Hasubanan type alkaloids from Stephania longa

Article abstract of DOI:10.1021/np0500833

Thirteen new hasubanan type alkaloids, stephalonines A-I (1-9), norprostephabyssine (15), isoprostephabyssine (16), isolonganone (18), and isostephaboline (21), as well as nine known ones, were isolated from the whole plants of Stephania longa, a well-known traditional Chinese medicine. Their structures were elucidated on the basis of spectroscopic data and chemical methods.

Full text of DOI:10.1021/np0500833

J. Nat. Prod. 2005, 68, 1201-1207
1201
Hasubanan Type Alkaloids from Stephania longa
Hua Zhang and Jian-Min Yue*
State Key Laboratory of Drug Research, Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese
Academy of Sciences, 555 Zu Chong Zhi Road, Zhangjiang Hi-Tech Park, Shanghai, 201203, People’s Republic of China
Received March 9, 2005
Thirteen new hasubanan type alkaloids, stephalonines A-I (1-9), norprostephabyssine (15), isoproste-
phabyssine (16), isolonganone (18), and isostephaboline (21), as well as nine known ones, were isolated
from the whole plants of Stephania longa, a well-known traditional Chinese medicine. Their structures
were elucidated on the basis of spectroscopic data and chemical methods.
The genus Stephania, belonging to the Menispermaceae
family, comprises about 60 species distributed mainly in
the tropical and subtropical areas of Asia and Africa.
Thirty-nine species and one variant occur in China, with
most species found in the south of China, especially in
Yunnan and Guangxi Provinces.1 Many plants of the
Stephania genus growing in China have a series of ap-
plications in Traditional Chinese Medicine (TCM) or folk-
lore herbs.2,3 The alkaloids from this genus are classified
into six major categories: hasubanan, aporphine, proapor-
phine, protoberberine, bisbenzylisoquinoline, and morphi-
nandienone types.3 More than 150 alkaloids have been
isolated from plants of the Stephania genus,3 and some
show important biological activities such as antitumor
activity and emetine type activity.4,5 Stephania longa Lour.
is a perennial herbaceous liana, and both its roots and the
whole plants are applied in TCM to treat fever, inflamma-
tion, and dysentery.2 There are several reports relevant to
the isolation of eight alkaloids and five nonalkaloids from
this plant.6 Recently, an extensive chemical study in our
laboratory has led to the isolation of 22 hasubanan type
alkaloids from the whole plants of S. longa. Among them,
13 alkaloids, stephalonines A-I (1-9), norprostephabys-
sine (15), isoprostephabyssine (16), isolonganone (18), and
isostephaboline (21), are new compounds. This paper
describes the isolation and structural elucidation of these
new alkaloids.
suggested that 1 was a hasubanan type alkaloid.8 Com-
parison of its NMR data (Tables 1 and 2) with those (Tables
1 and 3) of the known alkaloid N-methylstephuline (11)9
showed that they were analogues possessing a hemiacetal
ether bridge between C-8 and C-10. The only difference was
the presence of a 2-methylbutyryl ester group at C-6 in 1.
Compared with compound 11, the deshielded proton at δH
5.31 (1H, m, H-6) of 1 caused by acylation effect supported
the location of the 2-methylbutyryl moiety at C-6. Metha-
nolysis of 1 with NaOMe-MeOH gave N-methylstephuline
(11). The 2S absolute configuration of the 2-methylbutyryl
moiety was determined by comparing the retention time
of methyl 2-methylbutyrate obtained from the methanolysis
of 1 with those of authentic samples (S-configuration and
a racemic mixture) on chiral GC analysis. Thus, the
structure of stephalonine A (1) was elucidated as (6â,7â,8â,-
10â)-8,10-epoxy-4,6-dihydroxy-3,7,8-trimethoxy-17-methyl-
hasubanan 6-((2S)-methylbutyrate) and was confirmed by
1
HSQC, HMBC, and H-1H COSY spectra.
Stephalonine B (2) had the molecular formula C29H33-
NO7 as determined by HREIMS. It was also a hasubanan
type alkaloid as compared with the spectroscopic data of
stephalonine A (1). The only difference between alkaloids
1 and 2 was the ester moiety at C-6. The ester group of 2
was determined to be a cinnamoyl group as judged by the
1H and 13C NMR data (Tables 1 and 2). The structure of 2
1
and the assignments for the H and 13C NMR data were
1
confirmed by HMQC, HMBC, and H-1H COSY spectra.
Results and Discussion
Stephalonine B (2) was therefore identified as (6â,7â,8â,-
10â)-8,10-epoxy-4,6-dihydroxy-3,7,8-trimethoxy-17-methyl-
hasubanan 6-cinnamate.
Stephalonine A (1) was obtained as a white powder. A
molecular formula of C25H35NO7 was assigned to 1 on the
basis of HREIMS at m/z 461.2411 [M]+ (calcd 461.2414),
indicating the presence of nine degrees of unsaturation. Its
IR spectrum showed the absorption bands of hydroxyl and
ester carbonyl groups at 3385 and 1732 cm-1, respectively.7
The NMR data (Tables 1 and 2) showed the presence of an
ester carbonyl (δC 177.0), six aromatic carbons (four
quaternary ones at δC 146.7, 143.5, 134.4, and 128.0 and
two aromatic methines at δC 115.9 and 106.5), three
oxygenated methines at δC 81.6, 77.0, and 67.0, three
O-methyls at δC 57.1, 55.7, and 51.6, an N-methyl at δC
38.6, and two aliphatic methyls at δC 15.2 and 11.1 in 1.
The aromatic ring and the carbonyl accounted for five
degrees of unsaturation, and the remaining four degrees
of unsaturation were attributed to the existence of four
more rings in 1.
Stephalonines C (3) and D (4) gave the same molecular
formula of C28H31NO7 as determined by HREIMS, which
was 14 mass units less than that of stephalonine B (2).
Comparison of the 1H and 13C NMR spectra (Tables 1 and
2) of 3 with those of 2 showed that compound 3 was the
N-demethyl product of 2, as judged by the absence of the
typical N-methyl group in both the 1H and 13C NMR
spectra. The structure of stephalonine C (3) was thereby
established as (6â,7â,8â,10â)-8,10-epoxy-4,6-dihydroxy-
3,7,8-trimethoxyhasubanan 6-cinnamate. Similarly, the 1H
and 13C NMR spectra (Tables 1 and 2) of 4 showed one
O-methyl less than compound 2, and the C-8 carbon signal
of 4 was shielded ca. ∆δC 2 ppm, suggesting the existence
of a hemiacetal group at C-8. Furthermore, comparison of
the NMR data of 4 with those (Tables 1 and 3) of longanine
12 indicated that compound 4 was a C-6 cinnamic ester of
12. Stephalonine D (4) was thus elucidated to be (6â,7â,8â,-
10â)-8,10-epoxy-4,6,8-trihydroxy-3,7-dimethoxy-17-methyl-
hasubanan 6-cinnamate.
The aforementioned spectroscopic data and the typical
EIMS fragmentation ions at m/z 360, 231, 230, and 229
* Corresponding author. Tel: +86-21-50806718. Fax: +86-21-50806718.
E-mail: jmyue@mail.shcnc.ac.cn.
10.1021/np0500833 CCC: $30.25
© 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/12/2005

1202 Journal of Natural Products, 2005, Vol. 68, No. 8
Zhang and Yue
Chart 1
Stephalonine E (5) showed a molecular formula of C29H31-
NO8 as deduced from HREIMS. The 1H and 13C NMR data
(Tables 1 and 2) of 5, closely related to those of stephalonine
B (2), showed the presence of a carbonyl at C-16 (δC 174.4)
replacing the C-16 methylene group in 2 to form an amide
functionality, which was confirmed by the HMBC correla-
tions of C-16 to the N-methyl protons and H2-15 at δH 3.07
(1H, d, J ) 17.1 Hz) and 2.46 (1H, d, J ) 17.1 Hz),
respectively. Comparing with compound 2, the deshielded
N-methyl protons at δH 3.06 (3H, s, H-17) and the shielded
carbon at δC 28.3 (C-17) were caused by the effects of the
C-16 amido group. Hence, the structure of stephalonine E
(5) was established as (6â,7â,8â,10â)-8,10-epoxy-4,6-dihy-
droxy-3,7,8-trimethoxy-17-methyl-16-oxohasubanan 6-cin-
namate.
data (Tables 1 and 2). Thus, the structure of stephalonine
G (7) was elucidated as (6â,7â,8â,10â)-8,10-epoxy-4,6-
dihydroxy-3,7,8-trimethoxy-17-methylhasubanan 6-(3,4-
dimethoxybenzoate). The structure of 7 and the assign-
ments of the proton and carbon signals were also confirmed
by HMQC and HMBC experiments.
Stephalonine H (8) was determined by HREIMS to have
a molecular formula of C28H33NO9, 14 mass units less than
1
that of 7. From the H and 13C NMR data (Tables 1 and
2), it was clear that stephalonine H (8) was a derivative of
7 by the loss of one methyl group in the acyl moiety, which
was identified as a 4-hydroxy-3-methoxybenzoyl group
(vanilloyl) in 8. The structure of stephalonine H (8) was
elucidated as (6â,7â,8â,10â)-8,10-epoxy-4,6-dihydroxy-3,7,8-
trimethoxy-17-methylhasubanan 6-vanillate. The structure
of 8 and the assignments for the proton and carbon signals
were also confirmed by HMQC and HMBC spectra.
Stephalonine I (9) exhibited a molecular formula of
C27H31NO9 as established by HREIMS at m/z 513.1996,
which was 14 mass units less than stephalonine H (8). The
1H and 13C NMR data (Tables 1 and 2) clearly indicated
that compounds 8 and 9 were similar, and the only
difference was the absence of the typical N-methyl group
in 9, suggesting that stephalonine I (9) was an N-demethyl
compound of 8. The structure of stephalonine I (9) was thus
established as (6â,7â,8â,10â)-8,10-epoxy-4,6-dihydroxy-
3,7,8-trimethoxyhasubanan 6-vanillate and was confirmed
by the HMBC spectrum.
Stephalonine F (6) had the molecular formula C27H31-
1
NO7 as determined by HREIMS. Analyses of the H and
13C NMR spectra (Tables 1 and 2) revealed that 6 was an
analogue of stephalonines A (1) and B (2), with the only
difference being the C-6 acyl group, which was defined as
1
a benzoyl group by the H and 13C NMR data (Tables 1
and 2). This was confirmed in the HMBC spectrum, in
which the H-6 at δH 5.61 (1H, m) correlated with C-7′ at
δC 166.3 to locate the benzoyl group. Therefore, stephalo-
nine F (6) was unambiguously assigned as (6â,7â,8â,10â)-
8,10-epoxy-4,6-dihydroxy-3,7,8-trimethoxy-17-methylha-
subanan 6-benzoate.
Stephalonine G (7) had a molecular formula of C29H35-
NO9 as established by HREIMS. Comparison of the 1H and
13C NMR data (Tables 1 and 2) with those of stephalonine
F (6) revealed that there were two additional O-methyl
groups in 7, and the proton chemical shifts (δH 3.85 and
3.86) indicated that they were attached to the aromatic ring
of the acyl group. The acyl moiety was identified as a 3,4-
Norprostephabyssine (15), isolated as white powder with
optical rotation [R]20 ) -80.4°, showed a molecular
D
formula of C18H21NO5 as assigned by HREIMS at m/z
331.1409 (calcd 331.1420). The most notable feature of its
1H and 13C NMR spectra (Tables 1 and 3) was that all the
proton and carbon signals were resolved in pairs as in the
case of prostephabyssine (14),6c suggesting that compound
1
dimethoxybenzoyl group on the basis of H and 13C NMR

Hasubanan Type Alkaloids from Stephania
Journal of Natural Products, 2005, Vol. 68, No. 8 1203

1204 Journal of Natural Products, 2005, Vol. 68, No. 8
Table 2. 13C NMR Data of Alkaloids 1-11
Zhang and Yue
1a
2a
3b
4a
5a
6a
7a
8c
9a
10a
11a
1
115.9
106.5
146.7
143.5
32.1
115.9
106.2
146.8
143.3
31.5
116.5
109.1
150.8
148.2
32.3
115.7
106.7
146.9
143.4
32.0
116.3
107.2
147.2
143.7
31.5
115.9
107.2
147.0
143.8
31.8
115.9
107.0
147.1
144.0
32.1
115.7
108.6
149.4
146.3
33.2
116.5
107.3
147.1
144.2
31.8
116.6
106.6
147.0
143.7
31.2
115.7
107.1
147.3
144.1
34.9
2
3
4
5
6
67.0
68.0
69.7
66.5
66.6
67.9
68.2
68.9
67.7
67.6
67.5
7
81.6
81.6
83.0
80.3
82.0
81.7
82.1
83.3
81.7
81.3
82.1
8
103.2
29.0
103.3
28.9
103.6
39.5
101.7
28.7
101.4
33.0
103.2
29.2
103.5
28.9
104.4
28.9
101.8
38.4
101.7
38.3
104.0
28.9
9
10
77.0
77.3
80.0
76.3
76.3
77.2
77.4
78.0
77.6
77.5
77.5
11
134.4
128.0
48.7
134.0
128.0
48.9
135.3
129.5
48.9
134.4
127.9
48.8
133.7
124.4
42.7
134.2
127.6
48.8
134.7
128.1
49.1
135.2
129.9
50.3
134.6
127.4
46.3
134.2
127.5
46.3
132.8
128.2
49.0
12
13
14
76.1
76.3
76.3
74.4
74.6
43.5
174.4
28.3
134.6
128.0
128.7
129.9
128.7
128.0
143.2
118.2
166.7
55.4
76.4
76.4
77.0
74.4
74.3
76.6
15
34.7
34.4
38.5
34.8
34.5
34.8
35.7
36.9
36.6
35.5
16
54.3
54.3
42.9
54.3
54.2
54.4
54.6
41.5
41.5
54.4
17
38.6
38.6
38.0
38.7
38.5
38.5
38.5
1′
177.0
40.0
134.7
128.0
128.6
129.7
128.6
128.0
142.6
118.6
167.0
55.1
136.5
129.7
130.4
131.7
130.4
129.7
145.3
119.5
168.7
56.3
134.7
128.0
128.6
129.8
128.6
128.0
143.0
118.4
166.7
55.5
129.8
129.9
127.3
132.0
127.3
129.9
166.3
124.7
112.3
148.1
152.6
109.6
128.1
166.4
122.4
113.9
147.7
152.5
115.5
125.7
166.7
122.0
111.8
145.7
149.8
113.2
125.3
166.3
128.4
113.2
145.6
148.1
110.3
121.5
142.5
116.7
167.2
55.4
2′
3′
26.2
4′
11.1
5′
15.2
6′
7′
8′
9′
3-OMe
7-OMe
8-OMe
3′-OMe
4′-OMe
55.7
57.1
51.6
55.8
57.6
51.6
55.9
57.7
51.5
56.1
56.2
55.9
58.1
51.7
56.0
55.9
57.4
51.8
56.2
55.7
56.8
51.8
57.1
51.6
58.0
52.6
57.6
57.4
52.0
56.9
51.9
55.9
a In CDCl3. b In CD3OD. c In C5D5N.
Table 3. 13C NMR Data of Alkaloids 12-21
12a
13a
14aa
14ba
15aa
15ba
16a
17a
18a
19a
20b
21b
1
115.3
107.0
147.4
144.0
35.2
116.5
107.3
147.4
144.3
34.9
117.1
108.6
145.8
142.9
32.3
116.9
107.6
146.9
143.8
29.6
117.9
109.1
145.8
143.0
29.6
117.4
107.9
147.1
144.3
29.6
121.1
109.2
146.3
142.9
28.8
116.1
107.6
147.0
143.8
44.6
204.2
88.6
107.0
29.0
77.1
115.5
107.6
147.0
143.9
41.9
207.4
81.6
105.4
29.3
76.2
116.4
107.9
147.2
144.0
31.1
115.4
107.5
147.3
143.6
27.8
115.6
107.8
147.5
143.8
23.1
25.8
69.7
99.8
30.6
76.1
133.5
125.3
50.0
74.4
33.3
54.7
39.3
55.7
2
3
4
5
6
66.0
67.2
113.5
151.4
196.6
29.2
92.1
114.4
148.6
199.7
35.7
92.3
113.5
151.6
193.4
28.8
33.0
28.7
7
79.9
81.9
151.3
98.3
29.3
75.9
151.4
97.1
38.7
76.7
207.3
98.5
30.4
77.6
71.7
8
102.4
28.8
102.4
38.3
102.3
29.9
9
10
76.4
77.6
65.7
66.2
67.7
75.3
11
133.0
128.2
48.7
132.8
127.8
46.4
131.8
129.5
48.2
133.7
126.2
48.7
132.1
128.1
46.6
134.0
126.2
46.5
130.8
128.1
48.4
134.0
124.3
52.3
134.0
125.0
52.2
134.3
125.1
50.4
134.0
125.8
50.6
12
13
14
74.2
74.5
70.6
73.0
70.8
71.4
71.3
76.9
74.2
79.2
74.6
15
36.0
36.9
32.9
34.1
32.3
36.4
33.2
34.7
35.3
33.6
33.6
16
54.4
42.0
51.4
54.6
41.6
41.9
53.1
54.3
54.1
54.4
54.4
17
38.0
36.0
38.9
37.4
38.6
38.2
38.3
38.7
3-OMe
7-OMe
8-OMe
55.7
55.8
56.5
52.1
56.1
55.9
56.2
55.1
56.0
54.5
56.1
55.8
55.8
56.0
55.6
56.7
55.0
54.4
55.1
59.4
58.6
51.8
47.6
a In CDCl3. b In CDCl3-CD3OD (5:1).
15 existed as two isomers in an equilibrium (a:b ) ca. 3:2)
Comparison of the 1H and 13C NMR data (Tables 1 and 3)
of the two alkaloids 14a and 16 showed that the major
differences were at C-10, suggesting that 16 was likely a
C-10 stereoisomer of 14a. The chemical shifts and coupling
constants of H-10 at δH 4.60 (1H, dd, J ) 4.0, 2.8 Hz) and
H-9 at δH 2.57 (1H, dd, J ) 14.9, 2.8 Hz) and 1.88 (1H, dd,
J ) 14.9, 4.0 Hz) in 16 were significantly different from
that of 14a, indicating that the 10-OH was R-oriented in
16. The NOESY (Figure 1) experiments for both compounds
14 and 16 were performed to verify the configuration, in
which the H-10 correlated with the N-methyl in 14a, while
no such correlation was observed in 16. The 3D structures
(Figure 1) of 14a and 16 were generated by computer
modeling with the molecular modeling program CS Chem3D
Ultra 8.0 (MM2 force field calculation was applied for
in CDCl3. The only difference between the two alkaloids
1
14 and 15 observed in their H and 13C NMR spectra was
the absence of N-methyl signals in 15. Accordingly, nor-
prostephabyssine (15) was established as (10â)-6,7-dide-
hydro-4,10-dihydroxy-3,7-dimethoxy-8-oxohasubanan (15a)
or (8â,10â)-6,7-didehydro-8,10-epoxy-4,8-dihydroxy-3,7-
dimethoxyhasubanan (15b). The structure of norprostepha-
byssine (15) and the assignments for the proton and carbon
signals were confirmed by HMQC and HMBC experiments.
Isoprostephabyssine (16) had the molecular formula
C19H23NO5 as determined by HREIMS, which was identical
with the molecular formula of prostephabyssine (14). The
spectroscopic analyses, especially the HMBC correlations,
implied that 16 had the same planar structure as 14a.

Hasubanan Type Alkaloids from Stephania
Journal of Natural Products, 2005, Vol. 68, No. 8 1205
Figure 1. Key NOESY correlations (dashed) and stereoviews generated from computer modeling of 14a and 16.
Silica gel H and neutral alumina (200-300 mesh) were used
energy minimization). The relative configuration and pre-
ferred conformation of 14a and 16 were consistent with
those of 14a and 16 assigned by NOESY experiments.
Consequently, isoprostephabyssine (16) was identified as
(10R)-6,7-didehydro-4,10-dihydroxy-3,7-dimethoxy-17-methyl-
8-oxohasubanan. This is the first report of a hasubanan
type alkaloid with a 10R-OH.
for column chromatography, and precoated silica gel GF254
plates (Qingdao Haiyang Chemical Company, Ltd.) were used
for TLC. Sephadex LH-20 (Amersham Biosciences), amino
silica gel (20-45 µm, Fuji Silysia Chemical Ltd.), RP-18 silica
gel (150-200 mesh, Merck), and MCI gel CHP20P (75-150
µm, Mitsubishi Chemical Industries, Ltd.) were also used for
column chromatography.
Plant Material. The plant material of S. longa was
collected from Guangxi Province of People’s Republic of China
in the summer of 2002 and was identified by Prof. Su-Hua
Shi of the Institute of Botany, School of Life Sciences, Zhong-
shan University. A voucher specimen was deposited in Shang-
hai Institute of Materia Medica (accession number: SL-2002-
1Y).
Isolonganone (18) had a molecular formula of C20H25NO6
as determined by HREIMS, which is identical with that of
1
longanone (17).6b An extensive comparison of the H and
13C NMR data (Tables 1 and 3) of the two alkaloids revealed
that they were stereoisomers at C-7. Alkaloid 17 is known
to have a C-7-â-OCH3; therefore alkaloid 18 was assigned
with a C-7-R-OCH3. Comparison of the IR absorption bands
for the C-6 carbonyls of 17 (1732 cm-1) and 18 (1720 cm-1
)
Extraction and Isolation. The powder of the whole plants
(5.0 kg) of S. longa was extracted with 95% EtOH at room
temperature (× 3, each for 5 days). After removal of the solvent
under reduced pressure, the residue (280 g) was suspended
in 2.0 L of acidic water (adjusted with 2.0 mol/L H2SO4 to pH
ca. 1-2). The acidified suspension was partitioned with EtOAc
to remove the nonalkaloids. The acidic solution was then
basified with 5% Na2CO3 to pH 6-7 and extracted with CHCl3
(3 × 1.0 L) to obtain the A1 alkaloids. The aqueous phase was
further adjusted with 5% Na2CO3 to pH 9-10 and extracted
with CHCl3 (3 × 1.0 L) again to give A2 alkaloids. TLC
monitoring showed that the A1 and A2 alkaloids did not differ
significantly and were thus combined to give 37.0 g of crude
alkaloids.
The crude alkaloids (37.0 g) were chromatographed on a
neutral alumina column (φ 6 × 45 cm, about 800 g) eluted with
gradient mixtures of Et2O-MeOH (from 100:1 to 1:1) to give
six major fractions (F1-F6). F1 (8.27 g) was subjected to a
silica gel column eluted with CHCl3-MeOH (100:1 to 40:1) to
afford three major subfractions, F1a-F1c. F1a was extensively
chromatographed on a silica gel column (CHCl3-MeOH, 70:
1) to obtain 1 (50 mg), 2 (155 mg), and 6 (18 mg). F1b was
purified on a silica gel column (petroleum ether-EtOAc-Et2-
NH, 7:1:0.3) and then preparative TLC (CHCl3-MeOH, 50:1)
to give 3 (86 mg). F1c was subjected to a silica gel column
(petroleum ether-EtOAc-Et2NH, 5:1:0.3) and then purified
on an amino silica gel column eluted with CHCl3 to get 7 (13
mg). F3 (6.02 g) was subjected to a silica gel column eluted
with a solvent mixture of petroleum ether-EtOAc-Et2NH (10:
1:0.3 to 2:1:0.3) to give three major fractions, F3a to F3c. F3a
was separated on a silica gel column eluted with CHCl3-
MeOH (50:1 to 20:1) to afford 14 (207 mg) and two major parts;
part 1 was purified by preparative TLC to give 16 (16 mg),
and part 2 was further separated on a RP-18 silica gel column
eluted with MeOH-H2O (1:1) to obtain 17 (41 mg) and 18 (18
mg). F3b was subjected to extensive silica gel column chro-
matography and eluted with CHCl3-MeOH (40:1 to 20:1) to
yield 4 (22 mg), 11 (20 mg), and 13 (15 mg). F3c was
chromatographed on a silica gel column eluted with CHCl3-
MeOH (30:1) to give 12 (102 mg) and 19, which was further
purified by preparative TLC (developed with CHCl3-MeOH,
20:1) to finally give 29 mg of 19. F5 (3.03 g) was chromato-
graphed on an MCI gel column eluted with MeOH-H2O (3:7
to 6:4) to obtain fractions F5a-F5d. F5a was chromatographed
also verified the configurational assignments for both
alkaloids, with a similar change reported in the case of two
C-7 isomers of hasubanan alkaloids.10 The structure of
isolonganone (18) was therefore elucidated as (7R,8â,10â)-
8,10-epoxy-4-hydroxy-3,7,8-trimethoxy-17-methyl-6-oxoha-
subanan.
Isostephaboline (21) had the same molecular formula of
C18H23NO5 as that of stephaboline (20),6c as determined by
HREIMS. Its 1H and 13C NMR data (Tables 1 and 3) were
very similar to those of 20, except for some minor chemical
shift differences for the carbons and protons around C-7.
This suggested that two compounds had the same planar
structure, and the only difference was the configuration
at C-7, which was confirmed by a 1H-1H COSY spectrum.
Alkaloid 20 possesses a C-7-â-OH; therefore compound 21
was assigned as a new isomer bearing a C-7-R-OH group.
Reduction of stephabyssine (19)6c with NaBH4 gave a
mixture of the alkaloids 20 and 21 (identified by ESIMS,
1H and 13C NMR spectra), which supported the structural
assignment. The structure of isostephaboline (21) was thus
established as (7R,8â,10â)-8,10-epoxy-4,7,8-trihydroxy-3-
methoxy-17-methylhasubanan.
The known alkaloids were identified as stephisoferuline
(10),9 N-methylstephuline (11),9 longanine (12),6a ste-
phuline (13),9 prostephabyssine (14),6c longanone (17),6b
stephabyssine (19),6c stephaboline (20),6c and cephatonine
(22)11 on the basis of ESIMS and 1H and 13C NMR spectra.
Except for 22, the 13C NMR data (Tables 2 and 3) of other
known compounds have been reported for the first time.
Experimental Section
General Experimental Procedures. IR spectra were
recorded on a Perkin-Elmer 577 spectrometer. Optical rota-
tions were determined on a Perkin-Elmer 341 polarimeter.
NMR spectra were measured on Bruker AM-400 (or 500) or
Varian Mercury-400 spectrometers. EIMS (70 eV) was carried
out on a Finnigan MAT 95 mass spectrometer. Chiral GC was
performed on a Perkin-Elmer Autosystem XL instrument with
a column of Rt-âDEXcst (30 m × 0.25 mm). All solvents used
were of analytical grade (Shanghai Chemical Company, Ltd.).

1206 Journal of Natural Products, 2005, Vol. 68, No. 8
Zhang and Yue
Stephalonine G (7): white powder; [R]2D0 -26.9° (c 0.29,
CHCl3); UV (MeOH) λmax (log ꢀ) 262 (3.92), 287 (3.68) nm; IR
(KBr) νmax 3431, 2937, 2837, 1703, 1603, 1512, 1464, 1269,
on a silica gel column eluting with petroleum ether-EtOAc-
Et2NH (3:1:0.3) to give 15 (30 mg) and 8 (26 mg). F5b was
chromatographed on a silica gel column (CHCl3-MeOH, 15:
1) to obtain a mixture of two major alkaloids, which were then
separated on an amino silica gel column (CHCl3-MeOH, 50:1
and 2:1) to give 10 (50 mg) and 9 (27 mg). F5d was purified by
TLC preparation (developed by CHCl3-MeOH, 20:1) to obtain
alkaloid 22 (43 mg). F6 (2.63 g) was first chromatographed on
an MCI gel column eluted with MeOH-H2O (3:7 to 6:4) to
enrich the alkaloids and was then separated on a silica gel
column eluted with petroleum ether-EtOAc-Et2NH (2:1:0.3)
to afford 20 (94 mg), 21 (10 mg), and 5, which was further
purified by preparative TLC (CHCl3-MeOH, 40:1) to obtain
5 mg.
Methanolysis of 1. To about 2 mL of MeOH solution
containing 8.0 mg of stephalonine A (1) was added a small
amount of NaOMe. The reaction was stirred overnight at about
40 °C. The reaction was quenched by addition of 2.0 mL of 0.1
N HCl and then partitioned with 2.0 mL of ether. The organic
phase, containing the methyl ester of 2-methylbutanoic acid,
was washed with 5% NaHCO3 and saturated NaCl solution
in turn and then dried with anhydrous Na2SO4 for the chiral
GC analysis. After basification and chloroform partition, the
aqueous phase gave 5 mg of N-methylstephuline (11).
Preparation of Authentic Samples. Methyl esters of
racemic 2-methylbutanoic acid and (S)-2-methylbutanoic acid
were prepared by methylation of the free acids with freshly
prepared CH2N2 in Et2O.
1225, 1045, 764 cm-1
;
1H NMR, see Table 1; 13C NMR, see
Table 2; EIMS 541 [M]+
(2), 360 (4), 231 (65), 230 (49), 229
(100), 198 (17), 196 (27); HIEIMS m/z 541.2312 (calcd for
C
29H35NO9, 541.2312).
Stephalonine H (8): white powder; [R]2D0 -100.7° (c 0.15,
C5H5N); UV (MeOH) λmax (log ꢀ) 264 (3.90), 288 (3.73) nm; IR
(KBr) νmax 3346, 2926, 2850, 1701, 1597, 1514, 1487, 1452,
1427, 1284, 1223, 1109, 1061, 881, 816, 764 cm-1; H NMR,
1
see Table 1; 13C NMR, see Table 2; EIMS m/z 527 [M]+ (5),
360 (6), 231 (76), 230 (58), 229 (100), 198 (14), 196 (20);
HREIMS m/z 527.2139 (calcd for C28H33NO9, 527.2156).
Stephalonine I (9): white powder; [R]2D0 -18.3° (c 0.115,
CHCl3); UV (MeOH) λmax (log ꢀ) 263 (3.91), 287 (3.74) nm; IR
(KBr) νmax 3361, 2951, 2868, 1699, 1597, 1510, 1489, 1448,
1421, 1369, 1279, 1219, 1182, 1117, 1057, 1034, 920, 766 cm-1
;
1H NMR, see Table 1; 13C NMR, see Table 2; EIMS m/z 513
[M]+ (2), 270 (3), 243 (4), 217 (18), 216 (37), 215 (100), 182
(15); HREIMS m/z 513.1996 (calcd for C27H31NO9, 513.1998).
Norprostephabyssine (15): white powder; [R]2D0 -80.4° (c
0.52, CHCl3); UV (MeOH) λmax (log ꢀ) 276 (3.45) nm; IR (KBr)
νmax 3425, 2937, 1674, 1639, 1487, 1441, 1277, 1225, 1138,
1080, 1053, 874, 812 cm-1; H NMR, see Table 1; 13C NMR,
1
see Table 3; EIMS m/z 331 [M]+ (33), 316 (17), 287 (20), 270
(13), 259 (83), 233 (47), 216 (35), 215 (100), 184 (39), 182 (45);
HREIMS m/z 331.1409 (calcd for C18H21NO5, 331.1420).
Chiral GC Analysis. GC analysis showed that the reten-
tion time (11.675 min) of the methyl ester of 2-methylbutanoic
acid prepared from 1 was consistent with that (11.668 min) of
the authentic sample methyl ester of (S)-2-methylbutanoic acid
(see Supporting Information: Figures S 80, S 81, and S 82).
Isoprostephabyssine (16): white powder; [R]2D0 -242.4°
(c 0.32, CHCl3); UV (MeOH) λmax (log ꢀ) 276 (3.68) nm; IR (KBr)
νmax 3423, 2935, 2839, 1672, 1641, 1489, 1452, 1279, 1225,
1097, 1059, 1032, 905, 810 cm-1 1H NMR, see Table 1; 13C
;
Stephalonine A (1): white powder; [R]2D0 +102.8° (c 0.68,
CHCl3); UV (MeOH) λmax (log ꢀ) 225 (3.69), 285 (2.97) nm; IR
(KBr) νmax 3385, 2958, 2939, 1732, 1616, 1487, 1464, 1441,
NMR, see Table 3; EIMS m/z 345 [M]+ (9), 327 (3), 317 (66),
302 (47), 299 (30), 284 (18), 247 (100), 229 (51), 214 (14), 196
(43), 168 (22); HREIMS m/z 345.1581 (calcd for C19H23NO5,
345.1576).
1
1275, 1196, 1101, 1047, 920, 814 cm-1; H NMR, see Table 1;
13C NMR, see Table 2; EIMS m/z 461 [M]+ (7), 446 (4), 360
(7), 231 (100), 230 (48), 229 (55), 198 (12), 196 (10); HREIMS
m/z 461.2411 (calcd for C25H35NO7, 461.2414).
Isolonganone (18): white powder; [R]2D0 +57.5° (c 0.57,
CHCl3); UV (MeOH) λmax (log ꢀ) 284 (3.31) nm; IR (KBr) νmax
3564, 3433, 2943, 2827, 1720, 1620, 1491, 1460, 1441, 1286,
Stephalonine B (2): white powder; [R]2D0 +7.7° (c 0.77,
CHCl3); UV (MeOH) λmax (log ꢀ) 277 (4.25) nm; IR (KBr) νmax
3406, 3012, 2941, 2837, 1713, 1641, 1489, 1441, 1371, 1313,
1273, 1178, 1097, 1043, 978, 920, 746 cm-1; 1H NMR, see Table
1; 13C NMR, see Table 2; EIMS m/z 507 [M]+ (4), 492 (2), 360-
(4), 231 (52), 230 (42), 229 (100), 198 (13), 196 (16); HREIMS
m/z 507.2239 (calcd for C29H33NO7, 507.2257).
1232, 1090, 1040, 968, 818 cm-1 1H NMR, see Table 1; 13C
;
NMR, see Table 3; EIMS m/z 375 [M]+ (4), 231 (19), 230 (31),
229 (100), 198 (18), 196 (14); HREIMS m/z 375.1684 (calcd for
C20H25NO6, 375.1682).
Isostephaboline (21): white powder; [R]2D0 +26.7° (c 0.30,
MeOH); UV (MeOH) λmax (log ꢀ) 226 (3.63), 285 (3.23) nm; IR
(KBr) νmax 3412, 3284, 3105, 2972, 1662, 1491, 1446, 1390,
Stephalonine C (3): white powder; [R]2D0 -4.2° (c 0.12,
MeOH); UV (MeOH) λmax (log ꢀ) 277 (4.21) nm; IR (KBr) νmax
3464, 2941, 2833, 1705, 1639, 1487, 1443, 1313, 1275, 1182,
1283, 1223, 1121, 1063, 1020, 982, 820, 619 cm-1; H NMR,
1
see Table 1; 13C NMR, see Table 3; EIMS m/z 333 [M]+ (11),
258 (8), 231 (100), 230 (35), 229, (20), 198 (37), 196 (18);
HREIMS m/z 333.1578 (calcd for C18H23NO5, 333.1576).
1
1063, 928 cm-1; H NMR, see Table 1; 13C NMR, see Table 2;
EIMS m/z 493 [M]+ (4), 270 (12), 231 (22), 229 (15), 216 (33),
215 (100), 184 (9), 182 (15); HREIMS m/z 493.2098 (calcd for
C28H31NO7, 493.2101).
Acknowledgment. The financial support of National
Natural Science Foundation (30025044) and the Foundation
from the Ministry of Science and Technology (2002CB512807)
is greatly acknowledged. We thank Prof. S.-H. Shi for the
collection and identification of the plant material.
Stephalonine D (4): white powder; [R]2D0 -17.9° (c 0.28,
CHCl3); UV (MeOH) λmax (log ꢀ) 277 (4.26) nm; IR (KBr) νmax
3431, 2937, 2839, 1703, 1639, 1487, 1450, 1313, 1277, 1178,
1
1067, 770 cm-1; H NMR, see Table 1; 13C NMR, see Table 2;
EIMS m/z 493 [M]+ (5), 345 (11), 259 (59), 247 (34), 231 (41),
230 (44), 229 (100), 198 (19), 196 (34); HREIMS m/z 493.2104
(calcd for C28H31NO7, 493.2101).
Supporting Information Available: 1D and 2D NMR, EIMS,
and IR spectra of all the new alkaloids; chiral GC analyses of methyl
ester of 2-methylbutanoic acid. This material is available free of charge
via the Internet at http://pubs.acs.org.
Stephalonine E (5): white powder; [R]2D0 +49.6° (c 0.115,
CHCl3); UV (MeOH) λmax (log ꢀ) 279 (4.25) nm; IR (KBr) νmax
3388, 3010, 2941, 2831, 1701, 1639, 1489, 1444, 1383, 1315,
1279, 1186, 1103, 1047, 952, 885, 812, 739 cm-1; 1H NMR, see
Table 1; 13C NMR, see Table 2; EIMS m/z 521 [M]+ (2), 390
(4), 243 (100), 242 (35), 212 (10); HREIMS m/z 521.2044 (calcd
for C29H31NO8, 521.2050).
References and Notes
(1) Liu, Y. H.; Luo, X. R.; Wu R. F.; Zhang, B. N. Delectis Florae
Reipublicae Popularis Sinicae Agendae Acacemiae Sinicae Edita.
Flora Republicae Popularis Sinicae; Science Press: Beijing, 1996;
Tomus 30 (1), p 41.
(2) State Administration of Traditional Chinese Medicine. Zhonghua
Bencao; Shanghai Science and Technology Press: Shanghai, 1999;
Fascicle 3, Vol. 8, pp 368-390.
(3) Wang, X. K.; Zhao, T. F. Zhongguo Yaoxue Zazhi 1990, 25, 3-6.
(4) Kuroda H.; Nakazawa, S.; Katagiri K.; Shiratori O.; Kozuka M.;
Fujitani K.; Tomita M. Chem. Pharm. Bull. 1976, 24, 2413-2420.
(5) Gupta R. S.; Krepinsky J. J.; Siminovitch L. Mol. Pharmacol. 1980,
18, 136-143.
Stephalonine F (6): white powder; [R]2D0 -11.9° (c 0.21,
CHCl3); UV (MeOH) λmax (log ꢀ) 282 (3.39) nm; IR (KBr) νmax
3419, 2939, 2835, 1709, 1487, 1452, 1281, 1122, 1059, 1045,
710 cm-1; 1H NMR, see Table 1; 13C NMR, see Table 2; EIMS
481 [M]+ (5), 360 (5), 231 (59), 230 (39), 229 (100), 198 (11),
196 (17); HREIMS m/z 481.2094 (calcd for C27H31NO7, 481.2100).

Hasubanan Type Alkaloids from Stephania
Journal of Natural Products, 2005, Vol. 68, No. 8 1207
(6) (a) Lao, A. N.; Gao, Y. L.; Tang, Z. J.; Xu, R. S. Yaoxue Xuebao 1980,
15, 696-698. (b) Lao, A. N.; Tang, Z. J.; Xu, R. S. Yaoxue Xuebao
1981, 16, 940-942. (c) Lao, A. N.; Gao, Y. L.; Tang, Z. J.; Wang, Y.
H.; Zhang, X. X.; Wang, C. G.; Xu, R. S. Huaxue Xuebao 1982, 40,
1038-1043. (d) Zhu, Z. Y.; Feng, Y. X.; He, L. Y.; Wang, Y. C. Yaoxue
Xuebao 1983, 18, 460-467. (e) Deng, J. Z.; Zhao, S. X. Phytochemistry
1993 33, 941-942. (f) Deng, J. Z.; Zhao, S. X. Zhongguo Yaoke Daxue
Xuebao 1993, 24, 73-75.
(8) Tomita, M.; Kato, A.; Ibuka, T. Tetrahedron Lett. 1965, 1019-1030.
(9) Kupchan, S. M.; Suffness, M. I. Tetrahedron Lett. 1970, 4975-4978.
(10) Matsui, M.; Yamamura, Y.; Takebayashi, T.; Iwaki, K.; Takami, Y.;
Kunitake, K.; Koga, F.; Urasaki, S.; Watanabe, Y. J. Nat. Prod. 1984,
47, 858-861.
(11) Kashiwaba, N.; Morooka, S.; Kimura, M.; Ono, M.; Toda, J.; Suzuki,
H.; Sano, T. J. Nat. Prod. 1996, 59, 476-480.
(7) Williams, D. H.; Fleming, I. Spectroscopic Methods in Organic
Chemistry, 5th ed.; Beijing World Publishing Corporation/McGraw-
Hill Publishing Company: Beijing, 1998; p 46.
NP0500833