Indole alkaloids from cultivated Vinca major

Article abstract of DOI:10.1016/j.tet.2014.09.026

Nine new indole alkaloids, vinmajines A-I (1-9), and 43 known indole alkaloids were isolated from cultivated Vinca major in Kunming. The new structures were elucidated by extensive spectroscopic and quantum theory analysis. In addition, the results also supported that types of indole alkaloids from V. major might be influenced significantly by the ecological environment.

Full text of DOI:10.1016/j.tet.2014.09.026

Tetrahedron 70 (2014) 8723e8729
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Indole alkaloids from cultivated Vinca major
,
,
Gui-Guang Cheng a b, Yun-Li Zhao a, Yu Zhang a, Paul-Keilah Lunga a c, Dong-Bao Hu a,
Yan Li a, Ji Gu a, Chang-Wei Song a, Wei-Bang Sun a, Ya-Ping Liu a , Xiao-Dong Luo
,
a,
*
*
a State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences,
Kunming 650201, PR China
b Yunnan Institute of Food Safety, Kunming University of Science and Technology, Kunming 650500, PR China
c
ꢀ
Department of Biochemistry, Laboratory of Phytobiochemistry and Medicinal Plants Study, Faculty of Science, University of Yaounde 1, P.O. Box 812,
Yaound, Cameroon
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 June 2014
Received in revised form 21 August 2014
Accepted 9 September 2014
Available online 16 September 2014
Nine new indole alkaloids, vinmajines AeI (1e9), and 43 known indole alkaloids were isolated from
cultivated Vinca major in Kunming. The new structures were elucidated by extensive spectroscopic and
quantum theory analysis. In addition, the results also supported that types of indole alkaloids from V.
major might be influenced significantly by the ecological environment.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Indole alkaloids
Cytotoxicity
Absolute configuration
Vinca major
1. Introduction
sarpagine, ajmaline, akuammine, and oxindole types. In our in-
vestigation, we found that the type of plant secondary metabolites
Plants of the Apocynaceae family are rich sources of structurally
diversified indole alkaloids, which originate from the condensation
of tryptophan with secologanin.1 Many of them, such as yohim-
bine,2 reserpine,3 and camptothecin4 are well known for their
pharmacological significance. We focused on antitumor indole al-
kaloids from the Alstonia, Melodinus, and Tabernaemontana genera
of Apocynaceae, and reported several novel and bioactive indole
and bisindole alkaloids recently.5 Particularly, the reported (19,20)-
E/Z-alstoscholarine,6 scholarisine A,7 melohenine B,8 and melote-
nine A9 have been total synthesized by other chemists, due to their
complex molecular architectures. The genus Vinca (Apocynaceae) is
distributed natively to Europe, Northwest Africa, and South-west
Asia. Pharmacological investigation on Vinca alkaloids and their
derivatives are mainly focused on anticancer activity, such as vin-
blastine, vincristine, vindesine, and vinorelbine.10 Vinca major is
a perennial evergreen herb with medicinal and ornamental values,
and have also been cultivated in China widely as an outside orna-
ment. Previous phytochemical investigation on this specie collected
from Turkey,11 Yugoslavia,12 Canada,13 Iran,14 India,15 and Japan,16
reported diverse alkaloidal patterns such as yohimbine,
would be influenced significantly by the ecological environment,17
which encouraged us to search for structurally unique and bi-
ologically active indole alkaloids from V. major in different habitats.
As a result, nine new indole alkaloids (Fig. 1) including four ajma-
line-type alkaloids, vinmajines AeD (1e4), two sarpagine-type al-
kaloids, vinmajines E and F (5e6), one oxindole-type alkaloid,
vinmajine G (7), two pyridino-indolo-quinolizidinone alkaloids,
vinmajines H and I (8e9) together with 43 known alkaloids were
isolated from V. major cultivated in Kunming botanical garden. The
new alkaloids were elucidated by means of spectroscopic methods.
To the best of our knowledge, the pyridino-indolo-quinolizidinone
alkaloids, vinmajines F/G, 19-O-acetylangustoline,18 angustoline,18
19-O-methylangustoline,19 and angustidine20 were first reported
from plants of the genus Vinca. All compounds were evaluated for
cytotoxicity against five human cancer cell lines. Reported herein
are the isolation, structural elucidation, and cytotoxic activities of
these compounds.
2. Results and discussion
Vinmajine A (1) was obtained as colorless oil and gave a positive
reaction with Dragendorff’s reagent, characteristic for an alkaloid.
Its molecular formula was established as C25H31N3O5 by the mo-
lecular ion at m/z 453.2269 [M]þ in the HREIMS, indicating twelve
* Corresponding authors. Tel.: þ86 871 65223177; e-mail addresses: liuyaping@
mail.kib.ac.cn (Y.-P. Liu), xdluo@mail.kib.ac.cn (X.-D. Luo).
http://dx.doi.org/10.1016/j.tet.2014.09.026
0040-4020/Ó 2014 Elsevier Ltd. All rights reserved.

8724
G.-G. Cheng et al. / Tetrahedron 70 (2014) 8723e8729
Fig. 1. Structures of vinmajines AeI (1e9).
degrees of unsaturation. The UV spectrum showed absorption
maxima characteristic of an indolenine chromophore (279, 222,
201 nm), which was supported by the characteristic chemical shift
of C-2 at dC 181.3 in the 13C NMR spectrum. The IR absorption at
1743 cmꢀ1 showed the presence of ester carbonyl group. In the 1H
NMR spectrum, three aromatic protons at dH 7.48, 6.99, and 6.87
form an ABX system with JAB¼8.5 Hz and JBx¼2.5 Hz revealed the
presence of a mono-substituted ring in a monoterpenoid indole
alkaloid.21 Besides substituent groups (two methoxyls and two
acetyls), compound 1 possessed 19 skeleton carbon resonances,
ascribable to one methyls, two sp3 methylenes, eight sp3 methines,
three sp2 methines, and four sp2 quaternary and one sp3 quaternary
carbons in the 13C NMR and DEPT spectra (Table 3). Comparison of
its NMR spectral data (Tables 1 and 3) and molecular formula with
those of 10-methoxyperakine11 suggested that 1 was an Ajmaline-
type indole alkaloid bearing an additional methoxyl group (dH 3.29,
dC 55.9) and an acetamido group (dH 2.06, dC 170.3, 23.5). Signal of
aldehyde group in 10-methoxyperakine11 was disappeared,
a downfield methine at dC 81.1 (d, C-21) was appeared as instead in
the 13C NMR spectrum of 1, which was supported by the HMBC
correlations of dH 2.57 (1H, t, J¼5.0 Hz, H-15) to dC 81.1 (d, C-21).
Furthermore, the HMBC correlations from the methine proton at dH
5.21 (1H, dd, J¼9.6, 7.8 Hz, H-21) to dC 55.9 (eOCH3) and 170.3
(eNCOCH3), and from dH 3.29 (eOCH3) and 2.06 (eNCOCH3) to dC
81.1 suggested that both the methoxyl (dH 3.29; dC 55.9) and the
acetamido group (dH 2.06; dC 170.3 and 23.5) were connected at C-
21 (Fig. 2).
In ROESY spectrum of 1, NOE correlations of dH 4.13 (H-3) with
2.14 (H-14a) and 2.82 (H-19), and of dH 1.28 (H3-18) with 3.59 (H-5)
and 1.36 (H-20) placed H-3, H-14a, and H-19 at the same side (
orientation), while H-20 at another side, because Ajmaline-type
alkaloids took -orientation of H-3 from their biosynthetic con-
a-
a
sideration. Furthermore, other chiral centers were identical to
those of 10-methoxyperakine supported by ROESY spectrum of 1
(Fig. 2). A quantum chemical calculation was applied to assign the
configuration of C-21 in 1. All DFT and TDDFT calculations were
carried out at 298.15 K in the gas phase with Gaussian 09. The
electronic circular dichroism (ECD) was investigated at the B3LYP/
6-311þþG(d,p) level.22 The CD spectrum generated was in agree-
ment with the experimental data of 1. The recorded and the com-
puted CD curves are illustrated in Fig. 3. All of the evidence from
NMR and ECD suggested that 1 has an absolute configuration of 3S,
5S, 7R, 15R, 16S, 17R, 19S, 20R, 21S.
The molecular formula of 2 was determined to C22H26N2O4 by
the positive HRESIMS ([MþH]þ at m/z 383.1964), and was the same
as that of 10-methoxyraucaffrinoline23 They have similar physical
data in the UV and IR spectra, assuming the existence of the same
functional groups. The 1H and 13C NMR spectra of 2 (Tables 1 and 3)
were similar to those of 10-methoxyraucaffrinoline except for
chemical shift of dH 1.99 (1H, dt, J¼13.5, 9.2 Hz, H-20) and dC 41.6 (d,
Table 1
1H NMR data of 1e3 in CDCl3
(d
in ppm and J in Hz)
No.
1a
2b
3a
2
3
5
6a
6b
9
3.10, overlap
3.57, dd (9.5, 5.0)
3.21, d (6.3, 5.0)
2.41, m
1.79, d (11.7)
6.73, d (2.6)
4.13, d (9.2)
3.59, m
2.71, dd (11.8, 4.9)
1.66, d (11.8)
4.10, d (8.6)
3.58, m
2.72, dd (11.9, 4.9)
1.61, d (11.9)
C-20). The difference was that the configuration of H-20 in 2 was a-
oriented, as supported by NOE correlations of dH 3.21 (H-19) with
4.10 (H-3) and 1.99 (H-20) in ROESY spectrum. Other parts of 2 were
identical to those of 10-methoxyraucaffrinoline, which was sup-
ported by detailed analysis of 2D NMR spectra of 2. Thus, com-
pound 2 was assigned as shown, and named vinmajine B.
Vinmajine C (3) gave the molecular formula C23H28N2O3 on the
basis of HREIMS. The 13C NMR and DEPT spectra of compound 3
displayed 23 carbon resonances ascribable to four methyls, three
methylenes, ten methines, and six quaternary carbons (Table 3).
These data were closely related to those of vincamedine,24 sug-
gesting that 3 was also an ajmaline-type alkaloid. The most notable
difference between the two alkaloids was the loss of eCOOCH3
group at C-16 in 3, as supported by the HMBC correlations of dH 2.13
(1H, d, J¼5.8 Hz, H-16) with dC 21.6 (t, C-14), 55.2 (s, C-7), and 77.5
(d, C-17). The E configuration of double bond at side chain was
indicated by the NOE correlations between dH 1.63 (CH3-18) and
3.11 (H-15). Furthermore, the NOE correlations of H-17 (dH 5.17)
6.99, d (2.5)
6.98, d (2.5)
10
11
12
14a
14b
15
6.87, dd (8.5, 2.5)
7.48, d (8.5)
2.14, overlap
1.51, dd (14.8, 4.8)
2.57, t (5.0)
2.31, t (6.0)
4.95, s
6.87, dd (8.5, 2.5)
7.47, d (8.5)
1.82, dd (13.6, 4.8)
1.76, m
2.56, d (4.4)
2.38, t (6.0)
6.70, dd (8.4, 2.6)
6.55, d (8.4)
2.46, dd (13.1, 3.9)
1.47, dd (13.7, 10.0)
3.11, overlap
2.13, t (5.8)
16
17
5.01, s
5.17, s
18
19
20
21a
21b
OCOCH3
NCOCH3
10-OCH3
21-OCH3
eNCH3
1.28, d (6.5)
2.82, m
1.36, t (8.3)
5.21, dd (9.6, 7.8)
1.19, d (7.4)
3.21, dd (9.2, 7.4)
1.99, dt (13.5, 9.2)
3.83, dd (10.9, 6.4)
3.70, t (10.2)
2.13, s
1.63, d (6.5)
5.24, q (6.5)
3.47, br s
1.94, s
2.14, s
2.06, s
3.79, s
3.29, s
3.81, s
3.74, s
2.58, s
with H-14a (dH 2.46), and H-15 (dH 3.11) suggested H-17 to be b-
a
oriented, identical to the configuration of vincamedine. Thus, the
structure of vinmajine C (3) was assigned as shown.
Recorded at 400 MHz.
Recorded at 500 MHz.
b

G.-G. Cheng et al. / Tetrahedron 70 (2014) 8723e8729
8725
Table 2
1H NMR data of 4e9a
(d in ppm and J in Hz)
No.
4b
5c
6c
7c
8c
9c
N1eH
3
10.49, s
4.21, br s
10.26, s
2.26,
overlap
3.16, m
11.0, s
11.90, s
3.64, d (8.6)
3.61, m
4.21, s
5a
3.05, overlap
3.10, d (15.0)
3.91, m
4.35, tdd
(13.5, 10.4, 6.2)
4.42, qt (13.4, 6.5)
3.14, m
5b
6a
3.83, d (4.0)
2.56, d (13.0)
2.21, m
2.13, dd
3.08, dd
3.07, m
(15.5, 4.9)
2.63, t (12.2)
6.88, d (1.8)
(12.3, 8.2)
1.77, m
6.93, d (7.7)
6.34, d (7.7)
6b
9
10
11
2.44, m
2.68, d (12.1)
6.65, d (2.1)
7.13, d (7.2)
6.73, t (7.2)
7.10, t (7.8)
7.53, d (8.0)
6.99, t (8.0)
7.13, t (8.0)
7.65, d (7.9)
7.12, t (7.9)
7.29, t (7.9)
6.52, dd
(8.5, 2.1)
7.05, d (8.5)
2.54, m
1.80, t (10.3)
6.78, dd
(8.4, 1.8)
7.21, d (8.4)
1.91, m
12
14a
14b
6.59, d (7.8)
2.48, m
1.71, dd
(13.4, 9.7)
3.48, d (4.9)
6.29, s
2.35, m
0.75, dd
(23.5, 11.6).
1.33, m
7.31, d (8.0)
7.04, s
7.50, d (7.9)
7.25, s
1.53, m
15
3.05, overlap
2.78, dd
(22.0, 7.1)
2.55, t (6.2)
9.79, s
16
2.55, t (6.2)
7.42, s
17a
17b
18
4.05, s
4.13, d (10.5)
4.05, d (10.5)
1.50, d (6.8)
5.36, q (6.8)
9.21, s
9.26, s
1.54, d (6.6)
5.17, q (6.6)
1.33, d (6.7)
3.28, dq
(13.6, 6.7)
2.06, m
1.35, d (6.1)
4.25, br s
1.51, d (6.7)
6.17, q (6.7)
1.50, d (6.5)
5.35, q (6.5)
19
20
21a
21b
NCH3
10-OCH3
eOH
OCOCH3
COOCH3
1.57, m
3.26, m
2.29, m
3.64, m
3.44, d (15.4)
2.54, s
3.56, m
9.77, s
8.58, s
1.99, s
8.78, s
3.84, s
3.54, s
9.45, s
8.58, s
1.93, s
2.92, s
5.61 d (4.1)
3.61s
a
Compounds 4 and 6 were measured in CDCl3, 5, 7, and 9 in DMSO-d6, 8 in acetone-d6.
Recorded at 400 MHz.
Recorded at 600 MHz.
b
c
Table 3
13C NMR spectral data of 1e9a
(
d
in ppm)
No.
1b
2c
3b
4b
5d
6d
7d
8d
9d
2
3
5
6
7
8
9
10
11
12
13
14
15
16
17
18
181.3C
56.9CH
51.2CH
37.4CH2
64.7C
181.4C
75.9CH
53.7CH
59.1CH
36.7CH2
55.2C
69.5CH
69.2CH
75.5CH
30.3CH2
55.8C
128.9C
124.9CH
119.3CH
128.4CH
109.4CH
154.0C
21.6CH2
29.1CH
60.7C
138.1C
49.7CH
57.0CH
23.7CH2
103.0C
126.9C
101.8CH
150.2C
110.4CH
111.3CH
130.8C
28.4CH2
29.0CH
48.6C
137.8C
180.7C
70.8CH
53.1CH2
34.5CH2
55.7C
128.7C
138.1C
41.2CH2
20.2CH2
116.4C
126.7C
120.5CH
121.0CH
125.5CH
112.7CH
139.7C
93.8CH
139.6CH
120.4C
151.2CH
22.3CH3
68.5CH
131.1C
127.7C
136.4C
40.2CH2
19.1CH2
114.5C
125.3C
119.6CH
119.8CH
124.4CH
111.9CH
138.4C
93.7CH
138.3C
118.8C
149.1CH
25.2CH3
63.8CH
134.8C
147.5CH
161.1C
56.8CH
51.1CH
38.1CH2
64.8C
52.1CH
44.4CH
27.5CH2
104.6C
127.9C
100.5CH
154.3C
111.7CH
111.9CH
131.5C
29.0CH2
24.9CH
53.2CH
202.2CH
19.1CH3
51.9CH
51.7CH
202.1CH
137.8C
110.9CH
157.8C
112.7CH
121.1CH
150.0C
22.2CH2
26.6CH
49.1CH
77.9CH
19.5CH3
52.2CH
48.2CH
81.1CH
137.9C
110.8CH
157.8C
112.7CH
120.9CH
149.8C
27.8CH2
26.0CH
43.4CH
78.4CH
14.0CH3
51.7CH
41.6CH
61.3CH2
131.2C
111.3CH
153.3C
112.4CH
109.3CH
148.4C
21.6CH2
25.9CH
47.2CH
77.5CH
12.9CH3
114.7CH
139.3C
55.7CH2
123.5C
124.7CH
108.2CH
157.3C
97.5CH
142.4C
30.1CH2
30.0CH
109.6C
154.6CH
18.4CH3
72.1CH
37.2CH
53.7CH2
73.4CH
12.5CH3
119.7CH
129.5C
69.9CH2
69.0CH2
12.5CH3
116.1CH
136.8C
54.4CH2
19
20
21
22
148.9CH
162.1C
OCOCH3
OCOCH3
COOCH3
COOCH3
NCOCH3
NCOCH3
10-OCH3
19-OCH3
NCH3
169.9C
21.1CH3
169.9C
21.0CH3
170.0C
21.2CH3
170.1C
170.4C
21.1CH3
170.7C
52.0CH3
171.6C
51.1CH3
166.7C
51.0CH3
23.5CH3
170.3C
55.6CH3
55.9CH3
55.5CH3
55.8CH3
34.8CH3
56.1CH3
34.4CH3
a
Compounds 1e4 and 6 were measured in CDCl3, 5, 7, and 9 in DMSO-d6, 8 in acetone-d6.
b
c
Recorded at 400 MHz.
Recorded at 500 MHz.
Recorded at 600 MHz.
d

8726
G.-G. Cheng et al. / Tetrahedron 70 (2014) 8723e8729
correlations indicated that the relative configuration of 7 was the
same as that of rumberine.
The mixture of vinmajine H (8) and 19-O-acetylangustoline was
obtained as yellow amorphous powder with the molecular formula
C
22H19N3O3, as established by HRESIMS based on the [MþH]þ peak
(m/z 374.1497), indicating fifteen degrees of unsaturation. The IR
spectrum displayed absorption bands due to eNH (3445 cmꢀ1),
ester (1720 cmꢀ1), and conjugated amide (1654 cmꢀ1) groups. The
13C NMR spectrum displayed indole ring signals [dC 128.7 (s, C-2),
116.4 (s, C-7), 126.7 (s, C-8), 120.5 (d, C-9), 121.0 (d, C-10), 125.5 (d,
C-11), 112.7 (d, C-12), 139.7 (s, C-13)], and signals for three sp2
methines (dC 93.8, 148.9, and 151.2), one sp3 methine (dC 68.5), two
sp3 methylenes (dC 41.2 and 20.2), two methyls (dC 21.1 and 22.3),
and six quaternary carbons (dC 120.4, 131.1, 138.1, 139.6, 162.1, and
170.4). These data indicated that it was a pyridino-indolo-quinoli-
zidinone alkaloid, and identical to those of 19-O-acetylangusto-
line.18 However, the optical rotation value indicated a racemate for
it. Then, the racemate was subsequently resolved by HPLC column
on a chiral stationary phase to afford the individual enantiomers,
(þ)-8, and (ꢀ)-19-O-acetylangustoline, showing opposite Cotton
effects in the CD spectra and opposite optical rotations. The abso-
lute configuration of 8 was also assigned using the quantum
method.22 The optical rotation was computed at the B3LYP/6-
311þþg(2d,2p)//B3LYP/6-311þþg(d,p) level. The calculated OR
value for the absolute configuration of 19S was þ55.9, which is
close to the experimental value of þ51.3. In addition, the ECDs
calculated at the B3LYP/6-311þþG(d,p) level, and showed agree-
ment with the experimented spectra with the experimental data of
8 (Fig. 4). Thus, the structure of vinmajine H (8) was determined as
19S-O-acetylangustoline.
Fig. 2. Key HMBC (
) and ROESY (
) correlations of 1.
Fig. 3. Calculated and experimental ECD spectra of 1.
Vinmajine D (4) was isolated as a white amorphous powder and
had a molecular ion peak [M]þ at m/z 382.1895 in its HREIMS,
identified as C22H26N2O4, 16 Da higher than that of vincamajine.25
Compound 4 was readily assumed as vincamajine-N(4)-oxide
from its 1H and 13C NMR spectral data, in particular the charac-
teristic downfield shifts of the carbon resonances at dC 69.2, 69.9,
and 75.5 for C-3, C-21, and C-5, respectively, with respect to those of
vincamajine. Other parts of 4 were identical to those of vincamajine
supported by its HSQC, HMBC, and ROESY spectra.
The molecular formula C23H26N2O5 of vinmajine E (5) was given
by HREIMS (m/z 410.1841 [M]þ). Its 1H and 13C NMR spectral data
showed that 5 had a structure similar to that of acetylakuammi-
dine,26 except for one more hydroxyl group (dH 8.58) in 5. The
hydroxyl was placed at C-10, which was supported by correlations
of dH 6.65 (1H, d, J¼2.1 Hz, H-9) with dC 150.2 (s, C-10), and 130.8 (s,
C-13); and of dH 7.05 (1H, d, J¼8.5 Hz, H-12) with dC 126.9 (s, C-8),
and 150.2 (s, C-10) in the HMBC spectrum of 5. Complete analysis of
2D NMR spectral data of 5 indicated that the other parts were the
same to those of acetylakuammidine.
Vinmajine F (6) had the molecular formula C22H22N2O3 as de-
termined by the positive HREIMS at m/z 338.1628 [M]þ. Its 13C NMR
spectrum showed close similarities to those of 19,20-
dihydroperaksine-17,21-al,27 with the presence of an additional
methoxyl group (dH 3.84; dC 56.1) in the aromatic region of 6 (Tables
2 and 3). The HMBC correlations of dH 3.84 (3H, s) with dC 154.3 (s,
C-10); and of dH 7.21 (1H, d, J¼8.4, H-12) with dC 127.9 (s, C-8) and
154.3 (s, C-10) allowed placement of the methoxyl group at C-10.
Thus, the structure of compound 6 was established as shown, and
all the signals were assigned on the basis of its 2D NMR spectra.
The HREIMS of vinmajine G (7) displayed its molecular ion peak
[M]þ at m/z 384.1690 (C21H24N2O5), identical to that of rumber-
ine.28 In addition, its NMR spectral data also suggested the simi-
larity of two compounds. Detailed comparison of 1H and 13C NMR
spectral data of the two compounds suggested that the hydroxyl
group was assigned at C-11 in 7, rather than at C-10, which was
further supported by the HMBC correlations of dH 9.45 (s, eOH)
with dC 157.3 (s, C-11), of dH 6.93 (1H, d, J¼7.4 Hz, H-9) with dC 55.7
(s, C-7), 142.4 (s, C-13), and 157.3 (s, C-11). Moreover, ROESY
Fig. 4. Calculated and experimental ECD spectra of 8.
The similar TLC behavior, UV, and IR spectral data of vinmajine I
(9) and 8 suggested both compounds have the same skeleton.
Vinmajine I (9) and angustoline were also separated in the form of
an enantiomeric mixture by HPLC on a Chiralpak IC column. The
HREIMS gave the molecular formula C20H18N3O2. However, the
optical rotation value of 9 [þ31.7 (c 0.10, MeOH)] was similar
magnitude, but of opposite sign to that of angustoline [ꢀ34
(CDCl3)].18 Hence, compound
angustoline.
9 was elucidated to be 19S-
In addition, other 43 known indole alkaloids were also isolated
from V. majorcultivatedinKunming, PRChina.Theknowncompounds
were identified as 19-O-acetylangustoline,18 angustoline,18 19-O-
methyl-angustoline,19 angustidine,20 perakine,29 10-methoxy
perakine,11 10-methoxyraucaffrinoine,23 vincamedine24 vincama-
jine,25 vincamajoreine,30 majoridine,30 10-methoxyvinorine,11

G.-G. Cheng et al. / Tetrahedron 70 (2014) 8723e8729
8727
vinorine,31 10-methoxyvellosimine,32 10-hydroxystrictamine,33 catha
foline,34 17-hydroxy-pseudoakuammigine,35 pseudoakuammigine,35
lanceomigine,35 17-methoxypseudoakuammigine,36 akuammine,37
strictosamide,38 ajmalicine,39 reserpinine,40 vallesiachotamine,30
isovallesiachotamine,41 vallesiachotamine lactone,42 majdine,43 iso-
majdine,43 vinerine,44 vineridine,44 herboxine,45 reserpinine oxin-
dole,46 carboxine B,28 tetraphylline pseudoindoxyl,47 vincadiff
ormine,48 3-oxo-tabersonine,49 lochnericine,50 3-oxo-lochnericine,50
vincamine,51 14,15-dehydrovincamine,52 akuammicine N(4)-oxide,53
and quebrachamine54 by comparison of their spectroscopic data
with those reported in the literature.
All alkaloids were evaluated for their cytotoxicity against five
human cancer cell lines using MTT method as reported pre-
viously.55 Compound 6 displayed stronger inhibitory effects against
A-549 with lower IC50 values than those of cisplatin. The com-
pounds 3-oxo-lochnericine, and 14,15-dehydrovincamine dis-
played moderate cytotoxicity against some of the cell lines, while
perakine, isomajdine, 10-methoxyvellosimine, and vincadifformine
exhibited weak cytotoxicity (Table S1). Other compounds were
810 spectrometer. Column chromatography (CC) was performed on
Silica gel (200e300 mesh, Qingdao Marine Chemical Ltd., Qingdao,
People’s Republic of China), RP-18 gel (20e45
mm, Fuji Silysia
Chemical Ltd., Japan), and Sephadex LH-20 (Pharmacia Fine
Chemical Co., Ltd., Sweden). Fractions were monitored by TLC (GF
254, Qingdao Haiyang Chemical Co., Ltd. Qingdao), and spots were
visualized by Dragendorff’s reagent. Medium pressure liquid
chromatography (MPLC) was employed using a Buchi pump system
coupled with C18-silica gel-packed glass column (15ꢁ230 and
26ꢁ460 mm). High performance liquid chromatography (HPLC)
was performed using an Agilent 1260 pump coupled with Agilent
semi-preparative and preparative C18 columns (150ꢁ9.4 and
250ꢁ21.2 mm, respectively). Chiral separation was performed by
HPLC on a Daicel Chiralpak IC column (250ꢁ10.0 mm).
4.2. Plant material
V. major was collected from Kunming Botanical Garden, Yunnan
province, PR China, and identified by Prof. Wei-Bang Sun, Kunming
institute of Botany. A voucher specimen (No. Sun20110820) has
been deposited at deposited in the State Key Laboratory of Phyto-
chemistry and Plant Resources in West China, Kunming Institute of
Botany, Chinese Academy of Sciences.
inactive (IC50 values >40 mM).
3. Conclusion
Monoterpenoid indole alkaloids (MIAs), including more than
2000 compounds, play a very important role in natural medicinal
history. Vinca alkaloids, one of the most notable examples of ter-
penoid alkaloids from strictosidine condensed by tryptamine and
secologanin, are characteristic of the genus Vinca and comprise five
main groups: eburna alkaloids, sarpagine alkaloids, ajmaline alka-
loids, akuammine alkaloids, and oxindole alkaloids. In this paper,
nine new indole alkaloids with 43 known alkaloids were isolated
from cultivated V. major. To the best of our knowledge, the alkaloids
with pyridino-indolo-quinolizidinone skeleton, including vinma-
jines F/G, 19-O-acetylangustoline, 19-O-methylangustoline, angus-
toline, and angustidine, were first reported from plants of the genus
Vinca, which have been isolated from genera of mitragyna, nauclea,
uncaria, and strychnos. Furthermore, three akuammine alkaloids,
4.3. Extraction and isolation
An air-dried and powdered sample (20 kg) was extracted with
MeOH (3ꢁ50 L) at room temperature and the solvent removed in
vacuo. The residue was dissolved in 0.3% aqueous hydrochloric acid
(v/v), and the solution was subsequently basified to pH 9e10, using
ammonia, then partitioned with EtOAc (3ꢁ10 L) to give an alka-
loidal extract. The extract was subjected to a silica gel column
(CHCl3/MeOH, 1:0e0:1) to afford fractions (IeVII). Fraction I (4.6 g)
was further applied to a silica gel column using a petroleum ether/
acetone gradient eluent (12:1e8:1) to yield alkaloids ajmalicine
(217 mg), vallesiachotamine lactone (47 mg), and vincadifformine
(64 mg). Fraction II (15.8 g) was separated by silica gel CC (petro-
leum ether/Me2CO, 8:1e2:1), then by RP-18 CC, eluted with MeOH/
H2O (5:5e10:0) to afford reserpinine (6.21 g), vallesiachotamine
(2.25 g), and a mixture (1.33 g). The mixture was chromatographed
on a silica gel column (petroleum ether/EtOAc, 6:1e2:1), then pu-
rified on a preparative C18 HPLC column with a gradient of MeOH/
H2O (65:35e70:30) to yield lochnericine (47 mg), 3-oxo-taber-
sonine (97 mg), and 3-oxo-lochnericine (43 mg). Fraction III (28.6 g)
was purified on a preparative C18 HPLC column with a gradient of
MeOH/H2O (40:60e70:30) to yield six subfractions III-1e6. Sub-
fraction III-1 (6.5 g) was further subjected to silica gel CC using
a petroleum ether/Me2CO gradient eluent (10:1e5:1) to yield
majdine (3.65 g), isomajdine (145 mg), and quebrachamine
(43 mg). Subfraction III-2 (130 mg) was separated on a preparative
C18 HPLC column with a gradient of MeOH/H2O (55:45e70:30) to
yield isovallesiachotamine (42 mg) and tetraphylline pseu-
doindoxyl (66 mg). Subfraction III-3 (210 mg) was further purified
on a C18 HPLC column with a gradient of MeOH/H2O (50:50e60:40)
to afford perakine (108 mg), herboxine (35 mg), and reserpinine
oxindole (21 mg). Subfraction III-4 (67 mg) was further purified on
a C18 HPLC column with a gradient MeOH/H2O (55:45e70:30) to
afford vincamine (17 mg) and 14,15-dehydro-16-epivincamine
(11 mg). Subfraction III-5 (1.9 g) was chromatographed on a silica
gel column (petroleum ether/Me2CO, 3:1e3:2), then purified on
a preparative C18 HPLC column with a gradient of MeOH/H2O
(55:45e65:45) to yield isovinerine (97 mg), and carboxine B
(16 mg). Fraction III-6 (5.3 g) was subjected to MPLC with RP-18 CC
(MeOH/H2O, 6:4e8:2), then followed by silica gel CC (petroleum
ether/Me2CO, 8:1e3:1) to yield 5 (15 mg), 10-methoxyperakine
(1.95 g), vincamajine (1.39 g), majoridine (121 mg), 10-
lanceomigine,
17-hydroxypseudoakuammigine,
and
17-
methoxypseudoakuammigine, two ajmalicine alkaloids, valle-
siachotamine lactone, and tetraphylline pseudoindoxyl, two aspi-
dospermine alkaloids, lochnericine, and 3-oxo-lochnericine, one
ajmaline alkaloids, perakine, one oxindole alkaloids. reserpinine
oxindole, one sarpagine alkaloid, cathafoline, and one strychnos
alkaoid, akuammicine N(4)-oxide, as well as 14,15-
dehydrovincamine were also obtained firstly from the genus
Vinca. In addition, twelve alkaloids, 10-methoxy-raucaffrinoline,
vinorine, 10-methoxyvinorine, vinerinine, vineridine, herboxine,
10-hydroxystrictamine, vallesiachotamine, isovallesiachotamine,
3-oxo-tabersonine, vincadifformine, and quebrachamine were first
reported in V. major, which have been isolated from other species of
Vinca.
4. Experimental section
4.1. General information
Optical rotations were measured with a Horiba SEPA-300 po-
larimeter. UV spectra were obtained using a Shimadzu UV-2401A
spectrometer. IR spectra were obtained by a Bruker FT-IR Tensor
27 spectrometer using KBr pellets. 1D and 2D spectra were run on
an AVANCE III-600 MHz, a Bruker DRX-500 MHz spectrometer, or
an AV-400 MHz spectrometer with TMS as an internal standard.
Chemical shifts (d) were expressed in parts per million with refer-
ence to solvent signals. HREIMS was recorded on a Waters Auto
Premier P776 spectrometer. HRESIMS was recorded on an API
QSTAR Pulsar 1 spectrometer. CD spectra were obtained on a JASCO

8728
G.-G. Cheng et al. / Tetrahedron 70 (2014) 8723e8729
methoxyvinorine (48 mg), and vinorine (36 mg). Fr. IV (8.2 g) was
purified by C18 MPLC with a MeOH/H2O gradient (40:60e80:20) to
yield subfractions IV-1e3. 10-Hydroxystrctamine (3.2 g) was crys-
tallized from subfraction IV-1. The mother liquid of this subfraction
(121 mg) was further separated on a preparative C18 HPLC column
with a gradient MeOH/H2O (55:45e70:30) to yield 3 (42 mg), 4
(20 mg), 6 (4 mg), and vincamedine (26 mg). Subfraction IV-2 was
further separated on the same column with MeOH/H2O (60:40) to
yield 7 (29 mg), cathafoline (11 mg), and akuammicine N(4)-oxide
(7 mg). Fraction V (91 g) was separated by silica gel CC (CHCl3/
MeOH, 15:1e10:1) to yield 10-methoxyvellosimine (14 mg), 17-
methoxypseudoakuammigine (780 mg), strictosamide (6.8 g), and
a mixture. Further separation of the mixture by a preparative C18
column gives subfractions V-1e5. Subfraction V-1 (1.2 g) was sep-
arated on a preparative C18 column with a gradient MeOH/H2O
(30:70e80:20), then separated on a semipreparative C18 HPLC
column (MeOH/H2O, 75%) to produce pseudoakuammigine
(11 mg), 17-hydroxypseudoakuammigine (6 mg), and akuammine
(6 mg). Subfraction V-2 (6.3 g) was purified by CC over silica gel
with CHCl3/Me2CO (9:1e4:1) to yield V-2-1 (11 mg), V-2-2 (10 mg),
and 19-O-methylangustoline (22 mg), HPLC separation of V-2-1
and V-2-2 with a Chiralpak column (CH2Cl2/MeOH, 5:95) led to two
pairs of enantiomers 8 (4 mg) and 19-O-acetylangustoline (4 mg),
as well as 9 (2 mg) and angustoline (2 mg). Subfraction V-3 (2.1 g)
was subjected to silica gel CC using CHCl3/Me2CO eluent (3:1) to
yield V-3-1e3. V-3-1 (130 mg) was further separated on a pre-
(4.44); IR (KBr) nmax 3434, 2928, 1714, 1458, 1381, 1233, 1109, 1035,
985 cmꢀ1; 1H (600 MHz) and 13C NMR (150 MHz) data (DMSO-d6),
see Tables 2 and 3; HREIMS m/z 410.1841 (calcd for C23H26N2O5
[M]þ,410.1842).
23
4.4.6. Vinmajine F (6). Colorless oil; [
a]
þ55.9 (c 0.101, MeOH);
D
UV (MeOH) lmax (log ε): 277 (3.85), 224 (4.34), 209 (4.34); IR (KBr)
nmax 3397, 2929, 1715, 1596, 1281, 1217, 1151, 1032 cmꢀ1 1H
;
(600 MHz) and 13C NMR (150 MHz) data (CDCl3), see Tables 2 and 3;
HREIMS m/z 338.1628 (calcd for C20H22N2O3 [M]þ, 338.1630).
22
4.4.7. Vinmajine G (7). White amorphous powder; [
a
]
ꢀ92.2 (c
D
0.098, MeOH); UV (MeOH) lmax (log ε): 217 (4.39), 194 (4.14); IR
(KBr) nmax 3370, 3289, 2947, 1704, 1629, 1440, 1314, 1211, 1151,
1086 cmꢀ1; 1H (600 MHz) and 13C NMR (150 MHz) data (DMSO-d6),
see Tables 2 and 3; HREIMS m/z 384.1690 (calcd for C21H24N2O5
[M]þ,384.1685).
22
4.4.8. Vinmajine H (8). Yellow amorphous powder; [
a]
þ51.3 (c
D
0.230, MeOH); UV (MeOH) lmax (log ε): 395 (3.59), 376 (3.59), 220
(3.54); IR (KBr) nmax 345, 1720, 1654, 1606, 1534, 1374, 1253, 1029,
740 cmꢀ1; 1H (600 MHz) and 13C NMR (150 MHz) data (acetone-d6),
see Tables 2 and 3; positive ion HRESIMS m/z 374.1497 (calcd for
C
22H20N3O3 [MþH]þ, 374.1505).
22
4.4.9. Vinmajine I (9). Yellow amorphous powder; [
a
]
þ31.7 (c
D
parative C18 HPLC column with
(50:50e70:30) to give a mixture, then chiral separated by HPLC on
Chiralpak IC column to afford (32 mg) and 10-
a
gradient MeOH/H2O
0.101, MeOH); UV (MeOH) lmax (log ε): 395 (4.03), 378 (4.14), 222
(4.14); IR (KBr) nmax 3440, 1605, 1501, 1453, 1384, 1325, 1117,
746 cmꢀ1; 1H (600 MHz) and 13C NMR (150 MHz) data (DMSO-d6),
see Tables 2 and 3; HREIMS m/z 332.1392 (calcd for C20H18N3O2
[M]þ, 332.1399).
a
2
methoxyraucaffrinoline (46 mg). V-3-2 (86 mg) was further sepa-
rated on a preparative C18 column with a gradient MeOH/H2O
(40:65e70:30) to afford 1 (7 mg) and vinerine (6 mg). V-3-3
(163 mg) was further separated on the same column with a gradi-
ent MeOH/H2O (55:45e65:35) to vincamajoreine (56 mg).
4.5. Computational methods
Conformational analysis is initially performed using Discovery
Studio 3.5 Client. The step provides a large number of MD mini-
mized geometries and an energy cutoff of 20 kcal/mol was applied
to reduce the number of energy minima to be subsequently re-
fined.56 The geometries and energies of the stationary points on the
potential energy surface were estimated using the DFT (B3LYP)
method in conjunction with the 6-31G(d). Vibrational analysis at
the B3LYP/6-311þþG(d,p) level of theory resulted in no imaginary
frequencies, confirming the considered conformers as real minima.
Free energy values were used to obtain the Boltzmann population
of conformers at 298.15 K. The theoretical calculation of ECD was
performed using time-dependent density-functional theory
(TDDFT) at B3LYP/6-311þþG(d,p) level methanol with PCM model
or in the gas phase. The specific rotation of a chiral molecule in
dilute solution calculated at B3LYP/6-311þþg(2d,2p)//B3LYP/6-
311þþg(d,p) with PCM model. All above calculations were carried
out with the Gaussian09 package.22 The calculated ECD spectra
were obtained by weighing the Boltzmann distribution rate of
lowest-energy conformers.57 The ECD Cotton effect is approxi-
mated by the Gaussian distribution.58
4.4. Spectroscopic data of the isolated compounds
4.4.1. Vinmajine A (1). Colorless oil; [
a
]
22 ꢀ23.2 (c 0.118, MeOH); UV
D
(MeOH) lmax (log ε): 279 (3.97), 222 (4.25), 201 (4.28); IR (KBr) nmax
3439, 2935, 1743, 1658, 1640, 1593, 1530, 1469, 1374, 1033 cmꢀ1; 1H
(400 MHz) and 13C NMR (100 MHz) data (CDCl3), see Tables 1 and 3;
HREIMS m/z 453.2269 (calcd for C25H31N3O5 [M]þ, 453.2264).
22
4.4.2. Vinmajine B (2). Colorless oil; [
a
]
D
þ45.5 (c 0.123, MeOH);
UV (MeOH) lmax (log ε): 279 (3.44), 223 (4.14), 202 (4.08); IR (KBr)
nmax 3429, 2938, 1742, 1622, 1592, 1469, 1220, 1029, 822 cmꢀ1 1H
;
(500 MHz) and 13C NMR (125 MHz) data (CDCl3), see Tables 1 and 3;
positive ion HRESIMS m/z 383.1964 (calcd for C22H27N2O4 [MþH]þ,
383.1971).
22
4.4.3. Vinmajine C (3). White amorphous powder; [
a
]
ꢀ36.7 (c
D
0.112, MeOH); UV (MeOH) lmax (log ε): 310 (3.60), 246 (4.01), 204
(4.47); IR (KBr) nmax 2947, 1737, 1481, 1373, 1250, 1213, 1157, 1119,
1033, 817, 714 cmꢀ1 1H (400 MHz) and 13C NMR (100 MHz) data
;
(CDCl3), see Tables 1 and 3; HREIMS m/z 380.2100 (calcd for
C
23H28N2O3 [M]þ, 380.2100).
4.6. Cytotoxicity assay
21
4.4.4. Vinmajine D (4). White amorphous powder; [
a
]
ꢀ15.2 (c
Five human cancer cell lines: human myeloid leukemia HL-60,
hepatocellular carcinoma SMMC-7721, lung cancer A-549, breast
cancer MCF-7 and colon cancer SW480 cells, were used in the cy-
totoxic assay. All the cells were cultured in RPMI-1640 or DMEM
medium (Hyclone, USA), supplemented with 10% fetal bovine se-
rum (Hyclone, USA) in 5% CO2 at 37 ꢂC. The cytotoxicity assay was
performed according to the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide) method in 96-well microplates.55
D
0.089, MeOH); UV (MeOH) lmax (log ε): 291 (3.44), 247 (3.86), 204
(4.47); IR (KBr) nmax 3417, 2950, 1735, 1611, 1465, 1293, 1245, 1139,
1079, 996, 759, 746 cmꢀ1 1H (400 MHz) and 13C NMR (100 MHz)
;
data (CDCl3), see Tables 2 and 3; HREIMS m/z 382.1895 (calcd for
C
22H26N2O4 [M]þ, 382.1893).
23
4.4.5. Vinmajine E (5). White amorphous powder; [
a
]
þ41.1 (c
D
0.106, MeOH); UV (MeOH) lmax (log ε): 279 (3.99), 225 (4.44), 207
Briefly, 100 mL adherent cells were seeded into each well of 96-

G.-G. Cheng et al. / Tetrahedron 70 (2014) 8723e8729
8729
21. Morfaux, A. M.; Mouton, P.; Massiot, G.; Le Men-Olivier, L. Phytochemistry 1990,
29, 3345e3349.
well cell culture plates and allowed to adhere for 12 h before drug
addition, while suspended cells were seeded just before drug
adding with initial density of 1ꢁ105 cells/ml. Each tumor cell line
was exposed to the test compound at concentrations of 0.064, 0.32,
22. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.;
Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Na-
katsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng,
G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.;
Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery,
J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K.
N.; Staroverov, V. N.; Keith, T.; Kobayashi, R.; Normand, J.; Raghavachari, K.;
Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J.
M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.;
Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.;
Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.;
Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman,
J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, Revision B.01; Gaussian:
Wallingford, CT, 2010.
1.6, 8, and 40 mM in triplicates for 48 h, with cisplatin (Sigma, USA)
as a positive control. After compound treatment, cell viability was
detected and cell growth curve was graphed. IC50 value was cal-
culated by Reed and Muench’s method.59
Acknowledgements
This project was supported by the National Natural Science
Foundation of China (81225024, 31170334), the National Science
and Technology Support Program of China (2013BAI11B02).
23. Boga, M.; Kolak, U.; Topcu, G.; Bahadori, F.; Kartal, M.; Farnsworth, N. R. Phy-
tochem. Lett. 2011, 4, 399e403.
ꢀ
24. Caron, C.; Yachaoui, Y.; Massiot, G.; Le Men-Olivier, L.; Pusset, J.; Sevenet, T.
Phytochemistry 1984, 23, 2355e2357.
Supplementary data
25. Jokela, R.; Lounasmaa, M. Planta Med. 1996, 62, 577e579.
26. Lyon, R. L.; Fong, H. H. S.; Farnsworth, N. R. J. Pharm. Sci. 1973, 62, 833e833.
27. Sun, L.; Ruppert, M.; Sheludko, Y.; Warzecha, H.; Zhao, Y.; Stockigt, J. Plant Mol.
Biol. 2008, 67, 455e467.
1D, 2D NMR (HSQC, HMBC, ROESY), and MS spectra of vinma-
jines AeI (1e9), CD curves of vinmajines A and I, and the cytotoxic
activities of the alkaloids are available. Supplementary data related
to this article can be found at http://dx.doi.org/10.1016/
j.tet.2014.09.026. These data include MOL files and InChiKeys of the
most important compounds described in this article.
28. Borges, J.; Manresa, M. T.; Ramon, J. L. M.; Pascual, C.; Rumbero, A. Tetrahedron
Lett. 1979, 3197e3200.
29. Batista, C. V. F.; Schripsema, J.; Verpoorte, R.; Rech, S. B.; Henriques, A. T.
Phytochemistry 1996, 41, 969e973.
30. Chatterjee, A.; Chakrabarty, M.; Ghosh, A. K.; Hagaman, E. W.; Wenkert, E.
Tetrahedron Lett. 1978, 3879e3882.
31. Meisel, H.; Doepke, W.; Gruendemann, E. Tetrahedron Lett. 1971, 1291e1293.
32. Edwankar, C. R.; Edwankar, R. V.; Deschamps, J. R.; Cook, J. M. Angew. Chem., Int.
Ed. 2012, 51, 11762e11765.
References and notes
33. Subhadhirasakul, S.; Takayama, H.; Aimi, N.; Ponglux, D.; Sakai, S.-I. Chem.
1. Hutchinson, C. R. Tetrahedron 1981, 37, 1047e1065.
Pharm. Bull. 1994, 42, 1427e1431.
2. Bader, F. E.; Dickel, D. F.; Schlittler, E. J. Am. Chem. Soc. 1954, 76, 1695e1696.
3. Muller, J. M.; Schlittler, E.; Bein, H. J. Experientia 1952, 8, 338e338.
4. Mattern, M. R.; Mong, S. M.; Bartus, H. F.; Mirabelli, C. K.; Crooke, S. T.; Johnson,
R. K. Cancer Res. 1987, 47, 1793e1798.
34. Kam, T.-S.; Iek, I.-H.; Choo, Y.-M. Phytochemistry 1999, 51, 839e844.
35. Vercauteren, J.; Massiot, G.; Le, M.-O. L.; Levy, J.; Prange, T.; Pascard, C. Tetra-
hedron Lett. 1981, 22, 2871e2874.
36. Vercauteren, J.; Massiot, G.; Le, M.-O. L.; Levy, J.; Delaude, C. Bull. Soc. Chim. Fr.
5. (a) Cai, X. H.; Du, Z. Z.; Luo, X. D. Org. Lett. 2007, 9, 1817e1820; (b) Feng, T.; Cai,
X. H.; Li, Y.; Wang, Y. Y.; Liu, Y. P.; Xie, M. J.; Luo, X. D. Org. Lett. 2009, 11,
4834e4837; (c) Cai, X. H.; Bao, M. F.; Zhang, Y.; Zeng, C. X.; Liu, Y. P.; Luo, X. D.
Org. Lett. 2011, 13, 3568e3571; (d) Bao, M. F.; Yan, J. M.; Cheng, G. G.; Li, X. Y.;
Liu, Y. P.; Li, Y.; Cai, X. H.; Luo, X. D. J. Nat. Prod. 2013, 76, 1406e1412; (e) Liu, Y.
P.; Li, Y.; Cai, X. H.; Li, X. Y.; Kong, L. M.; Cheng, G. G.; Luo, X. D. J. Nat. Prod. 2012,
75, 220e224; (f) Feng, T.; Li, Y.; Cai, X. H.; Gong, X.; Liu, Y. P.; Zhang, R. T.; Zhang,
X. Y.; Tan, Q. G.; Luo, X. D. J. Nat. Prod. 2009, 72, 1836e1841; (g) Feng, T.; Cai, X.
H.; Liu, Y. P.; Li, Y.; Wang, Y. Y.; Luo, X. D. J. Nat. Prod. 2010, 73, 22e26.
6. Gerfaud, T.; Xie, C.; Neuville, L.; Zhu, J. Angew. Chem., Int. Ed. 2011, 123,
4040e4043.
1982, 291e296.
ꢀ
37. Lewin, G.; Le Menez, P.; Rolland, Y.; Renouard, A.; Giesen-Crouse, E. J. Nat. Prod.
1992, 55, 380e384.
38. Dhooghe, L.; Mesia, K.; Kohtala, E.; Tona, L.; Pieters, L.; Vlietinck, A. J.; Apers, S.
Talanta 2008, 76, 462e468.
39. Martin, S. F.; Hunter, J. E.; Benage, B.; Geraci, L. S.; Mortimore, M. J. Am. Chem.
Soc. 1991, 113, 6161e6171.
40. Hu, C.; Wang, P. Shanghai Diyi Yixueyuan Xuebao 1982, 9, 64e65.
41. Waterman, P. G.; Zhong, S. Planta Med. 1982, 45, 28e30.
42. Solis, P. N.; Wright, C. W.; Gupta, M. P.; Phillipson, J. D. Phytochemistry 1993, 33,
1117e1119.
7. (a) Adams, G. L.; Carroll, P. J.; Smith, A. B. J. Am. Chem. Soc. 2012, 134,
4037e4040; (b) Adams, G. L.; Carroll, P. J.; Smith, A. B. J. Am. Chem. Soc. 2013,
135, 519e528; (c) Watanabe, T.; Kato, N.; Umezawa, N.; Higuchi, T. Chem.dEur.
J. 2013, 19, 4255e4261; (d) Smith, M. W.; Snyder, S. A. J. Am. Chem. Soc. 2013,
135, 12964e12967.
43. Yagudaev, M. R.; Yunusov, S. Y. Chem. Nat. Compd. 1980, 16, 170e176.
44. Khalmirzaev, M. M.; Malikov, V. M.; Seitanidi, K. L.; Yagudaev, M. R.; Yunusov,
S. Y. Khim. Prir. Soedin. 1977, 718e719.
45. Chkhikvadze, G.; Khalmirzaev, M.; Vachnadze, V.; Malikov, V.; Yunusov, S.
Chem. Nat. Compd. 1976, 12, 201e202.
8. Lancefield, C. S.; Zhou, L.; Lebl, T.; Slawin, A. M. Z.; Westwood, N. J. Org. Lett.
2012, 14, 6166e6169.
9. Zhao, S.; Sirasani, G.; Vaddypally, S.; Zdilla, M. J.; Andrade, R. B. Angew. Chem.,
46. Yagudaev, M. R. Khim. Prir. Soedin. 1966, 2, 185e192.
47. (a) Seguin, E.; Hotellier, F.; Koch, M.; Sevenet, T. J. Nat. Prod. 1984, 47, 687e691;
(b) Cancelieri, N. M.; Curcino Vieira, I. J.; Mathias, L.; Braz-Filho, R. Magn. Reson.
Chem. 2003, 41, 287e290.
Int. Ed. 2013, 52, 8309e8311.
10. (a) Ehrhardt, H.; Schrembs, D.; Moritz, C.; Wachter, F.; Haldar, S.; Graubner, U.;
Nathrath, M.; Jeremias, I. Blood 2011, 118, 6123e6131; (b) Banerji, A.; Chakra-
barty, M. Phytochemistry 1974, 13, 2309e2312; (c) Baguley, B. C.; Holdaway, K.
M.; Thomsen, L. L.; Zhuang, L.; Zwi, L. J. Eur. J. Cancer 1991, 27, 482e487; (d)
Pauwels, O.; Kiss, R.; Pasteels, J. L.; Atassi, G. J. Pharm. Pharmacol. 1995, 47,
870e875; (e) Jean-Decoster, C.; Brichese, L.; Barret, J.-M.; Tollon, Y.; Kruczynski,
A.; Hill, B. T.; Wright, M. Anti-Cancer Drugs 1999, 10, 537e543.
11. Atta Ur, R.; Sultana, A.; Nighat, F.; Bhatti, M. K.; Kurucu, S.; Kartal, M. Phyto-
chemistry 1995, 38, 1057e1061.
48. Wenkert, E.; Cochran, D. W.; Hagaman, E. W.; Schell, F. M.; Neuss, N.; Katner, A.
S.; Potier, P.; Kan, C.; Plat, M. J. Am. Chem. Soc. 1973, 95, 4990e4995.
49. Achenbach, H.; Benirschke, M.; Torrenegra, R. Phytochemistry 1997, 45,
325e335.
50. Cai, X. H.; Li, Y.; Liu, Y. P.; Li, X. N.; Bao, M. F.; Luo, X. D. Phytochemistry 2012, 83,
116e124.
51. Bombardelli, E.; Bonati, A.; Gabetta, B.; Martinelli, E. M.; Mustich, G.; Danieli, B.
Tetrahedron 1974, 30, 4141e4146.
52. Zhang, Y. W.; Yang, R.; Cheng, Q.; Ofuji, K. Helv. Chim. Acta 2003, 86, 415e419.
53. Salim, A. A.; Garson, M. J.; Craik, D. J. J. Nat. Prod. 2004, 67, 1591e1594.
54. Wenkert, E.; Hagaman, E. W.; Kunesch, N.; Wang, N.-y.; Zsadon, B. Helv. Chim.
Acta 1976, 59, 2711e2723.
12. Farnsworth, N. R.; Fong, H. H.; Blomster, R. N.; Draus, F. J. J. Pharm. Sci. 1962, 51,
217e224.
13. Balsevich, J.; Constabel, F.; Kurz, W. G. Planta Med. 1982, 44, 91e93.
14. Chatterjee, A.; Banerji, A.; Chakrabarty, M. Planta Med. 1975, 28, 109e111.
15. Mukhopadhyay, G.; Mukherjee, B.; Patra, A.; Chatterjee, A.; Ghosh, R.; Roy-
chowdhury, P.; Kawazura, H. Phytochemistry 1991, 30, 2447e2449.
16. Sakushima, A.; Nishibe, S. Phytochemistry 1988, 27, 915e919.
17. Liu, Y. P.; Lai, R.; Yao, Y. G.; Zhang, Z. K.; Pu, E. T.; Cai, X. H.; Luo, X. D. Org. Lett.
2013, 15, 4940e4943.
18. Au, T. Y.; Cheung, H. T.; Sternhell, S. J. Chem. Soc., Perkin Trans. 1 1973, 13e16.
19. Lin, L. Z.; Cordell, G. A. Phytochemistry 1990, 29, 2744e2746.
20. Abreu, P.; Pereira, A. Heterocycles 1998, 48, 885e891.
55. Mosmann, T. J. Immunol. Methods 1983, 65, 55e63.
56. Mazzanti, A.; Casarini, D. WIREs Comput. Mol. Sci. 2012, 2, 613e641.
57. (a) Stephens, P. J.; Harada, N. Chirality 2010, 22, 229e233; (b) Bruhn, T.;
€
Schaumloffel, A.; Hemberger, Y.; Bringmann, G. SpecDis Version 1.53; University
€
€
of Wurzburg: Wurzburg, Germany, 2012.
58. (a) Lu, T.; Chen, F. J. Comput. Chem. 2012, 33, 580e592; (b) Humphrey, W.;
Dalke, A.; Schulten, K. J. Mol. Graphics 1996, 14, 33e38.
59. Reed, L. J.; Muench, H. Am. J. Hyg. 1938, 27, 493e497.