Alkaloid constituents from flower buds and leaves of sacred lotus (Nelumbo nucifera, Nymphaeaceae) with melanogenesis inhibitory activity in B16 melanoma cells

Article abstract of DOI:10.1016/j.bmc.2012.11.038

Methanolic extracts from the flower buds and leaves of sacred lotus (Nelumbo nucifera, Nymphaeaceae) were found to show inhibitory effects on melanogenesis in theophylline-stimulated murine B16 melanoma 4A5 cells. From the methanolic extracts, a new alkaloid, N-methylasimilobine N-oxide, was isolated together with eleven benzylisoquinoline alkaloids. The absolute stereostructure of the new alkaloid was determined from chemical and physicochemical evidence. Among the constituents isolated, nuciferine, N-methylasimilobine, (-)-lirinidine, and 2-hydroxy-1-methoxy-6a,7-dehydroaporphine showed potent inhibition of melanogenesis. Comparison of the inhibitory activities of synthetic related alkaloids facilitated characterization of the structure-activity relationships of aporphine- and benzylisoquinoline-type alkaloids. In addition, 3-30 μM nuciferine and N-methylasimilobine inhibited the expression of tyrosinase mRNA, 3-30 μM N-methylasimilobine inhibited the expression of TRP-1 mRNA, and 10-30 μM nuciferine inhibited the expression of TRP-2 mRNA.

Full text of DOI:10.1016/j.bmc.2012.11.038

Bioorganic & Medicinal Chemistry 21 (2013) 779–787
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Alkaloid constituents from flower buds and leaves of sacred lotus
(Nelumbo nucifera, Nymphaeaceae) with melanogenesis inhibitory
activity in B16 melanoma cells q
Seikou Nakamura a, Souichi Nakashima a, Genzo Tanabe b, Yoshimi Oda a, Nami Yokota a,
Katsuyoshi Fujimoto a, Takahiro Matsumoto a, Rika Sakuma a, Tomoe Ohta a, Keiko Ogawa a, Shino Nishida a,
Hisako Miki a, Hisashi Matsuda a, Osamu Muraoka b, Masayuki Yoshikawa a,
⇑
a Department of Pharmacognosy, Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan
b School of Pharmacy, Kinki University, 3-4-1 Kowakae, Higashi-osaka, Osaka 577-8502, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Methanolic extracts from the flower buds and leaves of sacred lotus (Nelumbo nucifera, Nymphaeaceae)
were found to show inhibitory effects on melanogenesis in theophylline-stimulated murine B16 mela-
noma 4A5 cells. From the methanolic extracts, a new alkaloid, N-methylasimilobine N-oxide, was isolated
together with eleven benzylisoquinoline alkaloids. The absolute stereostructure of the new alkaloid was
determined from chemical and physicochemical evidence. Among the constituents isolated, nuciferine,
N-methylasimilobine, (ꢀ)-lirinidine, and 2-hydroxy-1-methoxy-6a,7-dehydroaporphine showed potent
inhibition of melanogenesis. Comparison of the inhibitory activities of synthetic related alkaloids facili-
tated characterization of the structure-activity relationships of aporphine- and benzylisoquinoline-type
Received 24 September 2012
Revised 12 November 2012
Accepted 16 November 2012
Available online 5 December 2012
Keywords:
Nelumbo nucifera
Melanogenesis inhibitor
N-Methylasimilobine N-oxide
Aporphine-type alkaloid
Benzylisoquinoline-type alkaloid
Medicinal flower
alkaloids. In addition, 3–30
nase mRNA, 3–30 M N-methylasimilobine inhibited the expression of TRP-1 mRNA, and 10–30
nuciferine inhibited the expression of TRP-2 mRNA.
lM nuciferine and N-methylasimilobine inhibited the expression of tyrosi-
l
lM
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
glycosides.2 However, detailed chemical and pharmacological
studies on the bioactive constituents of the flower buds and leaves
of N. nucifera have yet to be reported.
The Nymphaeaceae plant Nelumbo (N) nucifera Gaertn. is widely
distributed throughout Asia and Oceania and has many common
names (e.g., sacred lotus, Indian lotus, Chinese water lily). Since an-
cient times, all parts of this plant have been used not only as a veg-
etable and food garnish but also as a traditional medicine. For
example, the flower buds of this plant are used both to treat blood
vomiting, bleeding due to internal and external injury and various
skin diseases, and as a sedative and an anti-inflammatory in tradi-
tional Chinese medicine. The flower buds are also used in Indian
Ayurvedic medicine for the treatment of premature ejaculation,
abdominal cramps and bloody discharge and as a cardiac tonic.
The leaves of N. nucifera are used to treat hematemesis, epistaxis,
hemoptysis, hematuria, hyperlipidemia, and obesity. As chemical
constituents of the flower buds of N. nucifera, many flavonol glyco-
sides have been isolated from the stamens, while the leaves are re-
ported to be rich in a number of alkaloids together with flavonol
We have been searching for inhibitors of melanin production
from natural medicines to develop new compounds for cosmetic
products and depigmenting agents.3–5 Recently, we reported the
isolation and structure elucidation of melanogenesis inhibitors
from the flower buds of Camellia japonica.6,7 As a continuation of
studies on melanogenesis inhibitors from medicinal flowers,8–18
we found that methanolic (MeOH) extracts and alkaloid fractions
from the flower buds and leaves of N. nucifera showed inhibitory
effects on melanogenesis.
In this paper, we describe the isolation of aporphine- and ben-
zylisoquinoline-type alkaloids with melanogenesis inhibitory
activity from the flower buds and leaves of N. nucifera, as well as
elucidation of the structure of a new alkaloid, N-methylasimilobine
N-oxide. In addition, the inhibitory effects of related commercial
and synthesized isoquinoline-type alkaloids on melanogenesis
were examined and their structure-activity relationships were
characterized. Lastly, the principal alkaloid constituents with
melanogenesis inhibitory activity were examined for their effects
on the expression of tyrosinase and TRP-1 and TRP-2 mRNA.
q
This paper is number 37 in our series ‘‘Medicinal Flower’’. For paper number 36,
see Ref. 1.
⇑
Corresponding author. Tel.: +81 75 595 4633; fax: +81 75 595 4768.
E-mail address: shoyaku@mb.kyoto-phu.ac.jp (M. Yoshikawa).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.11.038

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S. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 779–787
2.2. Structure of N-methylasimilobine N-oxide (1)
2. Results and discussion
N-Metylasimilobine oxide (1) was obtained as a pale yellow oil
2.1. Isolation of melanogenesis inhibitors from the flower buds
and leaves of N. nucifera
with negative optical rotation (½a D24
ꢀ56.3°). The IR spectrum of 1
ꢁ
showed absorption bands at 3400 and 1509 cmꢀ1 ascribable to hy-
droxyl and aromatic groups. In the EI-MS spectrum of 1, a molec-
ular ion peak was observed at m/z 297 (M+) and the molecular
formula of 1 was determined to be C18H19NO3 by high-resolution
(HR) MS measurement. The 1H (CD3OD) and 13C NMR (Table 4)
spectra of 1, which were assigned by various NMR experiments,42
showed signals due to two methyls, [d 3.39 (O N-CH3), 3.56
(OCH3)], three methylenes, a methane, and five aromatic protons.
The proton and carbon signals in the 1H and 13C NMR data of 1
were superimposable on those of N-methylasimilobine (5), except
for those around the N-oxide, whereas the proton and carbon sig-
nals in the N-oxide part were similar to those of nuciferine N-oxide
(3).26 The relative stereostructure of the N-oxide region of 1 was
clarified by NOESY experiment, which showed a NOE correlation
between the 6a-proton and the N-methyl group. Next, oxidation
of N-methylasimilobine (5) with m-chloroperbenzoic acid (m-
CPBA)26 yielded 1. In addition, O-methylation of 5 with trimethysi-
lyldiazomethane (TMSCH2N2) yielded nuciferine (2), whose abso-
lute configuration has been determined by synthesis.25
Consequently, the absolute configuration of the 6a position in 1
and 5 was elucidated to be R. On the basis of these findings, the
absolute stereostructure of 1 was determined to be as shown in
Figure 2.
Melanocytes can be stimulated by many effectors including
ultraviolet radiation19 and
a
-melanocyte-stimulating hormone
-MSH).20 It is generally accepted that the cAMP pathway plays
a key role in the regulation of melanogenesis and that cAMP is in-
volved in
-MSH-stimulated signal transduction.21,22 In the pres-
(a
a
ent study, we used a phosphodiesterase inhibitor, theophylline,
to stimulate B16 melanoma 4A5 cells. As shown in Table 1, the
MeOH extract from the flower buds of N. nucifera cultivated in
Thailand significantly inhibited melanogenesis with an IC50 value
of 20 lg/mL in theophylline-stimulated murine B16 melanoma
4A5 cells. The MeOH extract of the leaves exhibited a moderate ef-
fect, whereas the inhibitory activity of the stamens and seeds was
weak. The extracts from the flower buds, stamens, and seeds
showed no cytotoxic effects and that of the leaves showed weak
cytotoxicity at a high concentration of 100 lg/mL (Table 1).
The MeOH extracts prepared under reflux from the flower buds
and leaves were suspended in H2O, and then extracted with ethyl
acetate (EtOAc) followed by 1-butanol (1-BuOH). The EtOAc- and
1-BuOH-soluble fractions from the flower buds strongly inhibited
melanogenesis with IC50 values of 9.5 and 10.1
The 1-BuOH-soluble fraction from the leaves also showed potent
inhibition (IC50 34.6 g/mL). These fractions showed weak cyto-
lg/mL, respectively.
l
toxic effects at a high concentration (Table 2).
2.3. Effects of alkaloids constituents (2–12) and related
alkaloids (19a, 19b, 23) on melanogenesis
The EtOAc- and 1-BuOH-soluble fractions from the flower buds
and leaves were subjected to ordinary- and reversed-phase silica
gel column chromatography, followed by HPLC to yield a new alka-
loid termed N-methylasimilobine N-oxide (1),⁄⁄ together with
nuciferine (2),⁄,⁄⁄,23-25 nuciferine N-oxide (3),⁄⁄,26,27 N-nornucifer-
ine (4),⁄,⁄⁄,25,28 N-methylasimilobine (5),⁄,⁄⁄,24,29 asimilobine
(6),⁄⁄,30 (ꢀ)-lirinidine (7, 5-demethylnuciferine),⁄,⁄⁄,31,32 dehy-
dronuciferine (8),⁄⁄,33,34 2-hydroxy-1-methoxy-6a,7-dehydroapor-
phine (9),⁄⁄,34 lysicamine (10),⁄,⁄⁄,35 D,L-armepavine (11),⁄⁄,36
Among the constituents isolated from the flower buds and
leaves of N. nucifera, the N-methylated aporphine-type alkaloids
nuciferine (2), N-methylasimilobine (5), (ꢀ)-lirinidine (7) and 2-
hydroxy-1-methoxy-6a,7-dehydroaporphine
(9)
significantly
inhibited melanogenesis with IC50 values of 15.8, 14.5, 19.3, and
13.3 M, respectively, whereas dehydronuciferine (8; IC50 ca.
85
M),43 and aporphine-type alkaloids lacking the N-methyl
l
pronuciferine (12),⁄,⁄⁄,37 kaempferol 3-O-b-
quercetin 3-O-b-
-glucopyranoside,⁄⁄,39 isorhamnetin 3-O-b-
glucopyranoside,⁄,40 quercetin 3-O-b- -galactpyranoside,⁄⁄,41 (+)-
D
-glucopyranoside,⁄,38
l
group, namely N-nornuciferine (4), asimilobine (6) and lysicamine
(10), did not show such potent inhibitory activity. The N-methyl-
ated benzylisoquinoline-type alkaloid, D,L-armepavine (11), also
showed activity equipotent to that of the N-methylated apor-
phine-type alkaloids. On the other hand, nuciferine N-oxide (3)
and an N-methylated proaporphine-type alkaloid, pronuciferine
(12), showed weak inhibitory activity. In addition, two alkaloids
(7, 10) were observed to have cytotoxic effects at a high concentra-
tion (100 lM) (Table 5). These results indicate that the N-methyl
group is essential for potent inhibitory activity, while the N-oxide
group markedly reduces this activity.
Next, to obtain further information on the structural require-
ments for potent inhibition of melanogenesis, the effects of related
commercial (13, 14) and synthesized isoquinoline-type alkaloids
D
D-
D
catechine,⁄⁄ b-sitsterol,⁄ and palmitic acid⁄ (⁄, from flower buds;
⁄⁄, from leaves) (Fig. 1). The flower buds of N. nucifera were ex-
tracted with MeOH at room temperature to give a MeOH extract
(rt), which was partitioned with a diethyl ether (Et2O)/1% aqueous
acetic acid (AcOH) mixture to yield an Et2O fraction and aqueous
AcOH phase. The aqueous AcOH phase was made basic with 10%
NH4OH solution, and then subjected to extraction with Et2O. The
Et2O-soluble fraction was washed with 1% aqueous NaOH to yield
an alkaloid fraction. The alkaloid fraction from the flower buds
showed potent inhibition of melanogenesis with an IC50 value of
4.2 lg/mL. Using a procedure to that described above, 2, 4, 5, 7,
10, and 12 were isolated from the alkaloid fraction (Table 3).
Table 1
Inhibitory effects of the MeOH extracts from several parts of N. nucifera on melanogenesis in B16 melanoma 4A5 cells
Concn (lg/mL)
Inhibition (%)
10
IC50 (lg/mL)
Control
1
3
30
100
Flower budsa
Stamena
Seedsa
0.0 2.7
0.0 3.6
0.0 2.5
0.0 2.7
14.3 1.2⁄⁄
8.0 3.2
ꢀ5.3 2.3
1.3 2.5
16.5 2.4⁄⁄
1.5 4.1
ꢀ0.3 2.9
6.8 1.8
29.8 3.2⁄⁄
62.1 1.6⁄⁄
8.8 5.1
91.7 1.8⁄⁄
12.9 2.3⁄⁄⁄
18.0 2.3
20.3
—
—
6.0 4.4
9.9 7.4
14.7 2.7
Leavesb
10.3 1.0⁄⁄
12.1 1.7⁄⁄
45.2 1.1⁄⁄
—
Each value represents the mean S.E.M., (n = 4).
Significantly different from the control group, ⁄p < 0.05, ⁄⁄p < 0.01.
a
The cell viabilities for the MeOH extracts from the flower buds, stamen, and seeds at 100 lM are more than 96%.
The cell viability for the MeOH extract from the leaves at 100 lM is 86.5%.
b

S. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 779–787
781
Table 2
Inhibitory effects of the EtOAc-, 1-BuOH- and H2O-soluble fractions from the flower buds and leaves of N. nucifera on melanogenesis in B16 melanoma 4A5 Cells
Concn (
l
g/mL)
Inhibition (%)
10
IC50 (lg/mL)
control
1
3
30
100
Flower buds
EtOAc fractiona,b
1-BuOH fractiona,c
H2O fractiona,b
0.0 3.3
0.0 5.5
0.0 4.2
4.0 6.4
13.4 3.1
13.8 3.0
26.9 4.4⁄⁄
46.9 1.6⁄⁄
81.8 2.5⁄⁄
75.7 1.1⁄⁄
18.0 1.4⁄⁄
93.3 3.0⁄⁄
73.5 2.7⁄⁄
22.9 0.3⁄⁄
9.5
10.1
—
ꢀ7.2 11.4
48.2 1.1⁄⁄
12.6 2.0⁄⁄
14.7 2.2
Leaves
EtOAc fractiona,b
1-BuOH fractiona,b
H2O fractiona,b
0.0 4.0
0.0 2.6
0.0 2.5
11.5 3.8
5.0 3.7
13.5 3.6⁄
12.7 4.5
17.3 2.6⁄⁄
14.0 0.7⁄⁄
24.1 1.1
2.9 22.1
42.8 3.9⁄⁄
27.6 1.9⁄⁄
11.4 1.6⁄⁄
83.5 0.4
—
34.6
—
12.5 3.6⁄
29.7 1.7⁄⁄
46.2 3.2⁄⁄
Each value represents the mean S.E.M., (n = 4).
Significantly different from the control group, ⁄p < 0.05, ⁄⁄p < 0.01.
a
The cell viabilities for all fractions at 30
l
M are more than 88%.
The cell viabilities for the EtOAc and H2O fractions from flower buds and the EtOAc, 1-BuOH, and H2O fractions from leaves at 100
The cell viability for the 1-BuOH fraction from flower buds at 100 M is 36.9%.
b
c
lM are more than 78%.
l
Figure 1. Alkaloids constituents from flower buds and leaves.
Table 3
Inhibitory effects of the MeOH extract (rt), Et2O-, aq AcOH-, and alkaloid fractions from the flower buds of N. nucifera on melanogenesis in B16 melanoma 4A5 cells
Concn (
l
g/mL)
Inhibition (%)
10
IC50 (lg/mL)
Control
1
3
30
100
MeOH extract (rt)a
Et2O fractionb
0.0 2.3
0.0 2.4
0.0 3.2
0.0 2.1
ꢀ13.3 1.7
1.7 3.1
ꢀ4.4 2.6
3.6 1.3
5.2 2.4
42.1 2.8⁄⁄
ꢀ0.3 3.6
82.3 0.3⁄⁄
17.1 3.8
86.2 0.5⁄⁄
—
35.7
—
25.9
4.2
5.2 2.3
aq AcOH fractiona
Alkaloids fractionc
ꢀ6.8 3.1
7.0 1.4
27.8 1.5⁄⁄
76.8 0.5⁄⁄
52.0 0.9⁄⁄
82.4 0.3⁄⁄
14.9 1.9⁄⁄
36.0 1.2⁄⁄
Each value represets the mean S.E.M., (n = 4).
Significantly different from the control group, ⁄p < 0.05, ⁄⁄p < 0.01.
a
The cell viabilities for the MeOH extract (rt) and its aq AcOH fraction at 30 and 100
lM are more than 86%.
b
c
The cell viability for the the Et2O fraction at 30 and 100
lM are 86.2% and 68.6%.
The cell viability for the the alkaloids fraction at 10 and 30
lM are 94.4% and 55.2%.
(19a, 19b, 23) were examined. Compounds 19a, 19b, and 23 were
synthesized according to previous reports (Fig. 3).44-51 As shown in
Table 5, a simple isoquinoline (13) and N-methylated isoquinoline
(14) did not show inhibitory activity. Thus, the benzylisoquinoline
or aporphine structure appears to be essential for inhibition. The
synthesized benzylisoquinoline- and aporphine-type alkaloids
(19a, 23) showed potent inhibition with IC50 values of 5.9 and
5.0 lM, respectively, and a N-methylated aporphine-type alkaloid

782
S. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 779–787
Table 4
(19b) with a methoxy group at the 2-position showed more potent
13C NMR (150 MHz, CD3OD) date for 1
activity than 19a and 23. These findings suggest that the N-methyl
group in the aporphine- or benzylisoquinoline-type skeleton is
essential for greater inhibition of melanogenesis, and an oxygen
group at the 2-position enhances this activity.
Position
1
Position
1
1
2
3
3a
4
5
6a
7
145.9
151.7
116.0
128.2
25.1
65.2
72.1
30.7
135.3
8
9
10
11
11a
11b
11c
N-CH3
O-CH3
129.4
128.9
128.4
128.7
132.6
128.3
121.9
58.0
2.4. Effects of nuciferine (2) and N-methylasimilobine (5) on the
expression of tyrosinase and TRP-1 and TRP-2 mRNA
Tyrosinase and tyrosinase-related protein 1 (TRP-1) and TRP-2
are known to catalyze the major steps in melanin synthesis.52 Re-
cently, we reported that several flavonoids inhibited the mRNA
expression of tyrosinase and TRP-1 and -2.3,4 In the present study,
to clarify the mechanism of action of the principal alkaloid constit-
uents 2 and 5 that had potent melanogenesis inhibitory activity,
we examined their effects on the mRNA expression of tyrosinase
and TRP-1 and -2 in B16 melanoma 4A5 cells. As a result, 2 and
5 were found to inhibit the expression of tyrosinase mRNA at 3–
7a
60.4
30
3–30
10 and 30
zyme activity of mushroom tyrosinase (less than 3% inhibition at
100 M). Collectively, these findings suggest that the inhibition
l
M. In addition, 5 inhibited the expression of TRP-1 mRNA at
M, whereas 2 inhibited the expression of TRP-2 mRNA at
M (Table 6). However, 2 and 5 did not inhibit the en-
l
l
l
of melanogenesis by 2 and 5 is partly mediated by the reduced
expression of tyrosinase and TRP-1 and TRP-2 mRNA, but the de-
tailed mechanism of action remains to be studied further.
3. Experimental
3.1. General experimental procedures
The following instruments were used to obtain physical data:
specific rotations,
a
Horiba SEPA-300 digital polarimeter
(l = 5 cm); IR spectra, a Shimadzu FTIR-8100 spectrometer; FABMS
Figure 2. Structure of N-methylasimilobine N-oxide (1).
Table 5
Inhibitory effects of alkaloid constituents, 2ꢀ14, 19a, 19b, and 23 from flower buds and leaves of N. nucifera on melanogenesis in B16 melanoma 4A5 cells
(
l
M) Inhibition (%)
10
32.1 1.5⁄⁄
IC50 (lM)
Control
1
3
30
100
2a,e
0.0 2.9
0.0 5.8
0.0 2.3
0.0 1.9
0.0 2.1
0.0 2.8
0.0 7.2
0.0 1.9
0.0 6.0
0.0 2.8
0.0 2.4
0.0 0.8
0.0 4.2
0.0 2.8
0.0 1.8
0.0 3.7
11.5 0.2⁄⁄
ꢀ4.8 4.2
5.8 1.7
16.5 1.7⁄⁄
ꢀ3.2 7.5⁄⁄
16.2 4.9
19.7 2.5⁄⁄
ꢀ7.4 3.4
11.1 2.0⁄⁄
4.8 2.3
72.3 2.8⁄⁄
33.7 2.5⁄⁄
17.6 1.8⁄⁄
70.6 1.0⁄⁄
39.7 1.8⁄⁄
65.9 0.7⁄⁄
8.0 5.1⁄⁄
87.4 5.0⁄⁄
65.3 0.9⁄⁄
50.4 1.9
89.3 0.5⁄⁄
88.4 0.8⁄⁄
91.9 1.9⁄⁄
90.5 1.2⁄⁄
36.2 1.8⁄⁄
—
15.8
ca. 43
62.9
14.5
>100
19.3
ca. 85
13.3
—
25.6
47.9
—
3a,c
ꢀ0.1 5.7
12.1 2.7⁄
37.9 1.7⁄⁄
12.1 4.5⁄
27.3 1.1⁄⁄
11.8 1.9
4a,c
5a,c
15.4 3.8⁄⁄
7.4 0.9
ꢀ2.6 2.0
14.5 4.2
10.9 1.3
4.8 3.7
6a,e
7a,f
8b,d
78.4 0.8⁄⁄
78.7 0.5⁄⁄
—
9a,e
13.2 3.3⁄
8.7 3.7⁄⁄
18.2 3.5
16.8 2.7⁄⁄
37.6 2.7⁄⁄
ꢀ1.5 2.0⁄⁄
34.0 1.5
10b,f
11a,c
12a,c
13a,e
14a,c
19aa,f
19bg
23a,d
7.5 1.3
4.0 4.1
80.3 1.2⁄⁄
66.4 0.6⁄⁄
26.1 3.4⁄⁄
15.9 0.8⁄⁄
22.3 3.0⁄⁄
—
18.3 2.6⁄⁄
40.3 0.9⁄⁄
2.8 2.6⁄⁄
16.5 2.0⁄⁄
72.2 0.4
ꢀ12.6 3.1
10.6 0.9⁄⁄
30.0 1.9
ꢀ41.5 4.4
9.7 3.5⁄⁄
42.7 2.7
ꢀ54.2 2.3
9.4 2.8⁄⁄
59.2 1.4
—
5.9
2.0
5.0
39.1 2.7⁄⁄
24.5 1.0⁄⁄
54.7 5.9⁄⁄
40.5 3.4⁄⁄
76.0 2.0⁄⁄
59.7 1.6⁄⁄
82.5 2.1⁄⁄
51.8 1.1⁄⁄
75.4 0.8⁄⁄
Control
0.0 1.4
10
30
100
300
1000
Arbutina
10.6 0.6⁄⁄
20.4 0.5⁄⁄
38.1 0.9⁄⁄
61.5 0.6⁄⁄
83.7 0.5⁄⁄
174
Each value represents the mean S.E.M., (n = 4).
Significantly different from the control group, ⁄p < 0.05, ⁄⁄p<0.01.
a
The cell viability at 30
The cell viability at 30
The cell viability at 100
The cell viability at 100
The cell viability at 100
The cell viabilities for 7, 10, and 19a at 100
The cell viabilities for 19b at 10 and 30 lM are 98.0% and 76.7%.
l
l
l
l
l
M is more than 86%.
M is more than 69%.
M is more than 82%.
M is more than 77%.
M is more than 46%.
b
c
d
e
f
l
M are 39.7%, 22.5%, and 41.1%.
g

S. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 779–787
783
Figure 3. Synthesis of isoquinoline-type alkaloids.
Table 6
Effects of
melanoma 4A5 cells
precoated TLC plates with Silica gel 60F254 (Merck, 0.25 mm) (or-
dinary-phase) and Silica gel RP-18 F254S (Merck, 0.25 mm) (re-
verse-phase); reversed-phase HPTLC, precoated TLC plates with
Silica gel RP-18 WF254S (Merck, 0.25 mm). Detection was achieved
by spraying with 1% Ce(SO4)2–10% aqueous H2SO4 followed by
heating.
2 and 5 on expression of tyrosinase and TRP-1 and 2 mRNA in B16
Concentration (lM)
0
3
10
30
Tyrosinase mRNA/b-actin mRNA
2
5
1.00 0.05
1.00 0.02
0.61 0.03⁄⁄
0.65 0.04⁄⁄
0.64 0.01⁄⁄
0.76 0.04⁄⁄
0.38 0.00⁄⁄
0.53 0.01⁄⁄
3.2. Plant material
TRP-1 mRNA/b-actin mRNA
The flower buds and leaves of N. nucifera, which was cultivated
in Khon Kaen province, Thailand, were purchased from Mae Chu
Co., Ltd (Nara, Japan) via Kwanjit Farm Co., Ltd (Nakhon Ratchas-
ima, Thailand) at 2010. The stamens and seed of N. nucifera were
collected by Dr. Y. Pongpiriyadacha (Rajamangala University of
Technology) in Nakhonsi Thammarat province, Thailand at 2010.
Voucher specimens (flower buds, leaves, stamens, and seeds) of
this plant are on file in our laboratory.
2
5
1.00 0.05
1.01 0.02
0.76 0.08
0.74 0.13
0.36 0.02⁄⁄
0.49 0.03⁄⁄
0.79 0.08⁄⁄
0.72 0.02⁄⁄
TRP-2 mRNA/b-actin mRNA
2
5
1.01 0.00
1.00 0.02
1.11 0.06
1.10 0.04
0.71 0.07⁄⁄
0.96 0.05
0.65 0.07⁄⁄
0.88 0.02⁄
Each value represents the mean S.E.M., (n = 4).
Significantly different from the control group, ⁄p < 0.05, ⁄⁄p < 0.01.
and HRFABMS, a JEOL JMS-SX 102A mass spectrometer; 1H NMR
spectra, JEOL JNM LA 500 (500 MHz) and JEOL JNM ECA 600
(600 MHz) spectrometers; 13C NMR spectra, JEOL JNM LA 500
(125 MHz) and JEOL JNM ECA 600 (150 MHz) spectrometers; and
HPLC, a Shimadzu RID-6A refractive index and SPD-10Avp UV–vis
detectors. A COSMOSIL 5C18-MS-II and YMC-pack ODS-A {[250 ꢂ
3.3. Extraction and isolation
(1) The flower buds (1.0 kg), leaves (0.9 kg), stamens (1.0 g) and
seeds (1.0 g) were powdered and extracted three times with
MeOH under reflux for 3 h. Evaporation of the solvent under
reduced pressure provided the MeOH extracts (155.0, 157.4,
0.13, 0.11 g, respectively). The MeOH extracts (145.0,
150.6 g) of the flower buds and leaves were partitioned into
on EtOAc–H2O (1:1, v/v) mixture to furnish the EtOAc-
soluble fractions (31.2, 39.9 g) and aqueous fraction. The
aqueous fractions were extracted with 1-BuOH to give the
1-BuOH- (41.0, 34.3 g) and H2O- (72.8 g, 76.4 g) soluble
fractions.
4.6 mm id (5
l
m) for analytical purposes] and [250 ꢂ 20 mm id
(5 m) for preparative purposes], Nacalai Tesque and YMC} column
l
was used. The following experimental conditions were used for
chromatography: ordinary-phase silica gel column chromatogra-
phy, Silica gel BW-200 (Fuji Silysia Chemical, Ltd, 150–350 mesh);
reverse-phase silica gel column chromatography, Chromatorex
ODS DM1020T (Fuji Silysia Chemical, Ltd, 100–200 mesh); TLC,

784
S. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 779–787
(2) The EtOAc-soluble fraction (31.2 g) from the flower buds
10-H), 7.37 (1H, d like, J = 7.6 Hz, 8-H), 8.31 (1H, d like, J = 7.6 Hz,
11-H); 13C NMR: given in Table 4; EIMS m/z 297 [M+]; HREIMS
m/z 297.1369 (Calcd for C18H19NO3 [M+], m/z 297.1365).
was subjected to normal-phase silica gel column chromatog-
raphy (1.0 kg, n-hexane?n-hexane-EtOAc?EtOAc?CHCl3–
MeOH–H2O) followed by reversed-phase silica gel column
chromatography (MeOH–H2O) and finally HPLC (column:
COSMOSIL 5C18-MS-II, eluent: MeOH–H2O) to give nucifer-
ine (2, 148 mg), N-nornuciferine (4, 11.2 mg), lysicamine
3.5. Oxidation of N-methylasimilobine (5)
Solution of N-methylasimilobine (5, 4.0 mg, 0.014 mmol) in
CHCl3 (1.0 mL) was treated with m-CPBA (75%, wet with H2O,
9.8 mg, 0.043 mmol), and the mixture was stirred at room temper-
ature for 2 h. Removal of the solvent from the reaction mixture un-
der reduced pressure furnished a residue, which was purified
by normal-phase silica gel column chromatography [CHCl3–
MeOH–H2O = 7:3:1, lower phase] and HPLC [YMC-pack ODS-A,
MeOH–H2O in 1% AcOH (50:50)] to give N-methylasimilobine
N-oxide (1, 1.9 mg). N-methylasimilobine N-oxide was identified
by comparison of the physical data (½aꢁD, 1H NMR, 13C NMR, and
MS) with that of natural form of N-methylasimilobine N-oxide.
(10,
36.5 mg),
kaempferol
3-O-b-D-glucopyranoside
(6.1 mg), isorhamnetin 3-O-b-
D-glucopyranoside (81.0 mg),
b-sitosterol (141 mg), and palmitic acid (96.3 mg). The
1-BuOH-soluble fraction (40.0 g) from the flower buds was
subjected to normal-phase silica gel column chromatogra-
phy (1.6 kg, CHCl3–MeOH–H2O) followed by preparative
TLC (silica-gel 60F, CHCl3–MeOH–H2O = 8:3:1) and HPLC
(COSMOSIL 5C18-MS-II, MeOH–H2O in 1% AcOH) to give
N-methylasimilobine (5, 6.6 mg), lysicamine (10, 102 mg),
and pronuciferine (12, 56.0 mg).
(3) The EtOAc-soluble fraction (36.9 g) from the leaves was sub-
jected to normal-phase silica gel column chromatography
(1.6 kg, CHCl3–MeOH?CHCl3–MeOH–H2O) followed by
reversed-phase silica gel column chromatography (MeOH–
H2O) and finally HPLC (YMC-pack ODS-A, MeOH–H2O in 1%
AcOH) to give N-methylasimilobine N-oxide (1, 3.3 mg),
nuciferine (2, 67.3 mg) nuciferine N-oxide (3, 40.7 mg), N-
nornuciferine (4, 2.3 mg), dehydronuciferine (8, 3.9 mg),
3.6. Methylation of N-methylasimilobine (5)
Solution of N-methylasimilobine (5, 5.0 mg, 0.018 mmol) in
MeCN (0.5 mL) and MeOH (0.5 mL) was treated with N,N-diisopyl-
ethylamine (0.006 mL, 0.036 mmol) and trimethylsilyldiazome-
thane (2 M in hexane, 0.018 mL, 0.036 mmol), and the mixture
was stirred at room temperature for 15 h. Removal of the solvent
from the reaction mixture under reduced pressure furnished a res-
idue, which was purified by normal-phase silica gel column chro-
matography [CHCl3–MeOH–H2O = 7:3:1, lower phase] to give
nuciferin (2, 4.0 mg). Nuciferin was identified by comparison of
the physical data ((½aꢁD, 1H NMR, 13C NMR, and MS) with that of
natural form of nuciferin.
lysicamine (10, 41.8 mg), quercetin 3-O-b-D-glucopyrano-
side (10.2 mg), quercetin 3-O-b- -galactopyranoside
D
(7.5 mg), and (+)-catechine (40.5 mg). The 1-BuOH-soluble
fraction (31.3 g) was subjected to normal-phase silica gel
column chromatography (1.3 kg, CHCl3–MeOH?CHCl3–
MeOH–H2O) followed by reversed-phase silica gel column
chromatography (MeOH–H2O) and finally HPLC (YMC-pack
ODS-A, MeOH–H2O in 1% AcOH) to give nuciferine (2,
83.0 mg), nuciferine N-oxide (3, 22.1 mg), N-methylasimilo-
bine (5, 282 mg), asimilobine (6. 149 mg), (ꢀ)-lirinidine (7,
7.2 mg), 2-hydroxy-1-methoxy-6a,7-dehydroaporphine (9,
2.9 mg), lysicamine (10, 3.0 mg), D,L-armepavine (11,
27.4 mg), and pronuciferine (12, 8.3 mg).
3.7. 1-[(2-Bromophenyl)methyl]-3,4-dihydro-1H-isoquinoline-
2-carboxaldehyde (17a, R = H)
According to the protocol reported,25,44 a mixture of 2-(bromo-
phenyl)-N-(2-phenylethyl)acetamide (15a, 2.9 g, 9.1 mmol) and
polyphosphoric acid (19 g) was heated at 150 °C for 12 h. After
being cooled, the reaction mixture was diluted with H2O (50 mL),
and the resulting mixture was neutralized with aqueous NaHCO3
and extracted with Et2O. The extract was washed with brine and
condensed to give a pale brown oil (2.8 g), to which was added
NaBH4 (830 mg, 22 mmol) in MeOH (10 mL), and the mixture was
stirred at 0 °C for 3 h. The reaction mixture was poured into brine
(50 mL) and extracted with CH2Cl2. The extract was washed with
brine and condensed to give 1-[(2-bromophenyl)methyl]-1,2,3,4-
tetrahydroisoquinoline (16a, 2.6 g) as a pale yellow oil. To the oil
was added pre-prepared acetic formic anhydride obtained by heat-
ing a mixture of Ac2O (8.1 mL) and formic acid (6.4 mL), and the
mixture was stirred at 70 °C for 3 h. After being cooled, the reaction
mixture was concentrated under reduced pressure. The residue was
diluted with H2O (20 mL) and the resulting mixture was extracted
with CHCl3. The extract was washed successively with aqueous
NaHCO3 and brine, and condensed to give a pale yellow oil
(3.0 g), which on normal-phase silica gel column chromatography
(CHCl3) gave title compound 17a (2.4 g, 80% from 15a).
(4) The flower buds (0.9 kg) were powdered and extracted four
times with MeOH at room temperature for 7 h followed by
evaporation under reduced pressure to produce the MeOH
extract (60.8 g). The MeOH extract (60.0 g) was partitioned
with Et2O-1% aqueous AcOH mixture and the 1% aqueous
AcOH fraction was made basic to litmus with 10% NH4OH
solution. The alkaline solution was extracted with Et2O and
the Et2O fraction was washed with 1% aqueous NaOH to fur-
nish the Et2O-soluble fraction (crude alkaloid fraction,
4.21 g). The crude alkaloid fraction (3.52 g) from the flower
buds was purified by ordinary and reversed-phase silica
gel column chromatographies followed by HPLC (COSMOSIL
5C18-MS-II, MeOH–H2O in 1% AcOH) to provide nuciferine (2,
183 mg), nornuciferine (4, 121 mg), N-methylasimilobine (5,
36.0 mg), (ꢀ)-lirinidine (7, 3.0 mg), lysicamine (10, 38.2 mg),
pronuciferine (12, 23.0 mg), and b-sitosterol (1.8 mg). The
known compounds were identified by comparison of their
physical data ([
reported values.
a
]D, 1H NMR, 13C NMR, and MS) with
Viscous oil; IR (neat): mmax 1668, 1431, 1292, 1196, 1157,
1026 cmꢀ1 1H NMR spectral analysis revealed the products to be
;
3.4. N-Methylasimilobine N-oxide
a ca. 3.5:1 mixture of two amide rotamers. 1H NMR (500 MHz,
CDCl3) d 2.83–2.90 (0.22 H, m, 4a-H), 2.87 (0.78 H, ddd-like, J = ca.
16.3, 4.6, 2.6, 4a-H), 2.92–2.98 (0.22 H, m, 4b-H), 2.98 (0.78H,
ddd, J = 16.3, 11.5, 6.3, 4b-H), 3.11 (0.78H, dd, J = 14.0, 10.9, benzylic
methylene), 3.17 (0.22H, dd, J = 14.0, 9.4, benzylic methylene), 3.27
(0.78H, ddd, J = 13.2, 11.5, 4.6, 3a-H), 3.37 (0.78H, dd, J = 14.0, 3.5,
benzylic methylene), 3.39 (0.22H, dd, J = 14.0, 4.9, benzylic methy-
lene), 3.68–3.72 (0.44H, m, 3a-H and 3b-H), 4.52 (0.78H, ddd,
A pale yellow oil; ½a D24
ꢀ56.3° (c 0.16, MeOH); IR (film): mmax
ꢁ
3400, 1509 cmꢀ1 1H NMR (600 MHz, CD3OD) d 2.76, 3.50 (2H, m,
;
4-H2), 3.20 (1H, dd, J = 4.0, 13.7 Hz, 7a-H), 3.28 (1H, m, 7b-H),
3.39 (3H, s, O N-CH3), 3.56 (3H, s, OCH3), 3.56, 3.73 (2H, m, 5-
H2), 4.44 (1H, dd, J = 4.0, 14.0 Hz, 6a-H), 6.71 (1H, s, 3-H), 7.25
(1H, dd like, J = 7.6, 7.6 Hz, 9-H), 7.30 (1H, dd like, J = 7.6, 7.6 Hz,

S. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 779–787
785
J = 13.2, 6.3, 2.6, 3b-H), 4.89 (0.78H, dd, J = 10.9, 3.5, H-1), 5.79
3.10. 1-[(2-Bromophenyl)methyl]-3,4-dihydro-6-methoxy-1H-
(0.22H, dd, J = 9.4, 4.9, H-1), 7.03–7.63 (8H, m, arom.), 7.50
(0.78H, s, CHO), 8.02 (0.22H, s, CHO); 13C NMR (125 MHz, CDCl3,
major rotamer/minor rotamor) d 28.1/29.7 (4-C), 34.3/40.2 (3-C),
43.4/41.9 (benzylic methylene), 56.8/51.1 (1-C), 126.6/126.9/
127.0/127.2/127.4/127.5/127.9/128.4/129.0/129.1/129.3/
131.4132.1/132.8/133.2 (d, arom.), 124.4/125.3/133.0/133.9/135.5/
135.6/136.6/137.0 (s, arom.), 161.3 (CHO).
isoquinoline-2-carboxaldehyde (17b, R = OCH3)
According to the protocol reported,25 a mixture of 2-(bromo-
phenyl)-N-[2-(2-methoxyphenyl)ethyl]acetamide (15b, 500 mg,
1.43 mmol) and phosphorous oxychloride (0.5 mL, 0.53 mmol) in
MeCN (7 mL) was heated under reflux for 12 h. After being cooled,
the reaction mixture was neutralized with aqueous NaHCO3, and
extracted with Et2O. The extract was washed with brine and con-
densed to give a pale brown oil (537 mg), to which was added with
NaBH4 (82 mg, 2.16 mmol) in MeOH (7 mL), and the mixture was
stirred at 0 °C for 3 h. The reaction mixture was poured into brine
(20 mL) and extracted with CH2Cl2. The extract was washed with
brine, and condensed to give 1-[(2-bromophenyl)methyl]-6-meth-
oxy-1,2,3,4-tetrahydroisoquinoline (16b, 478 mg) as a pale yellow
oil. To the oil was added pre-prepared acetic formic anhydride ob-
tained by heating a mixture of Ac2O (1.3 mL) and formic acid
(1.0 mL), and the mixture was stirred at 70 °C for 1 h. After being
cooled, the reaction mixture was concentrated under reduced pres-
sure. The residue was diluted with H2O (20 mL) and the resulting
mixture was extracted with CHCl3. The extract was washed succes-
sively with aqueous NaHCO3 and brine, and condensed to give a
pale yellow oil (438 mg), which on normal-phase silica gel column
chromatography (n-hexane–EtOAc = 10:1?5:1) gave title com-
pound 17b (385 mg, 96%).
3.8. 5,6,6a,7-Tetrahydro-4H-dibenzo[de,g]quinoline-6-
carboxaldehyde (18a, R = H)
According to the protocol reported,25 a mixture of compound
17a (1.0 g, 3.0 mmol), K2CO3 (836 mg, 6.1 mmol), di-tert-butylm-
ethylphosphonium tetrafluoroborate (226 mg, 0.9 mmol), and
Pd(OAc)2 (137 mg, 0.6 mmol) in DMA (5 mL) was heated at
150 °C for 12 h. The reaction mixture was concentrated under re-
duced pressure, and the residue was diluted with CHCl3. The
CHCl3-insoluble material was filtered off, and the filtrate was con-
densed to give a brown oil (893 mg), which on normal-phase silica
gel column chromatography (CHCl3) gave title compound 18a
(350 mg, 46%).
Pale brown solid; mp 133–135 °C; IR (neat): mmax 1662, 1427,
1392, 1256, 1242, 1184, 1123, 1041 cmꢀ1 1H NMR analysis re-
;
vealed the products to be a ca. 2:1 mixture of two amide rotamers.
1H NMR (500 MHz, CDCl3) d 2.83 (0.66H, dd, H = 14.3, 14.1, 7a-H),
2.86 (0.66H, br d-like, J = ca. 15.8, 4a-H), 2.81–2.91 (1.02H, m, 4a-H,
4b-H and 7a-H), 2.96 (0.66H, ddd-like, J = ca. 15.8, 12.6, 4.6, 4b-H),
3.16 (0.34H, J = 12.6, 10.3, 4.0, 5a-H), 3.19 (0.34H, dd, J = 14.6, 14.6,
7b-H), 3.27 (0.66H, dd, J = 14.1, 4.6, 7b-H), 3.43 (0.66H, ddd,
J = 12.6, 12.6, 2.9, 5a-H), 3.87 (0.66H, ddd, J = 12.6, 4.6, 2.0, 5b-H),
4.50 (0.34H, ddd, J = 12.6, 4.3, 3.5, 5b-H), 4.70 (0.34H, dd, J = 14.6,
4.6, 6a-H), 5.13 (0.66H, dd, J = 14.3, 4.6, 6a-H), 7.08–7.81 (6H, m,
arom.), 8.29 (0.66H, s, CHO), 8.42 (0.34H, s, CHO); 13C NMR
(125 MHz, CDCl3, major rotamer/minor rotamor) d 31.0/29.6 (4-
C), 33.0/37.1 (7-C), 42.1/36.1 (5-C), 49.3/53.1 (6a-C), 122.7/122.8/
123.7/124.0/127.3/127.5/127.6/127.7/127.9/128.1/128.2/128.6/
129.1 (d, arom.), 130.9/131.4/133.5/133.7/133.8/134.2/134.3/
134.5/134.6/134.9 (s, arom.), 162.2/162.0 (CHO); positive-ion FAB-
MS: m/z 250 [M+H]+.
Viscous oil; IR (neat):
mmax 1670, 1612, 1504, 1431, 1284, 1257,
1234, 1157, 1022 cmꢀ1 1H NMR analysis revealed the products to
;
be a ca. 4:1 mixture of two amide rotamers. 1H NMR (500 MHz,
CDCl3) d 2.79–2.84 (0.2 H, m, 4a-H), 2.83 (0.8 H, ddd-like, J = ca.
16.3, 5.1, 2.6, 4a-H), 2.88–2.95 (0.2 H, m, 4b-H), 2.95 (0.8H, ddd,
J = 16.3, 11.5, 6.3, 4b-H), 3.07 (0.8H, dd, J = 14.0, 10.6, benzylic
methylene), 3.13 (0.2H, dd, J = 14.0, 9.5, benzylic methylene),
3.26 (0.8H, ddd, J = 13.2, 11.5, 5.1, 3a-H), 3.33 (0.8H, dd, J = 14.1,
3.5, benzylic methylene), 3.34 (0.2H, dd, J = 14.0, 4.9, benzylic
methylene), 3.64–3.69 (0.4H, m, 3a-H and 3b-H), 3.79 (0.6H, s,
OCH3), 3.81 (2.4H, s, OCH3), 4.50 (0.8H, ddd, J = 13.2, 6.3, 2.6, 3b-
H), 4.83 (0.8H, dd, J = 10.6, 3.5, 1-H), 5.73 (0.22H, dd, J = 9.5, 4.9,
1-H), 6.72–7.63 (7H, m, arom.), 7.49 (0.8H, s, CHO), 8.01 (0.2H, s,
CHO); 13C NMR (125 MHz, CDCl3, major rotamer/minor rotamor)
d: 28.5/30.0 (4-C), 34.1/40.2 (3-C), 43.5/42.0 (benzylic methylene),
55.3/55.2 (OCH3), 56.4/50.1 (1-C), 112.9/113.0/113.4/113.5/127.2/
127.8/128.0/128.4/128.5/129.0/131.5/132.1/132.7/133.1 (d, arom.),
124.4/125.3/127.7/127.8/134.4/135.3/136.7/137.1/158.3/158.6/ (s,
arom.), 161.31/168.28 (CHO).
3.9. 6-Methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline
(19a, R = H)
A mixture of compound 18a (70 mg, 0.28 mmol) and ca. 0.9 M
solution of THF-boran complex (1 mL, 0.9 mmol) in THF (4 mL)
was heated under reflux for 6 h. The reaction was quenched with
2 N HCl (1 mL), and the mixture was made alkaline by addition
of 1 N aqueous NaOH (30 mL). The resulting mixture was then
heated under reflux for 4 h, and extracted with EtOAc. The extract
was washed with brine and condensed to give a pale brown oil,
which on normal-phase silica gel column chromatography (CHCl3)
gave title compound 19a (47 mg, 71%).
3.11. 2-Methoxy-5,6,6a,7-tetrahydro-4H-
dibenzo[de,g]quinoline-6-carboxaldehyde (18b, R = OCH3)
In a similar manner used for the preparation of 18a, compound
18b (370 mg, 1.0 mmol) was subjected to coupling reaction to give
a pale yellow oil (356 mg), which on normal-phase silica gel col-
umn chromatography (n-hexane-Et2O, 2:1) gave title compound
18b (131 mg, 46%).
Pale yellow oil; The 1H NMR spectroscopic properties of 19a
were in accord with those reported,46 and signals were unambigu-
ously assigned in the present study. 1H NMR (500 MHz, CDCl3) d
2.54 (1H, ddd-like, J = ca. 11.5, 11.5, 3.8, 5a-H), 2.56 (3H, s,
NCH3), 2.70 (1H, dd, J = 14.0, 14.0, 7a-H), 2.76 (1H, br dd, J = ca.
15.0, 3.8, 4a-H), 3.07 (1H, ddd, J = 11.5, 5.8, 1.5, 5b-H), 3.17 (1H,
dd, J = 14.0, 4.6, 7b-H), 3.17–3.23 (1H, m, 4b-H), 3.23 (1H, dd,
J = 14.0, 4.6, 6a-H), 7.07 (1H, d-like, J = 7.5, arom.), 7.21–7.28 (3H,
m, arom.), 7.31 (1H, ddm, J = ca. 7.8, 7.0, arom.), 7.76 (1H, d,
J = 7.8, arom.), 7.71 (1H, d, J = 7.6, arom.); 13C NMR (125 MHz,
CDCl3) d 29.1 (4-C), 34.1 (7-C), 44.0 (NCH3), 53.4 (5-C), 62.0 (6a-
C), 121.8/123.7/126.8/127.3/127.5/128.0/128.4 (d, arom.), 133.4/
133.5/133.8/134.3/135.3 (s, arom.).
Pale yellow solid; mp 171–173 °C; IR (KBr): mmax 1662, 1608,
1427, 1400, 1319, 1246, 1195, 1161, 1049 cmꢀ1 1H NMR spectral
;
analysis revealed the products to be a ca. 2:1 mixture of two amide
rotamers; 1H NMR (500 MHz, CDCl3) d 2.79 (0.66H, dd, H = 14.0,
14.0, 7a-H), 2.79–2.85 (0.66H, m, 4a-H), 2.85–2.87 (0.68H, m, 4a-
H and 4b-H), 2.89 (0.34H, dd, J = 14.3, 4.6, 7a-H), 2.95 (0.66H,
ddd-like, J = ca. 16.0, 12.3, 4.6, 4b-H), 3.14 (0.34H, dd, J = 14.6,
14.3, 7b-H), 3.18 (0.34H, J = 12.9, 10.6, 4.0, 5a-H), 3.27 (0.66H, dd,
J = 14.0, 4.6, 7b-H), 3.43 (0.66H, ddd, J = 12.3, 12.3, 2.6, 5a-H),
3.85 (0.66H, ddd, J = 12.3, 4.6, 1.8, 5b-H), 3.86 (3H, s, OCH3), 4.47
(0.34H, ddd, J = 12.9, 4.6, 2.9, 5b-H), 4.66 (0.34H, dd, J = 14.6, 4.6,
6a-H), 5.07 (0.66H, dd, J = 14.0, 4.6, 6a-H), 6.63–7.77 (6H, m,
arom.), 8.29 (0.66H, s, CHO), 8.42 (0.33H, s, CHO); 13C NMR

786
S. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 779–787
(125 MHz, CDCl3, major rotamer/minor rotamor) d: 31.3/30.0 (4-C),
33.3/37.4 (7-C), 42.2/36.2 (5-C), 49.1/52.8 (6a-C), 109.0/109.1/
112.6/112.8/123.7/124.0/127.4/127.9/128.2/128.3/128.7/129.2 (d,
arom.), 123.4/123.8/133.5/133.7/134.5/135.1/135.2/135.5/135.90/
135.94/158.8/159.2 (s, arom.), 162.3/162.0 (CHO).
The melanoma cells (2.0?104 cells/400
lL/well) were seeded into
24-well multiplates. After 24 h of culture, a test compound and
theophylline 1 mM were added and incubated for 72 h. The cells
were harvested by incubating with PBS containing EDTA 1 mM
and 0.25% trypsin, and then the cells were washed with PBS. The
cells were treated with NaOH 1 M (120
lL/tube, 80 °C, 30 min) to
3.12. 6-Methyl-2-methoxy-5,6,6a,7-tetrahydro-4H-
dibenzo[de,g]quinoline (19b, R = OCH3)
yield a lysate, an aliquot (100 L) of the lysate was transferred to
l
a 96-well microplate, and the optical density of each well was
measured with a microplate reader (Model 550, Bio-Rad Laborato-
ries) at 405 nm (reference: 655 nm). The test compound was dis-
solved in DMSO, and the final concentration of DMSO in the
medium was 0.1%. The production of melanin was corrected based
on cell viability. Inhibition (%) was calculated using the following
formula, and IC50 values were determined graphically.
In a similar manner used for the preparation of 19a, compound
18b (70 mg, 0.25 mmol) was reduced with THF-boran complex to
give a pale yellow oil (72 mg), which on normal-phase silica gel
column chromatography (n-hexane–acetone = 2:1?1:1) gave title
compound 19b46,47 (46 mg, 69%).
Pale yellow oil; IR (neat): mmax 1608, 1454, 1453, 1357, 1319,
Inhibitionð%Þ ¼ ½ðA ꢀ BÞ=Aꢁ=ðC=100Þ ꢂ 100
1246, 1123, 1072 cmꢀ1 1H NMR (500 MHz, CDCl3) d 2.54 (1H,
;
where A and B indicate the optical density of vehicle- and test com-
pound-treated groups, respectively, and C indicates cell viability (%).
ddd-like, J = ca. 11.7, 11.7, 4.0, 5a-H), 2.56 (3H, s, NCH3), 2.69
(1H, dd, J = 13.5, 13.5, 7a-H), 2.73 (1H, br dd, J = ca. 15.0, 4.0 4a-
H), 3.08 (1H, ddd, J = 11.7, 6.1, 1.5, 5b-H), 3.16 (1H, dd, J = 13.5
4.6, 7b-H), 3.19 (1H, dd, J = 13.5, 4.6, 6a-H), 3.21 (1H, ddd-like,
J = ca. 15.0, 11.7, 6.1, 4b-H), 3.85 (3H, s, OCH3), 6.63 (1H, d,
J = 2.6, arom.), 7.12 (1H, d, J = 2.6, arom.), 7.22–7.34 (3H, m, arom.),
7.69 (1H, dd, J = ca. 7.5, arom.); 13C NMR (125 MHz, CDCl3) d: 29.7
(4-C), 34.4 (7-C), 43.7 (NCH3), 53.4 (5-C), 55.3 (OCH3), 61.7 (6a-C),
108.2/112.5/123.7/127.3/127.7/128.5 (d, arom.), 126.4/134.1/
134.8/134.9/135.6/158.6 (s, arom.). Positive-ion FABMS: m/z 266
[M+H]+, FABHRMS m/z 266.1548 (Calcd for C18H20NO [M+H]+, m/
z 266.1545).
3.16. Cell viability
The cell viability was assessed by our previous report3 with a
slight modification. The melanoma cells (5.0?103 cells/100
lL/
well) were seeded into 96-well microplates and incubated for
24 h. After 70-h incubation with theophylline 1 mM and a test
compound, 10 lL of WST-8 solution (Cell Counting Kit-8™) was
added to each well. After a further 2 h in culture, the optical den-
sity of the H2O-soluble formazan produced by the cells was mea-
sured with a microplate reader (Model 550, Bio-Rad Laboratories)
at 450 nm (reference: 655 nm). The test compound was dissolved
in dimethylsulfoxide (DMSO), and the final concentration of DMSO
in the medium was 0.1%. Cell viability (%) and inhibition (%) were
calculated using the following formula, and IC50 values were deter-
mined graphically.
3.13. 1-Phenylmethy-3,4-dihydro-1H-isoquinoline-2-
carboxaldehyde (22)
In a similar manner used for the preparation of 16a, the Bisch-
ler–Napieralski reaction of N-(2-phenylethyl)benzeneacetamide
(20,48 460 mg, 1.93 mmol) by using polyphosphoric acid, and sub-
sequent NaBH4 reduction of the resulting dihydroispquinoline
derivative gave 1-phenylmethyl-1,2,3,4-tetrahydroisoquinoline
(21),49 to which was then acylated with acetic formic anhydride
to give title compound 22 (490 mg), which was used for the next
reaction without further purification. The 1H NMR spectral proper-
ties were in accord with those reported.50,51
Cell viabilityð%Þ ¼ A=B ꢂ 100
where A and B indicate the optical density of vehicle- and test com-
pound-treated groups, respectively.
3.17. Expression of tyrosinase, TRP-1, and TRP-2 mRNA
The melanoma cells (1.0 ꢂ 105 cells/2 mL/well) were seeded
into 6-well multiplates and cultured for 24 h. The cells were then
incubated with the test compound and theophylline 1 mM for
72 h. Total RNA was extracted from the cells using an RNeasy™
mini Kit (Qiagen) according to the manufacturer’s instructions.
The concentration and purity of the RNA were determined by mea-
suring the absorbance at 260 nm and determining the ratio of the
3.14. 2-Methyl-1-phenylmethyl-1,2,3,4-tetrahydroisoquinoline
(23)
According to the literature,51 a mixture of the crude compound
22 (100 mg) and LiAlH4 (30 mg, 0.8 mmmol) in THF (1 mL) was
heated under reflux for 1 h. Work-up gave a pale brown oil
(84 mg), which on normal-phase silica gel column chromatography
(CHCl3–MeOH = 100:1) gave title compound 2351 (74 mg, 79% from
20).
readings at 260 and 280 nm. cDNAs were synthesized from 1 lg to-
tal RNA using iScript™ cDNA Synthesis kit (Bio-Rad Laboratories)
according to the manufacturer’s instructions. Template cDNA thus
obtained was incubated with gene-specific primers and with iQ™
SYBRÒ Green Supermix (Bio-Rad Laboratories) in a Mini Opticon
(Bio-Rad Laboratories). The abundance of each gene product was
calculated by relative quantification, with values for the target
genes normalized to ?-actin mRNA. The thermal cycling program
had initial denaturation (95 °C for 2 min) and then 40 cycles of
denaturation (95 °C for 30 s), annealing (58 °C for 30 s) and exten-
sion (72 °C for 30 s). The primer pairs were: tyrosinase primers, 50-
CAGATCTCTGATGGCCAT-30 and 50-GGATGACATAGACTGAGC-30;
TRP-1, 50-CTTTCTCCCTTCCTTACTGG-30; 50-TGGCTTCATTCTTGGT
GCTT-30; TRP-2, 50-TGAGAAGAAACAAAGTAGGCAGAA-30 and 50-
CAACCCCAAGAGCAAGACGAAAGC-30; and b-actin primers, 50-
ATGGGTCAGAAGGACTCCTACG-30 and 50-AGTGGTACGACCAGAGGC
ATAC-30.
Pale yellow oil; 1H NMR (500 MHz, CDCl3) d 2.66 (1H, ddd,
J = 16.3, 4.9, 4.9, 4a-H), 2.76 (1H, ddd, J = 12.6, 5.2, 4.9, 3a-H),
2.88 (1H, ddd, J = 16.3, 8.6, 5.2, 4b-H), 2.89 (1H, dd, J = 13.8, 6.3,
benzylic methlene), 3.15 (1H, J = 13.7, 5.7, benzylic methylene),
3.20 (1H, J = 12.6, 8.6, 4.9, 3b-H), 3.81 (1H, dd, J = 6.3, 5.7, 6a-H),
6.75 (1H, d, J = 7.4, arom.), 7.00–7.26 (8H, m, arom.); 13C NMR
(125 MHz, CDCl3,) d 25.9 (4-C), 41.5 (benzylic methylene), 42.8
(NCH3), 47.0 (3-C), 65.1 (1-C), 125.3/125.9/126.0/127.9/128.0/
128.7/129.6 (d, arom.), 134.3/137.8/140.0 (s, arom.).
3.15. Melanogenesis
Screening test for melanogensis using B16 melanoma 4A5 cells
was performed as described previously3 with slight modifications.

S. Nakamura et al. / Bioorg. Med. Chem. 21 (2013) 779–787
787
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