Alpneumines A-H, new anti-melanogenic indole alkaloids from Alstonia pneumatophora

Article abstract of DOI:10.1016/j.bmc.2010.04.086

Eight new indole alkaloids, alpneumines A-H (1-8) were isolated from the Malaysian Alstonia pneumatophora (Apocynaceae) and their structures were determined by MS and 2D NMR spectroscopic methods. Alpneumines E and G (5 and 7), vincamine, and apovincamine showed anti-melanogenesis in B16 mouse melanoma cells.

Full text of DOI:10.1016/j.bmc.2010.04.086

Bioorganic & Medicinal Chemistry 18 (2010) 4415–4421
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Alpneumines A–H, new anti-melanogenic indole alkaloids from Alstonia
pneumatophora
Koichiro Koyama a, Yusuke Hirasawa a, Takahiro Hosoya a, Teh Chin Hoe b, Kit-Lam Chan b, Hiroshi Morita a,
*
a Faculty of Pharmaceutical Sciences, Hoshi University, Ebara 2-4-41, Shinagawa-ku, Tokyo 142-8501, Japan
b School of Pharmaceutical Sciences, University Sains Malaysia, 11800 Penang, Malaysia
a r t i c l e i n f o
a b s t r a c t
Article history:
Eight new indole alkaloids, alpneumines A–H (1–8) were isolated from the Malaysian Alstonia pneumato-
phora (Apocynaceae) and their structures were determined by MS and 2D NMR spectroscopic methods.
Alpneumines E and G (5 and 7), vincamine, and apovincamine showed anti-melanogenesis in B16 mouse
melanoma cells.
Received 7 April 2010
Revised 23 April 2010
Accepted 24 April 2010
Available online 29 April 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Alkaloids
Alstonia pneumatophora
Alpneumines A–H
Anti-melanogenesis
1. Introduction
acid. Water-soluble materials, which were adjusted to pH 9 with sat-
urated Na2CO3, were extracted with CHCl3. CHCl3-soluble materials
Alstonia species, which is widely distributed in the tropical re-
gions of Africa and Asia, are a well-known rich source of unique
heterocyclic alkaloids having a monoterpene indole skeleton and
have attracted great interest from biogenetic1 and biological points
of view2 such as anticancer, antibacterial, anti-inflammatory, anti-
tussive, and antimalarial properties. Previous investigations have
indicated that the existence of monoterpene indole alkaloids is re-
lated to plant inhabitability. For example, picrinine-type indole
alkaloids have been isolated generally in Alstonia species from In-
dia, Pakistan, and Thailand, whereas those belonging to the angu-
stilobine skeleton exist predominantly in the plants from
Indonesia and Philippine.3 In our search for structurally and bioge-
netically interesting indole alkaloids, eight new alkaloids, alpneu-
mines A–H (1–8) were isolated from Alstonia pneumatophora
(Apocynaceae) collected in Malaysia. In this paper, we describe
the isolation and structure elucidation of 1–8, and anti-melanogen-
esis with B16 mouse melanoma cells of 5 and 7, vincamine, and
apovincamine.
weresubjectedto a silicagelcolumn(EtOAc/MeOH1:0?0:1). MeOH
eluted fractions were purified by C18 HPLC (MeOH/H2O/TFA solvent
system) to afford alpneumines A (1, 1.0 mg, 0.0005%), B (2, 2.4 mg,
0.0012%), C (3, 1.0 mg, 0.0005%), D (4, 1.6 mg, 0.0008%), E (5,
1.0 mg, 0.0005%), F (6, 1.8 mg, 0.0009%), G (7, 3.4 mg, 0.0017%),
and H (8, 0.8 mg, 0.0004%) together with known alkaloids, vinca-
mine,4,5 apovincamine,5 alstilobanine B,6 akuammidine,7 scholar-
ine,8 echitamidine,9 and actinophyllic acid.10
Alpneumine A {1, ½a D20
þ 101 (c 0.1, MeOH)} showed the pseudo-
ꢀ
molecular ion peak at m/z 371 (M+H)+ in the LC–ESIMS, and the
molecular formula, C20H22N2O5, was established by LC–HRESIMS
[m/z 371.1603 (M+H)+,
D
+0.2 mmu]. 13C NMR data (Table 2)
revealed 20 carbon signals due to five sp2 quaternary carbons, three
sp2 methines, one ketone, one ester carbonyl, one sp3 quaternary
carbon, three sp3 methines, four sp3 methylenes, one methyl, and
one methoxy group. Among them, two methylenes (dC 66.6; dH
4.08, 4.11 and dC 60.1; dH 3.78, 3.85) and one methine (dC 75.5, dH
4.83) were ascribed to those bearing an N-oxide, while one methyl
(dC 51.8; dH 3.77) and one quaternary carbon (dC 143.6) were those
bearing an oxygen atom.
1.1. Isolation and structures of alpneumines A–H (1–8)
Partial structures a (C-9–C-11), b (C-5 and C-6), and c (C-3, C-14–
C-15, and C-20–C-21) were deduced from detailed analysis of 1H–1H
COSY spectrum of 1 (Fig. 1). The presence of a scholaricine skeleton
was deduced from the HMBC correlations. Then, the HMBC cross-
peaks of H3-18 to C-19 and C-20 indicated the connection between
C-20 and C-18 through C-19. In addition, chemical shifts of C-3,
C-5, and C-21 implied the presence of an N-oxide at N-4.11 Thus,
The leaves of A. pneumatophora were extracted with MeOH, and
the MeOH extract was partitioned between EtOAc and 3% tartaric
* Corresponding author. Tel./fax: +81 3 5498 5778.
E-mail address: moritah@hoshi.ac.jp (H. Morita).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.086

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K. Koyama et al. / Bioorg. Med. Chem. 18 (2010) 4415–4421
Table 1
1H NMR data [dH (J, Hz)] of alpneumines A–H (1–8) in CD3OD at 303 K
1
2
3
4
5
3
5a
4.83 (m)
4.11 (m)
4.35 (br s)
3.45 (m)
4.53 (br s)
3.84 (2H, m)
4.41 (br s)
3.50 (m)
4.33 (br s)
3.42 (m)
5b
6a
4.08 (m)
2.92 (m)
3.20 (m)
3.02 (m)
3.30 (m)
2.75 (m)
3.24 (m)
2.65 (m)
2.80 (m)
6b
9
2.37 (m)
2.06 (m)
2.19 (m)
7.41 (d, 7.2)
2.14 (m)
2.08 (m)
7.06 (d, 7.6)
6.99 (dd, 8.0, 7.6)
6.89 (d, 8.0)
6.88 (d, 7.5)
6.83 (dd, 7.9, 7.5)
6.72 (d, 7.9)
6.88 (dd, 7.4, 0.9)
6.84 (dd, 7.8, 7.4)
6.75 (dd, 7.8, 0.9)
7.31 (d, 7.5)
6.95 (dd, 7.9, 7.5)
7.20 (dd, 7.9, 7.7)
7.00 (d, 7.7)
2.11 (ddd, 14.5, 3.2, 3.2)
1.32 (br d, 14.5)
3.47 (m)
2.34 (3H, s)
3.21 (m)
3.54 (m)
10
11
12
14a
14b
15
18
20
21a
21b
23
6.98 (dd, 7.7, 7.2)
7.21 (dd, 7.7, 7.5)
6.99 (d, 7.5)
2.58 (ddd, 14.2, 3.5, 2.5)
1.59 (br d, 14.2)
3.74 (m)
2.38 (3H, s)
3.54 (ddd, 12.5, 3.8, 3.1)
3.80 (m)
2.58 (br d, 14.4)
1.71 (br d, 14.4)
3.83 (m)
2.46 (3H, s)
3.73 (br d, 12.6)
3.85 (m)
2.21 (br d, 13.8)
1.60 (br d, 13.8)
3.64 (br s)
2.35 (3H, s)
3.16 (m)
3.13 (m)
3.02 (m)
3.71 (3H, s)
2.08 (ddd, 14.6, 3.3, 3.1)
1.38 (br d, 14.6)
3.51 (m)
2.36 (3H, s)
3.25 (m)
3.63 (dd, 13.8, 6.0)
3.11 (dd, 13.8, 5.7)
3.85 (3H, s)
3.78 (m)
3.77 (3H, s)
3.41 (m)
3.72 (3H, s)
3.05 (dd, 13.9, 5.6)
3.83 (3H, s)
6
7
8
3a
3b
5ª
5b
3.28 (m)
3.04 (br d, 13.9)
3.72 (m)
3.62 (m)
2.75 (m)
3.54 (m)
3.28 (m)
4.17 (m)
3.96 (m)
3.23 (2H, m)
3.40 (dd, 13.5, 2.7)
2.87 (m)
3.10 (3H, s)
6a
6b
2.51 (m)
9
10
11
12
7.62 (d, 7.9)
7.15 (dd, 7.9, 7.4)
7.21 (dd, 7.9, 7.4)
7.43 (d, 7.9)
2.43 (m)
7.52 (d, 7.7)
7.15 (dd, 7.7, 7.0)
7.19 (dd, 8.1, 7.0)
7.24 (d, 8.1)
2.43 (m)
1.61 (m)
2.02 (m)
1.64 (m)
7.64 (d, 7.8)
7.15 (dd, 7.8, 7.1)
7.20 (dd, 7.9, 7.1)
7.40 (d, 7.9)
2.60 (dddd, 14.9, 14.4, 4.1, 4.0)
1.56 (br d, 14.9)
2.89 (br s)
14a
14b
15a
15b
17a
17b
18a
18b
19a
19b
20
2.29 (m)
2.92 (br d, 5.7)
4.40 (d, 8.0)
3.72 (d, 8.0)
2.40 (d, 15.0)
2.25 (d, 15.0)
1.03 (3H, t, 7.5)
4.33 (d, 12.1)
4.01 (d, 12.1)
3.68 (ddd, 12.6, 3.5, 3.4)
3.62 (dd, 12.6, 12.4)
2.07 (m)
3.22 (m)
2.41 (m)
2.06 (m)
1.15 (m)
3.73 (m)
21a
21b
23
5.48 (d, 7.4)
3.92 (3H, s)
4.80 (m)
4.27 (br s)
3.78 (3H, s)
3.79 (3H, s)

K. Koyama et al. / Bioorg. Med. Chem. 18 (2010) 4415–4421
4417
Table 2
13C NMR data (dC) of alpneumines A–H (1–8) in CD3OD at 303 K
1
2
3
4
5
2
3
168.3
75.5
171.2
62.3
168.6
75.7
168.4
62.0
168.4
61.5
5
66.6
53.9
68.0
53.7
53.8
6
40.2
42.5
40.4
42.7
44.0
7
53.8
56.7
54.0
57.4
57.8
8
9
134.5
112.3
124.2
117.6
143.6
132.5
27.9
30.3
98.1
29.6
207.7
46.0
135.9
112.3
123.8
117.0
143.3
132.7
31.0
30.9
97.6
29.6
209.3
49.0
133.8
120.9
122.8
130.2
111.9
145.6
28.1
30.9
97.8
29.7
208.6
46.5
135.1
112.6
123.9
117.3
143.4
132.8
25.5
28.2
102.1
28.4
210.5
48.6
46.7
168.4
52.0
134.3
121.3
122.5
129.8
111.6
145.9
26.0
28.5
102.1
28.7
210.9
48.9
47.0
168.4
51.8
10
11
12
13
14
15
16
18
19
20
21
22
23
60.1
168.0
51.8
46.7
168.5
51.6
60.9
168.0
51.7
Figure 2. Selected NOESY correlations (arrows) and relative stereochemistry for
alpneumine A (1).
6
7
8
2
3
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
138.4
47.0
50.7
127.2
58.6
69.7
139.3
59.5
58.9
Alpneumine B (2) showed the pseudomolecular ion peak at m/z
355 (M+H)+ in ESIMS, and the molecular formula, C20H22N2O4, was
establishedbyHRESIMS[m/z355.1651(M+H)+,
D
ꢁ0.1 mmu], which
26.1
20.5
was smaller than that of 1 by a 16 Da. IR spectrum suggested the
presence of hydroxyl (3434 cmꢁ1) group. The 1H and 13C NMR data
(Tables 1 and 2) of 2 disclosed 20 carbon signals that were similar
to those of 1 except for the chemical shifts of C-3, C-5, and C-21.
These results showed that 2 did not possess an N-oxide moiety. A de-
tailed analysis of 2D NMR data (1H–1H COSY, HSQC, and HMBC) sug-
gested that the gross structure of 2 was the same as that of 1 except
for an N-oxide group. The relative stereochemistry of 2 was eluci-
dated by the NOESY correlations. NOESY correlation of H-14a to H-
103.4
128.3
118.2
121.4
123.9
112.6
137.5
19.7
106.4
128.4
119.7
122.0
124.2
112.9
137.0
17.4
25.9
83.2
44.4
8.3
31.6
38.4
72.8
173.8
54.1
106.8
128.4
118.9
121.4
123.5
112.7
138.5
27.0
38.1
56.2
79.0
70.2
33.4
34.6
73.7
173.1
53.0
55.9
58.6
74.8
20 indicated that an acetyl group at C-20 was a-configuration as that
of 1. Treatment of alpneumine A (1) with Na2SO3 in aqueous MeOH
afforded thereductivederivative, whosespectroscopic data and spe-
cific rotation were identical with alpneumine B (2). Thus, the config-
uration of 2 was assigned as the same as 1.
53.4
107.8
62.4
171.5
53.5
Alpneumine C (3) showed the pseudomolecular ion peak at m/z
355 (M+H)+ in ESIMS, and the molecular formula, C20H22N2O4, was
established by HRESIMS [m/z 355.1653 (M+H)+,
D+0.1 mmu], which
was smaller than that of 1 by a 16 Da. The 1H and 13C NMR data
(Tables 1 and 2) of 3 were analogous to those of 1 except for 1H
and13CchemicalshiftsaroundtheC-12 sp2 methine. Adetailedanal-
ysis of 2D NMR data suggested that the gross structure of 3 was the
same as that of 1 except for the presence of a hydroxyl group at C-12.
The conformation of a piperidine ring (C-3, C-14–C-15, C-20–C-21,
and N-4) was deduced to be a chair form by a NOESY correlation of
H-14a to H-20 and 3J coupling constant (3J20,21a = 12.5 Hz). Thus,
alpneumine C was assigned as 3.
Alpneumine D (4) showed the pseudomolecular ion peak at m/z
355 (M+H)+ in ESIMS, and the molecular formula, C20H22N2O4, was
Figure 1. Selected 2D NMR correlations for alpneumine A (1).
established by HRESIMS [m/z 355.1653 (M+H)+,
D +0.1 mmu]. The
NMR data of 4, when compared with those of 2, the similar chemical
the gross structure of alpneumine A was elucidated to be 1 possess-
ing a scholaricine skeleton with an acetyl group at C-20 and an N-
oxide at N-4. The relative stereochemistry of 1 was elucidated by
NOESY correlations as shown in computer-generated 3D drawing
shift pattern except for those of C-14 and C-15 was observed. Fur-
3
thermore, 3J coupling constants (3J20,21a = 6.0 Hz, J20,21b = 5.7 Hz)
was difference from those of 2. This phenomenon indicated that
the relative stereochemistry at C-20 was different from 2. As a result
of comparison of the chemical shifts of C-14 and C-15 of 4 with those
of 2 and 19-epischolaricine,3c 4 showed similar 13C chemicalshifts to
those of 19-epischolaricine (dC14 28.2, dC15 27.9). Furthermore, a
NOESY correlation between H-14a and H-20 was not observed. From
these results, alpneumine D was an epimer at C-20 of alpneumine B.
Alpneumine E (5) showed the pseudomolecular ion peak at m/z
339 (M+H)+ in ESIMS, and the molecular formula, C20H22N2O3, was
(Fig. 2). The presence of an acetyl group with a-configuration at C-
20 was elucidated by NOESY correlations between H-20 and H-
14a. The NOESY correlation of H-15/H-20 and coupling pattern of
H-14a (br d, 14.4 Hz) indicated that both H-3 and H-15 were equa-
torially oriented in piperidine ring (C-3, C-14–C-15, C-20–C-21,
and N-4). The conformation of cis-octahydroindolizine ring was sup-
ported by the NOESY correlation of H-6a/H-21a. Thus, the relative
stereochemistry of 1 was assigned as shown in Figure 2.

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K. Koyama et al. / Bioorg. Med. Chem. 18 (2010) 4415–4421
established by HRESIMS [m/z 339.1703 (M+H)+,
D
0.0 mmu]. 1H
and 13C NMR data (Tables 1 and 2, respectively) suggested that
the structure of 5 was the analogue to 4 except for that around
C-12. Chemical shift at C-12 of 5 shifted upfield as compared with
4 and this indicated that C-12 of 5 did not possess hydroxyl moiety.
The relative stereochemistry at C-20 of 5 was identical with that of
4, judging by comparing of chemical shifts around C-20 and no
NOESY correlation between H-14a and H-20.
Alpneumine F (6) showed the pseudomolecular ion peak at m/z
355 (M+H)+ in ESIMS, and the molecular formula, C20H22N2O4, was
established by HRESIMS [m/z 355.1648 (M+H)+,
D
ꢁ0.4 mmu],
which was larger than that of actinophyllic acid10 by a CH2 unit.
1H and 13C NMR data of 6 were analogous to those of actinophyllic
acid, although a methoxy signal (dH 3.92) correlating with C-22 (dC
171.5) in HMBC spectrum was observed. Therefore, the structure of
6 was assigned to be C-22 methyl ester of actinophyllic acid.
Alpneumine G (7) showed the pseudomolecular ion peak at m/z
371 (M+H)+ in ESIMS, and the molecular formula, C21H26N2O4, was
Figure 4. Selected NOESY correlations (arrows) and relative stereochemistry for
alpneumine H (8).
established by HRESIMS [m/z 371.1967 (M+H)+,
D
+0.2 mmu]. 1H
and 13C NMR data (Tables 1 and 2, respectively) suggested that
the structure of 7 was the analogue to vincamine.4 Comparing
chemical shifts of 7 and vincamine suggested to be an N-oxide of
vincamine. Treatment of alpneumine G (7) with Na2SO3 in aqueous
MeOH afforded the reductive derivative, whose spectroscopic data
and specific rotation were identical with vincamine.
mentation can be controlled at many different stages of melanogen-
esis. Especially, tyrosinase inhibitors and antioxidants can be used
for inhibition of cellular pigmentation17 since tyrosinase is a key en-
zyme18 to produce melanin, and a copper-containing enzyme that
Alpneumine H (8) showed the pseudomolecular ion peak at m/z
357 (M+H)+ in ESIMS, and the molecular formula, C20H24N2O4, was
can catalyze two different reactions: the hydroxylation of L-tyrosine
to DOPA; and the conversion of DOPA to DOPA quinones.19 These
quinones lead to polymeric pigmented materials by enzymatic and
non-enzymatic reactions, resulting in the pigment deposition on
skin.20 Inhibitionof melaninsynthesisis importantfor thetreatment
of skin pigmentations, whitening reagent as a cosmetics.21
established by HRESIMS [m/z 357.1803 (M+H)+,
D
ꢁ0.6 mmu]. The
NMR data suggested that the structure of 8 coincided with alstiloba-
nine B with a rare uleine skeleton like undulifoline,12 which has been
isolated from Alstonia undulifolia, except for chemical shifts around
N-4.6 The chemical shifts of H2-3 and H-21 of 8 were observed at
higher field in comparison with those of alstilobanine B, which
was indicated that a shielding effect by N-4-oxide in alstilobanine
B was stronger than those of 8 (Fig. 3). Furthermore, the presence
of NOESY correlations of H3-5/H-9 and H-21 indicated that 8 and
alstilobanine B were stereoisomers at an N-4-oxide (Fig. 4).
B16 melanoma cells stimulated by IBMX, melanin inducer (3-iso-
butyl-1-methylxanthine), were cultured in the presence of isolated
alkaloids at 6.3–100 lM for 4 days. Figure 4 showed the melanin
content and the cell viability treated with each compound. After
the treatment for 4 days, melanin content in B16 melanoma cells
was determined by measuring OD at 400 nm, and the cell viability
also evaluated by MTT method. Alpneumine G (7), vincamine, and
apovincamine belonging to an eburnane type skeleton markedly de-
creased the melanin content (<50%) in B16 cells at high cell viability
Biogenetic relationship among monoterpene indole alkaloids
such as stemmadenine and 5-nor-indole derivatives such as valles-
amine and apparicine have been discussed.13 Alpneumines F (6)
and H (8) possessing an uleine skeleton might be generated
through N-oxidation of a pericine-type indole alkaloid such as
stemmadenine and pericine by Polonovski-type fragmentation,14
while alpneumines A–E (1–5) possessing a scholaricine skeleton8
might be derived from pre-acuamicine. Alpneumine G (7) might
be produced by rearrangement from an aspidosperma skeleton.15
dose dependently (IC50 58.3
lM for 7, 68.9 lM for vincamine, and
49.8 M for apovincamine). There was no tendency between oxida-
l
tive stages of the eburnane type skeleton about melanogenesis
inhibitory activity on B16 melanoma cell. Although alpneumine G
(7) showed the efficient activity on anti-melanogenesis, it showed
low cytotoxicity at high dose. Among alpneumines A–E (1–5)
belonging to the scholaricine type skeleton, alpneumine E (5) only
1.2. Anti-melanogenesis activity
showed moderate inhibition of melanogenesis (IC50 71.4 lM). It
was thefirst report to find this activityfor eburnanetypeindolealka-
loids such as alpneumine G (7) and vincamine.
Inhibition of melanogenesis can be achieved by antioxidation
and inhibition of tyrosinase. The effect of these alkaloids on mela-
nogenesis by tyrosinase was examined using in vitro mushroom
tyrosinase assay.22,23 However, none of them showed the tyrosi-
Skin pigmentation is the result of melanogenesis that occurs in
melanocytesand/or melanoma cells.16 The regulationof cellular pig-
nase inhibition at even high dose (IC50 >100 lM). To determine
the effect on cellular tyrosinase protein level in B16 melanoma
cells, we performed Western blotting analysis on cell lysate ob-
tained from B16 melanoma cells after treatment of hit compounds.
Alpneumine G (7) was incubated with 0.1 mM IBMX in B16 mela-
noma cells. As shown in Figure 5, alpneumine G decreased the
tyrosinase expression levels in B16 cells at 50 and 75 lM, respec-
tively. Therefore, alpneumine G (7) decreased the tyrosinase
protein expression, resulting in the reduction of melanin content
in B16 melanoma cells. The mode of action of 7 might be due to
inhibition of tyrosinase expression in B16 melanoma cells (Fig. 6).
Figure 3. Chemical shifts of alpneumine H (8) and alstilobanine B. Left: alpneumine
H, Right: alstilobanine B.

K. Koyama et al. / Bioorg. Med. Chem. 18 (2010) 4415–4421
4419
O
H
H
N
N
N
N
H3COOC
H3COOC
OH
OH
CH3
CH3
alpneumine G (7: IC50 58.3 µM)
vincamine (IC50 68.9 µM)
N
H
H
H
N
N
O
CH3
N
H
H
H3COOC
COOCH3
CH3
apovincamine (IC50 49.8 µM)
alpneumine E (5: (IC50 71.4 µM)
Figure 5. Melanin content in B16 melanoma cells and cell viability (–j–) treated with alpneumine E and G (5 and 7), vincammine, and apovincamine with IBMX.
1
experiments were optimized for JCH = 145 Hz and HMBC experi-
n
ments for JCH = 8 Hz. High resolution positive-mode ESI mass
spectra were obtained on a Thermo Scientific LTQ Orbitrap XL
spectrometer.
2.2. Material
The leaves of A. pneumatophora were collected at Malaysia in
2006. A voucher specimen (FR050802) has been deposited in the
herbarium of Hoshi University.
Figure 6. Effect of alpneumine G (7) on tyrosinase protein expression in B16
melanoma cells.
2.3. Extraction and isolation
The leaves of A. pneumatophora (200 g) were crushed and
extracted with MeOH. The MeOH extract (57.8 g) was treated with
3% tartaric acid (pH 2) and then partitioned with EtOAc. The aque-
ous layer was treated with saturated Na2CO3 (aq) to pH 9 and ex-
tracted with CHCl3 to give alkaloidal fraction (1.2 g). The alkaloidal
fraction was purified by SiO2 column (AcOEt/MeOH 1:0?0:1) and
the fraction eluted by MeOH was purified by ODS HPLC (MeOH/
H2O/TFA, 10:90:0.1?55:45:0.1?100:0:0.1; flow rate, 3 mL/min;
2. Experimental section
2.1. General methods
1H and 2D NMR spectra were recorded on a Bruker Avance 600
spectrometer and chemical shifts were reported using residual
CD3OD (dH 3.31 and dC 49.0) as internal standards. HSQC

4420
K. Koyama et al. / Bioorg. Med. Chem. 18 (2010) 4415–4421
UV detection at 254 nm) to afford alpneumines A (1, 0.0005%), B (2,
0.0012%), C (3, 0.0005%), D (4, 0.0008%), E (5, 0.0005%), F (6,
0.0009%), G (7, 0.0017%), and H (8, 0.0004%) together with known
alkaloids, vincamine (0.0055%),4 apovincamine (0.0030%),5 alsti-
2.4. Chemical conversion of alpneumine A (1) into alpneumine
B (2)
To a solution of alpneumine A (1, 0.2 mg) in aqueous MeOH
(0.2 mL) was added Na2SO3 (1.0 mg) and the mixture was kept at
rt for 30 min. After evaporation, the residue was applied to a silica
gel column (CHCl3/MeOH, 4:1) to give a compound (0.15 mg),
lobanine
B
(0.0005%),6 akuammidine (0.0020%),7 scholarine
(0.0009%),8 echitamidine (0.0010%),9 and actinophyllic acid
(0.0002%).10
whose spectroscopic data and [a]D value were identical with those
of natural alpneumine B (2).
2.3.1. Alpneumine A (1)
Colorless amorphous solid; ½a D20
ꢀ
þ 101 (c 0.1, MeOH); IR (film)
2930) and
2.5. Chemical conversion of alpneumine G (7) into vincamine
mmax 3434 and 1638 cmꢁ1; UV (MeOH) kmax 335 (
e
233 nm (e
4170); 1H and 13C NMR data (Table 1 and Table 2);
To a solution of alpneumine G (7, 0.2 mg) in aqueous MeOH
(0.2 mL) was added Na2SO3 (1.0 mg) and the mixture was kept at
rt for 30 min. After evaporation, the residue was applied to a silica
gel column (CHCl3/MeOH, 4:1) to give a compound (0.15 mg),
whose spectroscopic data and [
of natural vincamine.
ESIMS m/z 371 (M+H)+; HRESIMS m/z 371.1603 [(M+H)+, calcd
for C20H23N2O5: 371.1601].
2.3.2. Alpneumine B (2)
a]D value were identical with those
Colorless amorphous solid; ½a D20
ꢀ
þ 20 (c 0.1, MeOH); IR (film)
3010) and
mmax 3434 and 1638 cmꢁ1; UV (MeOH) kmax 335 (
e
232 nm (e
3490); 1H and 13C NMR, see Table 1; ESIMS m/z 355
2.6. Anti-melanogenesis assay
(M+H)+; HRESIMS m/z 355.1651 [(M+H)+, calcd for C20H23N2O4:
355.1652].
B16 cells were cultured in DMEM medium supplemented with
10% FBS and penicillin/streptomycin. B16 cells at 5000 cells in
2.3.3. Alpneumine C (3)
100 lL per well were seeded onto 96-well microtiter plate, and
were preincubated for 24 h at 37 °C in a humidified atmosphere
containing 5% CO2. The cells were cultured in the medium contain-
ing IBMX (100 lM) with or without the test sample of different
Colorless amorphous solid; ½a D20
ꢀ
þ 25 (c 0.1, MeOH); IR (film) mmax
2300), 292 ( 1970),
3418 and 1645 cmꢁ1; UV (MeOH) kmax 327 (
e
e
and 226 nm (e
2860); 1H and 13C NMR, see Table 1; ESIMS m/z 355
(M+H)+; HRESIMS m/z 355.1653 [(M+H)+, calcd for C20H23N2O4:
concentrations for 48 h, and then the medium were replaced the
355.1652].
same condition fresh medium, further incubation for 48 h. The
cells were dissolved in 50 lL of 1 N NaOH, and incubated at 90 °C
for 1 h. Relative melanin content was determined spectrophoto-
metrically by absorbance at 400 nm. Arbutin was used as a positive
control.
2.3.4. Alpneumine D (4)
Colorless amorphous solid; ½a D20
ꢀ
þ 21 (c 0.1, MeOH); IR (film)
3460) and
mmax 3434 and 1644 cmꢁ1; UV (MeOH) kmax 334 (
e
233 nm (e
4160); 1H and 13C NMR, see Table 1; ESIMS m/z 355
(M+H)+; HRESIMS m/z 355.1653 [(M+H)+, calcd for C20H23N2O4:
2.7. Cell viability assay
355.1652].
The cell viability was determined by MTT assay. 15 lL of MTT
solution (5 mg/mL) was added into each well of the cultured med-
ium. After further 2 h incubation, the medium was removed, and
then 50 lL of DMSO were added to resolve the formazan crystals.
The optical density measurements were made using a microplate
reader equipped with a two wavelengths system at 550 nm and
700 nm. In each experiment, three replicates were prepared for
each sample. The ratio of the living cells was determined on the ba-
sis of the difference of the absorbance between those of samples
and controls.
2.3.5. Alpneumine E (5)
Colorless amorphous solid; ½a D20
þ 13 (c 0.1, MeOH); IR (film)
ꢀ
mmax 3360, 2951, 2926, 1681, and 1603 cmꢁ1; UV (MeOH) kmax
326 (e 2870), 291 (e 2310), and 225 nm (e
4330); 1H and 13C
NMR, see Table 1; ESIMS m/z 339 (M+H)+; HRESIMS m/z
339.1703 [(M+H)+, calcd for C20H23N2O3: 339.1703].
2.3.6. Alpneumine F (6)
Colorless amorphous solid; ½a D20
ꢀ
þ 73 (c 0.1, MeOH); IR (film)
1670) and
mmax 3417 and 1675 cmꢁ1; UV (MeOH) kmax 281 (
e
2.8. Western blotting
217 nm (e
7130); 1H and 13C NMR, see Table 1; ESIMS m/z 355
(M+H)+; HRESIMS m/z 355.1648 [(M+H)+, calcd for C20H23N2O4:
Cells treated with sample were lysated in lysis buffer, and ex-
tracts were separated on a 7.5% SDS–PAGE. Proteins were trans-
ferred to an Immuno-Blot polyvinylidene difluoride membrane
(PVDF, Bio-Rad) and blocked for 1 h with 5% skim milk. Blotted
membranes were incubated with primary antibodies diluted
1:2000 for anti-tyrosinase (Santa Cruz) for overnight, 1:2000 for
b-actin (Sigma) for 1 h, followed by horseradish peroxide–conju-
gated rabbit or mouse IgG (Amersham). Protein bands were visual-
ized with ECL solution (GE Healthcare).
355.1652].
2.3.7. Alpneumine G (7)
Colorless amorphous solid; ½a D20
þ 109 (c 0.1, MeOH); IR (film)
ꢀ
mmax 3649, 2953, 1748, and 1684 cmꢁ1; UV (MeOH) kmax 267 (
e
1070) and 221 nm (e
4540); 1H and 13C NMR, see Table 1; ESIMS
m/z 371 (M+H)+; HRESIMS m/z 371.1967 [(M+H)+, calcd for
C21H27N2O4: 371.1965].
2.3.8. Alpneumine H (8)
Acknowledgements
Colorless amorphous solid; ½a D20
ꢀ
þ 106 (c 0.1, MeOH); IR (film)
1220)
mmax 3735, 3648, and 1682 cmꢁ1; UV (MeOH) kmax 281 (
e
This work was partly supported by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports, Science,
and Technology of Japan, and a grant from the Open Research
Center Project.
and 219 nm (e
5150); 1H and 13C NMR, see Table 1; ESIMS m/z 357
(M+H)+; HRESIMS m/z 357.1803 [(M+H)+, calcd for C20H25N2O4:
357.1809].

K. Koyama et al. / Bioorg. Med. Chem. 18 (2010) 4415–4421
4421
6. Koyama, K.; Hirasawa, Y.; Zaima, K.; Hoe, T. C.; Chan, K. L.; Morita, H. Bioorg.
Med. Chem. 2008, 16, 6483.
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