Cytotoxic bisbenzylisoquinoline alkaloids from Stephania epigaea

Article abstract of DOI:10.1021/np400084t

Six new bisbenzylisoquinoline alkaloids (1-6) and seven known compounds (8-14) were isolated from the tubers of Stephania epigaea, in addition to the major alkaloid, cepharanthine (7). The structures of 1-6 were elucidated by combined spectroscopic data analysis and chemical methods, with their configurations determined from their optical rotation values and confirmed using circular dichroism. Compounds 1-6 belong to the oxyacanthine type of bisbenzylisoquinoline alkaloids and have a rare methylenedioxy substituent. Compound 1, a dimer composed of benzylisoquinoline and seco-aristolactam units, represents a new type of bisbenzylisoquinoline alkaloid, while compounds 3-6 are bisbenzylisoquinoline N-oxides. These compounds were evaluated for their in vitro cytotoxicities against six human cancer cell lines (A-549, ECA109, HL-60, MCF-7, SMMC-7721, and SW480). Cepharanthine (7), the major component of S. epigaea, exhibited cytotoxicity against all of these cancer cell lines except ECA109, while its known analogue, 10, displayed cytotoxicity against all six cancer cell lines.

Full text of DOI:10.1021/np400084t

Article
pubs.acs.org/jnp
Cytotoxic Bisbenzylisoquinoline Alkaloids from Stephania epigaea
†
,‡
†
†
†
†
§
†
Jun-Jiang Lv, Min Xu, Dong Wang, Hong-Tao Zhu, Chong-Ren Yang, Yi-Fei Wang, Yan Li,
,†
and Ying-Jun Zhang*
†State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of
Sciences, Kunming 650201, People’s Republic of China
‡
University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China
§
Guangzhou Jinan Biomedicine Research and Development Center, Guangzhou 510632, People’s Republic of China
*
S Supporting Information
ABSTRACT: Six new bisbenzylisoquinoline alkaloids (1−6)
and seven known compounds (8−14) were isolated from the
tubers of Stephania epigaea, in addition to the major alkaloid,
cepharanthine (7). The structures of 1−6 were elucidated by
combined spectroscopic data analysis and chemical methods,
with their configurations determined from their optical
rotation values and confirmed using circular dichroism.
Compounds 1−6 belong to the oxyacanthine type of
bisbenzylisoquinoline alkaloids and have a rare methylenedioxy substituent. Compound 1, a dimer composed of
benzylisoquinoline and seco-aristolactam units, represents a new type of bisbenzylisoquinoline alkaloid, while compounds 3−
6
are bisbenzylisoquinoline N-oxides. These compounds were evaluated for their in vitro cytotoxicities against six human cancer
cell lines (A-549, ECA109, HL-60, MCF-7, SMMC-7721, and SW480). Cepharanthine (7), the major component of S. epigaea,
exhibited cytotoxicity against all of these cancer cell lines except ECA109, while its known analogue, 10, displayed cytotoxicity
against all six cancer cell lines.
he genus Stephania (Menispermaceae), containing 60
species, is distributed mainly in the warmer parts of Asia
elucidated on the basis of detailed spectroscopic analysis and
chemical methods. The isolated compounds 3−14 were
evaluated for their cytotoxicity against six human cancer cell
lines (A-549 human lung carcinoma, ECA109 human
esophagus cancer, HL-60 human myeloid leukemia, MCF-7
human breast adenocarcinoma, SMMC-7721 hepatocellular
carcinoma, and SW480 colon cancer), and the results obtained
are discussed herein.
T
and Africa, with about two-thirds of the number of this genus
1
growing in mainland China. These species have been utilized
in folk medicine for the treatment of asthma, cancer, dysentery,
fever, hyperglycemia, intestinal complaints, inflammation, sleep
2
disturbances, and tuberculosis. Several chemical studies on
Stephania spp. have been carried out over the past five decades,
which have led to the identification of more than 200
3
4
5
hasubanan, aporphine, protoberberine, and bisbenzylisoqui-
RESULTS AND DISCUSSION
6
■
noline alkaloids as the major constituents. Among these,
The alkaloid portion from the tubers of S. epigaea was subjected
to repeated column chromatography over silica gel, followed by
preparative thin-layer chromatography on silica gel (GF254)
and recrystallization, to afford the main component cepha-
ranthine (7), together with 13 bisbenzylisoquinoline alkaloids
cepharanthine (7) was reported as a main bisbenzylisoquinoline
alkaloid having various biological activities, such as antitumor
7
8
activity, suppression of cytokine production, and induction of
9
apoptosis.
Stephania epigaea H. S. Lo (Menispermaceae) is a herbaceous
liana mainly growing in the southwest and southeast of Yunnan
Province, People’s Republic of China. Its tubers have been used
by local people to treat fever and for sedation. Previous studies
showed that it produces cepharanthine (7) and the other
alkaloids cydeanine, delavaine, isochondodendrine, (−)-norcy-
(
1−6, 8−14). All showed a positive reaction to Dragendorff’s
reagent. The known compounds (7−14) (see Supporting
Information) were identified as cepharanthine (7), secoce-
pharanthine (8), cepharanoline (9), (+)-2-norcepharan-
thine (10), cepharanthine-2′β-N-oxide (11), 3′,4′-dihydros-
tephasubine (12), homaromoline (13), and fangchinoline
(
6
11
12
6
6
13
14
10
cleanine, and runanine. In order to explore a new source and
further investigate the bioactivities of cepharanthine (7) and its
analogues, a detailed chemical investigation on the tubers of S.
epigaea was carried out. This led to the identification of 13
minor bisbenzylisoquinoline alkaloids (1−6, 8−14), in addition
to the main component, cepharanthine (7). Compounds 1−6
are new cepharanthine analogues, and their structures were
15
14), respectively, using authentic samples and by compar-
ison of their spectroscopic and physical data with literature
values.
Received: January 28, 2013
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Journal of Natural Products
Article
of the ABX-coupled aromatic proton at δ 6.80 (dd, J = 1.8, 8.2
H
Hz, H-14) with C-12 (δ 147.1)/C-α (δ 37.7, CH ), H-1 (δ
C
C
2
H
3
.63) with C-9, and H-α with C-1 revealed the connectivity of
the 1,3,4-trisubstituted benzene C-ring to C-1 of the B-ring
through a methylene group (C-α). Cross-peaks of the aromatic
proton at δ 6.99 (H-11′) with C-9′ (δ 134.9), both H-10′
H
C
(
δ 7.41) and H-14′ (δ 7.10) with C-12′ (δ 153.8), both H-
H
H
C
α′ (δ 2.55, 3.56) and H-1′ (δ 4.75) with C-9′, and H-1′ with
H
H
C-8′a (δ 130.9) confirmed the connection of the para-
C
disubstituted benzene C′-ring via another methylene group, C-
α′, to the heteroatom-bearing methine (δ 64.4, C-1′), which
C
was connected to the C-8′a position of the A′-ring. The above
key HMBC correlations were used to construct the
cepharanthine skeleton in 1. Also, HMBC correlations of the
downfield shifted methylenedioxy protons (δ 5.69 and 5.74)
H
with C-6′ and C-7′ and of the two methoxy groups (δ 3.69
H
and 3.84) with C-6 (δ 149.1) and C-12 (147.1), respectively,
C
could be observed. These, together with the ROESY
correlations (Figure 1) of the methoxy group signals at δH
3
.69 with H-5 and of the other methoxy group signal at δ 3.84
H
with H-13, revealed the locations of the methylenedioxy group
and two methoxy groups in 1, which were the same as those in
7
. Furthermore, HMBC correlations of both H-5′ and 2′-N-
CH (δ 3.24) with the carbonyl carbon (δ 168.8, C-3′), and
3
H
C
2
1
′-N-CH also with C-1′, were used to determine the B′-ring in
3
−1
. This was suppported by the IR band at 1687 cm , produced
by the characteristic absorption of a secondary amide.
In the EIMS of 1, a fragment ion peak at m/z 380
corresponded to the upper half of the molecule, as a result of
the cleavage of two benzylic bonds, C-1/C-α and C-1′/C-α′.
These data confirmed that compound 1 is a head-to-head
Compound 1 was obtained as a white, amorphous powder.
Its molecular formula was established as C H N O , on the
3
6
34
2
7
+
basis of the positive HREIMS (m/z 606.2379 [M] , calcd for
16
bisbenzyl-isoquinoline alkaloid. It was concluded that the
C H N O , 606.2366), corresponding to 21 degrees of
3
6
34
2
7
1
3
unsaturation. In the C NMR and DEPT spectra of 1
Table 1), 36 carbon signals were observed, assigned as four
diphenyl ether bridge occurs between C-11/C-12′ and C-7/C-
2
5
(
8′. The positive optical rotation value of 1 {[α]
+172.8 (c
D
methyls, of which two are methoxy groups (δ 55.5, 56.2), five
1.1, MeOH)} and a circular dichroism (CD) curve (Supporting
C
methylenes, including one bearing a heteroatom (δ 51.0) and
Information) similar to that of 7 indicated the 1R, 1′S
C
15
one bearing two oxygen atoms (δC 102.3), two aliphatic
configurations in 1, the same as those of cepharanthine (7).
methines bearing heteroatoms (δ 64.4, 64.5), a carbonyl (δC
On the basis of the above evidence, the structure of compound
1 was elucidated as 3′-nor-4′-oxocepharanthine, which is a
dimer consisting of benzylisoquinoline and seco-aristolactam
units.
C
1
68.8), and 24 aromatic carbons arising from four benzene
1
rings. The H NMR spectrum (Table 2) showed the presence
of one para-disubstituted benzene ring [δ 7.41, 6.99 (each 1H,
H
dd, J = 2.5, 8.3 Hz), 7.10, 6.39 (each 1H, brd, J = 8.4 Hz)], a
Compound 2, a white, amorphous powder, gave a molecular
1
,3,4-trisubstituted benzene ring [δ 6.76 (d, J = 8.2 Hz), 6.80
formula of C36
(m/z 589.2338 [M + H] , calcd for C36H N O , 589.2338),
33 2 6
H N O , as deduced by the positive HREIMS
32 2 6
H
+
(
[
dd, J = 1.8, 8.2 Hz), 5.64 (brs)], three aromatic singlet protons
δH 6.41, 6.67, 6.92 (each 1H, s)] due to two benzene rings,
four heteroatom-bearing singlet methyls [δ 2.53, 3.24, 3.84,
implying 22 degrees of unsaturation. The NMR data were
6
closely related to those of cepharanthine (7), except for the
H
3
1
.69], and one methylenedioxy group [δ 5.69 and 5.74 (each
H, d, J = 1.2 Hz)]. These NMR spectroscopic features of 1
signals arising from the B- or B′-ring. Instead of two aliphatic
H
methylenes (δ
and two N-CH
aromatic methines (δ
aromatic quaternary carbon (δ
(δ 41.9) group were observed in 2, indicating that the B- or
28.9, 51.2), one N-bearing methine (δ 64.2),
C C
6
were closely related to those of cepharanthine (7). However,
3
(δ
C
42.0, 43.9) groups in 7, signals for two
120.6, 140.6), one downfield shifted
160.5), and only one N-CH
instead of six aliphatic methylenes (δ 51.2, 45.9, 41.2, 38.6,
C
C
6
2
3
8.6, 25.9) in 7, only four methylene signals at δ 51.0, 38.6,
C
3
C
7.7, and 28.2, together with an additional carbonyl carbon at
C
1
δC 168.8, were observed for 1. All these characteristics
suggested that compound 1 is a norcepharanthine analogue.
In the HMBC spectrum of 1 (Figure 1), three aromatic
singlet protons were assigned as H-5 (δ 6.41), H-8 (δ 6.67),
B′-ring in 2 is an aromatic ring. On comparison with 7, the H
NMR spectrum of 2 displayed two additional mutually coupled
aromatic protons at δ 8.18 and 7.53 (each 1H, d, J = 5.9 Hz).
H
In the HMBC spectrum, correlations of the signal at δ 7.53
H
H
H
and H-5′ (δ 6.92) of the A and A′ aromatic rings, respectively,
with C-5 (δ 107.6) and C-8a (δ 123.2), of δ 8.18 with C-4a
H
C
C
H
based on the correlations of δ 6.41 (s) with C-4/C-8a/C-6/C-
(137.7), and of both aromatic protons at δ 8.18 and 7.67 (H-
H
H
7
, δ 6.67 (s) with C-6/C-4a/C-7, and δ 6.92 (s) with C-6′/
8) with the downfield shifted aromatic quaternary carbon at δC
160.5 confirmed that the B- or B′-ring in 2 is dehydrogenated
to form a pyridine ring. Thus, the aromatic proton signals at δH
8.18 and 7.53 were assigned at H-3 and H-4, respectively.
H
H
C-7′/C-8′a, and from the correlations of H-8 with δ 64.5 (CH,
C
C-1), H-5 with δ 28.2 (CH , C-4), and the N-CH (δ 2.53)
C
2
3
H
group with δ 51.0 (CH , C-3). These observations allowed the
C
2
B-ring of 1 to be constructed. In addition, HMBC correlations
Furthermore, the signal at δ 160.5 was assigned to C-1 based
C
B
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Journal of Natural Products
Article
Table 1. 13C NMR Spectroscopic Data for Compounds 1−6 in CD OD (δ in ppm)
3
a
5
a
,
c
a
b
b
b
position
1
2
3
4
a
b
6
1
64.4, CH
44.1, CH3
51.0, CH2
28.2, CH2
133.1, C
160.5, C
160.6, C
169.3, C
78.2, CH
58.7, CH3
60.3, CH2
25.2, CH2
128.1, C
81.4, CH
60.2, CH3
68.0, CH2
28.1, CH2
129.1, C
64.8, CH
43.5, CH3
50.7, CH2
28.0, CH2
132.6, C
2
3
4
4
5
6
7
8
8
-N-CH3
141.6, CH
120.6, CH
137.7, C
141.7, CH
120.8, CH
138.0, C
47.0, CH2
26.5, CH2
138.4, C
a
111.8, CH
149.1, C
107.6, CH
156.5, C
107.8, CH
156.1, C
112.5, CH
155.6, C
112.6, CH
152.0, C
112.6, CH
152.9, C
112.9, CH
150.6, C
141.5, C
146.7, C
146.1, C
143.8, C
144.1, C
145.4, C
143.5, C
119.2, CH
128.1, C
119.6, CH
123.2, C
119.9, CH
120.7, C
123.5, CH
121.4, C
120.0, CH
124.2, C
121.4, CH
127.8, C
119.9, CH
127.8, C
a
α
37.7, CH2
130.9, C
42.4, CH2
132.8, C
42.5, CH2
132.7, C
42.0, CH2
130.6, C
39.0, CH2
128.3, C
35.2, CH2
133.9, C
38.8, CH2
131.4, C
9
1
1
1
1
1
1
2
3
4
5
4
6
7
8
8
0
117.3, CH
148.7, C
122.5, CH
150.3, C
122.7, CH
150.2, C
124.6, CH
150.4, C
117.1, CH
150.2, C
125.4, CH
149.8, C
117.3, CH
149.7, C
1
2
147.1, C
150.5, C
150.8, C
151.5, C
149.9, C
152.6, C
148.6, C
3
111.4, CH
124.4, CH
64.5, CH
28.2, CH3
168.8, C
114.5, CH
125.0, CH
61.1, CH
41.9, CH3
45.3, CH2
24.3, CH2
105.0, CH
127.0, C
114.7, CH
131.9, CH
75.5, CH
56.8, CH3
59.9, CH2
27.1, CH2
105.0, CH
125.0, C
114.7, CH
125.3, CH
75.4, CH
56.7, CH3
60.1, CH2
27.3, CH2
104.7, CH
124.8, C
112.9, CH
124.3, CH
62.7, CH
42.0, CH3
45.7, CH2
25.9, CH2
103.9, CH
126.8, C
115.3, CH
126.3, CH
60.4, CH
42.7, CH3
45.4, CH2
24.5, CH2
104.4, CH
128.3, C
112.8, CH
125.3, CH
77.2, CH
58.5, CH3
58.8, CH2
25.8, CH2
103.2, CH
125.1, C
4
′
′-N-CH3
′
′
′
′a
′
′
′
′a
97.7, CH
126.6, C
150.5, C
149.8, C
151.3, C
151.2, C
149.2, C
149.4, C
149.5, C
139.2, C
135.5, C
136.2, C
136.1, C
134.7, C
134.9, C
135.0, C
137.2, C
139.8, C
138.9, C
139.4, C
139.0, C
140.7, C
139.2, C
130.9, C
122.5, C
120.8, C
121.0, C
123.6, C
121.6, C
121.0, C
α′
38.6, CH2
134.9, C
42.2, CH2
136.8, C
38.9, CH2
135.0, C
38.8, CH2
135.0, C
41.2, CH2
139.3, C
41.4, CH2
134.8, C
42.7, CH2
136.6, C
9
1
1
1
1
1
′
0′
1′
2′
3′
4′
129.2, CH
122.5, CH
153.8, C
131.5, CH
122.5, CH
158.0, C
132.2, CH
122.3, CH
158.8, C
132.3, CH
122.5, CH
159.2, C
129.7, CH
122.9, CH
153.3, C
133.3, CH
120.7, CH
160.6, C
133.6, CH
122.6, CH
154.4, C
121.7, CH
132.7, CH
102.3, CH2
55.5, CH3
56.2, CH3
122.4, CH
132.1, CH
102.5, CH2
56.6, CH3
56.8, CH3
122.7, CH
131.9, CH
103.4, CH2
56.8, CH3
56.9, CH3
122.1, CH
131.8, CH
103.1, CH2
56.2, CH3
56.9, CH3
121.7, CH
133.6, CH
102.1, CH2
55.6, CH3
56.5, CH3
120.3, CH
132.0, CH
102.1, CH2
55.5, CH3
57.1, CH3
123.7, CH
128.8, CH
102.3, CH2
55.5, CH3
56.7, CH3
OCH O
2
6
1
-OCH3
2-OCH3
a
b
c
Data were recorded at 150 MHz. Data were recorded at 100 MHz. Data were detected in CD OD + CDCl (1:1).
3
3
on the HMBC correlations of H-α (δ 4.52 and 4.11) with δC
those of 2, except for the significantly downfield chemical shifts
H
1
60.5, C-9 (δ 132.8), and C-10 (δ 122.5). Other HMBC,
of C-1′, 2′-N-CH , C-3′, and C-4′ with Δδ of 14.4, 14.9, 14.6,
C
C
3
1
1
H− H COSY, and ROESY correlations (Figure 1) were used
to confirm the planar structure of 2. In the EIMS of 2, an ion
and 2.8 ppm, respectively, suggesting that N-2′ in 3 is
oxygenated. This was confirmed by the EIMS, in which a
+
+
peak at m/z 482 [M − 106] , together with a corresponding
weak molecular ion peak at m/z 604 [M] (25%) and a major
+
+
base peak at m/z 481 [M − 107] due to the loss of a C′-ring,
fragment ion peak at m/z 588 [M − 16] (100%) were
indicated the characteristics of a bisbenzylisoquinoline with C-
observed, accompanied by the base peak at m/z 587, due to the
loss of oxygen. A somewhat weak ion peak at m/z 379
corresponded to the upper half of 3. On comparing with
16
7
/C-8′ and C-11/C-12′ diphenyl ether bridges. A weak ion
peak at m/z 362 (3%) corresponded to the upper half of
compound 2. By comparing with 1,2-dehydro-2-norlimacusine
compound 7, the proton signals of H-1′ (δ 4.96) and 2′-N-
17
[α]25 −94 (c 0.2, MeOH)}, with the same C-7/C-8′ and C-
H
{
1
D
CH (δ 3.31) were shifted downfield by 0.35 and 0.70 ppm,
3
H
1/C-12′ diphenyl ether bridge linkages, the negative optical
2
5
respectively, suggesting a trans relationship between the N-
rotation value of 2 {[α] −13.5 (c 1.1, MeOH)} was used to
D
16
oxygen and H-1′ in 3. The ROESY correlation of 2′-N-CH
3
confirm the 1′R configuration. Therefore, compound 2 was
elucidated as (−)-1,3,4-dehydrocepharanthine.
with H-1′ (Figure 1) also supported the opposite orientation of
H-1′ with the N-oxygen in 3. The 1′S configuration of 3 was
determined by its same positive [α] value (+40.7) to that of
Compound 3 was obtained as a white, amorphous powder.
Its molecular formula was established as C H N O according
25
D
3
6
32
2
7
20
18
+
to the positive HREIMS (604.2198 [M] , calcd for
C H N O , 604.2210), 16 Da more than that of 2. The H
NMR and C NMR spectroscopic data were very similar to
(+)-coclobine {[α] +130 (c 0.5, CHCl )} and confirmed
D 3
1
by the different CD spectrum of 3 with that of (−)-1,3,4-
dehydrocepharanthine (2) (Supporting Information). There-
3
6
32
2
7
1
3
C
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Journal of Natural Products
Article
1
Table 2. H NMR Spectroscopic Data for Compounds 1−6 in CD OD (δ in ppm)
3
a
,
c
a
b
b
a
a
b
position
1
2
3
4
5a
4.54 brs
3.45 s
5b
6
1
3.63 m
2.53 s
4.25 m
2.98 s
3.86 m
2.63 s
2
3
4
5
8
-N-CH3
2.43 brd, 2.77 m
2.45 m
8.18 d (5.9)
7.53 d (5.9)
7.23 s
8.20 d (6.0)
7.54 d (6.0)
7.23 s
3.61 m
2.67 m
6.76 s
7.21 s
3.00 m, 3.08 m
2.36 m, 3.23 m
6.64 s
3.69 m, 3.75 m
3.16 m
2.56 m, 2.78 m
2.39 m, 2.57 m
6.56 s
6.41 s
6.72 s
6.67 s
7.67 s
7.69 s
6.79 s
6.93 s
6.75 s
α
2.87 dd (3.8, 14.7) 4.11 d (12.7)
4.14 d (13.8)
4.51 d (13.8)
7.20 brs
3.23 m
2.70 m
2.95 m
3
.12 brd
4.52 d (12.7)
7.15 d (1.6)
6.94 d (8.6)
3.46 m
4.14 brd (13.6)
7.06 brs
3.18 dd (15.0, 3.5)
5.40 brs
1
1
1
1
2
3
4
5
0
5.64 brs
7.10 brs
6.94 brd (8.5)
5.28 brs
3
4
′
6.76 d (8.2)
6.80 dd (1.8, 8.2)
4.75 brd (11.0)
6.88 d (8.6)
6.72 m
7.16 d (8.7)
7.31 brd (8.7)
6.87 m
7.03 dd (1.6, 8.6) 7.01 dd (1.6, 8.6)
7.03 dd (2.0, 8.5) 6.89 m
6.87 m
4.61 m
4.96 m
4.87 m
4.26 m
4.38 dd (2.8, 11.1) 4.84 brd (6.8)
′-N-CH3 3.24 s
′
′
2.61 s
3.31 s
3.23 s
2.49 s
2.49 s
3.69 s
3.03 m, 3.51 m
2.89 m, 3.04 m
7.23 s
3.60 m, 3.93 m
3.26 m, 3.37 m
6.56 s
3.52 m, 3.87 m
3.20 m, 3.33 m
2.90 m, 3.22 m
2.73 m, 3.03 m
6.39 s
2.90 m, 3.21 m
2.87 m, 2.96 m
3.47 m
2.95 m, 3.46 m
6.47 s
′
6.92 s
6.53 s
6.40 s
d
d
d
α′
2.55
m
2.95 m
2.77 dd (11.4, 12.6) 2.71
m
2.80 m
2.84
3.19
m
m
3.04 dd (6.8, 15.2)
3.60 brd (15.2)
7.10 dd (2.0, 8.5)
6.44 dd (2.0, 8.5)
6.92 dd (2.0, 8.5)
7.57 dd (2.0, 8.5)
5.60 brs
d
3.56 dd (1.2, 14.2) 3.36 m
4.43 brd (12.6)
4.40 brd (12.5)
7.51 brd (8.5)
3.35 brd (12.0)
7.39 brd (8.3)
1
1
1
1
0′
7.41 dd (2.5, 8.3)
6.99 dd (2.5, 8.3)
6.39 brd (8.4)
7.10 brd (8.4)
5.69 d (1.2)
7.05 dd (8.3, 1.8) 7.52 dd (8.6, 2.4)
7.19 dd (8.3, 2.5) 6.60 dd (8.6, 2.4)
6.63 dd (8.5, 2.5) 7.19 m
7.28 brd (8.8)
6.55 brd (8.8)
1′
3′
4′
6.65 dd (2.5, 8.5) 6.79 m
7.14 dd (2.5, 8.0) 6.49 dd (2.3, 8.2) 6.97 dd (2.4, 8.3)
7.40 dd (8.5, 1.8) 7.03 dd (8.5, 2.4)
6.92 dd (2.5, 8.0) 7.10 brd (8.2)
6.57 brd (8.3)
5.43 brs
OCH O
5.49 brs
5.66 brs
3.77 s
5.51 brs
5.69 brs
3.75 s
5.56 brs
5.72 brs
3.63 s
5.55 brs
5.56 brs
3.74 s
2
5
.74 d (1.2)
3.69 s
2-OCH3 3.84 s
5.60 brs
5.61 brs
6
-OCH3
3.52 s
3.71 s
1
3.85 s
3.81 s
3.86 s
3.84 s
3.93 s
3.84 s
a
b
c
d
Data were recorded at 600 MHz. Data were recorded at 500 MHz. Data were detected in CD OD + CDCl (1:1). Overlapped with singlet 2-N-
3
3
CH signal.
3
1
13
fore, compound 3 was determined to be (+)-1,3,4-dehydroce-
pharanthine-2′β-N-oxide.
ranthine (7). The H and C NMR spectra of 5 displayed two
sets of signals with an integral ratio of 6.5:3.5, implying the
occurrence of a pair of compounds, 5a (major) and 5b (minor).
The protons and their corresponding carbons of 5a and 5b
were fully separated and assigned on the basis of detailed
The molecular formula of compound 4 was assigned as
+
C H N O , according to the HREIMS (m/z 606.2366 [M] ,
3
6
34
2
7
calcd for C H N O , 606.2366), with 21 degrees of
3
6
32
2
7
13
1
13
unsaturation. The C NMR spectrum of 4 was very close to
that of 1,3,4-dehydrocepharanthine-2′β-N-oxide (3), except
that the aromatic methines of C-3 and C-4 in 3 were replaced
analysis of the 1D- and 2D-NMR spectra. The H and
NMR features of 5a and 5b were closely related to those of
cepharanthine (7), except for the chemical shifts arising from
C
by two aliphatic methylenes at δ 47.0 and 26.5 in 4. Their
the B-ring. On comparing to those of 7 (δ 64.5, 44.1, and
C
C
corresponding mutually coupled proton signals were at δ 3.61
51.0), C-1, 2-N-CH , and C-3 of 5a and 5b were downfield
H
3
and 2.67, respectively. In addition, the chemical shift of C-1 in 4
was shifted downfield to δ 169.3 (δ 160.6 for 3). The EIMS
shifted to δ 78.2/81.4, 58.7/60.2, and 60.3/68.0, respectively,
C
suggesting that both 5a and 5b are N-oxides of 7. This was
supported by the reduction of 5 with zinc power and HCl at
room temperature, which yielded only cepharanthine (7) as the
product. The N-oxide positions for 5a and 5b were both
determined to be at the 2-N position from the HMBC
C
C
+
of 4 exhibited a fragment ion peak at m/z 588 [M − 16]
(100%), suggesting the presence of a bisbenzylisoquinoline N-
oxide functionality. The two aliphatic methylenes at δ 47.0
C
and 26.5 were assigned at C-3 and C-4, respectively, due to the
HMBC correlation of H-5 (δH 6.76, s) with δ 26.5 (C-4),
correlations of H-8 (δH 6.79/6.93) and 2-N-CH (δH 3.45/
C
3
while the downfield shifted carbon signal at δC 169.3 was
2.98) with δ 78.2/81.4 (C-1), H-5 (δH 6.64/6.72) with δC
C
assigned at C-1, based on its HMBC correlations with H-8 (δ
25.2/28.1 (C-4), H-1 (δ 4.54/4.25) with C-α (δ 39.0/35.2),
H
H
C
1
1
7
.21) and H-3 (δ 3.61). Other H− H COSY and HMBC
and H-α (δ 3.23/2.70, 3.46/4.14) with C-9 (δ 128.3/133.9)
H
H
C
1
1
correlations (Figure 1) were used to confirm the structure of 4.
The trans relationship between the 2′-N-oxygen and H-1′ was
and C-10 (δ 117.1/125.4). Other H− H COSY and HMBC
C
correlations (Figure 1) helped confirm the same planar
structures of 5a and 5b. The only difference between 5a and
5b was the oxygen orientation at the 2-N position. In the H
revealed by the ROESY correlation of 2′-N-CH with H-1′. The
3
2
5
1
similar positive [α]
(+40.7) and CD Cotton effects
D
(
Supporting Information) to those of 3 supported the 1′S
NMR spectrum, the chemical shifts of 2-N-CH for 5a and 5b
3
configuration in 4. Consequently, compound 4 was deduced as
were downfield shifted by 0.89 and 0.42 ppm, respectively,
compared with that of 7, suggesting that 5a is cepharanthine-
(+)-1-dehydrocepharanthine-2′β-N-oxide.
19
Compound 5 was obtained as a white, amorphous powder
2α-N-oxide and 5b is cepharanthine-2β-N-oxide. This was
confirmed by the weak ROESY correlation of H-1 with 2-N-
CH in 5b, but no correlation between H-1 and 2-N-CH was
and gave a molecular formula of C H N O , as deduced from
the positive HREIMS (m/z 623.2757 [M + 1] , calcd for
3
7
38
2
7
+
3
3
C H N O , 623.2757), 16 Da more than that of cepha-
observed in 5a (Figure 1). The large positive optical rotation
3
6
33
2
7
D
dx.doi.org/10.1021/np400084t | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 1. Key HMBC and ROESY correlations of compounds 1−5.
value of [α]25 +204.2 (c 1.0, MeOH) together with the
The isolated compounds 3−14 were evaluated for their
cytotoxicity against human lung carcinoma (A-549), human
esophagus cancer (ECA109), human myeloid leukemia (HL-
60), human breast adenocarcinoma (MCF-7), hepatocellular
carcinoma (SMMC-7721), and colon cancer (SW480) cell
lines. Tanespimycin (17-AAG) and cisplatin were used as
positive control substances. Among these, compounds 4, 7, 10,
13, and 14 showed cytotoxic potency against the above six
human cancer cell lines (Table 3), and the other compounds
tested were inactive (IC50 > 10 μM). It is noted that
cepharanthine (7) as the major component of S. epigaea
exhibited inhibitory activity against all cancer cell lines except
ECA109. The known analogue (+)-2-norcepharanthine (10)
also showed cytotoxicity against all six cancer cell lines, with
D
chemical reduction of 5 with zinc power yielding only 7 implied
the same 1R, 1′S configurations in both compounds 5a and 5b
as those in 7. Therefore, 5 was determined to be a mixture of
cepharanthine-2α-N-oxide (5a) and -2β-N-oxide (5b).
Compound 6 was obtained as a white, amorphous powder.
Its molecular formula was determined as C H N O due to
3
7
38
2
7
+
the positive HREIMS (m/z 622.2387 [M] , calcd for
C H N O , 622.2679), which was also 16 Da more than
3
6
32
2
7
1
13
that of cepharanthine (7). Comparison the H and C NMR
spectroscopic data (Table 2) with those of cepharanthine-2′β-
N-oxide revealed that compound 6 has a similar structure.
However, the downfield shifted H-1′ [δ 4.84 (6), δH 4.63
H
(
11)] and 2′-N-CH [δ 3.69 (6), δH 3.31 (11)] signals
3
H
suggested that compound 6 is a 2′α-N-oxide of cepharanthine
Table 3. Cytotoxicities of Compounds 4, 7, 10, 13, and 14
(7). The proton signals at δ 4.84 and 3.69 were assigned to H-
H
1
′ and 2′-N-CH , respectively, on the basis of their HMBC
3
IC50 (μM)
correlations with C-4′a (δ 125.1)/C-8′ (δ 139.2)/C-9′ (δ
1
C
C
C
A-
HL-
60
SMMC-
7721
36.6) and C-3′ (δ 58.8)/C-1′ (δ 77.2). Since no NOE
C
C
compound
549 ECA109
MCF-7
SW480
effects between H-1′ and 2′-N-CH were observed, this proved
3
4
>10
5.0
10.0
>10
3.2
>10
9.2
>10
2.9
>10
9.9
>10
4.7
indirectly that H-1′ is oriented on the same side of the molecule
as the oxygen of N-oxide. The large positive optical rotation
7
1
0
3.7
2.8
2.3
4.8
3.7
value of 6 ([α]25 +229.0 (c 1.0, MeOH)) and its similar CD
D
13
>10
>10
7.3
>10
>10
>10
>10
1.2
9.5
>10
>10
6.7
>10
>10
>10
ND
spectrum to that of cepharanthine (7) implied that compound
6
with zinc power and HCl at room temperature yielded
cepharanthine (7). Consequently, compound 6 was determined
to be cepharanthine-2′α-N-oxide.
1
4
5.0
has the same 1R, 1′S configurations as 7. The reduction of 6
a
cisplatin
ND
1.1
>10
ND
tanespimycin
ND
ND
ND
a
Not determined.
E
dx.doi.org/10.1021/np400084t | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
−
1 1
IC50 values ranging from 2.3 to 4.8 μM. In turn, the new
compound (+)-1-dehydrocepharanthine-2′β-N-oxide (4) dis-
played selective cytotoxicity for the ECA109 cell line (Table 3).
Both 4 and 10 are cepharanthine analogues bearing only one
3424, 2925, 1629, 1509, 1274, 1126, 1068 cm ; H NMR (CD
OD,
3
5
2
6
00 MHz) and 13C NMR (CD OD, 125 MHz) data, see Tables 1 and
; EIMS m/z 604 [M] , 588, 574, 481, 379, 295; HREIMS m/z
04.2198 [M] (calcd for C H N O , 604.2210).
3
+
+
36
32
2
7
(
+)-1-Dehydrocepharanthine-2′β-N-oxide (4): white, amor-
N-CH in their respective structures.
25
3
phous powder; [α] +40.7 (c 1.0, MeOH); UV (MeOH) λ (log
D
max
ε) 207 (4.73), 280 (3.99) nm; IR (KBr) ν 3431, 2925, 1626, 1509,
max
−1
1
13
EXPERIMENTAL SECTION
1270, 1126, 1069 cm ; H NMR (CD OD, 500 MHz) and C NMR
■
3
(
1
CD OD, 125 MHz) data, see Tables 1 and 2; EIMS m/z 605 [M −
General Experimental Procedures. Optical rotations were
performed on a P-1020 polarimeter (JASCO, Tokyo, Japan). IR
spectra was measured on a Bruker Tensor 27 spectrometer with KBr
pellets. 1D- and 2D-NMR spectra were run on Bruker AM-400, DRX-
3
+
+
] , 590, 547, 483, 295; HREIMS m/z 606.2380 [M] (calcd for
C H N O , 606.2366).
36 32
2
7
Cepharanthine-2α-N-oxide (5a) and Cepharanthine-2β-N-
oxide (5b): white, amorphous powder; [α]25 +204.2 (c 1.1, MeOH);
D
5
00, and AVANCE III-600 NMR spectrometers operating at 400, 500,
1
13
UV (MeOH) λmax (log ε) 204 (4.82), 280 (3.83) nm; IR (KBr) νmax
and 600 MHz for H and 100, 125, and 150 MHz for C, respectively.
Coupling constants are expressed in Hz, and chemical shifts are given
on a ppm scale with tetramethylsilane as internal standard. The MS
data were recorded on a VG Auto Spec-3000 spectrometer (VG,
Manchester, U.K.) with glycerol as the matrix. HREIMS was recorded
on an API Qstar Pulsa LC/TOF spectrometer. Silica gel (200−300
mesh, Qingdao Haiyang Group Co., Ltd., Qingdao, People’s Republic
of China) was used for column chromatography. TLC and preparative
TLC were carried out on precoated silica gel GF254 plates, which
were visualized by spraying with Dragendorff’s reagent or immersing in
I2 vapor.
−
1 1
3
4
2
1
426, 2930, 1625, 1512, 1272, 1127, 1065 cm ; H NMR (CD OD,
3
00 MHz) and 13C NMR (CD OD, 100 MHz) data, see Tables 1 and
; EIMS m/z 622 [M − 1] , 606, 379; HREIMS m/z 623.2748 [M +
3
+
+
] (calcd for C H N O , 623.2757).
36
33
2
7
Cepharanthine-2′α-N-oxide (6): white, amorphous powder;
25
[
(
1
(
6
α]
+229.0 (c 0.9, MeOH); UV (MeOH) λ
(log ε) 208
max
D
4.77), 283 (3.84) nm; IR (KBr) ν 3423, 2926, 1628, 1511, 1272,
max
−1
1
13
127, 1071 cm ; H NMR (CD OD, 400 MHz) and C NMR
3
+
CD OD, 100 MHz) data, see Tables 1 and 2; EIMS m/z 622 [M] ,
06, 379, 365, 190, 174, 145; HREIMS m/z 622.2687 [M] (calcd for
3
+
C H N O , 622.2679).
36
32
2
7
Plant Material. The tubers of S. epigaea was collected in October
Reduction of Compounds 5 and 6. Compounds 5 (i.e., 5a and
b) (20 mg) and 6 (10 mg) were separately dissolved in 10% HCl (30
2
009 in Dali City, Yunnan Province, People’s Republic of China, and
identified by one of the authors (C.R.Y.). A voucher specimen (KUN
432112) has been deposited in the herbarium of Kunming Institute
5
mL), and then zinc powder (100 mg) was added. After being stirred at
room temperature for 2 h, the reaction mixture was extracted with
0
of Botany, Chinese Academy of Sciences.
CHCl three times. The organic layer was subjected to preparative
3
Extraction and Isolation. The air-dried tubers of S. epigaea (350
kg) were extracted with 1% hydrochloric acid solution (700 L × 3) at
room temperature. The extract was adjusted to pH 10 with 5% NaOH
to give a precipitate (72.8 kg). The precipitate was refluxed with
ethanol to obtain a total alkaloid portion (1.8 kg), which was subjected
to passage over a silica gel column, eluting with CHCl −CH OH
TLC (CHCl −CH OH−NH ·H O, 20:1:0.07) to afford cepharan-
3
3
3
2
thine (7) (4 and 7 mg from 5 and 6, respectively).
Cytotoxicity Assay. The six cancer cell lines (A-549 lung cancer,
ECA109 human esophagus cancer, HL-60 human myeloid leukemia,
MCF-7 breast cancer, SMMC-7721 hepatocellular carcinoma, and
SW480 colon cancer) were cultured in RPMI 1640 medium
containing 10% fetal bovine serum and 100 U/mL penicillin/
3
3
(
20:1), to afford four major fractions. Fraction 1 (9.0 g) was
chromatographed on a silica gel column (EtOAc−CH OH, 15:1−7:1),
3
streptomycin in a humidified incubator in a 5% CO atmosphere at
2
followed by preparative TLC, to afford 1 (3 mg, petroleum ether−
3
3
7 °C. Cells (5 × 10 /well) were plated in 96-well plates in 100 μL of
acetone−diethylamine (2:1:0.02) and 8 (19 mg, EtOAc−CH OH
3
medium, in which the test samples were added at various
concentrations. After 48 h incubation, MTS [3-(4,5-dimethylthiazol-
(
10:1)). Fraction 2 (1.4 kg) was recrystallized four times in methanol
to give 7 (895 g). Fraction 3 (12 g) was applied to repeated column
2
-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]
chromatography over silica gel (CHCl −CH OH, 10:1) to give 2 (2
3
3
solution (5 mg/mL in phosphate-buffered saline) was added (20 μL/
well), while MTT [[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide] was added instead of MTS for the ECA109 cancer cell
line. The incubation was continued for another 4 h to give a formazan
product. In each well, 100 μL of 20% SDS was added after 100 μL of
medium was removed and then incubated overnight for the formazan
product to dissolve completely. The absorbance of the solution was
measured at 570 nm using a Bio-Rad 680 instrument. Compound
concentrations inhibiting 50% of cell growth (IC values) were
mg, EtOAc−CH OH, 5:1−3:1), 9 (506 mg, EtOAc−CH OH−
3
3
NH ·H O, 15:1:0.15), 10 (210 mg, CHCl −CH OH−NH ·H O,
3
2
3
3
3
2
2
1
1
0:1:0.07), 12 (30 mg, EtOAc−petroleum ether−diethylamine,
0:1:0.1), 13 (250 mg, EtOAc−CH OH−NH H O, 30:1:0.3), and
3
3·
2
4 (33 mg, EtOAc−CH OH−NH ·H O, 30:1:0.3). Fraction 4 (40 g)
3
3
2
was chromatographed on a silica gel column (EtOAc−CH OH−
3
NH ·H O, 4:1:0.1), followed by preparative TLC, to give 3 (70 mg,
3
2
CHCl −CH OH−NH ·H O, 10:1:0.1), 4 (17 mg, EtOAc−CH OH−
3
3
3
2
3
50
diethylamine, 4:1:0.01), a mixture of 5a and 5b (700 mg, EtOAc−
2
0
calculated by the Reed and Muench method. Tanespimycin (17-
AAG) was used as the positive control for the ECA109 cell line, and
cisplatin was used as the positive control for the other cancer cell lines.
CH OH−NH H O, 4:1:0.1), 6 (70 mg, EtOAc−CH OH−NH ·H O,
3
3·
2
3
3
2
4
:1:0.02), and 11 (1.0 g, CHCl −CH OH−NH ·H O, 10:1:0.07).
3
3
3
2
3
-Nor-4-oxocepharanthine (1): white, amorphous powder;
2
5
[α]
+172.8 (c 1.1, MeOH); UV (MeOH) λmax (log ε) 203
D
ASSOCIATED CONTENT
(
1
(
4
4.52), 281 (3.76) nm; IR (KBr) ν 3427, 2924, 1688, 1629, 1511,
max
■
−1
1
13
270, 1128, 1067 cm ; H NMR (CD OD, 600 MHz) and C NMR
S
3
* Supporting Information
+
CD OD, 150 MHz) data, see Tables 1 and 2; EIMS m/z 606 [M] ,
3
Structures of the known compounds 8−14 from S. epigaea and
+
32, 380, 225, 195; HREIMS m/z 606.2379 [M] (calcd for
1
13
1
1
the H and C NMR, HSQC, HMBC, H− H COSY, ROESY,
EIMS, and CD spectra for compounds 1−6 are available free of
charge via the Internet at http://pubs.acs.org.
C H N O , 606.2366).
3
6
34
2
7
(
−)-1,3,4-Dehydrocepharanthine (2): white, amorphous pow-
25
der; [α]
−13.5 (c 1.1, MeOH); UV (MeOH) λmax (log ε) 202
D
(
1
(
4.58), 235 (4.49) nm; IR (KBr) ν 3427, 2926, 1629, 1505, 1274,
126, 1065 cm ; H NMR (CD OD, 600 MHz) and C NMR
3
max
−1
1
13
AUTHOR INFORMATION
Corresponding Author
Tel: +86-871-6522-3235. Fax: +86-871-6522-3235. E-mail:
zhangyj@mail.kib.ac.cn (Y.-J. Zhang).
■
*
+
CD OD, 150 MHz) data, see Tables 1 and 2; EIMS m/z 588 [M] ,
3
+
4
5
81, 294; HREIMS m/z 589.2333 [M + 1] (calcd for C H N O ,
36 33 2 6
89.2338 [M + 1]).
(
+)-1,3,4-Dehydrocepharanthine-2′β-N-oxide (3): white,
25
amorphous powder; [α]
+40.7 (c 1.1, MeOH); UV (MeOH)
Notes
D
λmax (log ε) 207 (4.76), 240 (4.58), 281 (3.89) nm; IR (KBr) νmax
The authors declare no competing financial interest.
F
dx.doi.org/10.1021/np400084t | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
ACKNOWLEDGMENTS
■
The authors are grateful to the members of the analytical group
and screening center for natural products at the State Key
Laboratory of Phytochemistry and Plant Resources in West
China, Kunming Institute of Botany, Chinese Academy of
Sciences, for measuring the spectroscopic and cytotoxicity data,
respectively. This work was supported by the 973 Program of
Ministry of Science and Technology of China
(
2011CB915503) and the Twelfth Five-Year National Science
and Technology Support Program (2012BAI29B06).
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