Macroline, akuammiline, sarpagine, and ajmaline alkaloids from Alstonia macrophylla

Article abstract of DOI:10.1016/j.phytochem.2013.11.014

A total of seventeen alkaloids, comprising six macroline (including alstofolinine A, a macroline indole incorporating a butyrolactone ring-E), two ajmaline, one sarpagine, and eight akuammiline alkaloids, were isolated from the stem-bark and leaf extracts

Full text of DOI:10.1016/j.phytochem.2013.11.014

Phytochemistry 98 (2014) 204–215
Contents lists available at ScienceDirect
Phytochemistry
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Macroline, akuammiline, sarpagine, and ajmaline alkaloids from Alstonia
macrophylla
Siew-Huah Lim a, Yun-Yee Low a, Saravana Kumar Sinniah b, Kien-Thai Yong b, Kae-Shin Sim b,
Toh-Seok Kam a,
⇑
a Department of Chemistry, University of Malaya, 50603 Kuala Lumpur, Malaysia
b Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 August 2013
Received in revised form 4 November 2013
Available online 13 December 2013
A total of seventeen alkaloids, comprising six macroline (including alstofolinine A, a macroline indole
incorporating a butyrolactone ring-E), two ajmaline, one sarpagine, and eight akuammiline alkaloids,
were isolated from the stem-bark and leaf extracts of the Malayan Alstonia macrophylla. The structure
and relative configurations of these alkaloids were established using NMR, MS and in several instances,
confirmed by X-ray diffraction analysis. Six of these alkaloids were effective in reversing multidrug-resis-
tance (MDR) in vincristine-resistant KB cells.
Keywords:
Alstonia macrophylla
Apocynaceae
Ó 2013 Elsevier Ltd. All rights reserved.
Indole alkaloids
NMR
X-ray crystallography
Reversal of multidrug-resistance
Introduction
the absolute configuration of perhentinine and macralstonine
(Lim et al., 2012). Reported herein are the isolation and structure
Plants of the genus Alstonia (Apocynaceae), which are shrubs or
trees, are distributed over tropical parts of Central America, Africa,
and Asia (Whitmore, 1973; Markgraf, 1974; Sidiyasa, 1998) and are
usually rich in alkaloids. A prominent feature of the Alstonia alka-
loids is the preponderance of the macroline unit, which abounds
in the alkaloids found in plants of the genus (Kam, 1999; Kam
and Choo, 2006). About six species occur in Peninsular Malaysia
and several of these (local name Pulai) are used in traditional med-
icine, for example, in the treatment of malaria and dysentery (Bur-
kill, 1966; Perry and Metzger, 1980). In Peninsular Malaysia, these
plants are mainly found in secondary and primary forest from sea
level to about 3000 m altitude, as well as in swampy areas (Whit-
more, 1973; Sidiyasa, 1998; Middleton, 2011). Recently the struc-
ture and absolute configurations of a number of new bisindoles
isolated from a sample of A. macrophylla Wall collected from the
western coast of Peninsular Malaya (Perak) were disclosed (Lim
et al., 2011, 2012, 2013). In addition, a potentially useful method
for the determination of the configuration at C-20 in E-seco macr-
oline–macroline bisindoles, such as perhentinine, seco-macralsto-
nine, and perhentidines AꢀC, was also reported. This was based
on comparison of the NMR chemical shifts of the bisindoles and
their acetate derivatives, in addition to X-ray determination of
determination of 17 new indole alkaloids (Fig. 1) from the leaf
and stem-bark extracts of this plant.
Results and discussion
Compound 1 was a minor alkaloid isolated from the leaf extract
of A. macrophylla. It was obtained as a light yellowish oil, with [a]
D
ꢀ104 (c 0.36, CHCl3). The UV spectrum showed two absorption
bands (227 and 285 nm) characteristic of an indole chromophore.
The IR spectrum showed a sharp band at 1769 cmꢀ1 due to a lac-
tone function. The EIMS of compound 1 had a molecular ion at
m/z 296, and high resolution measurements yielded the molecular
formula C18H20N2O2. Other notable fragment peaks observed at m/
z 197, 182, 181, 170, and 144, are typical of macroline derivatives
(Mayerl and Hesse, 1978), while the mass fragment at m/z 281 can
be attributed to loss of a CH3. The 13C NMR spectrum (Table 1) dis-
played a total of 18 carbon resonances, corresponding to two
methyl, three methylene, eight methine and five quaternary car-
bons. The presence of the lactone functionality, and an oxymethyl-
ene carbon, was supported by the observed carbon signals at d
181.0 and d 70.7, respectively. The 1H NMR spectrum (Table 2)
showed the presence of an unsubstituted indole moiety (d 7.11–
7.49), two methyl groups corresponding to N1-Me at d 3.64 and
N4-Me at d 2.42, and two downfield resonances at d 4.42 (H-17b,
⇑
Corresponding author. Tel.: +60 3 79674266; fax: +60 3 79674193.
E-mail address: tskam@um.edu.my (T.-S. Kam).
t, J = 8 Hz) and 4.52 (H-17a, dd, J = 11, 8 Hz) due to the geminal
0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2013.11.014

S.-H. Lim et al. / Phytochemistry 98 (2014) 204–215
205
Fig. 1. Structures of compounds 1–29.
hydrogens of an oxymethylene corresponding to C-17. The COSY
and HSQC data disclosed partial structures that are characteristic
of a macroline-type skeleton, such as NCHCH2 and NCHCH2-
CHCHCH2O, corresponding to the N-4–C-5–C-6 and N-4–C-3–C-
14–C-15–C-16–C-17–O fragments, respectively. The NMR spectro-
scopic data were suggestive of a macroline derivative, such as
alstonerine (2). (Ratnayake et al., 1987; Ghedira et al., 1988; Kam
m/z 339, which analyzed for C21H26N2O2. The UV spectrum showed
absorption maxima at 228 and 286 nm, which are characteristic of
an indole chromophore.
The 1H and 13C NMR spectroscopic data (Tables 1 and 2) showed
the presence of an unsubstituted indole moiety (dH 7.09–7.50, dC
108.7–120.9), two N-methyl signals (N4-Me, dC 41.8, dH 2.29; N1-
Me, dC 29.1, dH 3.61), a methyl ketone function (dC 208.6; dC 28.4,
dH 2.12), an oxymethylene, characteristic of C-17 in macroline alka-
loids (dC 68.6, dH 3.72, and 3.95), and another oxymethylene signal at
dC 64.3 (dH 3.86 and 4.18). The NMR signals, assigned with the aid of
COSY and HSQC, indicated that 3 is a macroline-type alkaloid. The
NMR spectroscopic data resembled those of alstonerine (2), which
was also isolated from the extract of this plant, except for the ab-
sence of signals associated with the trisubstituted C-20ꢀC-21 dou-
ble bond, such as the olefinic carbon resonances at C-20 (d 126.5)
and C-21 (d 157.4), and the signal due to the vinylic H-21 in the 1H
NMR spectrum (d 7.52). These resonances have in 3 been replaced
by a methine at C-20 (dC 51.9, dH 1.97, m) and a methylene at C-21
(dC 64.3; dH 3.86, dd, J = 12.5, 3 Hz, dH 4.18, d, J = 12.5 Hz), consistent
with saturation of the C-20ꢀC-21 double bond in 3. Less substantial
changes were observed for the signals of carbons b to both carbons
(C-20, C-21) in the 13C NMR spectrum. The configuration at C-20
can be deduced from the observed NOEs, viz., H-20/H-14b, H-18,
et al., 1999), except for the absence of the typical a,b unsaturated
ketone group and the associated vinyl-H, in the ring E of compound
1. In addition, both H-17 signals in 1 were shifted downfield to dH
4.42 and 4.52, when compared to those in alstonerine (2) (dH 4.16
and 4.40), as well as other typical macroline alkaloids (Kam et al.,
2004a; Kam and Choo, 2004a). Furthermore, the observed coupling
constants for the H-17 resonances in the case of 1 differed signifi-
cantly when compared to those in 2 (1: H-17b, t, J = 8 Hz; H-17
a,
dd, J = 11 and 8 Hz; 2: H-17b, ddd, J = 11, 4, 2 Hz; H-17 , t,
a
J = 11 Hz), indicating changes in ring E of 1 (J17-17 = 8 Hz) compared
to normal macrolines with a six-membered ring E (J17-17 = 11 Hz)
as exemplified by alstonerine (2). The presence of a lactone func-
tionality as a part of ring E (a butyrolactone moiety) was deduced
from the observed three-bond correlations from H-14
a, H-14b, and
H-17 , to the lactone carbonyl C-18 in the HMBC spectrum (Fig. 2).
a
The relative configurations at the various stereogenic centers of 1
were established by NOESY, and were similar to those in other
macroline alkaloids. Alstofolinine A (1) is the first example of a
H-21a
; H-21a/H-14a, H-20, H-21b, which indicated the orientation
of H-20 is
a
. Compound 3 is, therefore, the 20,21-dihydro derivative
macroline indole alkaloid incorporating a
in ring E.
c
-butyrolactone moiety
of alstonerine (2), which, while previously encountered as an inter-
mediate compound in synthesis (Zhang and Cook, 1990), is here
encountered as an optically active natural product for the first time.
Macrocarpine D (4) was obtained as a light yellowish oil, with
Compound 3 was isolated in small amounts as a light yellowish
oil, with [a] –31 (c 0.11, CHCl3). The IR spectrum showed a band
D
at 1710 cmꢀ1 due to a ketone function. The presence of a ketone
function was confirmed by the observed resonance at d 208.6 in
the 13C NMR spectrum. The ESIMS of 3 showed an [M+H]+ ion at
[
a
]
D ꢀ43 (c 0.89, CHCl3). It was isolated from the stem-bark extract
of A. macrophylla, as well as A. angustifolia (Tan, 2011). The IR spec-
trum indicated the presence of hydroxyl and secondary amine

206
S.-H. Lim et al. / Phytochemistry 98 (2014) 204–215
Table 1
13C (100 MHz) NMR spectroscopic data for compounds 1, 3, 4, 6, 9, 11, 13, 15–17, 19–22, 24, 26, and 28.a
C
1
3
4
6
9
11
13
15
16
17
19
20
21
22
24
26
28
2
3
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
132.4
52.0
52.4
23.6
106.8
127.3
119.0
120.0
122.3
109.8
138.1
29.5
34.1
43.2
70.7
181.0
–
–
–
–
–
–
–
–
30.1
42.2
–
–
–
–
–
–
–
–
–
–
–
–
–
133.0
53.6
54.8
22.4
107.0
126.6
118.4
118.9
120.9
108.7
137.1
30.3
24.6
39.3
68.6
28.4
208.6
51.9
64.3
–
–
–
–
–
29.1
41.8
–
–
–
132.2
55.0
55.1
22.5
107.7
127.1
118.0
119.4
121.3
110.9
135.5
26.1
27.1
43.6
67.7
20.3
70.5
46.9
61.7
–
–
–
–
–
–
41.6
–
–
–
132.7
53.5
54.9
22.6
106.7
126.4
118.2
119.2
121.1
108.9
137.0
32.0
26.7
37.0
64.6
23.8
105.1
44.1
32.2
108.6
70.1
27.9
23.0
64.0
30.1
42.2
–
–
–
–
–
–
–
–
–
–
–
–
–
183.0
62.9
61.7
41.0
56.8
120.8
125.8
106.4
160.2
96.6
145.5
33.1
26.2
41.6
65.9
31.0
208.7
47.1
–
–
–
–
–
–
26.4
–
–
–
183.1
63.4
61.8
40.8
57.0
121.1
125.1
106.6
160.2
96.5
145.6
34.2
26.9
40.7
65.3
24.9
65.5
42.5
–
–
–
–
–
–
26.4
–
–
–
139.8
49.2
54.9
27.3
103.4
127.4
118.2
118.9
120.9
108.8
137.4
34.5
34.9
44.1
65.4
12.6
117.0
136.9
53.9
–
–
–
–
–
29.4
–
–
–
–
80.7
85.3
51.5
30.8
46.3
140.0
120.9
119.4
127.3
109.8
153.8
42.3
36.5
52.0
–
13.6
119.1
137.5
55.0
–
–
–
–
–
37.6
–
51.7
173.0
–
–
–
–
–
–
–
–
–
–
–
70.1
48.7
46.5
28.7
42.9
137.8
123.3
119.5
127.5
109.1
152.6
25.2
32.5
49.6
–
12.9
119.5
138.1
56.2
–
–
–
–
–
34.0
–
51.0
172.8
–
–
–
–
–
–
–
–
–
–
–
70.7
48.9
46.6
28.7
43.2
139.4
110.6
153.8
112.3
109.7
146.8
25.2
32.6
49.6
–
13.0
119.9
138.0
56.3
–
–
–
–
–
34.9
–
51.1
172.9
56.0
–
–
–
–
–
97.4
20.8
55.0
41.0
57.0
137.0
123.5
116.9
127.8
105.3
149.0
26.2
34.8
50.6
–
13.5
122.4
138.9
58.3
–
–
–
–
–
27.3
–
51.5
173.8
–
–
–
–
–
–
–
–
–
–
–
102.5
17.5
66.3
35.1
54.1
135.5
122.6
119.4
129.0
107.8
149.7
25.3
33.8
49.3
–
13.7
128.3
132.0
74.0
–
–
–
–
–
32.9
–
52.1
172.6
–
–
–
–
–
–
–
–
–
–
–
98.2
20.8
55.2
41.4
57.4
128.0
111.9
140.9
150.1
92.3
144.5
26.6
34.9
51.5
–
13.9
123.1
139.0
58.6
–
–
–
–
–
102.7
17.1
66.1
34.9
54.3
136.6
110.6
153.6
112.8
107.9
143.9
25.2
33.8
49.3
–
13.6
128.4
131.9
73.7
–
–
–
–
–
33.3
–
52.1
172.5
56.1
–
–
–
–
–
109.3
49.7
87.2
40.5
50.8
136.5
112.1
154.3
112.6
108.8
144.2
25.9
31.3
51.6
–
12.8
120.6
136.0
46.4
–
–
–
–
–
30.0
–
51.7
172.5
56.0
–
–
–
–
–
69.8
69.8
76.0
30.3
56.1
128.9
128.8
109.7
124.6
119.6
154.3
22.2
29.5
60.6
74.0
12.6
119.6
130.1
70.8
–
70.0
69.7
75.9
31.6
55.5
127.1
123.6
119.7
129.0
109.8
153.9
22.1
29.7
59.8
74.9
12.6
120.4
129.5
71.0
–
–
–
–
–
–
–
–
–
N1-Me
N4-Me
CO2Me
CO2Me
10-OMe
11-OMe
10
28.4
–
34.8
–
52.3
170.8
–
–
–
–
–
–
–
–
–
–
–
34.6
–
52.5
169.9
–
51.9
174.0
58.2
56.4
–
–
–
–
–
–
–
–
–
–
55.7
–
–
–
–
–
–
–
–
55.6
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
121.5
111.9
148.8
153.4
110.4
123.4
56.1
56.1
163.7
20
30
40
50
–
–
–
–
–
–
–
–
–
–
–
–
60
30-OMe
40-OMe
C@O
–
–
–
–
–
–
–
–
–
–
–
–
–
a
Measured in CDCl3.
orientation of H-19 and H-20 in 4, were deduced to be
a
and b,
respectively, from the observed NOEs for H-19/H-14
a and H-20/
H-15, H-16.
Macrodasine H (6) was obtained as a light yellowish oil, with
[
a
]
ꢀ11 (c 0.14, CHCl3). The UV spectrum had two absorption
D
maxima at 233 and 288 nm, indicating the presence of an indole
chromophore. The IR spectrum displayed
a broad band at
3423 cmꢀ1 due to a hydroxyl function. The ESIMS of 6 showed an
[M+H]+ peak at m/z 439, and high-resolution measurements
yielded the molecular formula C26H34N2O2. The 13C NMR spectrum
(Table 1) had a total of 26 carbon signals comprising three methyl,
seven methylene, ten methine and six quaternary carbons, in
agreement with the molecular formula. The observed carbon reso-
nance at d 70.1 was due to an oxymethine carbon, while the carbon
resonances at d 64.6 (C-17) and 64.0 (C-26) were due to two oxy-
methylene carbons. In addition, two low-field quaternary carbon
signals were observed at d 108.6 and 105.1, each of which was at-
tached to two oxygen atoms. The 1H NMR spectrum (Table 2)
showed the presence of an unsubstituted indole moiety (d 7.11–
7.50), three methyl groups corresponding to N1-Me, N4-Me, and
18-Me, at d 3.62, 2.33, and 1.62, respectively, and two sets of res-
onances due to two oxymethylenes at d 3.75, 4.05; and at d 3.39,
3.84.
Fig. 2. Selected HMBCs and NOEs of 1.
functions at 3395 and 3292 cmꢀ1, respectively, while the UV spec-
trum indicated an indole chromophore (kmax 231 and 286 nm). The
ESIMS of 4 showed an [M+H]+ peak at m/z 327, and HRESIMS mea-
surements established the molecular formula as C20H26N2O2, 15
mass units less than that of macrocarpine B (5) (Kam et al.,
2004a). Comparison of the NMR spectroscopic data of 4 with those
of 5 (Tables 1 and 2) indicated replacement of the N1-Me group by
an NH function in 4. The NMR data also indicated that the relative
configurations at the various stereogenic centers of 4 follow those
in the macroline-type alkaloids. In common with compound 5, the
The COSY spectrum disclosed some partial structures, which are
characteristic of a macroline-type skeleton, such as NCHCH2,
CHCH2, and NCHCH2CHCHCH2O, corresponding to the N-4–C-5–
C-6, C-20–C-21, and N-4–C-3–C-14–C-15–C-16–C-17-O fragments,

S.-H. Lim et al. / Phytochemistry 98 (2014) 204–215
207
Table 2
1H (400 MHz) NMR spectroscopic data for compounds 1, 3, 4, 6, 9, 11, 13, 15, and 16.a
H
1
3
4
6
9
11
13
15
16
2
3
–
–
–
–
–
–
–
2.89 s
–
3.25 d (4)
3.98 m
3.91 br s
3.97 m
3.95 m
3.93 m
3.19 m
3.20 m
4.14 dd
(10, 2)
2.73 t (6)
5a
5b
6b
6a
3.06 d (6)
–
2.83 d (7)
–
2.94 d (7)
–
2.96 d (7)
–
3.86 m
–
3.90 m
–
2.77 dd
(13, 6.5)
4.02 td
(13, 5)
2.58 dd
(14, 7)
3.45 td (14, 6)
–
2.47 br d (16) 2.52 dd (16)
2.46 d (16) 2.41 d (16)
2.08 d (14)
2.06 d (14)
2.60 br d (15) (b) 1.32 dd
(15, 5)
3.03 dd
(15, 5) (a)
7.41 d (8)
7.08 td (8, 1)
2.07 dd
(14, 6)
3.35 m
3.27 dd
(16, 6)
7.49 d (8)
7.11 td
(8, 1)
3.24 dd
(16, 7)
7.50 d (8)
7.09 td (8, 1)
3.27 dd
(16, 7)
7.49 d (7.5) 7.50 d (8)
7.11 t (7.5) 7.11 t (8)
3.26 dd
(16, 7)
2.39 dd
(14, 8)
7.76 d (9)
6.70 dd (9, 2)
2.38 dd
(14, 8)
7.39 d (8)
6.60 dd
(8, 2)
2.87 m
9
10
6.91 dd (8, 1)
6.68 td (8, 1)
6.98 d (7)
6.64 t (7)
11
7.22 td
(8, 1)
7.18 td (8, 1)
7.15 t (7.5) 7.20 t (8)
–
–
7.19 td (8, 1)
7.09 td (8, 1)
7.01 t (7)
12
14b
7.31 d (8)
2.10 m
7.27 d (8)
1.42 dt (12, 3) 1.62 dt
(13, 4)
2.44 dd
(12, 4)
2.35 m
2.12 m
7.32 d (7.5) 7.30 d (8)
6.45 d (2)
1.69 m
6.45 d (2)
1.72 m
7.29 d (8)
1.53 m (b)
6.61 br d (8)
1.90 dd (14, 3) (b) 1.99 dt (15, 4) (a)
6.51 d (7)
1.50 m
2.41 m
14a
2.13 m
2.28 td
(13, 4)
1.86 dd
(13, 5)
2.97 m
1.69 m
1.82 m
2.04 td (12, 2) (a) 2.20 dd (14, 3) (a) 2.13 dd (15, 3) (b)
15
16
2.17 m
2.54 m
2.01 m
1.89 dt
(12, 4)
1.84 m
2.09 m
2.69 m
1.72 m
2.23 br s
1.62 m
3.61 m
2.90 d (4)
3.39 m
2.72 d (4)
17b
4.42 t (8)
3.72 dd
(11.5, 5)
3.74 dd
(12, 5)
4.08 t (12)
1.16 d (6)
3.50 m
1.50 m
3.75 dd
(12, 5)
4.05 t (12)
1.62 s
3.81 m (a)
3.90 m
3.47 m
–
–
17a
18
19
4.52 dd (11, 8) 3.95 t (11.5)
4.01 d (12) (b) 4.00 dd (11, 1) 3.47 m
2.20 s
–
–
–
–
–
–
2.12 s
–
1.97 m
1.29 d (6)
3.90 m
1.57 d (7)
5.28 q (7)
–
1.49 dd (7, 2)
5.41 br q (7)
–
1.46 dd (7, 2)
5.41 q (7)
–
–
20a
1.96 dd
(12.5, 8)
–
1.81 dd
(12.5, 8) (a)
2.16 t
(12.5) (b)
3.51 dd
(10, 5) (ax)
1.37 m (ax)
2.05 m (eq)
1.56 m
1.56 m
3.39 td
(10, 4.5) (ax)
3.84 m (eq)
–
2.69 dd (18, 5) 1.53 m
20b
21a
–
–
–
–
2.80 dd (18, 8) 1.86 m
–
–
–
3.86 dd
(12.5, 3) (a)
4.18 d
(12.5) (b)
–
3.34 dd
(11, 8)
3.50 dd
(11, 5)
–
–
–
–
–
–
–
3.63 m
2.98 m
2.98 d (16)
21b
23
–
–
3.63 m
–
4.10 br d (17)
–
3.95 d (16)
–
24a
24b
25
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
26a
26b
NH
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
7.89 br s
N1-Me
N4-Me
CO2Me
11-
3.64 s
2.42 s
–
–
3.61 s
2.29 s
–
–
–
3.62 s
2.33 s
–
–
3.16 s
–
–
3.17 s
–
–
3.61 s
2.95 s
–
3.78 s
–
2.68 s
–
3.53 s
–
2.34 s
–
–
–
–
–
3.84 s
3.83 s
OMe
a
Measured in CDCl3.
respectively. An additional fragment, viz., OCHCH2CH2CH2O, was
also indicated from the COSY spectrum which, taken with the other
NMR spectroscopic data, indicated affinity to the macrodasine
group of alkaloids reported recently (Tan et al., 2011). These macr-
oline alkaloids incorporate additional fused spirocyclic tetrahydro-
6 differs from macrodasine F (7), by the absence of the OH substi-
tuent at C-25.
The ring junction stereochemistries between rings C, D, E, and F
were deduced to be similar to those of macroline alkaloids, as well
as macrodasines (A–G) from the NOE data. The reciprocal NOEs ob-
served for H-23/H-21b, H-25a and H-24a/H-26a suggested a chair
conformation adopted by the tetrahydropyran ring G, as shown in
Fig. 3, in which H-24a, H-25a, and H-26a are all axially oriented. As
in the case of macrodasine F (7), a NOE was not observed between
the C-26 hydrogens and 18-Me, suggesting that the configuration
of the spirocyclic C-22 is S, i.e., the C-26 hydrogens and 18-Me
are directed away from each other (Tan et al., 2011).
furan–tetrahydrofuran (macrodasines
A (8), B, C, G) and
tetrahydrofuran–tetrahydropyran (macrodasines D, E, F (7)) rings.
The OCHCH2CH2CH2O partial structure noted from the COSY spec-
trum suggested that compound 6 belonged to the latter group of
macrodasine alkaloids (macrodasines D, E, and F (7)) characterized
by incorporation of fused 5/6 (F/G) spirocyclic rings (Tan et al.,
2011). This was further supported by the following correlations
observed in the HMBC spectrum, viz., H-26b, H-24 to C-22, H-24
to C-26, H-25 to C-23, and, H-21 to C-15, C-23. The proposed
structure is consistent with the full HMBC data (Fig. 3). Compound
Alstonoxine C (9) was isolated as a light yellowish oil, with [a]
D
ꢀ30 (c 0.39, CHCl3), and subsequently crystallized from CH2Cl2–
hexane as colorless block crystals. The UV spectrum showed
absorption maxima at 216, 266, and 291 nm, indicative of an oxin-

208
S.-H. Lim et al. / Phytochemistry 98 (2014) 204–215
Fig. 3. Selected HMBCs and NOEs of 6.
dole chromophore. The IR spectrum showed the presence of lactam
and ketone carbonyl (1694 cmꢀ1), NH (3295 cmꢀ1), and hydroxyl
(3390 cmꢀ1) functions, while the carbon resonances at d 183.0,
208.7, 65.9 confirmed the presence of lactam (oxindole), ketone,
and oxymethylene groups, respectively. The ESIMS showed an
[M+H]+ peak at m/z 359, analyzing for C20H26N2O4. The 1H NMR
spectroscopic data (Table 2) showed features typical of macroline
oxindole alkaloids. Notable features include presence of an
N1-Me (dH 3.16 s), a methyl ketone (dH 2.20 s; dC 31.0, C-18), and
an oxymethylene (dH 3.80 m, and 4.01 br d, J = 12 Hz; dC 65.9;
C-17). In addition, signals due to the presence of a methylene
Fig. 4. X-ray crystal structure of 9.
mixture of the epimeric alcohol products in approximately equal
amounts, which were separated by preparative centrifugal TLC
(SiO2, 1% MeOH/CHCl3, NH3-saturated). The NMR data indicated
that the slower eluting compound corresponded to compound
11. With sufficient amounts obtained in this manner, the configu-
ration at C-19 could be determined by Horeau’s procedure (see
Experimental) (Horeau and Kagan, 1964; Barnekow and Cardellina,
1989), which showed that the C-19 configuration in 11 is S (the fas-
ter eluting compound 11a therefore corresponded to the 19R epi-
mer). The structure and relative configuration of alstonoxine B
(12) was previously reported from Alstonia angustifolia var. latifolia
(Kam and Choo, 2000), based on analysis of the NMR and MS data
which at the time were insufficient to assign the configuration at
C-19. Alstonoxine B (12) was also isolated in the present study,
and since suitable crystals were obtained from CH2Cl2–hexane
group
a to a carbonyl group were observed at d 2.69 (dd, J = 18,
5 Hz) and 2.80 (dd, J = 18, 8 Hz). These features are reminiscent
of the C-18ꢀC-19ꢀC-20 side-chain in the E-seco macroline oxin-
dole alstonoxine A (10). The NMR spectroscopic data (Tables 1
and 2) of 9 did in fact show a close resemblance to those of alsto-
noxine A (10) (Kam and Choo, 2000), except for the absence of an
aromatic hydrogen resonance at C-11, which was replaced in 9 by a
3H singlet at d 3.84 due to an aromatic methoxy substituent. The
placement of the latter at C-11 was further confirmed by
the observed NOEs between OMe and H-10, as well as H-12. The
configuration of the spirocyclic C-7 was assigned as S from the
observed reciprocal NOEs between H-9 and H-15. In view of
the availability of suitable crystals (crystals were obtained from
hexane–CH2Cl2), an X-ray diffraction analysis was carried out
which provided confirmation of the structure and relative configu-
ration of alstonoxine C (9) deduced from the spectroscopic data
(Fig. 4). The X-ray structure of alstonoxine C (9) also provided
additional support for the original assignment of the structure
and relative configuration of alstonoxine A (10).
Alstonoxine D (11) was isolated as a light yellowish oil, with
[a] –16 (c 0.23, CHCl3). The UV and IR spectra of 11 were similar
D
to those of alstonoxine B (12) (Kam and Choo, 2000), while the
ESIMS showed an [M+H]+ peak at m/z 361 (C20H28N2O4 + H). The
1H and 13C NMR spectroscopic data (Tables 1 and 2) of 11 were
generally similar to those of alstonoxine B (12), except for the pres-
ence of an additional aromatic methoxy singlet at d 3.83, and the
absence of one aromatic-H signal. Placement of the methoxy sub-
stituent at C-11 was supported by the observed coupling behaviour
of the aromatic hydrogens, as well as from the observed three-
bond correlations from H-9 to C-11 and C-13, in the HMBC spec-
trum. This assignment was further supported by the observed
NOEs between 11-OMe and H-10, H-12. The configuration of the
spirocenter C-7 was assigned as S from the observed NOEs between
H-9 and H-15. The difference between alstonoxine D (11) and the
previous compound 9 resides in the C-18–C-19–C-20 side-chain.
Whereas C-19 in 9 is a ketone carbonyl, the C-19 in 11 is an oxy-
methine associated with a secondary alcohol functionality. This
difference was also reflected in the respective NMR spectroscopic
data of 9 and 11. The NMR data of 11 were, however, insufficient
to establish the stereochemistry of C-19. Towards this end,
alstonoxine C (9) was treated with NaBH4/MeOH, which gave a
Fig. 5. X-ray crystal structure of 12.

S.-H. Lim et al. / Phytochemistry 98 (2014) 204–215
209
Fig. 6. Selected HMBCs and NOEs of compound 15.
solution in this instance, X-ray diffraction analysis was carried
which established the relative configuration of C-19 in alstonoxine
B (12) as S (Fig. 5). The X-ray structure of 12 also provided
additional support for the determination of the C-19 configuration
of 11 as 19S, using Horeau’s procedure (vide supra), since the NMR
spectroscopic data of 11 and 12 indicated that the non-aromatic
portion of 11 was virtually identical to the non-aromatic portion
of 12. Therefore the configuration of C-19 in 11 can be assumed
to be similar to that of alstonoxine B (12).
Fig. 8. X-ray crystal structure of compound 16.
ous stereogenic centers were deduced from the NOESY data which
indicated similarity with those in affinisine. The observed H-16/H-
6b NOE indicated that the configuration of C-16 is R. This left the
geometry of the C-19–C-20 double bond as a possible point of
departure between the two compounds. This was confirmed by
the observed reciprocal NOEs for H-19/H-15 and H-21/H-18, which
established the geometry of the 19,20-double bond as Z. Com-
pound 13 is therefore the 19,20-Z isomer of affinisine (19,20-E).
Compound 15 (2(R)-3-hydroxycathafoline) was isolated as a
Compound 13 was obtained as a light yellowish oil, [a] +8 (c
D
0.45, CHCl3). The UV spectrum showed absorption maxima at
229, 254, and 284 nm, indicative of an indole chromophore. The
IR spectrum had a broad band at 3372 cmꢀ1 due to an OH function.
The ESIMS of 13 showed an [M+H]+ peak at m/z 309, which ana-
lyzed for C20H24N2O. The 13C NMR spectrum (Table 1) displayed
a total of 20 resonances, comprising two methyl, four methylene,
nine methine, and five quaternary carbon atoms, in agreement
with the molecular formula. Analysis of the 1H NMR data (Table 2)
established the presence of an unsubstituted indole moiety (d
7.08–7.41), an N1-Me (d 3.61), an aminomethylene (d 3.63), an
oxymethylene associated with a hydroxymethyl group (d 3.47),
and an ethylidene side-chain (d 1.57, 5.28). The COSY spectrum
yielded a fragment consistent with a sarpagine-type compound,
viz. NCHCH2CHCH(CH2O)CHCH2. Analysis of the 2-D NMR spectro-
scopic data indicated that 13 possessed the same molecular con-
nectivity as affinisine (14) (Clivio et al., 1991), which was also
isolated. The 1H and 13C NMR spectroscopic data (Tables 1 and 2)
of 13 were generally similar to those of 14 except for notable dif-
ferences in the chemical shifts of C-15 in the 13C NMR spectrum,
and H-15 in the 1H NMR spectrum. The configurations at the vari-
light yellowish oil, with [a] –48 (c 0.24, CHCl3). The IR spectrum
D
showed a band at 1739 cmꢀ1 due to an ester group. The UV spec-
trum showed typical dihydroindole absorptions at 202, 252, and
295 nm. The ESIMS of 15 showed an [M+H]+ peak at m/z 355,
which analyzed for C21H26N2O3. The 13C NMR spectrum (Table 1)
displayed a total of 21 resonances, comprising three methyl, four
methylene, eight methine, and six quaternary carbon atoms. The
1H NMR spectrum (Table 2) showed the presence of an unsubsti-
tuted indole moiety (d 6.61–7.09), an N1-Me (d 2.95), a methyl es-
ter (d 3.78), an aminomethylene (dH 2.98, 4.10; dC 55.0), and an
ethylidene side-chain (d 1.49, 5.41). The COSY spectrum yielded
the following fragments, viz., NCH2CH2 and CH2CHCH. The 13C
NMR spectrum (Table 1) showed the presence of two downfield
resonances, a methine at d 80.7, and an oxygenated quaternary car-
bon at d 85.3. The former resonance was characteristic of C-2 of
cathafoline alkaloids with a H-2b orientation (Das et al., 1977),
while the latter resonance at d 85.3 was characteristic of a carbi-
nolamine moiety, suggesting the presence of hydroxy-substitution
at C-3. This was further supported by the observed three bond cor-
relations from H-5 and H-21 to the oxygenated C-3 in the HMBC
spectrum (Fig. 6). The 1H and 13C NMR spectroscopic data of 15
showed a close correspondence with those of cathafoline (18)
(Atta-ur-Rahman et al., 1988), except for the downfield shift of
the C-3 signal in the 13C NMR spectrum (from d 47.2 in 18 to d
85.3 in 15) due to substitution by the OH group. The configurations
at the various stereogenic centers of 15 were similar to those of
cathafoline (18) as indicated by the observed NOEs from the NOESY
spectrum (Fig. 6). The observed NOE between H-16 and H-9 indi-
cated that the configuration of C-16 is R (H-16 directed towards
the indole moiety). The observed NOE between H-16 (d 2.90) and
the H-14 signal at d 1.90 allowed the attribution of this signal to
H-14b. The NOE between this H-14b and H-2 indicated that the ori-
Fig. 7. Selected NOEs of compound 16.

210
S.-H. Lim et al. / Phytochemistry 98 (2014) 204–215
Table 3
1H (400 MHz) NMR spectroscopic data for compounds 17, 19–22, 24, 26, and 28.a
H
17
19
20
21
22
24
26
28
2
3
3.21 d (4)
3.92 m
–
2.55 dd
(14, 7)
3.43 td (14, 6)
–
–
–
–
–
3.86 d (5)
3.76 m
3.96 m
3.93 m
3.93 m
–
1.68 m (b)
2.32 m (a)
2.73 br t (11) (b)
1.73 m
2.90 m
3.42 dd
(11, 9)
3.91 m
1.68 m (b)
2.32 m (a)
2.75 br t (10.5) (b)
1.71 m
2.85 m
3.35 br t (10)
3.72 m
–
4.70 d (3)
5a
5b
6a
6b
2.53 m
4.13 d (4)
3.33 td
(11, 9) (a)
1.97 ddd
(14, 9, 1) (a)
2.50 ddd
3.38 m (a)
3.94 m
–
2.78 br d (13)
–
2.02 dd
(14, 6)
3.28 m
2.06 dd
(15, 9)
2.81 m
1.97 dd (14, 8) (a)
2.41 m (b)
2.06 dd (15, 9)
2.74 m
2.17 dd (14, 3)
3.37 br d (14)
–
–
2.62 m
3.00 br d (13)
(14, 11, 9) (b)
7.22 dd (7.5, 1)
6.58 td (7.5, 1)
9
10
6.72 d (2)
–
7.11 d (8)
6.69 td
(8, 1)
7.02 s
–
6.76 d (2.5)
–
6.76 d (2.5)
–
7.16 m
6.80 t (8)
6.82 m
6.51 m
11
6.66 dd (9, 2)
7.06 td (7.5, 1)
7.13 td
(8, 1)
–
6.67 dd (8.5, 2.5)
6.69 dd (9, 2.5)
7.18 m
7.07 m
12
14a
14b
6.52 br d (9)
1.97 m (a)
2.13 dd
6.27 br d (7.5)
1.76 m
1.76 m
6.47 d (8)
1.85 m
1.92 m
5.96 s
1.77 m
1.77 m
6.37 br d (8.5)
1.83 m
1.91 m
6.54 br d (9)
1.79 br d (14) (b)
2.11 m (a)
6.66 d (8)
1.82 td (10, 3)
2.55 m
6.63 br d (8)
1.96 m
2.81 dd (14, 5)
(14, 3) (b)
3.36 m
2.71 d (4)
–
15
16
17
18
3.61 m
2.83 br s
–
3.65 m
2.80 br s
–
3.62 m
2.79 br s
–
3.64 m
2.77 br s
–
3.27 br s
2.41 d (4)
–
3.56 m
–
4.13 s
3.61 m
–
5.76 br s
1.53 d (7)
1.43 dd (7, 2)
1.59 dd (7, 2)
1.60 dd
1.60 dd (7, 2)
1.58 d (7)
1.48 dd (7, 2)
1.60 dd (7, 1)
(7, 2)
19
21a
21b
5.38 br q (7)
2.96 br d (16)
3.94 m
2.61 s
3.70 s
–
3.52 s
–
–
–
–
–
5.40 q (7)
3.00 br d (15)
3.81 m
2.63 s
–
–
5.73 q (7)
3.91 br d (15)
4.20 m
3.07 s
–
–
5.41 q (7)
3.02 br d (15)
3.84 m
2.63 s
3.78 s
3.86 s
3.80 s
–
–
–
–
–
5.69 q (7)
3.85 br d (15)
4.18 br d (15)
3.02 s
3.69 s
–
3.77 s
–
–
–
–
–
5.39 q (7)
3.06 br d (18)
3.75 m
2.90 s
3.64 s
–
3.70 s
–
–
–
–
–
5.29 br q (7)
3.72 m
3.96 m
2.61 s
–
–
5.35 q (7)
3.87 m
4.25 br d (16)
2.62 s
–
–
N1-Me
10-OMe
11-OMe
CO2Me
20
3.79 s
3.79 s
3.70 s
3.38 s
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
7.30 d (2)
6.84 m
7.49 dd (9, 2)
50
60
30-OMe
40-OMe
3.84b
3.89b
s
s
a
Measured in CDCl3.
Assignments may be reversed.
b
entation of H-2 is b as in cathafoline (2R), which was also consis-
tent with the observed C-2 resonance at d 79.1. Furthermore, in
the cathafoline alkaloids, the rigid architecture of the molecule re-
stricts the orientation of any substituent at C-3 (including H) to be
CHCHCH2CHCH, which differed from the previous compound by
the replacement of CH2CHCH with a CHCHCH2CHCH fragment. Fur-
thermore, the downfield resonance at ca. d 85 in the 13C NMR spec-
trum, associated with the carbinolamine moiety in 15, was not
observed in the spectrum of 16. Comparison of the 1H and 13C
NMR spectroscopic data (Tables 1 and 2) indicated a general simi-
larity with those of cathafoline (18), except for the more notable
differences in the chemical shifts of C-2 and C-14 in the 13C NMR
spectrum, and H-2 in the 1H NMR spectrum. This observation, as
well as the fact that 16 is isomeric with cathafoline (18) as shown
by the MS data (vide supra), suggested that 16 is a stereoisomer of
18. The relative configurations at the various stereogenic centers of
16 were similar to those of cathafoline (18) as deduced from the
NOESY data, except for the configuration at C-2. In the case of 16,
a NOE was not observed between H-2 and H-14b (which was the
case in 15), but was instead observed between H-2 and H-6a,
N1-Me, and H-3. These NOEs were different from those observed
in the 2R cathafoline alkaloids, and indicated that the relative con-
figuration of C-2 in 16 is S (H-2a) (Fig. 7), i.e. an inference which
was also consistent with the observed resonance of C-2 at d 70.1
(Das et al., 1977; Massiot et al., 1983). Since suitable crystals of
16 were obtained from CH2Cl2–hexane, an X-ray diffraction analy-
sis was carried out which confirmed the assignment of configura-
tion of C-2 as S based on the NMR spectroscopic data (Fig. 8).
Compound 16 is therefore the C-2 epimer of cathafoline (18).
Compound 17 (2(S)-10-methoxycathafoline) was obtained as a
a
only and as such, the orientation of the C-3ꢀOH is therefore by
necessity, . This is the first isolation of a naturally occurring
a
3-hydroxycathafoline, although a compound previously assigned
as 3-hydroxycathafoline has been encountered in transformations
of the echitamine alkaloids (Massiot et al., 1983; the 13C NMR data
of the reported compound at C-2, C-3, C-14, and C-21, however,
were different from those of 15).
Compound 16 (2(S)-cathafoline) was isolated as a light yellow-
ish oil, with [a]D –175 (c 0.22, CHCl3), and was subsequently crys-
tallized from CH2Cl2–hexane as colorless block crystals. The UV
spectrum showed absorption maxima at 208, 246, and 308 nm,
consistent with a dihydroindole chromophore. The IR spectrum
showed a band at 1737 cmꢀ1 due to an ester function, which was
confirmed by the observed carbon shift of the carbonyl function
at d 172.8 in the 13C NMR spectrum. The ESIMS showed an
[M+H]+ peak at m/z 339, which analyzed for C21H26N2O2.
The 13C NMR spectrum (Table 1) showed a total of 21 reso-
nances, comprising three methyl, four methylene, nine methine,
and five quaternary carbon atoms. The 1H NMR spectrum (Table 2)
showed many features which were also present in 15 indicating
the presence of similar groups, such as an unsubstituted indole
moiety, an N1-Me, a methyl ester, an aminomethylene, and an
ethylidene side-chain. The COSY spectrum, however, showed the
presence of the following partial structures, viz., NCH2CH2 and
light yellowish oil, with [
spectra were similar to those of compound 16. The ESIMS showed
a
]
ꢀ138 (c 0.47, CHCl3). The UV and IR
D

S.-H. Lim et al. / Phytochemistry 98 (2014) 204–215
211
loids. The NMR spectroscopic data (Tables 1 and 3) indicated a
close resemblance to those of vincorine (23) (Das et al., 1974;
Morfaux et al., 1992), except for the absence of the methoxy group
at C-10. The relative configuration of C-16 and the geometry of the
19,20-double bond, were similar to those of vincorine (23) as
shown by the NOE data (Fig. 9).
Compound 20 (10-demethoxyvincorine N(4)-oxide) was ob-
tained as a yellowish oil, with [a]D –62 (c 0.5, CHCl3). The UV spec-
trum (208, 247, and 299 nm) showed absorption maxima
characteristic of a dihydroindole chromophore, while the IR spec-
trum showed the presence of an ester carbonyl (1734 cmꢀ1) func-
tion. The ESIMS of 20 showed an [M+H]+ peak at m/z 355, which
analyzed for C21H26N2O3, 16 mass units higher than that of 19.
Compound 20 was readily identified as the N4-oxide of 10-
demethoxyvincorine from its NMR spectroscopic data (Tables 1
and 3), in particular, the characteristic downfield shifts of the car-
bon resonances for C-2, C-5, and C-21, when compared with those
of 10-demethoxyvincorine (19).
Fig. 9. Selected HMBCs and NOEs of compound 19.
an [M+H]+ peak at m/z 369, which is 30 mass-units more than 16.
The NMR spectroscopic data of 16 and 17 (Tables 1 and 3) were
generally similar, except for the presence of a methoxy group in
17. The substitution of the methoxy group at C-10 was deduced
from the coupling behaviour of the aromatic hydrogens, the ob-
served three-bond correlations from H-12 and 10-OMe to C-10 in
the HMBC spectrum, and the following observed NOEs: H-9/10-
OMe; H-11/10-OMe, H-12; and, H-12/H-11, N1-Me. In view of
Compound 21 (11-methoxyvincorine) was obtained as a light
yellowish oil, [
a]
ꢀ86 (c 0.27, CHCl3). The UV spectrum showed
D
dihydroindole absorption maxima at 208, 256 and 317 nm, while
the IR spectrum indicated the presence of an ester carbonyl
(1733 cmꢀ1) function. The ESIMS of 21 had an [M+H]+ peak at
m/z 399, corresponding to the molecular formula C23H30N2O4, dif-
fering from vincorine (23) by addition of 30 mass units. The 1H and
13C NMR spectroscopic data (Tables 1 and 3) were similar in all re-
spects to those of 23, except for the aromatic resonances and the
presence of an additional aromatic methoxy substituent at dH
3.86 (dC 56.4). In the 1H NMR spectrum of 21, two aromatic singlets
were observed at d 7.02 and 5.96, which were assigned to H-9 and
H-12, respectively based on the NOE data (NOEs observed for H-9/
10-OMe, H-16; H-12/NMe, 11-OMe). These features are consistent
with 10,11-dimethoxy-substitution on the indole moiety. Com-
pound 21 is therefore assigned as 11-methoxyvincorine.
the diagnostic NOEs observed for H-2/H-6
observed C-2 resonance at d 70.7, the configuration at C-2 in 17
is also (H-2 ). Compound 17 is therefore, 2(S)-10-
methoxycathafoline.
Compound 19 (10-demethoxyvincorine) was isolated as a light
yellowish oil, with [ –127 (c 0.48, CHCl3). The UV spectrum
a, N1-Me, H-3, and the
S
a
a]
D
showed absorption bands at 205, 256, and 308 nm, characteristic
of a dihydroindole chromophore. The IR spectrum showed a band
at 1736 cmꢀ1 due to an ester carbonyl function (dC 173.8). The
ESIMS of 19 gave an [M+H]+ ion at m/z 339 and high-resolution
measurements gave the formula C21H26N2O2. The 1H NMR spectro-
scopic data (Table 3) displayed a number of features, which were
characteristic of akuammiline type alkaloids and which were also
common with those present in the previous compounds 15–17,
such as the presence of an unsubstituted indole moiety, an N1-
Me, a methyl ester, an aminomethylene, and an ethylidene side-
chain. A downfield quaternary carbon resonance was observed at
d 97.4 in the 13C NMR spectrum (Table 1), which was assigned to
C-2, from the observed three-bond correlation from N1-Me, H-
14, and H-21 to C-2 in the HMBC spectrum (Fig. 9). The downfield
shift of this carbon was consistent with it being linked to two
nitrogen atoms, which is a characteristic of the vincorine type alka-
Compound 22 (vincorine N(4)-oxide) was obtained as a yellow-
ish oil, with [a]D –84 (c 0.4, CHCl3). The UV spectrum (211, 250 and
323 nm) was characteristic of a dihydroindole chromophore, while
the IR spectrum showed the presence of an ester (1734 cm–1) func-
tion. The ESIMS of 22 had an [M+H]+ at m/z 385, which analyzed for
C22H28N2O4, 16 mass units higher than that of vincorine (23).
Compound 22 was readily identified as the N4-oxide of vincorine
Table 4
Cytotoxic effects of compounds 1, 3, 4, 6, 9, 11, 13, 15–17, 19, 21, and 24.
Compound
IC50
,
l
g/mL
KB/VJ300a
KB/Sa
KB/VJ300(+)b
Alstofolinine A (1)
20,21-Dihydroalstonerine (3)
Macrocarpine D (4)
Macrodasine H (6)
Alstonoxine C (9)
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
0.015
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
5.88
>25
>25
20.77
>25
13.2
>25
>25
Alstonoxine D (11)
19,20-Z-Affinisine (13)
2(R)-3-Hydroxycathafoline (15)
2(S)-Cathafoline (16)
2(S)-10-Methoxycathafoline (17)
10-Demethoxyvincorine (19)
11-Methoxyvincorine (21)
11-demethoxyquaternine (24)
Vincristine
7.47
>25
23.58
8.14
11.23
4.60
6.60
–
Kopsamine
6.38 (5.6)c
a
KB/S and KB/VJ300 are vincristine-sensitive and vincristine-resistant human
oral epidermoid carcinoma cell lines, respectively.
b
With added vincristine, 0.1
VJ300 cells.
l
g/mL, which did not affect the growth of the KB/
c
With added vincristine, 0.25
VJ300 cells (Kam et al., 1998b).
lg/mL, which did not affect the growth of the KB/
Fig. 10. Selected NOEs of compound 24.

212
S.-H. Lim et al. / Phytochemistry 98 (2014) 204–215
from its NMR spectroscopic data (Tables 1 and 3), in particular the
characteristic downfield shifts of the carbon resonances for C-2, C-
5, and C-21, when compared with those of vincorine (23).
Compound 24 (11-demethoxyquaternine) was obtained as a
found to reverse multidrug resistance in vincristine-resistant KB
(VJ300) cells (Table 4).
The structures of several new macroline oxindole alkaloids
from various Alstonia species were previously reported (Kam
and Choo, 2000). In view of a systematic error involving the
labeling of the configuration (R or S) at the spirocyclic carbon
(C-7) in these alkaloids, a list of these alkaloids is now included
with the correct R/S labeling for C-7: isoalstonisine (reported
7S, corrected 7R) (Kam and Choo, 2000), macrogentine
(reported 7S, corrected 7R) (Kam and Choo, 2000),
light yellowish oil, [
a
]
ꢀ10 (c 0.21, CHCl3). The UV spectrum
D
had absorption maxima at 208, 241, 307 nm, suggesting the pres-
ence of a dihydroindole chromophore, and the IR spectrum dis-
played an ester carbonyl band at 1736 cmꢀ1 (dC 172.5). The
ESIMS showed an [M+H]+ peak at m/z 383, which analyzed for
C
22H26N2O4. The 1H and 13C NMR spectroscopic data (Tables 1
and 3) established the presence of a substituted indole moiety,
an ethylidene side-chain, and three 3H singlets at dH 3.70, 3.64,
and 2.90, due to a methyl ester (dC 172.5, 51.7), an aromatic meth-
oxy (dC 56.0), and an NMe (dC 30.0) group, respectively. In addition,
a deshielded methine was observed as a doublet at dH 4.70
(J = 3 Hz), with the corresponding carbon resonance observed at
dC 87.2. The observed downfield 1H and 13C shifts shown by this
methine, is characteristic of a carbon, which is adjacent to a nitro-
gen and an oxygen atom. The NMR spectroscopic data indicated a
similarity to those of quaternine (25) (Abe et al., 1994a), which was
also present in the same plant. The main difference noted from the
NMR spectroscopic data was the absence of one of the aromatic
methoxy groups (two aromatic methoxy groups were present in
quaternine). The aromatic doublet at d 6.76 was assigned to H-9
alstonoxine A (reported 7R, corrected 7S) (Kam and Choo,
2000), alstonoxine B (reported 7R, corrected 7S) (Kam and Choo,
2000), alstofoline (reported 7R, corrected 7S) (Kam and
Choo, 2000), N1-demethylalstonisine (reported 7R, corrected
7S) (Kam and Choo, 2000), affinisine oxindole (reported 7R,
corrected 7S) (Kam and Choo, 2004b).
Concluding remarks
The present investigation reports the presence of 17 new indole
alkaloids and completes the study of the alkaloids of this particular
sample of A. macrophylla. The new indole alkaloids found were
mainly of the macroline, sarpagine, akuammiline/vincorine and
ajmaline types. It is noted that there is some variation in the alka-
loid composition between the indole alkaloids found in the present
sample collected on the western coast of Peninsular Malaysia (Per-
ak), compared to a sample collected from the eastern coast of Pen-
insular Malaysia (Terengganu) (Kam and Choo, 2004a). Although a
number of alkaloids from the various subtypes were common in
both samples, nevertheless, alkaloids which were found in one
sample, were absent in the other, and vice versa. For instance, sar-
pagine and ajmaline alkaloids found in the present sample were
not found in the previous sample. There is also a greater predom-
inance of the akuammiline alkaloids, as well as bisindole alkaloids
(Lim et al., 2011, 2012, 2013) in the present sample. Among the
alkaloids tested, the akuammiline alkaloids, 11-methoxyvincorine
(21) and 11-demethoxyquaternine (24), were the most active in
reversing multi-drug resistance in vincristine-resistant KB cells.
from its NOE with H-6a (Fig. 10), while the other aromatic doublet
at d 6.54 was assigned to H-12 from the observed NOE between
this hydrogen and N1-Me. The observed NOEs for OMe/H-9, H-11
confirmed the placement of the aromatic methoxy group at C-10.
The relative configuration of C-16 was assigned as R (H-directed to-
wards the indole moiety), from the observed NOEs between H-16
and H-14b, H-15 (Fig. 10).
Compound 26 (vincamajine N(4)-oxide) was obtained as a yel-
lowish oil, with [a]D –29 (c 0.04, CHCl3). The UV spectrum (210,
231 and 282 nm) was characteristic of a dihydroindole chromo-
phore, while the IR spectrum showed the presence of OH and ester
groups (3400 and 1737 cm–1). The presence of an ester function
was further confirmed by the observed signals at d 52.3 and
170.8 in the 13C NMR spectrum. The ESIMS of 26 showed an
[M+H]+ at m/z 383 (C22H26N2O4), which was 16 mass units higher
than that of vincamajine (27). The NMR spectroscopic data (Table 1
and 3) indicated an alkaloid of the ajmaline type and were in fact
similar to those of vincamajine (27) (Cherif et al., 1989), which
was also isolated, except that the resonances of C-3, C-5, and
C-21, were shifted downfield in 26. Based on these observations,
compound 26 was readily identified as the N4-oxide of
vincamajine.
Experimental
General
Melting points were determined on a Mel-Temp melting point
apparatus and are uncorrected. Optical rotations were determined
on a JASCO P-1020 digital polarimeter. IR spectra were recorded on
a Perkin-Elmer Spectrum 400 spectrophotometer. UV spectra were
obtained on a Shimadzu UV-3101PC spectrophotometer. 1H and
13C NMR spectra were recorded in CDCl3 using TMS as internal
standard on JEOL JNM-LA 400 and JNM-ECA 400 spectrometers at
400 and 100 MHz, respectively. NOESY experiments were carried
out on a Bruker Avance III 600 spectrometer at 600 MHz. ESIMS
and HRESIMS were obtained on an Agilent 6530 Q-TOF or on a JEOL
AccuTOF-DART mass spectrometer.
Compound 28 (vincamajine 17-O-veratrate N(4)-oxide) was ob-
tained as a yellowish oil, with [a]D –75 (c 0.94, CHCl3). The UV
spectrum (209, 254 and 292 nm) was characteristic of a dihydroin-
dole chromophore, while the IR spectrum displayed the presence
of an ester (1737 cm–1) function. The ESIMS of 28 had an [M+H]+
at m/z 547, which analyzed for C31H34N2O7. The 1H NMR (Table 3)
spectrum showed similar features as those shown by vincamajine
(27), except for the presence of additional signals due to the acid
residue (30,40-dimethoxybenzoic acid or veratric acid) associated
with an ester group at C-17 (vincamajine 17-O-veratrate (29))
(Abe et al., 1994b), and the downfield shifts of the carbon reso-
nances for C-3, C-5, and C-21, when compared with those of vinca-
majine 17-O-veratrate (29). Compound 28 (measured mass was 16
mass units higher than that of 29) is therefore readily identified as
the N4-oxide of vincamajine 17-O-veratrate.
Plant material
All thenewcompounds testedshowed no appreciablecytotoxicity
against drug-sensitive and vincristine-resistant KB cells (IC50 >25 lg/
ml in all cases). However, 11-methoxyvincorine (21), 11-demethoxy-
quaternine (24), 19,20-Z-affinisine (13), 2(S)-10-methoxycathafoline
(17), 10-demethoxyvincorine (19), and macrodasine H (6), were
A. macrophylla was collected in Perak, Malaysia and identifica-
tion was confirmed by Dr. Richard C. K. Chung, Forest Research
Institute, Malaysia, and Dr. K. T. Yong, Institute of Biological Sci-
ences, University of Malaya. Herbarium voucher specimens
(K671) are deposited at the Herbarium, University of Malaya.

S.-H. Lim et al. / Phytochemistry 98 (2014) 204–215
213
Extraction and isolation
Alstonoxine C (9)
Light yellowish oil and subsequently colorless block crystals
25
The leaf (7 kg) and stem-bark (9 kg) were exhaustively ex-
tracted with EtOH at room temperature and the concentrated EtOH
extract was then partitioned with dilute acid (3% tartaric acid) fol-
lowed by basification of the aqueous fraction with concentrated
NH4OH solution and extraction of the liberated alkaloids with
CHCl3. The alkaloids were isolated by initial silica gel column chro-
matography (CC) using CHCl3 with increasing proportions of
MeOH, followed by further chromatography of the appropriate
partially resolved fractions using centrifugal preparative TLC. The
solvent systems used for centrifugal preparative TLC were Et2O–
hexane (4:1; NH3-saturated), Et2O (NH3-saturated), Et2O–MeOH
(100:1; NH3-saturated), Et2O–MeOH (20:1; NH3-saturated), Et2O–
MeOH (10:1; NH3-saturated), EtOAc–hexane (1:5; NH3-saturated),
EtOAc–hexane (1:4; NH3-saturated), EtOAc–hexane (1:1; NH3-sat-
urated), EtOAc–hexane (2:1; NH3-saturated), EtOAc–hexane (3:1;
NH3-saturated), EtOAc (NH3-saturated), EtOAc–MeOH (10:1;
NH3-saturated), CHCl3–hexane (1:1; NH3-saturated), CHCl3–hex-
ane (1:2; NH3-saturated), CHCl3 (NH3-saturated), CHCl3–MeOH
(100:1; NH3-saturated), and CHCl3–MeOH (50:1; NH3-saturated).
The yields (mg kgꢀ1) of the alkaloids from the leaf extract were
as follows: 1 (0.9), 9 (7.9), 13 (4.0), 15 (5.0), 16 (4.7), 17 (6.9), 19
(1.0), 20 (1.8), 21 (0.7), 22 (20.6), 24 (13.2), 26 (0.2), and 28 (1.9).
The yields (mg kgꢀ1) of the alkaloids from the stem-bark extract
were as follows: 3 (0.2), 4 (3.5), 6 (0.3), 11 (2.2), 13 (4.3), and 16
(0.1).
from CH2Cl2ꢀhexane; mp 130–132 °C; [
a
]
D ꢀ30 (c 0.39, CHCl3);
UV (EtOH) kmax (log
e
) 219 (5.17), 266 (4.33) and 291 (4.24) nm; IR
(dry film) mmax 3390, 3295, 1694 cmꢀ1; for 1H NMR and 13C NMR
spectroscopic data, see Tables 2 and 1, respectively; ESIMS m/z
359 [M+H]+; HRESIMS m/z 359.1979 (calcd for C20H27N2O4,
359.1971).
Alstonoxine D (11)
25
Light yellowish oil; [
a
]
ꢀ16 (c 0.23, CHCl3); UV (EtOH) kmax
D
(log
e
) 224 (4.62), 274 (3.80) and 286 (3.80) nm; IR (dry film) mmax
3391, 3298, 1695 cmꢀ1; for 1H NMR and 13C NMR spectroscopic
data, see Tables 2 and 1, respectively; ESIMS m/z 361 [M+H]+; HRE-
SIMS m/z 361.2125 (calcd for C20H29N2O4, 361.2127).
19,20-Z-Affinisine (13)
25
Light yellowish oil; [
(log
a]
+8 (c 0.45, CHCl3); UV (EtOH) kmax
D
e
) 229 (5.37), 254 (4.92), 284 (4.73) nm; IR (dry film) mmax
3372 cmꢀ1; for 1H NMR and 13C NMR spectroscopic data, see
Tables 2 and 1, respectively; ESIMS m/z: 309 [M+H]+; HRESIMS
m/z: 309.1973 [M+H]+ (calcd for C20H25N2O, 309.1967).
2(R)-3-Hydroxycathafoline (15)
25
Light yellowish oil; [
a
]
ꢀ48 (c 0.24, CHCl3); UV (EtOH) kmax
D
(log
e
) 202 (4.64), 252 (4.11), 295 (3.69) nm; IR (dry film) mmax
1739 cmꢀ1; for 1H NMR and 13C NMR spectroscopic data, see
Tables 2 and 1, respectively; ESIMS m/z: 355 [M+H]+; HRESIMS
m/z: 355.2028 [M+H]+ (calcd for C21H27N2O3, 355.2022).
Characterization data
Alstofolinine A (1)
2(S)-Cathafoline (16)
25
Light yellowish oil; [
(log
a]
D ꢀ104 (c 0.36, CHCl3); UV (EtOH) kmax
Light yellowish oil and subsequently colorless needles from
25
e
) 227 (4.10), 285 (3.43) nm; IR (dry film)
m
max 1769 cmꢀ1; for
CH2Cl2-hexanes; mp 121–123 °C; [
(EtOH) kmax (log
film)
a
]
ꢀ175 (c 0.22, CHCl3); UV
D
1H NMR and 13C NMR spectroscopic data, see Tables 2 and 1,
respectively; EIMS m/z 296 [M]+ (91), 281 (7), 265 (4), 240 (5),
227 (40), 212 (5), 197 (78), 182 (23), 181 (98), 170 (24), 167
(36), 154 (12), 144 (7), 119 (11); HREIMS m/z 296.1528 (calcd for
e) 208 (4.68), 246 (4.38), 308 (4.06) nm; IR (dry
m
max 1737 cmꢀ1; for 1H NMR and 13C NMR spectroscopic data,
see Tables 2 and 1, respectively; ESIMS m/z: 339 [M+H]+; HRESIMS
m/z: 339.2071 [M+H]+ (calcd for C21H27N2O2, 339.2073).
C18H20N2O2, 296.1525).
2(S)-10-Methoxycathafoline (17)
25
Light yellowish oil; [
a]
D ꢀ138 (c 0.47, CHCl3); UV (EtOH) kmax
20,21-Dihydroalstonerine (3)
(log
e
) 208 (4.85), 245 (4.60), 307 (4.17) nm; IR (dry film) mmax
25
Light yellowish oil; [
a]
ꢀ31 (c 0.11, CHCl3); UV (EtOH) kmax
D
1737 cmꢀ1; for 1H NMR and 13C NMR spectroscopic data, see
Tables 3 and 1, respectively; ESIMS m/z: 369 [M+H]+; HRESIMS
m/z: 369.2172 [M+H]+ (calcd for C22H29N2O2, 369.2178).
(log
e
) 228 (4.50), 286 (3.79) nm; IR (dry film) mmax 1710 cmꢀ1
;
for 1H NMR and 13C NMR spectroscopic data, see Tables 2 and 1,
respectively; EIMS m/z 338 [M]+ (100), 307 (8), 295 (11), 264
(10), 251 (6), 238 (8), 223 (5), 210 (14), 197 (78), 182 (19), 170
(13), 158 (10), 119 (4), 84 (12), 70 (25), 57 (6), 49 (10); HREIMS
m/z 338.1993 (calcd for C21H26N2O2, 338.1994); ESIMS m/z 339
[M+H]+; HRESIMS m/z 339.2080 (calcd for C21H27N2O2, 339.2073).
10-Demethoxyvincorine (19)
25
Light yellowish oil; [
a] D –127 (c 0.48, CHCl3); UV (EtOH) kmax
(log
e
) 205 (5.21), 256 (4.78), 308 (4.29) nm; IR (dry film) mmax
1736 cmꢀ1; for 1H NMR and 13C NMR spectroscopic data, see Ta-
bles 3 and 1, respectively; ESIMS m/z: 339 [M+H]+; HRESIMS m/z:
339.2072 [M+H]+ (calcd for C21H27N2O2, 339.2073).
Macrocarpine D (4)
25
Light yellowish oil; [
a]
ꢀ43 (c 0.89, CHCl3); UV (EtOH) kmax
D
(loge) 231 (5.16), 286 (4.48) nm; IR (dry film) mmax 3395,
10-Demethoxyvincorine N(4)-oxide (20)
3292 cmꢀ1; for 1H NMR and 13C NMR spectroscopic data, see
Tables 2 and 1, respectively; EIMS m/z 324 [M]+ (100), 306 (9),
295 (18), 277 (28), 240 (55), 212 (29), 184 (53), 145 (60), 117
(54), 95 (27); ESIMS m/z 327 [M+H]+; HRESIMS m/z 327.2079
(calcd for C20H27N2O2, 327.2073).
25
Light yellowish oil; [
(log
a]
–62 (c 0.5, CHCl3); UV (EtOH) kmax
D
e
) 208 (5.43), 247 (5.19), 299 (4.70) nm; IR (dry film) mmax
1734 cmꢀ1; for 1H NMR and 13C NMR spectroscopic data, see
Tables 3 and 1, respectively; ESIMS m/z: 355 [M+H]+; HRESIMS
m/z: 355.2019 [M+H]+ (calcd for C21H27N2O3, 355.2022).
Macrodasine H (6)
11-Methoxyvincorine (21)
25
25
Light yellowish oil; [
(log
a]
ꢀ11 (c 0.14, CHCl3); UV (EtOH) kmax
Light yellowish oil; [
a
]
–86 (c 0.27, CHCl3); UV (EtOH) kmax
D
D
e
) 233 (4.22), 288 (3.55) nm; IR (dry film) mmax 3423 cmꢀ1
;
(loge) 208 (4.16), 256 (3.75), 317 (3.54) nm; IR (dry film) mmax
for 1H NMR and 13C NMR spectroscopic data, see Tables 2 and 1,
respectively; ESIMS m/z 439 [M+H]+; HRESIMS m/z 439.2598 (calcd
for C26H35N2O4, 439.2591).
1733 cmꢀ1; for 1H NMR and 13C NMR spectroscopic data, see
Tables 3 and 1, respectively; ESIMS m/z: 399 [M+H]+; HRESIMS
m/z: 399.2078 [M+H]+ (calcd for C23H31N2O4, 399.2084).

214
S.-H. Lim et al. / Phytochemistry 98 (2014) 204–215
Vincorine N(4)-oxide (22)
and refined with full-matrix least-squares on F2 (SHELXL-97). All
25
Light yellowish oil; [
(log
a
]
–84 (c 0.4, CHCl3); UV (EtOH) kmax
non-hydrogen atoms were refined anisotropically and all hydrogen
atoms were placed in idealized positions and refined as riding
atoms with the relative isotropic parameters. Crystallographic data
for compounds 9, 12, and 16 have been deposited with the Cam-
bridge Crystallographic Data Centre. Copies of the data can be ob-
tained, free of charge, on application to the Director, CCDC, 12
Union Road, Cambridge CB2 1EZ, UK (fax: +44 0 1223 336033, or
e-mail: deposit@ccdc.cam.ac.uk).
D
e
) 211 (4.88), 250 (4.51), 323 (4.05) nm; IR (dry film) mmax
1734 cmꢀ1; for 1H NMR and 13C NMR spectroscopic data, see
Tables 3 and 1, respectively; ESIMS m/z: 385 [M+H]+; HRESIMS
m/z: 385.2130 [M+H]+ (calcd for C22H29N2O4, 385.2127).
11-Demethoxyquaternine (24)
25
Light yellowish oil; [
a
]
–10 (c 0.21, CHCl3); UV (EtOH) kmax
D
(log
e
) 208 (4.64), 241 (4.35), 307 (3.96) nm; IR (dry film) mmax
Crystallographic data of alstonoxine C (9): Colorless block crys-
1736 cmꢀ1; for 1H NMR and 13C NMR spectroscopic data, see
Tables 3 and 1, respectively; ESIMS m/z: 383 [M+H]+; HRESIMS
m/z: 383.1978 [M+H]+ (calcd for C22H27N2O4, 383.1971).
tals,
C20H26N2O4.H2O, Mr = 376.44, orthorhombic, space group
P212121, a = 7.3753(2) Å, b = 12.6375(3) Å, c = 19.5260(4) Å,
a
= b = c =
= 90°, V = 1819.93(8) Å3, T = 100 K, Z = 4, Dcalcd
1.374 g cmꢀ3, crystal size 0.16 ꢁ 0.18 ꢁ 0.24 mm3, F(000) = 704.
Vincamajine N(4)-oxide (26)
Light yellowish oil; [
(log
The final R1 value is 0.0383 (wR2 = 0.0898) for 3750 reflections
25
a]
–29 (c 0.04, CHCl3); UV (EtOH) kmax
[I > 2
Crystallographic data of alstonoxine B (12): colorless block crys-
tals, 19H26N2O3, Mr = 330.42, monoclinic, space group P21,
a = 10.7388(4) Å, b = 10.5321(3) Å, c = 15.2354(5) Å, = 90°,
r(I)]. CCDC number: 935820.
D
e
) 210 (4.94), 231 (4.70) and 282 (4.30) nm; IR (dry film) mmax
3400 and 1737 cmꢀ1; for 1H NMR and 13C NMR spectroscopic data,
see Tables 3 and 1, respectively; ESIMS m/z: 383 [M+H]+; HRESIMS
m/z: 383.1980 [M+H]+ (calcd for C22H27N2O4, 383.1971).
C
a = c
b = 92.851(2), V = 1721.02(10) Å3, T = 100 K, Z = 4, Dcalcd
=
1.275 g cmꢀ3, crystal size 0.21 ꢁ 0.41 ꢁ 0.49 mm3, F(000) = 712.
Vincamajine 17-O-veratrate N(4)-oxide (28)
The final R1 value is 0.0382 (wR2 = 0.1076) for 7729 reflections
25
Light yellowish oil; [
(log
a]
–75 (c 0.94, CHCl3); UV (EtOH) kmax
[I > 2r(I)]. CCDC number: 935819.
Crystallographic data of 2(S)-cathafoline (16): Colorless block
crystals, C21H26N2O2, Mr = 338.44, monoclinic, space group P21,
D
e
) 209 (5.75), 254 (5.40) and 292 (5.07) nm; IR (dry film) mmax
1737 cmꢀ1; for 1H NMR and 13C NMR spectroscopic data, see
Tables 3 and 1, respectively; ESIMS m/z: 547 [M+H]+; HRESIMS
m/z: 547.2443 [M+H]+ (calcd for C31H35N2O7, 547.2444).
a = 7.0991(3) Å, b = 8.6382(4) Å, c = 14.4902(6) Å,
a
=
c
= 90°,
b = 101.237(4),
V = 871.55(7) Å3,
T = 100 K,
Z = 2,
Dcalcd
=
1.290 g cmꢀ3, crystal size 0.08 ꢁ 0.26 ꢁ 0.37 mm3, F(000) = 364.
NaBH4 reduction of alstonoxine C (9)
The final R1 value is 0.0367 (wR2 = 0.0928) for 2138 reflections
To a mixture of compound 9 (12.4 mg, 0.035 mmol) in MeOH
(5 ml) at 0 °C was added NaBH4 (6.5 mg, 0.17 mmol). The solution
was stirred at 0 °C for 1 h. Saturated Na2CO3 (5 ml) solution was
added, and the product was extracted with CH2Cl2 (3 ꢁ 10 ml).
The combined organic extract was dried (Na2SO4), filtered, and
concentrated in vacuo, and the residue was purified by centrifugal
preparative TLC (SiO2, 1% MeOH:CHCl3, NH3-saturated) to afford
[I > 2r(I)]. CCDC number: 936523.
Cytotoxicity assays
Cytotoxicity assays (Mosmann, 1983) were carried out follow-
ing essentially the same procedure as described previously (Kam
et al., 1998a, 2004b). Human oral epidermoid carcinoma cells
(KB) and vincristine-resistant KB cells (VJ-300) were maintained
in Eagle’s MEM, supplemented with 10% fetal bovine serum and
2% penicillin/streptomycin. The cells were cultured at 37 °C under
a humidified atmosphere in a CO2 incubator. The cells were then
seeded in a 96-well microtiter plate (Nunc, Germany) at a concen-
tration of 70,000 cells/mL, and incubated in a CO2 incubator at
37 °C. After 24 h, the cells were treated with samples at six differ-
11 (5.1 mg, 41%) and 11a (4.8 mg, 39%). Compound 11a: colorless
25
oil; [a
]
ꢀ33 (c 0.24, CHCl3); 1H NMR (CDCl3, 400 MHz) d 7.69
D
(1H, d, J = 8.3 Hz), 6.58 (1H, dd, J = 8.3, 2.2 Hz), 6.45 (1H, d,
J = 2.2 Hz), 4.02 (1H, m), 3.99 (1H, m), 3.96 (1H, m), 3.92 (1H, m),
3.83 (3H, s), 3.24 (1H, m), 3.17 (3H, s), 2.76 (1H, m), 2.38 (1H, dd,
J = 8.4, 2.3 Hz), 2.06 (1H, d, J = 8.4 Hz), 1.94 (1H, m), 1.77 (1H, m),
1.72 (1H, m), 1.58 (1H, m), 1.53 (1H, m), 1.29 (1H, d, J = 6 Hz); HRE-
SIMS m/z 327.1718 (calcd for C19H23N2O3, 327.1703).
ent concentrations (0.1, 0.3, 1, 3, 10 and 30 lg/ml) and incubated
for 72 h. Wells containing untreated cells (without addition of
sample) were regarded as negative controls. DMSO was used to di-
lute the samples and the final concentration of DMSO in each well
was not in excess of 0.5% (v/v). No adverse effect due to presence of
Determination of the C-19 configuration of compound 6 by Horeau’s
method
To a solution of compound 11 (5 mg, 0.038 mmol) and anhy-
drous pyridine (1 ml), was added, racemic 2-phenylbutyric anhy-
dride (0.1 ml). The resulting mixture was stirred for 24 h at rt.
H2O (3 ml) was then added and the mixture was allowed to stand
for 30 min. The pH of the solution was adjusted to pH 9 by drop-
wise addition of NaOH (0.1 M), after which the solution was ex-
tracted with CH2Cl2 (3 ꢁ 20 ml). The aqueous layer was acidified
to pH 3 using 1.0 M HCl and extracted with CH2Cl2 (3 ꢁ 10 ml).
Evaporation of the solvent from the organic phase gave the unre-
acted 2-phenylbutyric acid. The optical rotation of the unreacted
2-phenylbutyric acid was found to be negative (R), indicating the
S configuration at C-19 in compound 11.
DMSO was observed. At the end of the incubation period, 20 ll of
MTT working solution (5 mg MTT in 1 ml phosphate-buffered sal-
ine) was added into each well and the 96-well microtiter plate was
incubated for another three hours at 37 °C. The medium was then
gently aspirated from each well and 200 ll of DMSO were added to
effect formazan solubilization. After agitation for 15 min, the
absorbance of each well was measured with a micro plate reader
(Emax, Molecular Devices, USA) at 540 nm with 650 nm. The cyto-
toxic activity of each sample was expressed as the IC50 value,
which is the concentration of the test sample that causes 50% inhi-
bition of cell growth. All the samples were assayed in three inde-
pendent experiments.
X-ray crystallographic analysis of alstonoxine C (9), alstonoxine B
(12), and 2(S)-cathafoline (16)
X-ray diffraction analysis was carried out on a Bruker SMART
APEX II CCD area detector system equipped with a graphite mono-
Acknowledgments
chromator and a Mo K
100 K. The structure was solved by direct methods (SHELXS-97)
a
fine-focus sealed tube (k = 0.71073 Å), at
We thank the University of Malaya and MOHE Malaysia (HIR-
F005) for financial support, and Dr. K. Komiyama (Center for Basic

S.-H. Lim et al. / Phytochemistry 98 (2014) 204–215
215
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cells.
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