SO . Filtration and concentration followed by silica gel chro-
matography (0e2% EtOAc in hexane) gave the title compound 6
ꢂ
2) and brine
(2 mL) was added TBAF trihydrate (0.12 g, 0.38 mmol). After being
ꢁ
stirred for 80 min at 0 C, saturated aqueous NH
4
Cl solution (30 mL)
was poured into the reaction mixture. The resultant mixture was
extracted with EtOAc (100 mL). The organic layer was washed with
Na
2
4
saturated aqueous NH
The aqueous layers were extracted with EtOAc (30 mL ꢂ 3), and the
combined organic layers were dried over Na SO . Filtration and
4
Cl solution (30 mL ꢂ 2) and brine (30 mL ꢂ 2).
1
(
0.70 g; cis:trans ¼ 3:2, as determined by H NMR; 100% from 4) as a
ꢀ
1 1
colorless solid. IR (film)
n
max 1164, 832, 781 cm . Cis-6; H NMR
2
4
0
0
(
6
400 MHz, CDCl
3
)
d
7.38 (m, 5H, Bn), 7.16 (d, J ¼ 8.6, 2H, H-2 , H-6 ),
concentration followed by silica gel chromatography (50e70%
0
0
.81 (d, J ¼ 8.6, 2H, H-3 , H-5 ), 6.47 (d, J ¼ 12.2, 1H, H-7), 6.39 (d,
EtOAc in hexane) gave the title compound 10 (37 mg, 51%) as a white
0
21
J ¼ 12.2, 1H, H-7 ), 6.35 (d, J ¼ 2.2, 2H, H-2, H-6), 6.18 (t, J ¼ 2.2, 1H,
solid. [
a
]
D
ꢀ0.21 (c 0.23, CHCl
3
). IR (film)
nmax 3405, 1755, 838,
ꢀ
1 1
0
0
H-4), 5.01 (s, 2H, Bn), 0.92 (s, 18H, TBS), 0.20 (s, 12H, TBS). Trans-6;
3
755 cm . H NMR (400 MHz, CDCl ) d
7.02 (d, J ¼ 8.6, 2H, H-2 , H-6 ),
1
0
0
0
0
H NMR (400 MHz, CDCl
3
)
d
7.38 (m, 7H, Bn, H-2 , H-6 ), 6.95 (d,
6.88 (d, J ¼ 8.6, 2H, H-3 , H-5 ), 6.17 (t, J ¼ 2.2,1H, H-4), 6.13 (d, J ¼ 2.2,

866
C. Oode et al. / European Journal of Medicinal Chemistry 87 (2014) 862e867
0
0
00
ꢁ
4
being stirred for 30 min at 0 C, saturated aqueous NH Cl solution
(30 mL) was poured into the reaction mixture. The resultant mixture
was extracted with EtOAc (100 mL). The organic layer was washed
2
5
H, H-2, H-6), 5.28 (t, J ¼ 9.1,1H, H-3 ), 5.23 (dd, J ¼ 7.6, 9.1,1H, H-4 ),
00
00
.16 (dd, J ¼ 7.5, 9.1, 1H, H-2 ), 5.05 (d, J ¼ 7.5, 1H, H-1 ), 4.25 (dd,
0
0
00
J ¼ 5.1, 12.2, 1H, H-6 ), 4.17 (dd, J ¼ 2.5, 12.2, 1H, H-6 ), 3.82 (ddd,
0
0
0
J ¼ 2.5, 5.1, 7.6,1H, H-5 ), 2.77 (m, 4H, H-7, H-7 ), 2.06 (s, 3H, Ac), 2.05
with saturated aqueous NH
4
Cl solution (30 mL ꢂ 2) and brine
13
(
d
s, 3H, Ac), 2.03 (s, 3H, Ac), 2.01 (s, 3H, Ac). C NMR (100 MHz, CDCl
3
)
(30 mL ꢂ 2). The aqueous layers were extracted with EtOAc
(30 mL ꢂ 3), and the combined organic layers were dried over
170.8 (Ac), 170.3 (Ac), 169.6 (Ac), 169.5 (Ac), 156.7 (C-3, C-5), 154.7
C-4 ), 144.4 (C-1), 136.6 (C-1 ), 129.6 (C-2 , C-6 ), 117.2 (C-3 , C-5 ),
08.1 (C-2, C-6),100.5 (C-4), 99.1 (C-1 ), 72.8 (C-5 ), 72.0 (C-3 ), 71.0
C-2 ), 68.2 (C-4 ), 61.9 (C-6 ), 37.8 (C-7), 36.6 (C-7 ), 20.72 (Ac),
0
0
0
0
0
0
(
1
(
2
2 4
Na SO . Filtration and concentration followed by silica gel chroma-
00
00
00
tography (50e55% EtOAc in hexane) gave the title compound 13
0
0
00
00
0
23
(90 mg, 100%) as a white solid. [
a
]
D
ꢀ25.2 (c 1.67, CHCl
3
). IR (film)
7.03 (d,
J ¼ 8.5, 2H, H-2 , H-6 ), 6.87 (d, J ¼ 8.5, 2H, H-3 , H-5 ), 6.18 (bs,1H, H-
þ
ꢀ1 1
0.65 (Ac), 20.62 (Ac), 20.60 (Ac). ESIHRMS m/z 561.1980 [MþH]
n
max 3441, 1755, 838, 755 cm . H NMR (400 MHz, CDCl ) d
3
0
0
0
0
(
calcd for C28 12, 561.1972).
33
H O
0
0
4
), 6.16 (bs, 2H, H-2, H-6), 5.21 (t, J ¼ 7.7,1H, H-3 ), 5.15 (dd, J ¼ 6.1, 7.7,
0
00
00
00
4.6. 4 -(
b
-D
-glucopyranosyl)-3,5-dihydroxybibenzyl (2)
1H, H-2 ), 5.11 (d, J ¼ 6.1, 1H, H-1 ), 4.99 (ddd, J ¼ 4.8, 7.7, 7.9, 1H, H-
0
0
00
4
), 4.20 (dd, J ¼ 4.8, 12.1, 1H, H-5 ), 3.49 (dd, J ¼ 7.9, 12.1, 1H, H-5 ),
13
ꢁ
0
To cold (0 C) and stirred solution of 10 (34 mg, 61
m
mol) in
L of NaOMe (5.2 M in MeOH,
.41 mmol) slowly. After being stirred for 1 h at room temperature,
2.75 (m, 4H, H-7, H-7 ), 2.06 (m, 9H, Ac). C NMR (100 MHz, CDCl
3
)
0
MeOH (4 mL) was added 79
0
m
d
170.4 (Ac),170.1 (Ac),169.9 (Ac),156.8 (C-3, C-5),154.6 (C-4 ),144.5
0 0 0 0 0
(C-1), 136.5 (C-1 ), 129.6 (C-2 , C-6 ), 116.9 (C-3 , C-5 ), 108.0 (C-2, C-
þ
00 00 00 00
6), 100.5 (C-4), 98.7 (C-1 ), 70.8 (C-3 ), 70.1 (C-2 ), 68.5 (C-4 ), 61.8
the resultant solution was neutralized with Amberlite IR-120 (H ).
Filtration and concentration followed by solid phase extraction
with Sep-Pak Plus C-18 cartridge (0e40% MeCN in H
crude compound that was further purified by preparative RP-HPLC
28% MeCN in H O, t
¼ 5.0 min). The title compound 2 (17 mg, 71%)
was obtained as a colorless solid. [
0
0
0
(C-5 ), 37.6 (C-7), 36.5 (C-7 ), 20.8 (Ac), 20.74 (Ac), 20.71 (Ac).
ESIHRMS m/z 489.1755 [MþH]þ (calcd for C25
2
O) gave the
29
H O10, 489.1761).
0
(
2
R
4.9. 4 -(
b
-D-xylopyranosyl)-3,5-dihydroxybibenzyl (2)
2
3
a]
D
ꢀ38.4 (c 0.59, MeOH). IR
1 1
ꢀ
ꢁ
(film)
n
max 3383, 2925, 1602, 1510, 1229, 1076, 837 cm ; H NMR
To cold (0 C) and stirred solution of 13 (78 mg, 0.16 mmol) in
MeOH (6 mL) was added 0.15 mL of NaOMe (5.2 M in MeOH,
0.78 mmol) slowly. After being stirred for 1 h at room temperature,
0
0
(
400 MHz, CD
3
OD)
d
7.08 (d, J ¼ 8.5 Hz, 2H, H-2 , H-6 ), 6.99 (d,
0
0
J ¼ 8.5 Hz, 2H, H-3 , H-5 ), 6.11 (bs, 2H, H-2, H-6), 6.07 (bs, 1H, H-4),
00
00
þ
4
(
.85 (d, J ¼ 7.3 Hz,1H, H-1 ), 3.89 (dd, J ¼ 11.8,1.6 Hz,1H, H-6 ), 3.69
the resultant solution was neutralized with Amberlite IR-120 (H ).
00
00
00
dd, J ¼ 11.8, 5.1 Hz, 1H, H-6 ), 3.43 (m, 4H, H-2 ꢀH-5 ), 2.80 (dd,
Filtration and concentration followed by solid phase extraction
0
13
J ¼ 9.2, 6.4, 2H, H-7 ), 2.70 (dd, J ¼ 9.2, 6.4, 2H, H-7). C NMR
with Sep-Pak Plus C-18 cartridge (0e40% MeCN in H
crude compound that was further purified by preparative RP-HPLC
(30%MeCN in H O, t
100%) was obtained as a colorless solid. [
IR (film)
(400 MHz, CD
2
O) gave the
0
(
100 MHz, CD
3
OD)
d
159.3 (C-3, C-5), 157.4 (C-4 ), 145.4 (C-1), 137.1
0
0
0
0
0
(
1
(
C-1 ), 130.4 (C-2 , C-6 ), 117.6 (C-3 , C-5 ), 108.1 (C-2, C-6), 102.5 (C-
), 101.2 (C-4), 78.1 (C-3 ), 78.0 (C-5 ), 74.9 (C-2 ), 71.4 (C-4 ), 62.5
C-6 ), 39.3 (C-7), 38.0 (C-7 ). ESIHRMS m/z 415.1368 [MþNa]
2
R
¼ 6.0 min). The title compound 2 (58 mg,
23
0
0
00
00
00
00
a
]
D
ꢀ3.48 (c 0.21, MeOH).
0
0
0
þ
ꢀ1 1
nmax 3355, 2927, 1601, 1510, 1226, 1039, 837 cm . H NMR
0
0
(
calcd for C20 Na, 415.1369).
H O
24 8
3
OD)
d
7.07 (d, J ¼ 8.2 Hz, 2H, H-2 , H-6 ), 6.94 (d,
0
0
J ¼ 8.2 Hz, 2H, H-3 , H-5 ), 6.11 (bs, 2H, H-2, H-6), 6.07 (bs, 1H, H-4),
0
00 00 00
00
00
4.7. 4 -(2 ,3 ,4 -Tri-O-acetyl-
b
-D
-xylopyranosyl)-3,5-di(tert-
4.80 (d, J ¼ 7.2 Hz, 1H, H-1 ), 3.89 (dd, J ¼ 11.4, 5.4 Hz, 1H, H-5 ),
0
0
00
00
butyldimethylsilyloxy)bibenzyl (12)
3.55 (m, 1H, H-4 ), 3.40 (m, 2H, H-3 , H-5 ), 2.79 (dd, J ¼ 9.5, 6.9,
0
13
2
CD
H, H-7 ), 2.69 (dd, J ¼ 9.5, 6.9, 2H, H-7); C NMR (100 MHz,
ꢁ
0
0
To a cold (ꢀ20 C) and stirredsolution of 7 (0.11 g, 0.24 mmol) and
3
OD)
d
159.3 (C-3, C-5), 157.2 (C-4 ), 145.4 (C-1), 137.1 (C-1 ),
0 0 0 0 00
2
,3,4-tri-O-acetyl-
a
-D
-xylopyranosyl trichloroacetimidate (11)
Cl (4 mL) was added 43 L of TMSOTf
, 4.7 mol). After being stirred for 40 min
L) and
130.4 (C-2 , C-6 ), 117.7 (C-3 , C-5 ), 108.1 (C-2, C-6), 103.1 (C-1 ),
0
0
00
00
00
(
0.20 g, 0.48 mmol) in CH
2
2
m
101.2 (C-4), 77.8 (C-3 ), 74.8 (C-2 ), 71.1 (C-4 ), 66.9 (C-5 ), 39.4 (C-
0
solution (0.11 M in CH
2
Cl
2
m
7), 38.0 (C-7 ). ESIHRMS m/z 385.1251 [MþNa]þ (calcd for
ꢁ
at ꢀ20 C, the resultant solution was quenched with TEA (15
m
19 22 7
C H O Na, 385.1263).
allowed to warm to room temperature. Concentration in vacuo,
followed by silica gel chromatography (20e40% EtOAc in hexane)
gave the title compound 12 (86 mg, 50% from 7) as a white solid.
4.10. Biological evaluation against melanogenesis
2
2
ꢀ1 1
[
a
]
D
ꢀ17.2 (c 2.42, CHCl
3
)
). IR (film)
d
n
max 1759,1162, 832, 781 cm . H
The assay was performed as reported previously with a few
modifications [18,19]. These modifications mainly involve preclud-
ing the use of antibiotics and hormones in the assay. Briefly, B16F0
0
0
NMR (400 MHz, CDCl
3
7.06 (d, J ¼ 8.6, 2H, H-2 , H-6 ), 6.87 (d,
0
0
J ¼ 8.6, 2H, H-3 , H-5 ), 6.24 (d, J ¼ 2.2, 2H, H-2, H-6), 6.14 (t, J ¼ 2.2,
00
00
4
1
(
1
7
H, H-4), 5,21 (t, J ¼ 7.9,1H, H-3 ), 5.15 (dd, J ¼ 6.1, 7.9,1H, H-2 ), 5.09
melanoma cells (ATCC CRL-6322), adjusted to 1.0 ꢂ 10 cells/well,
0
0
00
d, J ¼ 6.1, 1H, H-1 ), 4.99 (dt, J ¼ 4.8, 7.9, 1H, H-4 ), 4.19 (dd, J ¼ 4.8,
were incubated in Dulbecco's modified Eagle's medium (DMEM)
0
0
00
2.0, 1H, H-5 ), 3.48 (dd, J ¼ 7.9, 12.0, 1H, H-5 ), 2.76 (m, 4H, H-7, H-
containing 5% fetal bovine serum (FBS) under 5% CO
2
atmosphere at
0
13
ꢁ
), 2.05 (m, 9H, Ac), 0.97 (m, 18H, TBS), 0.16 (m, 12H, TBS). C NMR
37 C for 24 h. The medium was replaced with DMEM containing 5%
FBS and an ethanolic solution of the investigated sample. After 72 h
of treatment under the conditions described above, the mediumwas
replaced again with the sample-containing medium. After 72 h,
trypsin was added to all the wells and the harvested cells were
pelleted by centrifugation. The cell pellets were washed with
phosphate buffer, 5% trichloroacetic acid, 33% ethanol in ether, and
ether. The pellet was then air dried and lysed with 1 M NaOH so-
(
100 MHz, CDCl
3
)
d
170.0 (Ac),169.9 (Ac),169.4 (Ac),156.3 (C-3, C-5),
54.8 (C-4 ),143.5 (C-1),136.5 (C-1 ),129.5 (C-2 , C-6 ),116.8 (C-3 , C-
), 113.7 (C-2, C-6), 109.8 (C-4), 98.8 (C-1 ), 70.8 (C-3 ), 70.2 (C-2 ),
8.5 (C-4 ), 61.8 (C-5 ), 37.8 (C-7), 36.8 (C-7 ), 25.7 (TBS), 18.2 (TBS),
0
0
0
0
0
1
5
6
2
0
00
00
00
0
0
00
0
0.8 (Ac), 20.73 (Ac), 20.69 (Ac), ꢀ4.4 (TBS). ESIHRMS m/z 717.3477
þ
[MþH] (calcd for C37
57 2
H O10Si , 717.3490).
0
00 00 00
ꢁ
4.8. 4 -(2 ,3 ,4 -Tri-O-acetyl-
b
-D
-xylopyranosyl)-3,5-
lution at 100 C for 10 min and the optical density (475 nm) of the
dihydroxybibenzyl (13)
resultant solution was measured. The melanin concentration was
determined by comparison with the standard curve (plotted using
commercially available melanin). Determination of the protein
concentration was performed by BCA protein assay method [20]
ꢁ
To a cold (0 C) and stirred solution of 12 (0.13 g, 0.18 mmol) in
THF (3 mL) was added TBAF trihydrate (0.24 g, 0.76 mmol). After

C. Oode et al. / European Journal of Medicinal Chemistry 87 (2014) 862e867
867
using the cell lysate prepared by 0.5% Triton X-100 treatment.
Negative and positive controls containing ethanol or kojic acid were
conducted in parallel. Cell viability was determined by MTT assay
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[
21]. All the assays were performed in triplicate (or more replicates)
[14] F. Orsini, F. Pelizzoni, B. Bellini, G. Miglierini, Synthesis of biologically active
polyphenolic glycosides (combretastatin and resveratrol series), Carbohydr.
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on separate occasions, and the Student's t-test was used to deter-
mine the level of significance between different groups.
[
[
[
15] W.M. Pearlman, Noble metal hydroxides on carbon nonpyrophoric dry cata-
lysts, Tetrahedron Lett. 8 (1967) 1663e1664.
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Acknowledgments
We are grateful to Drs. Michinori Karikomi and Yoichi Yamada
Utsunomiya University) for technical assistance with measurement
(
[
[
of the optical rotations and the NMR spectra. This study was sup-
ported in part by JSPS KAKENHI Grant Number 23510252. C. O.
thanks Saito Yutaka Scholarship of Utsunomiya University Foun-
dation for graduate fellowship.
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Appendix A. Supplementary data
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