Unusual spirocyclic macroline alkaloids, nitrogenous derivatives, and a cytotoxic bisindole from Alstonia

Article abstract of DOI:10.1016/j.tet.2004.03.031

The bark extract of the Malayan A. macrophylla provided several novel indoles with unprecedented carbon skeletons, an unusual nitrogenous compound, a cytotoxic bisindole, several new macroline alkaloids, in addition to other known alkaloids. The structures of the new compounds were established by spectroscopic analysis.

Full text of DOI:10.1016/j.tet.2004.03.031

Tetrahedron 60 (2004) 3957–3966
Unusual spirocyclic macroline alkaloids, nitrogenous derivatives,
and a cytotoxic bisindole from Alstonia
a,*
a
Toh-Seok Kam, Yeun-Mun Choo and Kanki Komiyama
b
a
Department of Chemistry, University of Malaya, Pantai Valley, 50603 Kuala Lumpur, Malaysia
The Kitasato Institute, 5-9-1 Shirokane, Minato-ku, Tokyo 108, Japan
b
Received 21 January 2004; revised 17 February 2004; accepted 11 March 2004
Abstract—The bark extract of the Malayan A. macrophylla provided several novel indoles with unprecedented carbon skeletons, an unusual
nitrogenous compound, a cytotoxic bisindole, several new macroline alkaloids, in addition to other known alkaloids. The structures of the
new compounds were established by spectroscopic analysis.
q 2004 Elsevier Ltd. All rights reserved.
1
. Introduction
six quaternary carbons) in agreement with the molecular
formula. In addition to the eight signals associated with the
1
3
The genus Alstonia is characterised by a preponderance of
1
indole moiety, the C NMR spectrum is notable for the
presence of two oxymethylenes (d 63.9, 64.3), two
oxymethines (d 77.7, 79.2), and two quaternary carbons
each of which are flanked by two oxygen atoms (d 105.5,
114.8), consistent with a highly oxygenated molecule as
–9
the macroline-type indole and oxindole alkaloids.
We
have previously reported the presence of new macroline
indoles as well as oxindoles from the Malayan species,
A. angustifolia var. latifolia, including isoalstonisine and
macrogentine, which represent the first macroline oxindoles
1
indicated by the molecular formula. The H NMR spectrum
3
,4
possessing the S configuration at the spirocyclic carbon.
of 1 (Table 1) showed the presence of an unsubstituted
indole chromophore, from the signals due to four aromatic
hydrogens, the presence of three methyl groups correspond-
ing to the N(1)–Me (d 3.63), N(4)–Me (d 2.33), and Me(18)
(d 1.59), and a hydroxymethyl group from the presence of a
pair of doublet of doublets at d 3.43 and 3.77 (corresponding
to the carbon resonance at d 63.9).
In continuation of our studies of Malaysian Alstonia, we
would like to report the structures of new alkaloids from
A. macrophylla, including novel macroline alkaloids
incorporating an unprecedented spiroketal unit, unusual
nitrogenous derivatives, and a cytotoxic bisindole.
9
The COSY spectrum disclosed some partial structures
which are characteristic of a macroline skeleton, such as
NCHCH and NCHCH CHCHCH O, corresponding to the
2
. Results and discussion
2
2
2
Macrodasine A 1 was obtained from the bark extract of
A. macrophylla as a colourless oil, with [a] ¼þ36 (c 0.36,
C(5)–C(6) and C(3)–C(14)–C(15)–C(16)–C(17) frag-
D
3
,4
ments. This is further supported by the observed hydrogen
chemical shifts and coupling behaviour for H(3), H(5),
H(16), H(17), as well as the three characteristic methyl
groups which are typical of a macroline compound (e.g.,
CHCl ). The UV spectrum was characteristic of an indole
3
chromophore with absorption maxima at 230 and 287 nm,
2
1
while the IR spectrum (3411 cm , broad) indicated the
presence of hydroxyl functions. The EIMS of 1 showed a
molecular ion at m/z 454, which analyzed for C H N O ,
requiring 11 degrees of unsaturation while the mass
fragments which were observed at m/z 197, 182, 181, 170,
3
alstonerine). At this stage, further analysis of the COSY
2
6 34 2 5
spectrum was complicated by overlap of some key signals.
Thus, two sets of partial structures can be proposed for the
remaining fragments, viz., CHCH and OCHCH CHCH O,
1
0
2
2
2
and 144 are typical of macroline derivatives and provided
1
3
versus CHCH CHO and CH CHCH O, which with the aid
2 2 2
early indication that 1 contained a macroline core. The
C
of the HMBC data led to two possible structures, 1 and 2,
respectively. Structure 1 is distinguished by the incorpor-
ation of a 1,6-dioxaspiro[4.4]nonane substructure fused
onto a macroline residue, while structure 2 on the other
hand, is distinguished by the incorporation of contiguously
fused tetrahydropyran and tetrahydrofuran rings onto the
NMR spectrum (Table 2) gave a total of 26 separate carbon
resonances (three methyls, six methylenes, 11 methines, and
Keywords: Alkaloids; NMR; Plants.
*
Corresponding author. Tel.: þ60379674266; fax: þ60379674193;
e-mail address: tskam@um.edu.my
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.03.031

1
Table 1. H NMR spectral data of 1, 3, 4, 5, 11, 12, 13, 14, 15, and 18
a
Position
1
3
4
5
11
12
13
14
15
18
Position
18
0
0
0
3
5
6
3.95 t (3)
2.98 d (7)
2.39 m
4.79 brs
3.94 t (3)
4.78 br s
3.96 m
3.98 t (3)
3.97 t (4)
2.91 d (7)
2.45 d (17)
3.82 br s
3.82 br s
4.09 dd (4, 2)
3.46 d (7)
2.54 m
3
3.79 t (3)
2.99 d (7)
2.28 m
3.32 m
6.90 s
3.56 d (7)
2.99 d (7)
3.57 d (7)
3.02 d (17)
2.87 d (7)
2.47 d (17)
2.91 d (7)
3.10 d (6)
2.48 d (16)
3.10 d (6)
2.48 d (16)
3.30 dd (16, 6)
7.33 d (8)
6.76 dd (8, 2)
—
5
3.04 d (18)
3.45 dd (18, 7)
7.56 br d (8)
7.23 td (8, 1)
7.35 td (8, 1)
7.40 br d (8)
2.41 d (17)
3.28 dd (17, 7)
7.50 br d (8)
7.12 td (8, 1)
7.21 td (8, 1)
7.31 br d (8)
1.56 m
2.43 d (17)
3.26 dd (17, 7)
7.49 br d (8)
7.10 td (8, 1)
7.18 td (8, 1)
7.29 br d (8)
6
3
.27 dd (17, 7)
3.46 dd (17, 7) 3.25 dd (17, 7)
3.27 dd (17, 7) 3.30 dd (16, 6)
3.08 m
0
9
7.50 br d (8)
7.12 br t (8)
7.21 td (8, 1)
7.31 br d (8)
7.55 br d (8)
7.23 td (8, 1)
7.35 td (8, 1)
7.40 br d (8)
1.79 dt (14, 5)
7.49 br d (8)
7.10 td (8, 1)
7.19 td (8, 1)
7.29 br d (8)
7.49 dd (8, 1)
7.09 td (8, 1)
7.17 td (8, 1)
7.27 dd (8, 1)
7.33 d (8)
6.76 dd (8, 2)
—
7.52 br d (8)
7.13 td (8, 1)
7.22 td (8, 1)
7.32 br d (8)
1.98 m
9
0
0
0
0
1
1
1
1
0
1
2
4
10
—
11
—
6.84 d (2)
6.84 d (2)
1.81 m
12
6.69 s
1.55 ddd (13, 5, 3) 1.78 br d (14)
1.42 ddd (13, 5, 2) 1.54 ddd (12, 4, 3) 1.39 dt (13, 4) 1.81 m
14
1.75 td (12, 3)
2
.04 m
0
2
.39 m
3.33 td (14, 4)
1.92 dt (14, 5)
2.33 dt (12, 5)
3.75 dd (12, 5)
2.42 m
3.35 td (14, 3)
1.96 dt (14, 5)
2.49 dt (13, 5)
2.50 td (13, 4)
2.06 dt (13, 5)
2.15 dt (11, 5)
2.29 m
2.26 td (13, 4) 2.13 m
2.13 m
2.41 m
2.14 m
1.57 m
15
2.54 m
0
0
1
1
1
5
6
7
1.85 m
1.84 dt (12, 5)
2.14 m
1.97 dt (13, 4)
1.86 dt (11, 4)
3.73 dd (11, 4)
1.86 m
1.86 m
2.64 dt (11, 5)
1.92 m
2.64 dt (11, 5)
1.92 m
16
1.84 dt (11, 4)
4.13 ddd
2.03 dt (12, 5)
3.70 dd (12, 5)
17
3.85 dd (12, 5)
3.88 dd (13, 5) 3.79 dd (11, 5)
3.74 dd (11, 4) 4.17 ddd (11, 4, 2) 4.19 ddd (11, 4, 2) 3.95 dd (11, 3)
(11, 4, 1)
4
.37 t (11)
0
0
4
.04 t (12)
4.82 t (12)
1.72 s
4.08 t (12)
1.54 s
—
4.92 t (13)
1.71 s
4.07 t (11)
4.06 t (11)
4.07 t (11)
1.13 d (6)
3.51 dq (10, 6)
1.69 m
3.83 d (7)
3.83 d (7)
1.68 s
—
4.45 t (11)
2.09 s
—
4.50 t (11)
2.17 s
—
4.01 dd (11, 2) 18
2.05 s
7.51 s
3.65 s
2.25 s
1
1
2
2
8
9
0
1
1.59 s
1.24 d (7)
1.15 d (6)
1.72 s
—
21
0
0
—
—
—
3.96 m
3.49 m
N(1)-Me
2.01 dd (12, 8)
1.85 m
2.07 m
2.02 m
2.02 m
2.10 m
2.10 m
1.07 m
1.46 m
—
—
3.32 m
2.41 m
3.08 m
—
N(4)-Me
0
1.85 dd (13, 8)
2.17 t (13)
5.21 d (4)
2.03 m
3.69 dd (11, 4)
3.31 dd (11, 8)
7.54 s
—
9.66 s
—
11 -OMe 3.87 s
2
.39 m
2.15 dd (13, 11) 2.10 m
3.81 dd (11, 6)
3.49 m
—
2
2
3
4
4.13 d (5)
1.85 m
—
—
—
—
—
2.50 dd (17, 7)
2.52 dd (17, 8)
2.28 dd (19, 7)
2.69 dd (19, 7)
4.69 tt (7, 4)
4.15 dd (12, 4)
4.30 dd (12, 3)
3.69 s
—
—
—
—
—
2
.39 m
2.14 td (9, 4)
—
—
—
—
—
—
2
2
5
6
4.42 m
4.37 dtd (9, 7, 4) 4.56 m
—
—
—
—
—
—
3.43 dd (12, 3)
3.98 dd (12, 7)
4.21 dd (12, 4)
3.69 s
3.61 dd (12, 4)
—
—
—
—
—
—
3
.77 dd (12, 2)
3.96 dd (12, 3)
—
—
—
—
—
—
N(1)–Me 3.63 s
N(4)–Me 2.33 s
3.63 s
2.34 s
—
3.62 s
2.31 s
—
3.62 s
2.30 s
—
3.60 s
2.34 s
—
—
—
3.55 s
2.34 s
—
2.88 s
2.88 s
2.36 s
—
2.36 s
—
2
2
1
3-OAc
6-OAc
1-OMe
—
—
—
2.02 s
—
2.06 s
—
2.02 s
—
—
—
—
—
—
—
—
—
—
—
3.84 s
3.84 s
—
a
3
CDCl , 400 MHz; assignments based on COSY and HMQC.

T.-S. Kam et al. / Tetrahedron 60 (2004) 3957–3966
3959
1
3
a
Table 2. C NMR spectral data of 1, 3, 4, 5, 7, 9, 10, 11, 12, 13, 14, 15 and 18
Position
1
3
4
5
7
9
10
11
12
13
14
15
18
Position
18
b
0
2
3
4
5
6
7
8
9
132.8
53.3
—
131.5
53.3
—
125.9
55.7
—
56.4
23.8
105.9
124.9
118.6
120.4
123.2
109.7
60.2
36.9
220.0
69.1
30.7
26.7
36.5
68.4
18.9
69.4
54.2
203.4
—
—
—
—
—
—
—
—
—
132.8
53.5
—
132.8
53.1
—
133.2
53.7
—
133.2
53.6
—
133.0
53.5
—
129.2
54.8
—
129.2
54.8
—
131.3
53.1
—
2
3
5
6
7
8
132.9
53.7
54.7
0
55.1
—
56.2
23.4
0
0
54.8
22.5
106.4
126.4
118.0
118.9
121.0
108.0
136.9
31.9
26.5
36.9
64.3
24.2
105.5
44.3
34.7
114.8
77.7
33.0
79.2
63.9
29.0
41.6
—
54.7
22.6
106.5
126.2
118.0
118.9
121.0
108.8
137.1
31.9
27.1
36.6
68.8
23.5
106.1
45.8
35.9
106.4
209.4
34.7
75.0
63.1
29.0
41.7
—
54.5
22.5
106.6
126.2
118.1
118.9
121.0
108.7
137.2
30.1
27.0
39.4
68.8
19.2
69.4
54.6
204.7
—
54.9
22.4
106.6
126.2
117.9
118.9
121.0
108.9
137.0
26.7
26.1
42.5
67.1
20.2
67.8
57.7
203.0
—
54.6
22.6
106.6
126.4
118.1
118.8
120.8
108.7
136.9
30.7
28.6
39.3
68.9
18.8
71.2
43.6
63.1
—
55.1
22.5
106.7
126.4
117.9
118.8
120.7
108.8
137.0
25.3
26.7
43.5
67.6
20.2
70.5
46.8
61.6
—
54.9
22.4
106.7
126.3
117.9
118.8
120.8
108.5
136.9
25.0
27.1
43.4
67.5
20.1
70.3
43.2
62.9
170.8
20.3
—
55.1
22.9
106.4
121.6
118.2
108.8
156.2
95.4
136.8
32.9
23.0
38.6
67.7
25.0
195.6
121.6
157.6
—
55.1
22.5
106.4
121.6
118.2
108.8
156.2
95.4
136.8
32.9
23.0
38.6
68.1
16.5
170.0
121.6
188.8
—
59.2
22.6
105.9
126.3
118.2
119.0
120.9
108.7
137.0
32.3
31.5
43.1
66.5
31.1
213.2
54.5
32.0
—
22.0
0
105.4
120.1
118.7
119.1
153.6
91.3
136.5
32.4
22.8
38.3
67.7
24.9
195.4
120.8
157.4
29.0
41.2
55.5
0
105.4
b
0
9
10
0
118.4
120.0
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
11
12
13
14
15
16
17
18
b
0
0
109.5
137.5
0
137.6
b
b
0
0
25.9
36.8
25.9
36.8
62.9
22.7
104.9
45.3
35.6
105.6
208.6
35.3
72.4
64.9
29.5
40.4
—
0
b
0
b
0
19
20
21
0
105.4
43.6
35.2
113.7
78.8
33.0
76.4
67.2
29.4
40.8
21.1
0
0
0
N(1)-Me
N(4)-Me
0
—
—
—
—
42.2
—
—
—
—
—
—
29.1
41.8
—
—
—
—
—
29.0
41.6
—
—
—
—
—
29.0
41.7
—
—
—
—
—
29.0
41.7
—
—
—
—
—
—
41.5
—
—
—
—
—
—
41.5
—
—
—
—
—
28.9
41.7
—
11 -OMe
—
—
28.9
41.6
—
N(1)–Me
N(4)–Me
2
2
1
3-OAc
6-OAc
1-OMe
170.0
20.9
170.8
—
—
—
—
—
20.7
170.6
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
55.8
55.8
a
CDCl , 100 MHz; assignments based on HMQC and HMBC.
Not detected.
3
b
same macroline unit. Both structures accommodate the
observed NMR chemical shifts as well as the HMBC
correlation data. To resolve the difficulty in distinguishing
splittings were due to coupling with the two H(21). Since
the stereochemistry of H(20) has been fixed as a, the 12 Hz
coupling must be due to coupling to H(21b). Irradiation of
H(23) causes NOE enhancement of H(25) and vice versa,
indicating that they are syn to each other. Aside from these,
further assignment of the remaining stereochemistry, such
as that of the spirocyclic centre at C(22), was precluded by
the unresolved signals of H(21) in 1, which were
fortuitously well resolved in the diacetate derivative 3.
Thus observation of the key NOE interaction between H(23)
and H(21b) in 3, not only allowed assignment of the
configuration at the spirocarbon as R, but also fixes the
stereochemistry of C(23) and C(25), respectively, as R, R.
the two structures, acetylation (Ac O, pyridine) was carried
2
out which yielded a single diacetylated derivative, provid-
ing cogent support for structure 1. Furthermore, conversion
1
to the acetylated derivative resulted in a better resolved H
NMR spectrum (Table 1), which removed the earlier
ambiguity associated with some of the key signals.
Specifically, the signals for H(21), H(23), H(24) and
H(25) were now sufficiently clear and well resolved in the
acetate derivative 3 {whereas H(24) and H(21) were
overlapping multiplets in 1}, and indicated the presence of
the key OCHCH CHCH O fragment, corresponding to the
2
2
C(23)–C(24)–C(25)–C(26) partial structure in 1. In
addition, the observed carbon resonance of d 114.8 for the
spirocyclic centre was in good agreement with that
previously noted for the spirocarbon in compounds contain-
Macrodasine B 4 was also obtained from the bark extract of
A. macrophylla, as a colourless oil, with [a] ¼þ149 (c
D
0.067, CHCl ). The UV spectrum was very similar to that of
3
1 with absorption maxima at 230 and 287 nm (log 1 3.95
and 3.23, respectively), characteristic of an unsubstituted
indole chromophore, while the IR spectrum showed in
1
1–14
ing a 1,6-dioxaspiro[4.4]nonane unit.
2
1
The ring junction stereochemistry between rings C, D, and
E, is assumed to follow that in the known macroline
addition to a broad OH band at 3435 cm , another band at
1765 cm2 , indicative of a five-membered cyclic ketone.
The EIMS of 4 showed a molecular ion at m/z 452, which
analyzed for C H N O , two mass units less than that of 1,
1
3
compounds (e.g., alstonerine) from the similarity of the
chemical shifts and coupling patterns observed for the ring
junction hydrogens, a supposition which is also in agree-
ment with the NOE and NOESY data. The observed NOE
between 18-methyl and H(17a) as well as H(20), fixes the
E/F ring junction stereochemistry as cis {18-Me and H(20)
both a}. The resonance for H(20) was a doublet of doublets
with J¼12, 8 Hz. Decoupling experiments indicated that the
2
6 32 2 5
and requiring 12 degrees of unsaturation. In common with 1,
the mass fragments which were observed at m/z 197, 182,
181, 170, and 144 are characteristic of macroline deriva-
1
0
tives, and indicated that 4 also contained a macroline-like
3
1
residue. The C NMR spectrum (Table 2) gave a total of 26
separate carbon resonances (three methyls, six methylenes,

3960
T.-S. Kam et al. / Tetrahedron 60 (2004) 3957–3966
1
0 methines, and seven quaternary carbons) in agreement
permitted NOE experiments to be carried out {NOE
9
between H(23) and H(21b)}, which is precluded in the
with the molecular formula, but differing from that of 1 by
the addition of a quaternary carbon at the expense of a
methine.
case of macrodasine B 4, where C(23) is now a ketone
carbonyl. The configuration at the spirocyclic C(22) and at
C(25) in 4 are therefore tentatively assigned as R and R,
respectively, on the grounds of a presumed close biogenetic
relationship with 1.
1
13
The H and C NMR spectral data share a number of
common features with that of a typical macroline (as well as
with 1), indicating that rings A–E are essentially
unchanged, but that substantial changes have occurred
Macrodasines A 1 and B 4, represent the first members of an
unusual class of macroline compounds which have
incorporated additional novel structural features, in the
form of fused spirocyclic tetrahydrofuran rings, incorporat-
ing an unprecedented spiroacetal moiety. The spiroketal
unit has been previously encountered in insect phero-
1
affecting rings F and G. Thus the H NMR spectrum
(
Table 1) showed the presence of three methyl groups
corresponding to the N(1)–Me (d 3.63), N(4)–Me (d 2.34),
and Me(18) (d 1.54), and a hydroxymethyl group, from the
presence of a pair of doublet of doublets at d 3.61 and 3.96
1
7–19
11–14,20,21
(
corresponding to the carbon resonance at d 63.1), which are
mones,
compounds,
marine natural products,
7,22–26
plant steroidal derivatives and various
microbial
1
17
similar to 1. The COSY spectrum of 4 showed in addition to
the NCHCH2, CHCH2, and NCHCH CHCHCH O
fragments, which are common to 1, a CH CHCH O
1
7
other plant secondary metabolites. It has however not been
found as a substructure in alkaloids. The macrodasines 1 and
4, thus represent the first instances of the incorporation of a
spiroketal unit in an indole alkaloid.
2
2
2
2
fragment, in place of the OCHCH CHCH O fragment
2
2
1
3
observed in 1. Comparison of the C NMR spectra of 1 and
, showed that the two oxymethylenes at d 68.8 and 63.1,
corresponding to C(17) and C(26), respectively, are intact,
4
A tentative proposal for a possible pathway to these unusual
4
,
as is the oxymethine corresponding to C(25) {d 75.0 c.f.
7
spiroacetal C(22), which was observed at d 106.4. However,
the other oxymethine at d 77.7 corresponding to C(23) in 1,
is absent in the spectrum of 4. Instead a ketone carbonyl
resonance at d 209.4 was observed in its place.
compounds is from the ring-opened form of alstonerine 6,
3
0
9.2 in 1}, and the quaternary carbon resonance due to the
which on alkylation by a six-carbon fragment at C(20),
followed by tandem intramolecular hemiketal formation
9
(Scheme 1), yields the ring system of the macrodasines.
An unusual nitrogenous compound, angustimalal 7 was also
obtained from this study. It was isolated as a colourless oil,
with [a] ¼þ78 (c 0.064, CHCl ). The IR spectrum showed
At this stage the structure of macrodasine B can be
assembled as shown in structure 4, which reveals it to be
the 23-oxo derivative of 1. The structure is consistent with
the HMBC data (Fig. 1), as well as the observed cyclic
ketone absorption at 1765 cm in the IR spectrum, which
is in excellent agreement with that of 3-oxacyclopentanones
D
3
2
1
two carbonyl bands, one at 1717 cm , corresponding to an
aldehyde carbonyl, and another at 1741 cm2 , indicative of
a five-membered ring ketone. The characterstic Fermi
1
2
1
21
doublets at 2767 and 2867 cm were clear in this instance,
1
and taken with the H NMR signal at d 10.0, confirmed the
2
1
(
(
1764 cm
)
15
versus that for 3-oxacyclohexanones
presence of the aldehyde function. The EIMS of 7 showed a
molecular ion at m/z 237, the odd mass indicating the
presence of a single nitrogen. HREIMS measurements gave
1725 cm2 ). In addition, the chemical shift and geminal
1
coupling constant for H(24) {d 2.50 dd, J¼17, 7 Hz; 2.52,
1
3
dd, J¼17, 8 Hz} are highly diagnostic of geminal hydrogens
the formula C H NO . The C NMR spectrum (Table 2)
1
3
19
3
4
,16
adjacent to a carbonyl carbon.
Reaction of 4 with Ac O/
2
showed a total of 13 peaks in agreement with the molecular
formula (two methyls, three methylenes, seven methines
and one quaternary carbon). Two methyl groups were
indicated, a CH CH (d 1.42) and an NCH (d 2.30). The
pyridine yielded the monoacetate derivative 5, in agreement
with the proposed structure. The NOESY and NOE data are
similar to those observed for 1 and confirmed the
stereochemistry of the E/F ring junction {18-Me and
H(20) both a}, in addition to the characteristic ring
junction stereochemistries for the C/D/E rings, which
correspond to that of a typical macroline. In the case of
macrodasine A 1, assignment of the configuration at the
spirocyclic C(22) was facilitated by the well-resolved H(21)
and H(23) signals in the diacetate derivative 3, which
3
3
quaternary carbon resonance at d 220.0 is due to a ketone
function while the methine at d 203.4 corresponds to the
aldehyde group. The COSY spectrum revealed the follow-
ing partial structures, NCHCH , NCHCH CHCHCH O, and
9
3
,4
2
2
2
CH CHCHCHvO. The former two fragments are
3
characteristic of macroline compounds and correspond to
the C(5)–C(6) and C(3)–C(14)–C(15)–C(16)–C(17)
fragments, respectively, of a macroline alkaloid, while the
latter fragment, together with the two methine resonances at
d 26.7 and 36.5, corresponds to the ring E portion of a
type-A macroline, such as talcarpine 9. The molecule can
therefore be assembled accordingly and requires only
insertion of a ketone function to complete the structure of
angustimalal as shown in 7, which is in perfect agreement
with the HMBC data (Fig. 2).
The ring junction stereochemistry was established from the
NOESY spectrum and was in agreement with that in a
typical macroline alkaloid (e.g., talcarpine 9). The stereo-
chemistries of the tetrahydropyran ring substituents were
Figure 1. Selected HMBC (H to C) of 4.

T.-S. Kam et al. / Tetrahedron 60 (2004) 3957–3966
3961
Scheme 1. (X¼O; or OH, H).
Three other new macroline indole derivatives were also
obtained from the bark extract, macrocarpines A 11, B 12,
and C 13. A common feature of these three alkaloids is that
they contain a saturated ring E, as exemplified by talcarpine
9
. Macrocarpine A 11 was obtained as a light yellowish oil,
[
a] ¼þ117 (c 0.11, CHCl ). The IR spectrum showed the
D
3
2
1
presence of a hydroxyl function (3400 cm ), while the UV
spectrum indicated an indole chromophore. The EIMS of 11
showed a molecular ion at m/z 340, which analysed for
Figure 2. Selected HMBC (H to C) of 7.
1
13
C H N O . Examination of the H and C NMR spectral
2
1 28 2 2
data (Tables 1 and 2, respectively) revealed a macroline
compound resembling talcarpine 9 in all respects except for
changes involving the substituents in the saturated E-ring,
viz., the replacement of the 20b-CHO substituent by a 20b-
hydroxymethyl substituent in 11. This is clearly indicated
by the presence of the hydroxymethyl signals (d 3.69, 3.81;
H
Figure 3. Selected NOE’s of 7.
dC 63.1) in place of the aldehyde signals of talcarpine. The
stereochemistry of the hydroxymethyl substituent at C(20)
is readily confirmed from the observed NOE interaction
between H(14b) and H(20) which is only possible if H(20)
is a. The assignment is also confirmed by chemical
correlation, by conversion of talcarpine 9 to 11 by NaBH4
reduction, and oxidation of 11 to talcarpine 9 by PCC.
Macrocarpine B 12 was obtained as a light yellowish oil,
also established on the basis of the NOESY spectrum
(
Fig. 3).
The stereochemistry of the two H(9) can be determined on
the basis of their respective coupling constants (see Section
2
1
3
). The a-oriented H(9) which is trans-diaxial with H(8) is
[a] ¼251 (c 0.34, CHCl ). The IR (OH, 3400 cm ), UV
D
3
þ
seen as a triplet, with J¼11 Hz. The observed NOE
interaction of H(9a) with H(11) indicated that the methyl
substituent is b. Similarly H(6a) can be distinguished from
H(6b) on the basis of their coupling interactions. The NOE
observed between H(6a)/H(11a), and between H(6b)/H(12)
confirmed the b-stereochemistry of the C(12) aldehyde
substituent (Fig. 3). This assignment is further vindicated by
comparison of the chemical shifts of the aldehyde-H in
angustimalal 7 (d 10.0) with the shifts observed for the
corresponding macroline alkaloids, talcarpine 9 (b-CHO, d
(indole), and EIMS (M m/z 340) spectral data were similar
to that of 11, as were the NMR spectral data (Tables 1 and
2), which were generally similar except for the noticeable
difference in the shifts of C(14) and C(16). These
similarities indicated that 12 is the C(20) epimer of 11
which is confirmed from the NOESY spectrum which
showed NOE interaction between H(20)/H(16). The assign-
ment was also confirmed by chemical correlation with N(4)-
methyl-N(4), 21-secotalpinine 10, via NaBH reduction.
4
Macrocarpine C 13 is readily shown to be the acetate
derivative of macrocarpine B 12 from the spectral data. The
9
9
.95) and N(4)-methyl-N(4), 21-secotalpinine 10 (a-CHO, d
.41) (see Section 3).
1
13
H and C NMR spectra (Tables 1 and 2) were similar to
that of 12 except for the presence of a methyl resonance at d
1.68 and the carbon signals at d 20.3 and 170.8. The
assignment was again supported by correlation with 12 via
The structure of angustimalal 7 shows that it retains all the
features of the non-indole portion of a type-A macroline
compound, except for the presence of an additional
oxygenated carbon. A similar compound, angustimaline 8
acetylation (Ac O/pyridine).
2
(corresponding to the non-indole portion of a type-B
macroline alkaloid in this case) has been encountered
once recently, from the bark extract of another Alstonia
species. The origin of such compounds remains enigmatic,
although a simple assumption (in the case of 7) is that it is
probably derived from fragmentation of a macroline-type
precursor, possibly talcarpine 9 (which also occurs in the
same plant), or its as yet unknown oxindole.
Two other new macroline derivatives 14 and 15 were
obtained as an inseparable mixture of type-B and type-A
forms (ratio 3:1, respectively), which co-eluted in column
chromatography and proved resistant to further attempts at
resolution by chromatography or fractional crystallization.
The H(18) {methyl} and H(21) {aldehyde-H for 15,
vinylic-H for 14} signals are clearly distinguishable in the
2
7
1
H NMR spectrum (Table 1), while the signals of H(17) are

3962
T.-S. Kam et al. / Tetrahedron 60 (2004) 3957–3966
partially overlapped. The rest of the hydrogen resonances of
1
NMR spectral data also revealed that this monomeric unit
corresponds to the new seco-macroline, alstomicine,
3
the two isomers are coincident. In the C NMR spectrum
Table 2), the majority of the signals are coincident with the
3
0
(
isolated from the leaf-extract of the same plant. The
point of branching in this second macroline unit must
therefore be from C(20). This leaves the methylene C(21)
exception of C(6), C(17), C(18), C(19), and C(21). This
behaviour has been observed previously in the case of the
macroline indoles, alstonerine (type-B) and alstonerinal
1
3
unaccounted for, which is observed at d 32.0 in the
NMR spectrum (d 2.41, 3.08). This methylene is directly
C
3
(
demethylalstonisine (type-B) and N(1)-demethylalstonal
type-A), and in the case of the macroline oxindoles, N(1)-
H
attached to C(20) of the seco-macroline unit, from the
3
4
(
type-A). In the event, the spectral data (Tables 1 and 2)
COSY spectrum, as well as the HMBC spectrum { J from
H(21) to C(19)}. In addition, the observation of another key
indicated that 14 and 15 are the N(1)-demethyl derivatives
of alstophylline 16 and alstophyllal 17, respectively. The
latter two compounds also occur as a pair of unresolvable
type-A and type-B isomers in both the stem and leaf
extracts.
0
three-bond correlation from H(9 ) to this C(21) provided
cogent support for the proposed structure, in which the two
macroline units are connected by a methylene bridge, as
shown in 18, although the configuration at C(20) could not
be established based on the present data. Perhentinine and
the other known bisindole obtained, villalstonine, both
showed moderate in vitro cytotoxicity towards the P388
murine leukemia cell line (IC50 12.3 and 4.4 mg/ml,
respectively) (the biological activity of Alstonia alkaloids
will be reported separately).
Two bisindole alkaloids were obtained, of which one was a
new natural product. Perhentinine 18 was obtained as a light
yellowish oil, [a] ¼261 (c 1.19, CHCl ). The IR spectrum
D
3
2
1
showed the presence of hydroxyl (3400 cm ), ketone
2
1
(
1
1701 cm ), and a,b-unsaturated ketone (1651,
1
616 cm2 ) functions, while the UV spectrum indicated
an indole chromophore, with charcteristic absorption
In addition to the above new alkaloids, 12 other known
alkaloids were also obtained from the bark extract of this
plant, as detailed in Section 3. A notable feature of the
alkaloidal composition, in addition to the novel structures
discussed above, is the predominance of the macroline
skeleton, which is a characteristic of Alstonia.
maxima at 231 and 298 nm. The LSIMS spectrum of 18
showed the MH ion at m/z 705, which analysed for
þ
C H N O . The H NMR spectrum (Table 1) showed
1
4
3 52 4 5
several clear features, inter alia, four aromatic hydrogen
signals associated with an unsubstituted indole moiety, two
aromatic singlets associated with another indole substituted
0
0
at positions 10 and 11 , a total of seven methyl singlets
corresponding to two N(1)-methyls, two N(4)-methyls, two
acetyls, and an aromatic methoxy group. Since only six
aromatic hydrogens are observed and both indolic nitrogens
are substituted, it is reasonable to conclude that the
bisindole is branched from one of the aromatic carbon
atoms of one monomer, with the adjacent position occupied
by the methoxy substituent. The low field region also
showed the presence of a vinylic singlet (d 7.51), which with
3. Experimental
3.1. General
UV spectra were recorded on a Shimadzu UV-3101PC
spectrophotometer. IR spectra were recorded on a Perkin–
Elmer RX1 FT-IR spectrophotometer. Optical rotations
were measured on a JASCO DIP-370 digital polarimeter or
an Atago Polax-D polarimeter. ESIMS was obtained on a
Perkin–Elmer API 100 instrument. HREIMS and
HRLSIMS measurements were carried out at Organic
Mass Spectrometry, Central Science Laboratory, University
0
the associated 18 -methyl singlet at d 2.05, indicated one
monomer to be a type-B macroline. This is supported by the
0
a ddd and a triplet at d 4.13 and 4.37, respectively. The
observation of the characteristic C(17 ) hydrogen signals as
,4
3
1
3
1
13
C NMR spectrum (Table 2) showed a total of 43 carbon
signals, comprising seven methyls, seven methylenes, 16
methines, and 13 quaternary carbon atoms.
of Tasmania, Tasmania, Australia. H and C NMR spectra
were recorded in CDCl using TMS as internal standard on a
3
JEOL JMN-LA400 spectrometer at 400 and 100 MHz,
respectively. Assignments are confirmed by COSY, HMQC,
HMBC, NOESY and NOE experiments. All solvents were
of analytical grade and were distilled before use.
Examination of the carbon spectrum revealed that one set of
signals showed a correspondence to a 10 , 11 -disubstituted
0
0
alstonerine. Furthermore, the observed low-field reson-
3
0
respectively, are characteristic of oxygenation at the
0
ances of both H(12 ) and C(12 ) at d 6.69 and 91.3,
3.2. Collection, extraction and isolation
0
28,29
0
adjacent C(11 ),
thus indicating position 11 as the site
0
Plant material was collected in Terengganu, Malaysia (June,
2000) and was identified by Dr. K. M. Wong, Institute of
Biological Sciences, University of Malaya, Kuala Lumpur,
Malaysia. Herbarium voucher specimens (K 659) are
deposited at the Herbarium of the University of Malaya.
Extraction of the ground bark material was carried out in the
usual manner by partitioning the concentrated EtOH extract
of methoxy substitution and position 10 as the site of
branching of the bisindole from this monomeric unit.
The second moiety constituting the bisindole was deduced
to be another macroline derivative from initial inspection of
the NMR spectral data. The C(17) hydogens are observed as
doublets of doublets at d 3.95 and 4.01 and the acetyl
hydrogens of C(18) are seen as a singlet at d 1.72, features
3
1
with dilute acid as has been described in detail elsewhere.
The alkaloids were isolated by initial column chromato-
graphy on silica gel using CHCl3 with increasing
proportions of MeOH, followed by rechromatography
of appropriate partially resolved fractions using
centrifugal TLC. Solvent systems used for centrifugal
4
which are characteristic of ring E-opened, seco-macrolines.
Further examination of the NMR spectral data indicated that
C(20), which is expected to be a methylene, is now
substituted, appearing as a methine at d 3.32 (d 54.5). The
C

T.-S. Kam et al. / Tetrahedron 60 (2004) 3957–3966
3963
1
13
TLC were Et O–petroleum ether (1:1; 2:1), Et O, CHCl –
2
Calcd for C H N O , 452.2311. H and C NMR spectral
26 32 2 5
2
3
MeOH (100:1), CHCl (NH -saturated), and EtOAc (NH -
3
saturated). The yields (g kg ) of the alkaloids were as
follows: 1 (0.004), 4 (0.0012), 7 (0.0012), 9 (0.0005), 10
data, see Tables 1 and 2, respectively.
3
3
2
1
3.2.3. Acetylation of macrodasine A 1. Macrodasine A 1
(11 mg) was added to a mixture of acetic anhydride/pyridine
(1:1; 2 ml) and the mixture stirred at room temperature for
2 h. The mixture was then poured into saturated Na CO
(0.010), 11 (0.002), 12 (0.031), 13 (0.028), 14 (0.0004), 15
(0.0002), 16 (0.027), 17 (0.016), 18 (0.0249), alstonisine
(0.028), alstonal (0.0085), N(4)-demethylalstophylline
2
3
oxindole (0.054), N(4)-demethylalstophyllal oxindole
0.035), villalstonine (0.393), pleiocarpamine (0.071),
fluorocarpamine (0.029), 16R,19E-isositsirikine (0.004),
and extracted with CH Cl . Removal of the solvent
2 2
(
followed by purification by centrifugal chromatography
over SiO (2% MeOH–CHCl ) afforded 5 mg (38%) of the
2
3
and 11-methoxyakuammicine (0.0012).
diacetate derivative 3 as a colourless oil; [a] ¼þ119
D
1
(
CHCl , c 0.06); IR (film) n
3
1739 and 1234 cm2 ; UV
(EtOH), lmax nm (log 1): 221 (4.17), 229 (4.22), 286 (3.56)
max
3
.2.1. Macrodasine A 1. [a] ¼þ36 (CHCl c 0.36); IR
D
3,
2
1
þ
281 (14), 253 (4), 207 (100), 197 (99), 182 (23), 167 (20),
(
dry film) n
3411 cm ; UV (EtOH), l
nm (log 1):
and 293 (3.52). EIMS, m/z (rel. int.): 538 [M ] (29), 465 (4),
max
max
þ
2
30 (3.88) and 287 (3.17). EIMS, m/z (rel. int.): 454 [M ]
1
13
(
(
78), 439 (4), 424 (44), 367 (7), 197 (100), 182 (27), 181
16), 170 (34), 144 (13), 70 (26), 57 (16) and 43 (36).
144 (8), 96 (9), 70 (23) and 55 (18). H and C NMR
spectral data, see Tables 1 and 2, respectively.
HREIMS found, m/z 454.2462, Calcd for C H N O ,
2
6 34 2 5
1
13
454.2468. H and
respectively.
C NMR: see Tables 1 and 2,
3.2.4. Acetylation of macrodasine B 4. Acetylation of
macrodasine B 4 (4 mg) with Ac O/pyridine as described
2
above gave the monoacetate derivative 5 as a colourless oil
(2 mg, 46%); [a] ¼þ147 (CHCl , c 0.02); IR (film) n
3
.2.2. Macrodasine B 4. [a] ¼þ149 (CHCl , c 0.07); IR
D
3
D
3
max
2
1
21
(
film) n
3435 and 1765 cm ; (EtOH), l
nm (log 1):
1767, 1739, and 1237 cm ; UV (EtOH), l nm (log 1):
max
max
max
þ
2
30 (3.95) and 287 (3.23). EIMS, m/z (rel. int.): 452 [M ]
222 (4.41), 228 (4.48), 285 (3.79) and 293 (3.75). EIMS, m/z
(rel. int.): 494 [M ] (30), 366 (8), 322 (14), 197 (100), 182
þ
(23), 170 (30), 158 (10), 144 (10) and 70 (33). H and
NMR spectral data, see Tables 1 and 2, respectively.
(
1
(
64), 437 (3), 421 (12), 366 (11), 322 (17), 293 (4), 237 (6),
97 (100), 182 (26), 181 (19), 170 (29), 144 (9), 85 (13), 70
19), 57 (16) and 40 (35). HREIMS found, m/z 452.2326,
1
13
C

3964
T.-S. Kam et al. / Tetrahedron 60 (2004) 3957–3966
3
.2.5. Angustimalal 7. [a] ¼þ78 (CHCl , c 0.06); IR
br s, H-5), 3.23 (1H, d, J¼7 Hz, H-2), 3.90 (1H, dd, J¼11,
D
3
2
1
(
film) n
1741 and 1711 cm ; UV (EtOH), l nm (log
): 211 (3.09) and 256 (2.42). EIMS, m/z (rel. int.): 237
5 Hz, H-9), 3.94 (1H, qd, J¼7, 2 Hz, H-11), 4.15 (1H, t,
max
max
13
1
J¼11 Hz, H-9), 10.0 (1H, d, J¼2 Hz, H-13); C NMR
þ
38 (13), 124 (28), 110 (32), 94 (65), 84 (97), 70 (26), 57
[
M ] (2), 209 (74), 194 (39), 180 (100), 166 (24), 150 (25),
spectral data, see Table 2.
1
(
C H NO , 237.1365. H (400 Hz; CDCl ; Me Si) d 1.42
50) and 42 (74). HREIMS found, m/z 237.1375, Calcd for
1
3.2.6. Talcarpine 9. [a] ¼226 (CHCl , c 0.12); UV
D
3
(EtOH), l nm (log 1): 209 (3.86), 226 (4.01), 277 (2.65),
285 (2.91) and 294 (2.65). ESIMS, m/z (rel. int.): 339
1
3
19
3
3
4
max
(
3H, d, J¼7 Hz, 10-Me), 1.67 (1H, m, H-6), 1.95 (1H, s,
H-12), 1.98 (1H, m, H-8), 2.06 (1H, d, J¼19 Hz, H-3), 2.11
1H, td, J¼13, 3 Hz, H-6), 2.30 (3H, s, N-Me), 2.44 (1H, dt,
J¼13, 6 Hz, H-7), 2.68 (1H, dd, J¼19, 7 Hz, H-3), 2.90 (1H,
þ
1
[MH ]. H (400 Hz; CDCl ; Me Si) d 1.30 (3H, d, J¼7 Hz,
3
4
(
18-Me), 1.45 (1H, ddd, J¼12, 4, 3 Hz, H-14), 1.79 (1H, br s,
H-20), 2.06 (1H, dt, J¼11, 5 Hz, H-16), 2.20 (1H, m, H-15),

T.-S. Kam et al. / Tetrahedron 60 (2004) 3957–3966
3965
1
13
2
.32 (3H, s, N(4)-Me), 2.45 (1H, d, J¼16 Hz, H-6), 2.50
382.2256. H and C NMR spectral data, see Tables 1 and
2, respectively.
(
(
(
1H, td, J¼12, 4 Hz, H-14), 2.90 (1H, d, J¼7 Hz, H-5), 3.27
1H, dd, J¼16, 7 Hz, H-6), 3.62 (3H, s, N(1)–Me), 3.89
1H, dd, J¼12, 5 Hz, H-17), 3.98 (2H, m, H-3 and H-19),
3.2.14. Acetylation of 12 to 13. Acetylation of 12 (27 mg)
with Ac O/pyridine as described above gave 13 (16 mg,
53%).
4
.14 (1H, t, J¼12 Hz, H-17), 7.10 (1H, td, J¼8, 1 Hz,
2
H-10), 7.19 (1H, td, J¼8, 1 Hz, H-11), 7.29 (1H, br d,
J¼8 Hz, H-12), 7.49 (1H, br d, J¼8 Hz, H-9), 9.95 (1H, d,
1
3
J¼3 Hz, H-21); C NMR spectral data, see Table 2.
3.2.15. N(1)-Demethylalstophylline 14 and N(1)-
þ
demethylalstophyllal 15. EIMS, m/z (rel. int.): 352 [M ]
3
.2.7. N(4)-Methyl-N(4), 21-secotalpinine 10. [a] ¼þ19
(74), 337 (6), 283 (12), 265 (4), 228 (12), 213 (79), 197 (28),
186 (100), 170 (19), 143 (12), 118 (5), 91 (6), 70 (40) and 40
(41). HREIMS found, m/z 352.1773, Calcd for C H N O ,
352.1787. H and C NMR spectral data, see Tables 1 and
2, respectively.
D
(
CHCl , c 0.45); UV (EtOH), l
nm (log 1): 205 (3.95),
28 (4.21), 280 (3.00), 285 (3.42) and 300 (3.12). ESIMS,
3
max
2
m/z (rel. int.): 339 [MH ]. H (400 Hz; CDCl ; Me Si) d
2
1 24 2 3
þ
1
1
13
3
4
1
.20 (3H, d, J¼7 Hz, 18-Me), 1.28 (1H, m, H-14), 1.93 (1H,
m, H-16), 2.31 (3H, s, N(4)-Me), 2.37 (3H, m, H-14, H-15,
H-20), 2.49 (1H, d, J¼16 Hz, H-6), 2.96 (1H, d, J¼7 Hz,
H-5), 3.30 (1H, dd, J¼16, 7 Hz, H-6), 3.58 (3H, s, N(1)-
Me), 3.75 (1H, dd, J¼12, 5 Hz, H-17), 3.93 (2H, m, H-3,
H-19), 4.06 (1H, t, J¼12 Hz, H-17), 7.13 (1H, td, J¼8,
3.2.16. Perhentinine 18. [a] ¼261 (CHCl , c 1.19); IR
D
3
(film) n
3400, 1701, 1651 and 1616 cm2 ; UV (EtOH),
lmax nm (log 1): 231 (4.25) and 298 (3.45). LSIMS, m/z (rel.
1
max
þ
(100). HRLSIMS found, m/z 705.4019, Calcd for
int.): 705 [MH ] (51), 661 (14), 379 (58), 239 (19) and 197
1
Hz, H-10), 7.21 (1H, td, J¼8, 1 Hz, H-11), 7.31 (1H, br d,
1
13
J¼8 Hz, H-12), 7.52 (1H, br d, J¼8 Hz, H-9), 9.41 (1H, br s,
[C H N O þH], 705.4016. H and C NMR spectral
4
3 42 4 5
1
3
H-21); C NMR spectral data, see Table 2.
data, see Tables 1 and 2, respectively.
3
.2.8. Macrocarpine A 11. [a] ¼þ117 (CHCl , c 0.11); IR
þ
09 (14), 226 (19), 197 (75), 182 (23), 170 (13) and 70 (18).
D
3
21
(
film) n
3400 cm ; UV (EtOH), l
nm (log 1): 230
max
max
Acknowledgements
(
4.15) and 286 (3.46). EIMS, m/z (rel. int.): 340 [M ] (100),
3
HREIMS found, m/z 340.2142, Calcd for C H N O ,
3
2
We would like to thank the University of Malaya and IRPA
for financial support of this work.
2
1 28 2 2
1
13
40.2151. H and C NMR spectral data, see Tables 1 and
, respectively.
3
.2.9. Reduction of 9 to 11. NaBH (27 mg) was added to a
4
References and notes
solution of 9 (24 mg) in MeOH (2 ml) at room temperature
and the mixture stirred for 5 h. Excess solvent was removed
under reduced pressure and water (5 ml) was then added.
The mixture was extracted with CHCl , dried (Na SO ), and
1. Hamaker, L. K.; Cook, J. M. Alkaloids: chemical and
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3
2
4
then chromatographed (SiO , centrifugal TLC, 1% MeOH–
2
CHCl ) to give macrocarpine A 11 (12 mg, 50%).
3
2. Kam, T. S. Alkaloids: chemical and biological perspectives;
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solution of 11 (12 mg) in CH Cl (2 ml) at room
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839–844.
2
2
temperature and the mixture stirred for 4 h. Water (5 ml)
was then added and the mixture was extracted with CHCl3,
dried (Na SO ), and then chromatographed (SiO2,
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2
4
centrifugal TLC, CHCl ) to give 9 (3 mg, 25%).
3
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.2.11. Macrocarpine B 12. [a] ¼251 (CHCl , c 0.34); IR
þ
25 (10), 309 (13), 226 (15), 197 (100), 182 (43), 170 (19),
D
3
21
(
film) n
3400 cm ; UV (EtOH), l
nm (log 1): 230
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max
max
(
4.34) and 288 (3.64). EIMS, m/z (rel. int.): 340 [M ] (75),
3
1
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1 28 2 2
found, m/z 340.2149, Calcd for C H N O , 340.2151. H
2
1
3
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787–8789.
3.2.12. Reduction of 10 to 12. Reduction of 10 (47 mg)
with NaBH (52 mg) as described above gave macrocarpine
B 12 (27 mg, 57%).
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.2.13. Macrocarpine C 13. [a] ¼235 (CHCl , c 1.55); IR
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D
3
21
(
film) n
1737 cm ; UV (EtOH), l
nm (log 1): 230
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max
max
(
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3
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2
3 30 2 3

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