Novel monoterpenoid indole alkaloids from Melodinus yunnanensis

Article abstract of DOI:10.1016/j.tet.2017.08.008

Five monoterpenoid indole alkaloids, namely meloyines A-B (1–2), meloyines II-III (3–4), and 10-O-glucosyl-scandine (5) together with thirty-four known alkaloids were isolated from leaves and twigs of Melodinus yunnanensis. Alkaloid 1 was characterized as an unprecedented skeleton with a 6/5/5/6/6/4 ring system, and alkaloids 3–4 were dimeric monoterpenoid quinolone alkaloids. Their structures were elucidated based on 1D and 2D- NMR, FTIR, UV, and MS spectroscopic data. The cytotoxic activity of new alkaloids were evaluated against three human cancer cell lines.

Full text of DOI:10.1016/j.tet.2017.08.008

Tetrahedron xxx (2017) 1e6
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Tetrahedron
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Novel monoterpenoid indole alkaloids from Melodinus yunnanensis
,
,
, b
Bing-Jie Zhang a b, Cheng Liu c, Mei-Fen Bao a, Xiu-Hong Zhong a, Ling Ni a b, Jing Wu a
,
,
*
Xiang-Hai Cai a
a State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201,
China
b University of Chinese Academy of Sciences, Beijing 100049, China
c Southwest China Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Five monoterpenoid indole alkaloids, namely meloyines A-B (1e2), meloyines II-III (3e4), and 10-O-
glucosyl-scandine (5) together with thirty-four known alkaloids were isolated from leaves and twigs of
Melodinus yunnanensis. Alkaloid 1 was characterized as an unprecedented skeleton with a 6/5/5/6/6/4
ring system, and alkaloids 3e4 were dimeric monoterpenoid quinolone alkaloids. Their structures were
elucidated based on 1D and 2D- NMR, FTIR, UV, and MS spectroscopic data. The cytotoxic activity of new
alkaloids were evaluated against three human cancer cell lines.
Received 24 April 2017
Received in revised form
3 August 2017
Accepted 8 August 2017
Available online xxx
© 2017 Published by Elsevier Ltd.
Keywords:
Melodinus yunnanensis
Apocynaceae
Monoterpenoid indole alkaloids
Structure elucidation
1. Introduction
venalstonine)11 and C17eC21 (pandine),12 were widely distributed
in Alstonia and Tabernaemontana genera. To discover novel and
Monoterpenoid quinolone alkaloids (MQAs) were a special kind
of natural products, which were proposed to arise by rearrange-
ment of monoterpenoid indole alkaloids (MIAs). A possible route
for MQAs biosynthesis was that the N1eC2 or C2eC7 bond cleaved
in the indole heterocyclic ring and then generated new amine and/
or keto functions. Then a new quinolone heterocycle would be
formed by this nucleophilic reaction. Natural MQAs mainly were
distributed in plants of Alstonia (corialstonine1 and scholarisine I-
II2), Melodinus (meloscandonine and scandine derivatives,3 rhazi-
mine,4 meloyunine C5) and Gardneria (gardquinolone6), Taber-
naemontana (voastrictine7 and voaharine8) genera among family
Apocynaceae. Pharmacological investigations on MQAs have
demonstrated their significant bioactivities. Additionally, famous
drugs, camptothecin from plants of both Camptotheca and Notha-
podytes genera, and quinine from Cinchona genus were belonged to
MQAs. Previous research indicated MQAs derived from
Aspidosperma-type MIAs are mainly alkaloids in plants of Melo-
dinus genus.5,9,10 Likewise, another small subtype in Aspidosperma
types, with CeC new bond such as C2eC18/19 (vindolinine and
bioactive alkaloids, the first studied on M. yunnanensis distributed
in south of Yunnan Province, China, led to ten new alkaloids.13 Since
the production of plant secondary metabolites were influenced by
variable environments,14 phytochemical research on same plant
from different place was undertaken again. Herein, this paper
described the isolation, structural determination, and cytotoxic
activities of new alkaloids (1e5) together with the thirty four
known ones, namely 15a
-hydroxy-meloscandonine (6),15 10-
hydroxyscandine (7),16 scandine (8),9 epimeloscine (9),17 melo-
scandonine (10),18 19-epimeloscandonine (11),19 tubotaiwine
(12),20 19R-hydroxytabersonine (13),21 11-methoxytabersonine
(14),22 tabersonine (15),23 11-methoxy-19-hydroxytabersonine
(16),24 11-hydroxytabersonine (17),25 pachysiphine (18),25 loch-
nericine (19),26 11-hydroxylochnericine (20),27 venalstonine (21),11
17
a b-
-hydroxyvenalstonine (22),28 venalstonidine (23),29 19
hydroxyvenalstonidine (24),30 kopsinine (25),31 kopsiloscine G
(26),32 19R-vindolinine (27),11 16 -hydroxy-19R-vindolinine (28),19
b
19S-vindolinine (29),11 19,20-dihydroakuammicine (30),33 stricti-
cine (31),34 rhazimol (32),35 scholarisin VII (33),36 picrinine (34),37
14-hydroxymeloyunine (35),13 14,15-dehydrovincamine (36),38 16-
hydroxymethylpleiocarpamine (37),39 akuammidine (38),40 voa-
phylline (39).41
* Corresponding author.
E-mail address: xhcai@mail.kib.ac.cn (X.-H. Cai).
http://dx.doi.org/10.1016/j.tet.2017.08.008
0040-4020/© 2017 Published by Elsevier Ltd.
Please cite this article in press as: Zhang B-J, et al., Novel monoterpenoid indole alkaloids from Melodinus yunnanensis, Tetrahedron (2017),
http://dx.doi.org/10.1016/j.tet.2017.08.008

2
B.-J. Zhang et al. / Tetrahedron xxx (2017) 1e6
2. Results and discussion
The MeOH extract of leaves and twigs of M. yunnanensis was
partitioned between H2O and EtOAc and column chromatography
was used to separate the alkaloidal fraction into 39 alkaloids.
Alkaloid (1) was isolated as a white powder. Its molecular for-
mula was determined as
C21H24N2O3 by the HRESIMS (m/z
353.1859 [M þ H]þ, calcd. 353.1865) in association with the 1H and
13C NMR spectroscopic data, indicating 11ꢀ of unsaturation. Its UV
spectrum showed the characteristic absorptions of the indoline
chromophore at 210, 248 and 289 nm,42 and IR absorption bands at
3423, 3356, 1710 and 1640 cmꢁ1 suggested the presence of NH, OH,
aromatic ring and ester carbonyl functionalities. The 13C NMR and
DEPT data suggested that alkaloid 1 possessed 21 carbons including
seven quaternary carbons, eight methines, four methylenes, one
methoxycarbonyl and one methyl (Table 1), close to tabersonine
skeleton. The 1H NMR spectrum of 1 displayed proton signals for an
unsubstituted indole A ring [dH 7.10 (d, J ¼ 7.8 Hz, H-9), 6.58 (t,
J ¼ 7.8 Hz, H-10), 6.92 (t, J ¼ 7.8 Hz, H-11), 6.63 (d, J ¼ 7.8 Hz, H-12)],
two olefin protons [dH 5.80 (dd, J ¼ 11.9, 4.3 Hz, H-14), 5.66 (d,
J ¼ 11.9 Hz, H-15)], one methoxyl (dH 3.49, s) and one methyl (dH
0.63, d, J ¼ 7.4 Hz, H-18) (Table 1). In the 13C NMR spectrum, the
downfield shifts at dC 50.0 (t), 53.5 (t) and 75.4 (d) were easy to
assigned as CH2-3/5 and CH-21, respectively. The characteristic
quaternary carbon at dC 52.0 was assigned as C-7 according to its
correlations from H-9 and H-5 in the HMBC spectrum (Fig. 2). In
addition, the HMBC correlations also indicated the connections of
C7eC6eC5, C3eC14eC15 and C15eC20eC21. The doublets methyl at dH
0.63 was assigned as C-18 on the basis of the cross peaks from H-18
to C-19 and C-20. The methylene signals (dH 2.36, 1.98) were
assigned as H2-6, which was supported by the correlations from dH
Fig. 1. Structures of alkaloids 1e5 from M. yunnanensis.
Fig. 2. The key HMBC and NOE correlations of alkaloid 1.
2.36 to C-8 and C-21. And the correlations from dH 2.32 to C-19 and
C-21 suggested the signals (dH 2.32, 2.09) were belonged to H2-17.
Furthermore, the correlations from H2-17 (dH 2.32, 2.09), H-18 (dH
0.63), and OH (dH 4.35) to signal of dC 89.0 and the correlations from
H-15 (dH 5.66), H2-17, OH (dH 4.35) to C-19 revealed the connection
of C16eC19. The chemical formula and the HMBC correlations from
NeH (dH 5.61), H2-6, H2-17 and H-21 (dH 2.13) to the signal at dC 60.5
(s) suggested that the quaternary carbon was C-2 which was
substituted by the methoxycarbonyl.
The configuration of alkaloid 1 was determined by NOE corre-
lations (Fig. 2). The absolute configurations at C-7, C-20, C-21 were
determined as R, R, S, for compound 1 was originated from
tabersonine-type alkaloid.23 The absolute configurations were then
determined as 2S, 7R, 16R, 19S, 20R, 21S, by the correlations be-
tween H-18/H-21, H-18/carbomethoxy and H-19/H-15, which was
further confirmed by ECD calculation method. The geometry was
optimized at B3LYP/6e31 þ g(d,p) level in methanol using the
continuum polarizable continuum model (CPCM). Harmonic vi-
bration frequencies were then calculated to confirm the stability of
these conformers. The theoretical ECD spectrum of (2S, 7R, 16R, 19S,
20R, 21S)-1 (1a) and (2R, 7R, 16R, 19S, 20R, 21S)-1 (1b) was calcu-
lated in methanol using Time-dependent Density functional theory
(TD-DFT) at the B3LYP/6e311 þ g(d,p) level of the Gaussian 09
program package.43 The experimental ECD spectrum of alkaloid 1
and that the calculated ECD spectrum for the molecular having 2S,
7R, 16R, 19S, 20R, 21S (1a) were in good agreement, which validated
the absolute configurations of 1 (Fig. 3). Thus, the structure of
alkaloid 1 was elucidated and alkaloid 1 was named subsequently
meloyine A (Fig. 1).
Table 1
1H and 13C NMR spectroscopic data from alkaloids 1e2 in acetone-d6
(
d
in ppm, J in
Hz).
No
1a
2b
dH
dC
dH
dC
NH
2
3
5.61, s
60.5 s
50.0 t
112.0 s
59.4 d
3.36, dd (16.4, 4.3)
3.11, d (16.4)
3.20, t (7.2)
2.96, overlap
2.36, m
3.35, overlap
5
6
53.5 t
37.4 t
3.52, m
2.64, m
2.82, m
2.20, m
49.2 t
24.2 t
1.98, m
7
52.0 s
50.7 s
8
9
125.9 s
126.7 d
117.4 d
127.3 d
113.8 d
143.4 s
130.6 d
127.8 s
128.5 d
118.9 d
128.4 d
115.6 d
144.5 s
28.3 t
7.10, d (7.8)
6.58, t (7.8)
6.92, t (7.8)
6.63, d (7.8)
7.26, d (8.0)
6.68, t (8.0)
6.95, t (8.0)
6.51, d (8.0)
10
11
12
13
14
5.80, dd (11.9, 4.3)
5.66, d (11.9)
2.29, m
1.93, m
3.57, m
15
16
17
129.1 d
89.0 s
42.6 t
37.8 d
58.1 s
85.0 d
2.32, d (10.1)
2.09, overlap
0.63, d (3H, 7.4)
2.27, q (7.4)
4.88, s
18
19
20
21
9.3 q
1.45, d (3H, 7.0)
5.32, q (7.0)
12.9 q
117.6 d
143.8 s
55.0 t
53.4 d
47.8 s
75.4 d
2.13, s
3.86, d (16.8)
2.88, d (16.8)
3.52, s (3H)
Alkaloid 2 possessed molecular formula of C21H24N2O4 as
established by HRESIMS ([M þ H]þ at m/z 369.1816) as well as the
NMR data. In the 1H-NMR spectrum (Table 1) of 2, an unsubstituted
indole ring [dH 7.26 (d, J ¼ 8.0 Hz), 6.95 (t, J ¼ 8.0 Hz), 6.68 (t,
J ¼ 8.0 Hz) and 6.51 (d, J ¼ 8.0 Hz)], one methyl ester group
COOCH3
COOCH3
OH
3.49, s (3H)
4.35, s
51.4 q
171.9 s
51.6 q
171.2 s
a
1H NMR recorded at 600 MHz, 13C NMR recorded at 150 MHz.
1H NMR recorded at 400 MHz, 13C NMR recorded at 100 MHz.
b
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B.-J. Zhang et al. / Tetrahedron xxx (2017) 1e6
3
Table 2
1H and 13C NMR spectroscopic data of alkaloid 3 in acetone-d6
(
d
in ppm, J in Hz)a.
No
dH
dC
No
dH
dC
NH
2
3
9.28, s
N0H
20
9.28, s
168.3 s
47.8 t
168.4 s
48.0 t
3.71, overlap
3.32, overlap
3.13, overlap
3.05, overlap
2.27, m
30
3.72, overlap
3.34, overlap
3.13, overlap
3.05, overlap
2.26, overlap
1.77, overlap
5
6
55.2 t
39.3 t
50
60
55.4 t
39.5 t
1.79, m
7
8
9
57.3 s
70
55.4 s
131.9 s
124.5 d
136.7 s
128.5 d
116.6 d
136.9 s
129.0 d
126.9 d
68.6 s
80
132.2 s
124.8 d
136.8 s
128.6 d
116.7 d
137.0 s
129.3 d
125.2 d
68.6 s
7.05, s
90
7.09, s
Fig. 3. Calculated ECD spectrum of (2S, 7R, 16R, 19S, 20R, 21S)-1a (red); (2R, 7R, 16R,
19S, 20R, 21S)-1b (blue); experimental ECD spectrum of 1 (black).
10
11
12
13
14
15
16
17
100
110
120
130
140
150
160
170
6.93, overlap
6.87, overlap
6.94, overlap
6.88, overlap
resonance signal at dH 3.52 (s) and a methyl resonance signal at dH
1.45 (d, J ¼ 7.0 Hz) were observed. In comparison of NMR spectra
(Table 1) with those of rhazimal,44 the formyl group at C-17 and
N1H of rhazimal were absent in 2, instead, appearance of a new
hemiacetal (dC 85.0, dH 4.88) displayed the linkage between C-17
and N-1. This new five-membered ring in 2 was confirmed by the
HMBC cross-peaks from H-17 (dH 4.88) to C-16 (dC 58.1), C-15 (dC
37.8), C-13 (dC 144.5), C-7 (dC 50.7) (Fig. 4). The additional hydroxyl
was located at C-2 in 2 through the HMBC correlations of H-3 (dH
3.35), H-6 (dH 2.82), H-14 (dH 1.93), and H-17 (dH 4.88) with qua-
ternary carbon dC 112.0 (C-2). In the ROESY spectrum, the NOE
correlations of H-18 (dH 1.45) with H-15 (dH 3.57) and of H-19 (dH
5.32) with H-21 (dH 2.88) suggested E configuration of the double
5.91, overlap
5.94, overlap
5.94, overlap
5.99, overlap
2.16, overlap
2.01, overlap
0.80, d (3H, 7.2)
2.30, q (7.2)
40.9 t
2.16, overlap
2.01, overlap
1.06, d (3H, 7.8)
1.73, q (7.8)
36.8 t
18
19
20
21
22
CH2
8.8 q
180
190
200
210
220
11.2 q
51.1 d
45.2 s
70.9 d
211.4 s
52.8 d
46.2 s
62.4 d
210.6 s
41.2 t
3.51, s
3.30, s
3.82, m (2H)
a
1H recorded at 600 MHz and 13C NMR recorded at 150 MHz.
bond C-19/20. The cross-peaks of H-17/H-15 revealed H-17 as
orientation. The molecular model of alkaloid 2 indicated that the 2-
OH in the rigid skeleton was -oriented. Thus, the structure of 2 was
a-
a
established and subsequently named meloyine B.
Alkaloid 3 was isolated as a white powder. The UV absorption at
212 and 254 nm showed the presence of quinolone rings,9 and the
IR spectrum indicated the presence of NH (3343 cmꢁ1), carbonyl
(1712 cmꢁ1), and benzene ring (1650 cmꢁ1). The molecular formula
of alkaloid 3 was determined as C41H40N4O4 by its HRESIMS and
NMR data. The 1H NMR spectrum (Table 2) displayed two NH sig-
nals [dH 9.28 (2H, s)], six aromatic protons [dH 6.94, 6.93, 6.88, 6.87,
7.05 and 7.09] and two doublet methyls (dH 0.80 and 1.06). Its 13C
and DEPT NMR spectra indicated that 3 possessed 41 carbons,
including two methyls, nine methylenes, 14 methines and 16 qua-
ternary carbons. Combination of HRESIMS at m/z 653.3125
(C41H41N4O4 [M þ H]þ) and NMR spectra suggested that 3 was a
dimeric alkaloid. The presence of three pairs of sp3 quaternary
carbons (dC 68.6, 68.6, 57.3, 55.4, 46.2, and 45.2) in the 13C and DEPT
spectra, suggesting that alkaloid 3 contained two meloscandonine
analogues (Fig. 1).17 In the 1H NMR spectra, the major difference
between two units was the chemical shifts of H-18/180, H-19/190, H-
21/210 (Table 1), suggesting that the two units were meloscando-
nine and its 19-epimer.19 The presence of an additional methylene
Fig. 5. Key HMBC correlations of alkaloids 3 and 5.
124.5), C-90 (dC 124.8), C-10 (dC 136.7), C-100 (dC 136.8), C-11 (dC
128.5) and C-110 (dC 128.6). The CH3-18/180 were determined as
a
and
b
orientation by their proton signal chemical shifts in 1H NMR
spectra19 and ROESY correlations of H-18/21 and H-190/210. Thus,
the structure of the dimeric alkaloid was elucidated and called as
meloyine II because meloyine I, a first dimer alkaloid was found
from same plant.13
Similar to alkaloid 3, the UV and IR spectra of 4 also suggested
the presence of the quinolone rings.9 Its 13C and DPET NMR data
showed a methyl, five methylenes, seven methines and seven
quaternary carbons, one more methylene than meloscandonine.
However, alkaloid 4 had a same molecular formula as 3 based on
the HRESIMS, indicating it was also a dimers with both same units.
Its chemical shift of CH3-18 (dH 0.68, dC 8.8) observed in alkaloid 4
supported the unit was 19-epimeloscandonine other than melo-
scandonine.19 Dimeric coupling via a methylene linkage at C-10 and
C-100 was supported by the HMBC correlations from eCH2 (dH 3.71)
to C-9/90 (dC 124.6), C-10/100 (dC 136.9) and C-11/110 (dC 128.5). Thus,
the structure of alkaloid 4 was established and named meloyine III.
The 1H-, 13C-NMR and DEPT spectra displayed a glucose unit on
basis of a proton signal at dH 4.78 (1H, d, J ¼ 7.5 Hz), an anomeric
carbon [dC 102.3 (d)], a methylene [dC 62.6 (t)], and other 4 methine
signals between dC 70 and dC 80. The singlet peak at dH 4.78 indi-
[
dC 41.2, dH 3.82 (2H, m)] connected two moieties at C-10/100 sup-
ported by the HMBC correlations (Fig. 5) from dH 3.82 to C-9 (dC
cated the
residue. The identification of the sugar residue was continued by
hydrolysis with 10% HCl to afford a -glucose which was confirmed
b-orientation of the anomeric proton of the glucosyl
D
Fig. 4. Key HMBC and NOE correlations of alkaloid 2.
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4
B.-J. Zhang et al. / Tetrahedron xxx (2017) 1e6
by comparison with authentic samples and determination of their
alkaloids 10 (137 mg) and 11 (158 mg). Fr. II (11 g) was purified on a
C18 MPLC column with a MeOHeH2O gradient from 40% to 80% to
yield five fractions II-1e5. II-1(2.2 g) was purified on a C18 MPLC
column with a gradient of MeOHeH2O from 20% to 40% to yield
alkaloids 8 (310 mg), 12 (530 mg) and 13 (543 mg). Fraction II-2
(2.0 g) was further purified on a C18 MPLC column with a
gradient of MeOHeH2O from 20% to 40% to afford fractions II-2-
1e3. II-2-3 was further purified on a C18 HPLC column with a
gradient of MeOHeH2O from 65% to 75% to afford alkaloid 16
(48 mg). Alkaloid 14 (215 mg) was crystallized from fraction II-3. II-
4 (1.5 g) was further purified on a C18 MPLC column with a gradient
MeOHeH2O 50%e70% to afford three fractions. II-4-1 was further
21
optical rotation values ([
a
]
D
¼ þ21ꢀ).45 The glycositatic position
was unambiguously determined to be at C-10 from the HMBC
correlations (Fig. 5) from H-1' (dH 4.78) and singlet H-9 (dH 7.02) to
C-10 (dC 154.4), and from H-9 (dH 7.02) to C-7 (dC 58.5). So alkaloid 5
was named as 10-O-glucosyl-scandine.
Moreover, other alkaloids (6e39) were identified by comparison
of their NMR spectroscopic data with the literature. Alkaloids 1e5
were evaluated for their cytotoxicity against three human cancer
cell lines. No one showed cytotoxicity under these conditions
(IC50 > 25 mM).
3. Experimental section
purified on C18 HPLC column with
(55:45e65:35, v/v) to yield alkaloid 15 (47 mg). II-4-2 was further
purified on C18 HPLC column with gradient MeOHeH2O
a gradient MeOHeH2O
3.1. General information
a
(55:45e65:35, v/v) to yield alkaloids 17 (51 mg) and 19 (17 mg). II-
4-3 was further purified on the silica gel CC with a petroleum ether-
acetone gradient (10:1, v/v) to afford alkaloid 18 (89 mg). Fraction III
(17.1 g) was purified on a C18 MPLC column with a MeOHeH2O
gradient eluent from 20% to 65% to yield fractions III-1e5. III-1
(5.5 g) was further purified on a C18 MPLC column with a MeO-
HeH2O gradient eluent from 20% to 65% to afford four fractions. III-
1-1 (1.1 g) was purified on a silica gel CC with a petroleum ether-
acetone gradient (8:1, v/v) to afford alkaloids 17 (361 mg) and 20
(220 mg). III-1-2 (1.0 g) was purified on a silica gel CC with a pe-
troleum ether-acetone gradient (8:1, v/v) to obtain alkaloids 20
(122 mg) and 23 (136 mg). III-1-3 was purified on a preparative C18
HPLC column with a gradient MeOHeH2O (55:45e65:35, v/v) to
yield alkaloid 21 (19 mg). III-1-4 (2.1 g) was purified on a silica gel
CC with a petroleum ether-acetone gradient (8:1, v/v) to obtain
alkaloids 22 (45 mg) and 24 (236 mg). III-2 (1.5 g) was further
purified on a C18 MPLC column with a MeOHeH2O gradient eluent
from 30% to 45% to afford three fractions. Each of the fractions was
purified on a C18 HPLC column with a gradient MeOHeH2O
(55:45e65:35, v/v) to afford alkaloids 20 (33 mg), 22 (18 mg) and
24 (52 mg). III-3 (6.2 g) was purified on a C18 MPLC column with a
MeOHeH2O gradient eluent from 30% to 65% to yield fractions III-3-
1e3. III-3-1 (0.8 g) was purified on a C18 HPLC column with a
gradient MeOHeH2O (35:65e45:55, v/v) to afford alkaloid 1
(3 mg). III-3-2 (2.5 g) was purified on a silica gel CC with a petro-
leum ether-acetone gradient (8:1, v/v) to afford alkaloids 27 (1.2 g)
and 28 (437 mg). III-3-3 (2.0 g) was further purified on a silica gel
CC with a petroleum ether-acetone gradient (8:1, v/v) to afford
alkaloids 25 (310 mg) and 26 (117 mg). III-4 (1.7 g) was purified on a
C18 MPLC column with a MeOHeH2O gradient eluent from 30% to
65% to yield two fractions. The fractions were purified on a C18 HPLC
column with a gradient MeOHeH2O (55:45e65:35, v/v) to afford
alkaloids 29 (56 mg) and 30 (21 mg). III-5 (2.3 g) was further pu-
rified on a silica gel CC with a CHCl3-acetone gradient (15:1, v/v) to
afford two fractions. III-5-1 was purified on a C18 HPLC column with
a gradient MeOHeH2O (60:40e70:30, v/v) to afford alkaloids 3
(12 mg) and 4 (15 mg). III-5-2 (98 mg) was purified on a LH-20 with
a MeOHeH2O eluent (70%) to afford III-5-2-1. III-5-2-1 was purified
on a C18 HPLC column with a gradient MeOHeH2O (55:45e65:35,
v/v) to afford alkaloid 7 (23 mg). Fraction IV (8.3 g) was purified on a
C18 MPLC column with a MeOHeH2O gradient eluent from 10% to
50% to yield fractions IV-1e2. IV-1 (0.5 g) was purified on a C18
MPLC column with a MeOHeH2O gradient eluent from 10% to 20%
to yield two fractions. Each of them was purified on a C18 HPLC
column with a gradient MeOHeH2O (25:75e35:65, v/v) to afford
alkaloids 2 (19 mg), 5 (8 mg), 32 (12 mg) and 33 (21 mg). IV-2 (1.0 g)
was purified on a silica gel CC with a CHCl3-acetone gradient (15:1,
v/v) to afford alkaloid 31 (126 mg). Fraction V (4.3 g) was purified
on a C18 MPLC column with a MeOHeH2O gradient eluent from 10%
to 50% to yield fractions V-1e2. V-1 (0.3 g) was purified on a C18
Optical rotations were measured with either a Horiba SEPA-300
polarimeter (Horiba Scientific, Kyoto, Japan) or JASCO DIP-370
digital polarimeter (Jasco International Co., Tokyo, Japan). UV
spectra were obtained using a Shimadzu UV-2401A spectropho-
tometer (Shimadzu Corp., Kyoto, Japan). Scanning IR spectroscopy
was performed on a Tenor 27 spectrophotometer using KBr pellets.
MS data were recorded on an Agilent G6230 TOF MS (Applied
Biosystems, Ltd., Warrington, UK). 1D- and 2D- NMR spectra were
obtained on Bruker Avance III-600, DRX-500, and AM-400 spec-
trometers (Bruker BioSpin GmBH, Rheinstetten, Germany) with
TMS as an internal standard. Column chromatography (CC) was
performed on silica gel (200e300 mesh, Qing-dao Haiyang
Chemical Co., Ltd, Qingdao, China) and C18-silica gel (20e45 mm,
Fuji Silysia Chemical Ltd.). Fractions were monitored by TLC on
silica gel plates (GF254, Qingdao Haiyang Chemical Co., Ltd.) and
spots visualized with Dragendorff's reagent spray. Medium pres-
sure liquid chromatography (MPLC) was employed using a Buchi
pump system coupled with C18-silica gel-packed glass column
(15 ꢂ 230 and 26 ꢂ 460 mm). High performance liquid chroma-
tography (HPLC) was performed using a Waters 600 pump (Waters
Corp., Milford, MA, USA) coupled with Sunfire analytical, semi-
preparative, or preparative C18 columns (150 ꢂ 4.6, 150 ꢂ 10 mm,
and 250 ꢂ 19 mm, respectively). The HPLC system employed a
Waters 2996 photodiode array detector and a Waters fraction col-
lector II (Waters Corp.).
3.2. Plant material
Leaves and twigs of M. yunnanensis Tsiang & P. T. Li were
collected in July, 2013 in Gengma, Yunnan Province, P. R. China, and
identified by Dr. Cheng Liu. A voucher specimen (Cai20130627) was
deposited in the State Key Laboratory of Phytochemistry and Plant
Resources in West China, Kunming Institute of Botany, Chinese
Academy of Sciences.
3.3. Extraction and isolation
13 kg of M. yunnanensis leaves and twigs were extracted with
MeOH (3 ꢂ 25 L) at room temperature for a week and the solvent
removed in vacuo. The residue was dissolved in 0.3% aqueous hy-
drochloric acid (v/v), basified with 5% aqueous ammonia to pH
9e10, and partitioned with EtOAc. The EtOAc phase (80 g) was
subjected to CC over silica gel (1.0 kg) and eluted with a CHCl3-
acetone gradient (from 1:0e0:1, v/v) to produce five fractions
(IeV). Fraction I (4.0 g) was purified on a C18 MPLC column with a
MeOHeH2O gradient from 40% to 80% to yield four fractions I-1e2.
I-1 (1.6 g) was further purified on the silica gel CC to afford alkaloids
8 (510 mg) and 9 (320 mg). I-2 (0.8 g) was purified on the silica gel
CC with a petroleum ether-acetone gradient (8:1, v/v) to afford
Please cite this article in press as: Zhang B-J, et al., Novel monoterpenoid indole alkaloids from Melodinus yunnanensis, Tetrahedron (2017),
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B.-J. Zhang et al. / Tetrahedron xxx (2017) 1e6
5
HPLC column with a gradient MeOHeH2O (20:80e30:70, v/v) to
afford alkaloids 6 (21 mg), 35 (31 mg) and 36 (17 mg). V-2 (1.2 g)
was purified on a C18 MPLC column with a MeOHeH2O gradient
eluent from 20% to 35% to afford two fractions. Alkaloids 34 (17 mg)
and 37 (43 mg) were purified on a C18 HPLC column with a gradient
MeOHeH2O (20:80e30:70, v/v) from V-2-1 and alkaloids 38
(26 mg) and 39 (20 mg) was isolated from V-2-2 with the same
condition.
(MeOH) lmax (log ε) 212 (3.89) and 254 (3.95) nm; IR (KBr) nmax
3358, 1678, 1644 cmꢁ1; The 1H and 13C NMR data (acetone-d6), see
Table 3; positive HRESIMS m/z 653.3125 [M þ H]þ (calcd for
C41H41N4O4, 653.3128).
20
10-O-glucosylscandine (5): white powder; [
a]
82 (c, 0.1,
D
MeOH); UV (MeOH) lmax (log ε) 212 (3.45) and 254 (3.07) nm; IR
(KBr) nmax 3483, 1686, 1668, 1598 cmꢁ1; The 1H and 13C NMR data
(acetone-d6), see Table 3; positive HRESIMS m/z 529.2184 [M þ H]þ
(calcd for C27H33N2O9, 529.2186).
3.4. Spectroscopic data
3.5. Cytotoxicity assay
20
Meloyine A (1): white powder; [
a]
117 (c, 0.1, MeOH); UV
D
Three human cancer cell lines, HeLa, SGC-7901 gastric cancer,
and A-549 lung cancer, were used for cytotoxic assays. Cells were
cultured in RPMI-1640 (Sigma-Aldrich, St. Louis, MO, USA) or
DMEM medium (Hyclone, Logan, UT, USA), supplemented with 10%
fetal bovine serum (Hyclone) in 5% CO2 at 37 ꢀC. Cytotoxicity assays
were performed according to the MTT (3-(4, 5-dimethylthiazol-2-
yl)-2, 5-diphenyl tetrazolium bromide) method in 96-well micro-
(MeOH) lmax (log ε) 210 (3.81), 248 (3.43) and 289 (3.12) nm; IR
(KBr) nmax 3423, 3356, 1710, 1640 cmꢁ1; The 1H and 13C NMR data
(acetone-d6), see Table 1; positive HRESIMS m/z 353.1859 [M þ H]þ
(calcd for C21H25N2O3, 353.1865).
20
Meloyine B (2): white powder; [
a
]
120 (c, 0.1, MeOH); UV
D
(MeOH) lmax (log ε) 221 (3.49) and 288 (3.10) nm; IR (KBr) nmax
1756, 1651, 1602 cmꢁ1; The 1H and 13C NMR data (acetone-d6), see
Table 1; positive HRESIMS m/z 369.1816 [M þ H]þ (calcd for
plates. Briefly, 100 mL of adherent cell types were seeded into each
well of 96-well cell culture plates and allowed to adhere for 12 h
before the addition of test compounds. Suspended cell types were
seeded at an initial density of 1 ꢂ 105 cells/mL just before drug
addition. Each tumor cell line was exposed to a test compound at
C
21H25N2O4, 369.1814).
Meloyine II (3): white powder; [
20
a]
55 (c, 0.1, MeOH); UV
D
(MeOH) lmax (log ε) 212 (3.42) and 254 (3.14) nm; IR (KBr) nmax
3343, 1712, 1650 cmꢁ1; The 1H and 13C NMR data (acetone-d6), see
Table 2; positive HRESIMS m/z 653.3125 [M þ H]þ (calcd. for
concentrations of 0.04, 0.20, 1.00, 5.0, and 25.0
mM in DMSO in
triplicate for 48 h, with cisplatin (Sigma-Aldrich) as the positive
control. After treatment, cell viability was assessed, cell growth
graphed, and IC50 values calculated by Reed and Muench's method.
C
41H41N4O4, 653.3128).
Meloyine III (4): white powder; [
20
a]
9 (c, 0.1, MeOH); UV
D
Acknowledgments
Table 3
1H and 13C NMR spectroscopic data of alkaloid 5 in acetone-d6
(
d
in ppm, J in Hz)a.
This project was supported in part by the National Natural Sci-
ence Foundation of China (31370377), the Young Academic and
Technical Leader Raising Foundation of Yunnan Province (No.
2010CI049).
No
4
5
dH
dC
dH
dC
NH
2
3
9.19, s
9.36, s
168.3 s
47.8 t
168.3 s
48.7 t
3.58, d (18.6)
3.21, m
3.05, m
2.96, m
2.12, m
3.17, m
2.91, m
3.05, m
2.87, m
2.24, m
1.78, m
Appendix A. Supplementary data
5
6
55.3 t
39.6 t
54.3 t
41.9 t
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.tet.2017.08.008.
1.71, m
7
57.3 s
58.5 s
8
9
131.9 s
124.6 d
136.9 s
128.5 d
116.7 d
136.9 s
129.3 d
126.9 d
68.6 s
130.6 s
116.6 d
154.4 s
116.5 d
116.4 d
131.0 s
124.4 d
132.0 d
64.6 s
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7.02, d (2.5)
10
11
12
13
14
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6.78, dd (8.7, 2.5)
6.71, d (8.7)
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48.3 s
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4.78, d (7.5)
3.38, m
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3.30, m
3.31, m
102.3 d
77.8 d
74.6 d
71.3 d
77.9 d
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Please cite this article in press as: Zhang B-J, et al., Novel monoterpenoid indole alkaloids from Melodinus yunnanensis, Tetrahedron (2017),
http://dx.doi.org/10.1016/j.tet.2017.08.008